XVIII Annual Congress of the European Society of Gene and Cell Therapy (ESGCT) October 22–25,2010 Milan,Italy

Or 41 Targeted integrations in stem cells

See supplement

Or 42 Mechanisms of retroviral silencing

Epigenetic processes play a central role in the regulation of gene expression, whether from endogenous loci or from exogenous vectors. KRAB-containing zinc finger proteins (KRAB-ZFPs) are tetrapod-restricted, sequence-specific DNA-binding transcriptional repressors that act by triggering the formation of heterochromain via their cofactor KAP1. While the functions of these regulators long remained unknown, recent evidence indicate that KRAB/KAP1-mediated transcriptional control is a master regulator of mammalian homeostasis, partaking in events as diverse as stem cell pluripotency, genomic imprinting, hematopoietic differentiation and control of behavioral stress. We discovered that KAP1 and KRAB-ZFPs mediate the early embryonic silencing of endogenous retroelements, and our newest data indicate that this system is also at play in modulating the expression of exogenous retroviruses such as HIV. I will discuss these results and other aspects of KRAB/KAP1-mediated control that seem particularly relevant for gene therapy.

Or 48

Abstract Withdrawn

Gregory Philip Sangamo BioSciences Inc Or 49

Editing the genome of stem cells with Zinc Finger Nuclease

The ability to engineer precise genetic modifications into human stem cells would both accelerate research and extend the range of potential therapeutic applications. This possibility is now being realized via the use of zinc finger nucleases (ZFNs). ZFNs are customizable, sequence-specific endonucleases that can be designed to introduce a discrete cleavage event at any user-chosen location within the stem cell genome. By adjusting conditions under which the cleavage event is subsequently repaired, one may efficiently and precisely disrupt or edit the targeted locus, or integrate a larger, gene-sized DNA fragment. This technology – which is portable to any eukaryote – has been used for diverse applications, including therapeutic gene modification in primary cells, trait stacking of producer cell lines for improved manufacture of biologics, and gene targeting in previously refractory species such as nematodes, rabbits and rats. The talk will focus on recent applications of this technology in human stem cells. Examples include gene tagging in embryonic stem cells, gene targeting in induced pluripotent stem cells, and gene addition at safe harbors in a variety of stem cell types. Preclinical proof-of-concept studies towards the development of autologous, CCR5-disrupted CD34 stem cells as a treatment for HIV will also be presented.

Lombardo A Mr 1 Cesana D Mrs 1 Genovese P Mr 1 Provasi E Mrs 2 Neri M Mrs 1 Di Stefano B Mr 3 Magnani Z Mrs 4 Pello O Mr 5 Holmes MC Mr 6 Gregory PD Mr 6 Broccoli V Mr 3 Gritti A Mrs 5 Bonini C Mrs 4 Naldini L Professor 1

1 San Raffaele Telethon Institute for Gene Therapy and Vita-Salute San Raffaele University, Milan, Italy 2 Experimental Hematology Unit and Vita-Salute San Raffaele University, Milan, Italy 3 Stem Cell and Neurogenesis Unit, San Raffaele Institute, Milan, Italy 4 Experimental Hematology Unit, San Raffaele Institute, Milan, Italy 5 San Raffaele Telethon Institute for Gene Therapy, San Raffaele Institute, Milan, Italy 6 Sangamo BioSciences Inc., Richmond, CA, United States Or 50 Combining Targeted Integration and Cassette Design for Robust and Benign Transgene Expression without Impacting Endogenous Gene Transcription

Site-specific integration may overcome major hurdles of current gene transfer approaches, namely insertional mutagenesis and unpredictable transgene expression. However, little is known about the impact of the insertion on the targeted locus and vice versa. Here we address this question by targeting a panel of transgene expression cassettes into the CCR5 gene or AAVS1 locus, and evaluate: i) permissiveness for transgene expression and ii) transcriptional perturbation of the locus. We found efficient zinc finger nucleases-mediated insertion in both sites across a panel of human primary cell types, including T lymphocytes, NSC and iPS cells. Transgene expression was always higher from the AAVS1 locus. Next, we measured the expression level of 26 genes present in the 400Kb region centered either on the gene-modified CCR5 or AAVS1 and found transcriptional up-regulation to be locus-dependent i.e. absent at AAVS1. Integration into AAVS1 targets intron 1 of the PPP1R12C gene with the expression cassettes added in the sense transcriptional orientation. Thus, insertion of new cis-acting elements could interfere with PPP1R12C expression. Indeed, analysis of cell clones containing mono- or bi-allelic insertion of the cassettes revealed that PPP1R12C transcription was negatively affected by the presence of splicing acceptor (SA) sites present in some of the cassettes. Eliminating these SA sites or inserting the cassettes in opposite orientation allowed for robust transgene expression while maintaining normal PPP1R12C expression. Overall, comparison of these different sites and cassette designs uncover essential features of the locus and the cassette that allow for benign targeted insertion in human primary cells.

Voigt K. Ms 1 Gogol-Döring A. Dr 2 Miskey C. Dr 2 Chen W. Dr 2 Izsvák Z. Professor 2 Cathomen T. Professor 3 Ivics Z. Dr 2

1 Max Delbrück Center for Molecular Medicine, Berlin, GermanyMax Delbrück Center for Molecular Medicine, Berlin, Germany 2 Max Delbrück Center for Molecular Medicine, Berlin, Germany 3 Hannover Medical School, Hannover, Germany Or 51 Retargeting Sleeping Beauty transposon insertions by engineered zinc finger DNA binding domains recognizing human L1 repeats

The Sleeping Beauty (SB) transposon is a nonviral, integrating vector system with proven efficacy in preclinical models. We mapped large numbers of SB insertions by a LAM-PCR protocol and Illumina sequencing. The data show a close-to-random insertion profile that could lead to genotoxic effects. We evaluated zinc finger (ZF) DNA binding domains to introduce a bias into SB's target selection properties. We chose to target human LINE1 (L1) repeats, because i) they are relatively AT-rich, and therefore represent attractive targets for SB, and ii) they are present in the human genome in large numbers, which could increase the chances of targeted transposition events. One 6-finger ZF protein recognizing the 3’-region of L1, ZFB, showed specific binding to an 18-bp site represented by >12,000 copies in the human genome. We generated >6000 SB insertions by the SB transposase and a ZFB/SB fusion. As random reference, a computer-generated dataset was used. An enrichment in transposon insertions in L1 elements was seen in the ZFB/SB dataset, which peaked at a ∼4-fold enrichment of transposon insertions in a 400-bp window around ZFB binding sites. Statistically relevant differences in transposon insertions in genes (39.4 % for ZFB/SB versus 42.1 % for SB) was also found. Thus, the data suggest that L1 repeats act as a sponge that retarget a fraction of SB insertions away from genes. Further improvements in ZF technology and a careful choice of targeted genomic regions may improve the safety profile of SB for future clinical applications.

Smith J Dr 1 Galetto R Dr 1 Poirot L Dr 1 Grosse S Dr 1 Schiffer-Mannioui S Dr 1 Arnould S Dr 1 Gouble A Dr 1 Duchateau P Dr 2 Desseaux C Dr 2 Pâques F Professor 3

1 Cellectis Genome Surgery, 102 avenue Gaston Roussel, 93235 Romainville Cedex, FRANCE 2 Cellectis, 102 avenue Gaston Roussel, 93235 Romainville Cedex, FRANCE 3 Cellectis and Cellectis Genome Surgery, 102 avenue Gaston Roussel, 93235 Romainville Cedex, FRANCE Or 52 Meganucleases for gene therapy

There is a growing interest in the use of targeted approaches that allow the precise repair of a deleterious mutation at its endogenous locus as well as the insertion of a functional coding sequence at a chosen locus. Such targeted approaches depend on specific tools, including sequence specific endonucleases that can induce high frequencies of homologous gene targeting in the vicinity of their cleavage site. Meganucleases, the most specific natural endonucleases, represent ideal tools for genome targeting. Natural as well as engineered meganucleases can be used to induce up to 20% of gene insertion into chosen human genes. Eventually, the use of these proteins for therapeutic applications will depend on their intrinsic properties (activity/specificity) as well as on the use or identification of appropriate vectorization methods. To assess the properties and potential of these proteins, we have evaluated their activity and toxicity in several assays. Meganucleases presenting a good activity/toxicity ratio could be used to induce targeted recombination or cleavage in different cell types, including stem cells. We have also developed an approach to select for potential safe harbors that could be used for targeted integration. The combination of an efficient and very specific meganuclease with an appropriate insertion site should provide a platform for addressing several different diseases affecting the same cell lineage.

Rahman SH Mr Gellhaus K Dr 2 Pars K Mr 2 Heilbronn R Professor 2 Cathomen T Professor

2 Institute of Virology (CBF), Charité Medical School, Berlin, Germany Or 53 A transient cell cycle arrest enhances AAV and ZFN-mediated gene targeting

Vectors based on adeno-associated virus (AAV) are efficient tools to modify genomes by gene targeting. Gene targeting is based on the homology-directed repair (HDR) pathway, which can be stimulated several 1000-fold by creating a DNA double strand break (DSB) in the target locus. Here, we aimed to assess the effect of the cell cycle on AAV and DSB-mediated HDR.

To this end, we established three human cell lines that carry a mutated eGFP gene flanked by recognition sites for the meganuclease I-SceI and zinc-finger nucleases (ZFNs). After treatment with different cytostatic drugs, cells were transduced with AAV vectors that encode the nuclease and a repair donor with the purpose of rescuing eGFP expression by HDR. The cell cycle profile, the extent of cytotoxicity and the frequency of HDR were assessed by flow cytometry. We show that a transient cell cycle arrest in the G2 phase increased AAV-mediated HDR up to 6-fold and we achieved targeted gene editing in up to 12% of transduced cells. A significant increase in HDR was also observed upon plasmid-based eGFP rescue, implying that drug-induced cell cycle arrest is a general means to augment DSB-mediated HDR. Noteworthy, the transient cell cycle arrest had only minor effects on the ratio of AAV vectors used for HDR vs. illegitimate vector integration.

In conclusion, the combined effects of site-specific DSBs with a transient cell cycle arrest allowed us to lower the vector dose - and hence illegitimate integration of the vectors - without compromising on the HDR frequency.

Seymour Len Department of Clinical Pharmacology, University of Oxford, Old Road Campus Research Building, OX3 7DQ

Or 54 Delivery issues for oncolytic viruses

While there are several examples where gene therapy approaches are looking promising, they all reflect situations where the gene vectors can be introduced efficiently into target cells – either by direct injection or by transduction ex vivo. Indeed, cancer gene therapy can be very effective if tumour-killing viruses are injected directly into tumours.

Unfortunately three quarters of people who develop cancer in the West go on to die from metastatic disease. In this situation it is not possible to inject gene therapy vectors into all the tumour nodules, and intravenous ‘systemic’ therapy is required. However the human bloodstream represents a very aggressive environment for most gene therapy vectors. Therapeutic microbes delivered via the bloodstream encounter many host defences and anatomical barriers that must be surmounted to enable their access to disseminated cancers. This is particularly true for adenovirus type 5 in humans, where most recipients have powerful pre-existing adenovirus-neutralising activity. We have recently shown that human (but not murine) erythrocytes provide an additional barrier by sequestering adenovirus onto the Coxsackie and Adenovirus Receptor and (via antibodies) complement receptor 1. Coating adenovirus with a layer of hydrophilic polymer can prevent this interaction and allow virus to circulate free in the plasma, showing passive targeting to disseminated tumours and mediating good anticancer efficacy. Entry of polymer-coated virus particles into the tumour mass is a product of fluid transfer, and is directly proportional to the area under the plasma concentration-time curve. Increasing extravasation of fluid through tumour-associated endothelium using permeability-enhancers such as Tumour Necrosis Factor alpha can improve virus particle entry into tumours over 100-fold, reaching as high as 10% injected dose (virus particles) per tumour. This provides the possibility for highly efficient targeting to tumours and good anticancer efficacy.

An alternative approach is to target agents to infect tumour-associated vasculature. However while this provides a vulnerable target to traditional gene therapy approaches, endothelial cells do not normally support ‘oncolytic’ viruses. One approach to overcoming this problem is to encode syncytium-forming proteins within the endothelial, to enable trans-complementation of virus replication by tumour-associated factors.

Jolly D Dr 1 Perez O Dr 2 Burnett R Dr 2 Gunzburg W Professor 3 Ibanez C Dr 2 Chen C Dr 2 Pettersson P Dr 2 Lin A Dr 2 Gruber H Dr 2 Pertschuk D Dr 2 Gessner D Ms 2 Hiraoka K Dr 4 Inagaki A Dr 4 Logg C Dr 4 Kashara N Professor 4 Ostertag D Dr 2 Robbins J Dr 2

1 Tocagen Inc. San Diego CA USA 2 Tocagen Inc. San Diego CA USA 3 Institute for Virology, Veterinary Medical University of Vienna, Austria; 4 Departments of Medicine and Molecular & Medical Pharmacology, UCLA, Los Angeles Or 55 Replicating Retrovirus Encoding a Prodrug Activator (Toca 511) Enters Clinic for the Treatment of Glioblastoma Multiforme (GBM)

A central problem for gene-mediated cancer therapies has been delivery of therapeutic genes to the tumor and not elsewhere. To address this issue, we are developing a replicating gammaretrovirus (amphotropic MLV) that delivers a therapeutic gene throughout a cancer mass by preferentially infecting and spreading through dividing cancer cells, without causing cell lysis. Toca 511 encodes an improved cytosine deaminase (CD), and is administered with the prodrug, 5-fluorocytosine (5-FC) that is converted locally to the anticancer drug 5-FU. In previous publications an earlier generation replicating vector preferentially infected intracranial human xenografts in nude mice (Tai et al. 2005 Mol Ther 12:842) and led to increased survival in treatment groups compared to controls. We improved the vector and CD gene, and manufactured and characterized concentrated purified vector lots. In two immune-competent mouse brain cancer rising-dose models (BALB/c-CT26 and B6C3F1-Tu2449), a single vector administration by intracranial injection, followed by systemic 5-FC, improved median survival up to at least 6 months (the duration of the experiment) while controls died within approximately 30 days. Toca 511 infected the brain tumor tissue efficiently after intracranial injection, expressed the CD gene and 5-FC was rapidly metabolized to 5-FU in vivo. Biodistribution studies in mice and dogs showed that there is almost no detectable spread or survival of viral vector over 6 months, outside of tumor, except in permissive BALB/c mice that lack the APOBEC3 MLV restriction factor. A phase I/II dose escalation study is now underway in patients with recurrent GBM.

Friedrich K Mrs 1 Münch R Mr 1 Jost C Dr 2 Peng K-W Professor 3 Plückthun A Professor 2 Cichutek K Professor 1 Muehlebach M Dr 1

1 Division of Medical Biotechnology, Paul Ehrlich Institut, Langen, Germany 2 Institute of Biochemistry, University of Zurich, Switzerland 3 Department of Molecular Medicine, Mayo Clinic, Rochester, MN, USA Or 56 DARPin-targeted oncolytic measles viruses effectively kill target tumour cells

In order to improve the safety while conserving tumour-killing efficiency of recombinant oncolytic viruses (OVs), we intended to exploit the binding specificity of Designed Ankyrin Repeat Proteins (DARPins), as new cell entry targeting domains for oncolytic measles virus (MV). DARPins exhibit a very high affinity and specificity in combination with an unusual protein stability and small molecular size. We previously utilized DARPins directed against HER2/neu as binding domains of receptor-blind MV hemagglutinin (H); these H-proteins allowed targeting of lentiviral vectors.

Insertion of a gene encoding a DARPin-H attachment protein into the MV genome allowed the generation of recombinant replicating MVs carrying one out of 6 different HER2/neu specific DARPins as targeting domain, respectively. All DARPin-targeted MV variants revealed HER2/neu-specific targeting, genomic stability, efficient viral replication and oncolytic activity in receptor-positive target cells in vitro. We found evidence that the target cell killing efficiency of the targeted viruses was dependent on the receptor density in target cells and the affinity of the DARPins to the target receptors. In receptor-high target cells, DARPin-targeted MVs displayed the cytotoxicity of parental, non-targeted oncolytic MV, killing more than 90% of target cells in three days. In vivo experiments analysing the oncolytic efficacy in appropriate Xenograft animal models are under way, as well as the side-by-side comparison of the DARPin-MV with published scFv-MV targeted against HER2/neu.

In conclusion, DARPins were found to be effective in targeting oncolytic MV and DARPin-targeted OVs will therefore complement existing targeting approaches.

Cawood R Mr 1 Seymour L Professor 1 Wong S L Miss 1

1 Department of Clinical Pharmacology, University of Oxford, UK Or 57 MicroRNA Regulation of Adenovirus Afford Control of Oncolytic Activity Without Changing Endogenous MicroRNA Levels

MicroRNA regulation of therapeutic viruses is an emerging field which allows the selective destruction of essential viral RNA molecules in sites of potential toxicity. We have previously demonstrated that the hepatic toxicity caused by intravenous injection of wild type adenovirus type 5 (Ad5WT) in mice can be prevented by incorporating 4 binding sites for the liver specific microRNA, mir122, into the 3’ UTR of E1A mRNA. This virus, termed Ad5mir122, is safe at doses 10-fold above the LD₅₀ of Ad5WT. Here we show that Ad5mir122 maintains wild type oncolytic potency and address the potential toxic effects of microRNA regulation on the host cell.

Complete microarray profiling of mRNA from Ad5WT-infected livers showed the levels of >3900 mRNAs were changed >2-fold, however, less than 600 were altered by infection with Ad5mir122. Comparison of these mRNAs with predicted mir122 targets showed no overlap, suggesting they might be affected by capsid proteins or residual viral gene expression. RT-QPCR for E1A 13S transcripts and western blot for E1A showed that both mRNA and protein were significantly knocked-down when compared to Ad5WT. RT QPCR for mir122 in infected livers showed that the quantity of mir122 was unchanged by Ad5mir122. In order to assess the anti-cancer activity of Ad5mir122, CD1 nude mice bearing HepG2 tumours were administered 2x10¹⁰ viral particles (X4, intravenous) and showed significantly reduced tumour growth and extended survival without toxicity. These results demonstrate that microRNA regulation can control viral replication without affecting endogenous mRNA targets, or the level of the microRNA.

Hemminki A Dr 1 Kangasniemi L Dr 2 Koski A Dr 1 Cerullo V Dr 1 Escutenaire S Dr 1 Rajecki M Dr 1 Diaconu I Dr 1 Joensuu T Dr 3 Kanerva A Dr 1 Pesonen S Dr 1

1 P.o.Box 63, 00014 Helsingin Yliopisto, Finland; 2 Oncos Therapeutics, Tukholmankatu 8 A, 00290 Helsinki, Finland; 3 International Comprehensive Cancer Center Docrates, Saukonpaadenranta 2, 00180 Helsinki, Finland Or 58 Personalized oncolytic adenovirus therapy for treatment of chemotherapy refractory solid tumors

Given the ease of construction of oncolytic adenoviruses with several modifications, the approach lends itself well to individually personalized medicine. This has been recognized also by legislators and patient-by-patient treatments are regulated by the EU Advanced Therapies directive, which has allowed us to treat more than 200 patients in an Advanced Therapy Access Program. Following extensive preclinical testing, 9 different viruses have been used. The optimal virus capsid, tumor specific promoter and arming device are selected based on preclinical and clinical data, taking into account the nature of the clinical problem in each patient (local vs systemic), while sero-switching has been utilized to enhance systemic delivery. Three schedules of low-dose cyclophosphamide have been used to reduce regulatory T-cells and induce TH2- >TH1 switch. Injections have been performed in ultrasound, visual or CT guidance, intratumorally, intracavitary and/or intravenously on an individual basis. Pretreatment tumor samples have been studied for selecting the optimal virus and for prediction of efficacy. Overall, treatments have been remarkably safe with no mortality. Serious adverse events are seen in circa 5% of treatments, while mild to moderate fever, flu-like symptoms, tumor pain and fatigue are common. Evidence of possible efficacy (radiological stable disease or better) has been seen in 48% of patients overall and up to 78% with the best virus and optimized schedule. With the best schedule, more than half survive for a year or longer which is unusual in this difficult patient population and compares well to historical controls. A clinical trial is in progress.

Dickson George

Or 59 Gene therapy for muscular dystrophy: current progress and future prospects

Crippa S Dr 1 Galli D Dr 2 Cassano M Dr 1 Janssens S Dr 3 Gijsbers R Dr 4 Debyser Z Dr 4 Cossu G Dr 5 Sampaolesi M Dr 6

1 Translational Cardiomyology, Stem Cell Research Institute, Catholic University of Leuven, Herestraat 49 B-3000 Leuven, Belgium; 2 Human Anatomy, University of Pavia, Via Forlanini 8, 27100 Pavia, Italy; 3 Cardiology Division, University Hospital Gasthuisberg, Leuven, Belgium 4 Molecular Medicine, Catholic University of Leuven, Belgium; 5 Division of Regenerative Medicine, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan Italy 6 Translational Cardiomyology, Stem Cell Research Institute, Catholic University of Leuven, Herestraat 49 B-3000 Leuven, Belgium; Human Anatomy, University of Pavia, Via Forlanini 8, 27100 Pavia, Italy Or 60 Shifting heart to skeletal muscle: miR669 functions as a cell fate switch between cardiac and skeletal muscle lineages

Resident progenitor cells in the heart are able to differentiate in one or more cardiac cell types. While surface marker expression and ability to generate cardiomyocytes have been characterized for several types of these progenitors^1–3, little is known about how cardiac differentiation is regulated. Recent works have highlighted an important role for miRNAs in regulating skeletal and cardiac myogenesis^4,5. In chronic cardiac diseases resident progenitors appear unable to counteract progressive degeneration, possibly because they may come exhausted in repeated attempts to regenerate the failing heart. Beta sarcoglycan (Sgcb) null mice are a model for limb girdle muscular dystrophy type 2E and undergo progressive dilated cardiomyopathy. In this study we isolated and characterized cardiac progenitors from Sgcb null hearts and unexpectedly observed their aberrant skeletal muscle phenotype both in vitro and in vivo. This is due to the lack of two key regulatory microRNAs, miR669a and miR669p, capable of suppressing skeletal myogenesis by directly targeting the MyoD 3'UTR. According to our findings, the absence of Sgcb gene abolished miR669p and reduced miR669a expression. miR669p, embedded in Sgcb gene, is absent in Sgcb-null progenitors due to the homologous recombination with neomycine cassette. miR669a, encoded by Sfmbt2 gene, is strongly downregulated due to a signal cascade involving intracellular calcium, calpain proteases and degradation of YY1, positive regulator for Sfmbt.

This finding opens a new scenario on the fate choice of mesoderm progenitors and makes the task of utilizing endogenous cardiac stem cells more complex in certain pathologies.

1) Anversa P. et al. J Am Coll Cardiol (2006)

2) Galvez G.B. et al. Cell Death Differ (2008)

3) Laugwitz K.L. et al. Nature (2005)

4) van Rooij E. et al. Science (2007)

5) van Rooij E. et al. Dev Cell (2009)

Schinkel S. Dr 1 Bauer R. Dr 1 Rutschow D. Dr 1 Stucka R. Dr 2 Lochmueller H. Dr 2 Kleinschmidt J. Dr 3 Katus H. Dr 1 Mueller O. Dr 1

1 Dept. of Cardiology, Angiology and Pneumology, University Hospital, INF 410, 69120 Heidelberg, Germany 2 Institute of Human Genetics, Newcastle University, International Centre for Life, Newcastle upon Tyne, NE1 3BZ, UK 3 Applied Tumorvirology, German Cancer Research Center, INF 242, 69120 Heidelberg, Germany Or 61 Prevention of cardiac function in mdx mice after AAV9-mediated microdystrophin gene transfer

Dystrophin plays an important role in muscle contraction linking the intracellular cytoskeleton to the extracellular matrix. Mutations within the dystrophin encoding sequence may lead to a complete loss of the protein causing Duchenne muscular dystrophy (DMD), frequently associated with severe cardiomyopathy. While clinical studies for gene therapy of skeletal myopathy are performed, therapies for cardiac muscles have barely been evolved.

Aim of our study was to establish an efficient long term treatment for DMD-associated cardiomyopathy in a mouse model for dystrophin-deficiency (mdx).

Mdx mice were treated with 1×10¹² viral genomes of AAV9, containing a microdystrophin-cDNA (μDys), transcriptionally controlled by either CMV or CMV enhanced myosin light chain (CMV-MLC) promoter. Mice were challenged by voluntary wheel exercise over 8 month while left ventricular fraction of shortening, a cardiac performance indicator, has been monitored. Finally, heart, quadriceps femoris muscle (MQF) and liver were dissected and analysed for expression of μDys. We found (1) an increased cardiac μDys expression with the CMV-MLC- in comparison to CMV-promoter, (2) a preserved fraction of shortening with the CMV-MLC- and CMV-promoter, indicating, that even a minor μDys expression leads to a physiological benefit, and (3) absence of μDys expression in MQF and liver. These findings demonstrate a high specificity and efficiency of the chosen delivery approach for cardiac muscle resulting in a sustained therapeutic effect.

In conclusion, we established an efficient and specific AAV9-mediated gene transfer of microdystrophin-cDNA in mdx hearts, which may become a tool to develop treatment strategies for DMD-associated cardiomyopathy.

Tedesco FS Dr 1 Hoshiya H Dr 2 D'Antona G Dr 3 Gerli M Dr 2 Messina G Dr 4 Tonlorenzi R Dr 2 Berghella L Dr 5 Torrente Y Dr 6 Kazuki Y Dr 7 Bottinelli R Professor 3 Oshimura M Professor 7 Cossu G Professor 4

1 Division of Regenerative Medicine, San Raffaele Scientific Institute and Vita-Salute San Raffaele/Open University, via Olgettina 58, 20132, Milan, Italy. 2 Division of Regenerative Medicine, San Raffaele Scientific Institute, via Olgettina 58, 20132, Milan, Italy. 3 Department of Physiology and Interuniversity Institute of Myology, University of Pavia, Via Forlanini 6, 27100, Pavia, Italy. 4 Division of Regenerative Medicine, San Raffaele Scientific Institute, via Olgettina 58, 20132, Milan, Italy and Department of Biology, University of Milan, via Celoria 26, 20133, Milan, Italy. 5 Institute of Cell Biology and Tissue Engineering, San Raffaele Biomedical Science Park, 00128, Rome 6 Stem Cell Laboratory, Department of Neurological Science, University of Milan, Fondazione IRCCS Policlinico Mangiagalli-Regina Elena, Milan, Italy. 7 Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, Japan Or 62 Rescue of dystrophic muscle by stem cell-mediated transfer of a HAC containing the dystrophin locus

Human artificial chromosomes (HACs) have many advantages over conventional gene therapy vectors, such as episomal maintenance and the ability to carry large genetic regions with their own regulatory elements. There is currently no evidence of efficacious therapy using HACs in any genetic disorder. We show here the rescue of a mouse model of Duchenne Muscular Dystrophy (DMD) by a novel strategy, combining HAC-mediated gene replacement with mesoangioblast (MABs: vessel-associated stem cells) transplantation. DMD is a devastating myopathy for which no therapy exists, despite many experimental and clinical trials. To this aim, we corrected MABs from dystrophic mdx mice (mdxMABs) with a HAC vector containing the large (2.4Mb) human dystrophin gene (DYS-HAC). DYS-HAC expressing mdxMABs robustly engrafted and produced many dystrophin positive fibers in dystrophic mice upon intra-muscular and intra-arterial transplantation, leading to significant morphological and functional amelioration of the dystrophic phenotype. These data provide the first functional evidence of HAC-mediated rescue in a pre-clinical model of a genetic disease and set the conditions for future clinical translation of this experimental strategy for Duchenne Muscular Dystrophy.

Moullier P Dr 1 Le Guiner C Dr 1 Montus M Dr 2 Servais L Dr 3 Garcia L Dr 3 Fromes Y Dr 4 Cherel Y Dr 5 Voit T Dr 3  + The « AFM-sponsored Duchenne Consortium » 6

1 GENETHON, Evry & INSERM UMR 649, Nantes - France 2 GENETHON, Evry - France 3 Institut de Myologie, Paris - France 4 GENETHON, Evry & Institut de Myologie, Paris - France 5 INRA UMR 703, Nantes - France 6 Genethon/Institut de Myologie/UMR703/UMR649 - France Or 63 Effective limb transduction with phenotypic correction after injection of rAAV8-U7snRNA in GRMD dogs

In the Duchenne Muscular Dystrophy (DMD) the selective removal by exon skipping of exons flanking an out-of frame mutation in the dystrophin messenger can result in in-frame mRNA transcripts that are translated into shorter but functionally active dystrophin.

The goal of our project is to determine in GRMD, the effective dose of our therapeutic product defined as a recombinant Adeno-Associated Virus serotype 8 (rAAV8) expressing a modified U7 snRNA specific for the exon skipping of the dystrophin transcript. The mode of delivery is the locoregional high-pressure intravenous (IV) injection in a forelimb.

Three groups of GRMD dogs were exposed to 3 different rAAV8-U7snRNA doses. Each dog was followed ≈3 months after injection. The primary outcomes are the restoration of dystrophin expression and the improvement of the tissue pathology in the injected limb compared to the controlateral. The secondary outcomes are the muscle strength correction, the biodistribution and shedding patterns as well as the immune response against rAAV8 capsid and dystrophin.

We built a unique network of laboratories with complementary skills to deliver a GLP-compliant set of preclinical data to further define the regulatory toxicology studies. The organization of our network and the results obtained in our GRMD dogs study will be presented.

This project is supported by AFM (Association Française contre les Myopathies) and by ADNA (Advanced Diagnostics for New Therapeutic Approaches), a program dedicated to personalized medicine, coordinated by Institut Mérieux and supported by research and innovation aid from the French public agency, OSEO.

Bendle G Dr 1 Linnemann C Mr 1 Hooijkaas A Ms 1 Bies L Ms 1 de Witte M Dr 1 Jorritsma A Dr 1 Kaiser A Dr 1 Pouw N Dr 2 Debets R Professor 2 Kieback E Dr 3 Uckert W Professor 3 Song J Dr 4 Haanen J Professor 1 Schumacher T Professor 1

1 Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands 2 Dept Medical Oncology, Erasmus MC-Daniel den Hoed Cancer Center, Rotterdam, The Netherlands 4 Division of Experimental Animal Pathology, The Netherlands Cancer Institute, Amsterdam, The Netherlands Or 64 Lethal Graft-versus-Host Disease in mouse models of T Cell Receptor Gene Therapy

The antigen-specificity of a T cell is solely determined by the T cell receptor (TCR) alpha- and beta-chains. Therefore, the transfer of TCR genes into patient T cells can be used to induce immune reactivity towards defined antigens to which the endogenous T cell repertoire is insufficiently reactive. This approach, which is called TCR gene therapy, is currently being developed to target tumors and pathogens and the clinical testing of this approach has commenced in cancer patients.

However, we have observed the occurrence of lethal cytokine-driven autoimmune pathology in mouse models of TCR gene therapy under conditions that closely mimic the (intended) clinical setting. This therapy-induced autoimmune process results in a fatal destruction of the hematopoietic compartment accompanied by more general aspects of Graft-versus-Host Disease. Autoimmune pathology is seen with a panel of different murine TCRs and when various strategies are used to promote in vivo T cell function. We have defined the molecular mechanism that underlies the development of this phenomenon, showing that the pairing of introduced and endogenous TCR chains in TCR gene modified T cells leads to the formation of self-reactive TCRs that are responsible for autoimmune pathology.

Our data show the development of strategies to limit the formation of self-reactive TCRs in TCR gene modified T cells will be essential for the safe clinical implementation of TCR gene therapy. To this end we demonstrate that adjustment in the design of gene therapy vectors and target T cell populations can be utilized to ameliorate the risk of TCR gene therapy-induced autoimmune pathology.

Pule M Dr

Or 65 T-cell engineering for cancer applications

Through the application of gene-therapy methods, it is now possible to generate large numbers of engineered T-cells with novel specificities and properties for cancer therapy. I will discuss three practical advances to this approach: (1) Administration of T-cells expressing antibody-derived Chimeric Antigen Receptors (CARs) against disialoganglioside has shown efficacy in refractory neuroblastoma. We have optimized our original retroviral cassette to build on this clinical success: We have refined the CAR by identifying the optimal format for surface expression and antigen recognition, replacing murine antigen-recognizing anti body single chains with a humanized version and introducing compound signalling endodomain. In addition, we have optimized co-expression of the iCasp9 suicide genes with this CAR, allowing deletion of transduced T-cells in the face of unwanted toxicity. This cassette is ready for clinical testing (2). A compact marker/suicide gene which relies on off-the-shelf reagents/pharmaceuticals would be of considerable practical utility. We have generated a highly compact (136 amino acid) marker/suicide gene construct by identifying and joining the minimal epitopes required for anti-CD34 mAb QBEND10 and Rituximab binding on a CD8 stalk. This allows facile detection of transduction efficiency, clinical grade sorting with Miltenyi CliniMACS beads. Exposure of transduced T-cells to rituximab and complement results in >95% killing (3) Finally, we have engineered several calcineurin mutants which engender tacrolimus and/or cyclosporine resistance in T-cells. We anticipate these will allow cellular immunotherapy of EBV driven lymphoma in the face of ongoing immunosuppression.

Pospori Constantina Xue Shao-An Holler Angelika Stauss Hans J. Department of Immunology, University College London, Royal Free Hospital,

Or 66 Adoptive immunotherapy with TCR-transferred lymphocytes

T cell receptor gene transfer has been used successfully to redirect the specificity of human and murine T cells. The first clinical studies have been completed and shown feasibility of this approach and persistence of TCRexpressing cells in patients. We have explored the possibility of using TCR gene transfer to generate antigen-specific regulatory T cells. Using a murine model system we found that antigen-specific regulatory T cells can be generated by TCR gene transfer into purified Treg, or by co-transfer of TCR and FoxP3 genes into purified CD4+T cells. Upon adoptive transfer, the gene-modified T cells were able to suppress arthritis in an antigen-specific fashion. This provides a rationale to use TCR gene transfer to control unwanted immune responses in vivo. Recently, we have started to explore if TCR gene transfer into haematopoietic stem cells will result in the generation of naïve and central memory T cells with specificity for a tumour-associated self-antigen. We have transferred the genes of an HLA-A0201-restrcited TCR with specificity for the tumourassociated Wilm's Tumour 1 (WT1) antigen into haematopoietic stem cells of HLA-A0201-transgenic mice followed by stem cell transplantation into syngeneic recipients. The results indicate that this experimental system provides a powerful model to study the thymic development, peripheral phenotype, persistence and function of WT1-specific T cells in vivo.

Small Eric J MD University of California, San Francisco

Or 67 Immunotherapy for Prostate Cancer: Current Indications and Future Prospects

Sipuleucel-T (Provenge) is an autologous active cellular immunotherapy product for the treatment of metastatic castrate resistant prostate cancer (CRPC). In the pivotal study, in men with asymptomatic or minimally symptomatic metastatic CRPC, treatment with Provenge improved overall survival compared with control, 25.8 months compared with 21.7 months (P = .032; HR = 0.775. The adverse events reported more frequently with Provenge treatment were chills, fever, headache, influenza-like illness, myalgia, hyperhidrosis, and groin pain. An integrated analysis of 3 randomized trials included 737 patients (488 sipuleucel-T: 249 placebo) revealed a significant sipuleucel-T treatment effect (HR = 0.735, 95% CI:0.613, 0.882, P < 0.001), and the treatment effect was found to be homogeneous across the 3 trials. A positive treatment effect was observed in subgroups, including those defined by age, race, ECOG performance status, number of bone metastases, and previous chemotherapy use. The mechanism of action of Sipuleucel-T is being studied in a pre-prostatectomy study.

CTLA-4 is an inhibitory molecule expressed on activated T cells. CTLA-4 blockade with ipilimumab augments T cell responses and anti-tumor immunity in animal models. Combination therapy with GM-CSF has demonstrated anti-cancer activity and a dose-response relationship between ipilimumab dose and effector T cell activation was observed. Future directions include a randomized phase 2 trial of Ipilimumab with and without GM-CSF, and a combination trial of Ipilimumab and Sipuleucel-T. An ongoing trial of radiation to a metastatic lesion with and without Ipilimumab is underway.

Provasi E Miss * 1 Genovese P * 2 Lombardo A 2 Magnani Z 1 Pei-Qi L 3 Reik A 3 Chu V 3 Kuball J 4 Bondanza A 1 Casorati G 5 Ciceri F 6 Bordignon C 7 Greenberg PD 4 Holmes MC 3 Gregory PD 3 Naldini L 2 Bonini C 1

1 Experimental Hematology Unit, Division of Regenerative Medicine Gene Therapy and Stem Cells, San Raffaele Hospital, Milan, Italy 2 San Raffaele Telethon Institute for Gene Therapy, San Raffaele Hospital, Milan, Italy 3 Sangamo Biosciences, Richmond, USA 4 Fred Hutchinson Cancer Research Center and University of Washington, Seattle, USA 5 Experimental Immunology Unit, Division of Immunology Transplantation and Infectious Diseases, San Raffaele Hospital, Milan, Italy 6 Hematology Clinical Unit, Division of Regenerative Medicine Gene Therapy and Stem Cells, San Raffaele Hospital, Milan, Italy 7 Vita Salute San Raffaele University, Milan, Italy Or 68 Editing Human Lymphocyte Specificity for Safe and Effective Adoptive Immunotherapy of Leukemia

T cell receptor (TCR) gene-transfer is an attractive strategy for the adoptive immunotherapy of tumors. However, the full potential of this approach is limited by the co-expression in one lymphocyte of endogenous and exogenous TCR chains, resulting in reduced expression of the introduced tumor-specific TCR, and in acquisition of two novel and unpredicted specificities due to TCR mispairing. To address these issues, we designed and transferred Zinc Finger Nucleases (ZFNs) targeting the constant region of the TCR alpha and beta chain genes by viral vectors into T lymphocytes, thus promoting the genetic disruption of the endogenous TCR. Lymphocytes targeted by each ZFNs set failed in expressing the CD3/TCR complex on cell surface. Once sorted, CD3^neg cells proved stable and permissive to lentiviral transduction. For a complete editing of T cell specificity, we selected a TCR specific for the WT1 tumor antigen and set up a protocol of sequential disruption of the TCR chains followed by lentiviral transfer of the WT1-specific TCR chains. At the end of the procedure we obtained a population of TCR-edited lymphocytes, carrying only the tumor-specific TCR that, in the absence of competition, was expressed at high and physiological levels. Accordingly, TCR-edited lymphocytes were superior to conventional TCR-transferred cells in promoting specific recognition of WT1^pos targets, including primary leukemias, and, most importantly, were devoid of residual reactivity including alloreactivity. These data demonstrate that genetic re-programming of T cell specificity in primary lymphocytes is feasible and functional and might improve the therapeutic outcome of cancer immunotherapy.

Roncarolo M.G Professor 1 Gregori S. Dr 2 Bacchetta R. Dr 2 Annoni A. Dr 2 Cantore A. Dr 2 Naldini L. Professor 1

1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Dpt.of Regenerative Medicine, Stem Cells, and Gene Therapy, and Vita-Salute San Raffaele University Via Olgettina 58, 20132 Milan, Italy 2 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Department of Regenerative Medicine, Stem Cells, and Gene Therapy, Via Olgettina 58, 20132 Milan, Italy Or 69 Strategies to overcome immunity against transgenes

Immune responses are a major hurdle to successful gene therapy. Existing approaches to overcome immune responses employ non-specific methods of immune suppression, including steroids, and compounds that block T-cell signaling, expansion, and function. Developing strategies for inducing antigen-specific tolerance can provide a way to overcome the limitations of the existing immune-suppressive agents.

Regulatory T (Tr) cells are a specialized subset of T cells that control immune responses and promote and maintain immune-tolerance. We established protocols to generate homogeneous populations of Tr cells using lentiviral vector (LV)-mediated over-expression of the tolerogenic molecules, IL-10 or FOXP3, into CD4⁺ T cells. Results demonstrate that stable over-expression of either FOXP3 or IL-10 in human CD4⁺ T cells confers Tr phenotype and functions. These results open the possibility to generate antigen-specific Tr cells for cellular therapy to promote specific tolerance in patients with immune-mediated diseases.

An alternative approach to modulate the immune response consists in de-targeting transgene expression from hematopoietic cells by miR142-regulated LV. Prevention of immune-mediated clearance of gene-modified cells and induction of robust tolerance to the encoded-antigen, mediated by transgene-specific Tr cells, was observed using miR142-LV in vivo. Not only integrase-competent LV but also integrase-defective LV (IDLV), despite low transgene expression, can promote immunological tolerance to foreign antigens.

These findings have important implications for developing future gene therapy strategies based on Tr cell therapy and on microRNA-regulated vector approach to restore/promote tolerance toward specific antigens/transgenes and prevent undesired immune responses.

Bosticardo M Dr Draghici E Ms Schena F Dr 2 Sauer AV Dr Fontana E Ms 3 Castiello MC Ms Locci M Dr Catucci M Mr Naldini L Professor Aiuti A Professor 4 Roncarolo MG Professor Poliani PL Dr 3 Traggiai E Dr 2 Villa A Dr 5

2 Laboratory of Immunology and Rheumatic Disease, IGG, Genoa, Italy 3 Department of Pathology, University of Brescia, Italy 4 University of Rome “Tor Vergata”, Rome, Italy 5 Istituto di Tecnologie Biomediche-CNR, Segrate, Milan, Italy Or 70 Gene transfer leads to B cell reconstitution in a murine model of Wiskott-Aldrich Syndrome

Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, eczema, autoimmunity and lymphomas. Transplantation of hematopoietic stem cells (HSC) from HLA-identical donors is curative, but not available to all patients. Therapy based on the transplant of genetically corrected autologous HSC could represent a valid alternative approach for patients lacking a suitable donor. We have developed a protocol of gene therapy (GT) for WAS using a lentiviral vector encoding for human WASp promoter/cDNA and demonstrated efficacy and safety of our gene transfer approach. Because of the importance of analyzing B cell reconstitution in an immunodeficiency with a strong autoimmune phenotype, we fully evaluated this aspect in a new series of experiments. We observed that WASp + B cells were present in all tissues isolated from GT treated mice, with the highest levels detected among splenic marginal zone B cells. These data were confirmed by the restoration of B cell follicle and marginal zone architecture in spleen of GT treated mice. To test functionality of B cells in GT treated mice, we performed a challenge with pneumococcal antigens four months after GT by injecting i.p. P23 vaccine. We could demonstrate an improved antibody response to challenge with pneumococcal antigens in GT treated mice. In addition, we observed the reduction of serum autoantibodies against dsDNA, which are present in majority of Was^-/- mice. These observations contribute to the implementation of our future clinical GT trial for WAS providing further evidence of the efficacy of our gene transfer approach.

Fransson M Ms 1 Piras E Ms 2 Wang H Mr 1 Duprez I Rasmussen Dr 1 LeBlanc K Professor 3 Brittebo E Professor 2 Loskog A Dr 1

1 Div of Clinical Immunology, Uppsala University 2 Dept of Pharmaceutical Biosciences, Uppsala University 3 Div of Clinical Immunology, Karolinska Institute and Hematology Center, Karolinska University Hospital Or 71 Genetically Engineered Cells Target CNS and Cure Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In the mouse model of MS (EAE) treatments with either T regulatory (Treg) cells or multipotent mesenchymal stromal cells (MSC) have proven beneficial. However, systemic administration of such cells may cause problems with infections. Further, few cells may reach the CNS to hamper the pathological immune reaction. We hypothesized that Tregs or MSCs can be targeted to the CNS using gene transfer of a CNS-targeting chimeric artificial receptor (CAR) to provide a local immunosuppression.

A scFv was cloned from the hybridoma (8.18C5) producing anti-human myelin oligodendrocyte glycoprotein (MOG) antibodies. The scFv was cloned into a CAR and transferred into a lentiviral vector. CD4⁺ T-cells were transduced with the CAR expressed in trans with murine FoxP3 which promotes Treg differentiation, while MSC were modified using the CAR only. Engineered Tregs and MSCs were analyzed for their suppressive ability. Further, engineered cells were stained with a fluorescent dye and injected into mice which were sacrificed and the cells were tracked in vivo. Finally, the engineered cells were used to treat EAE mice.

By inserting the genes for FoxP3 and CAR we developed Tregs that potently suppressed T-cells in vitro. Similar results were obtained with the CAR engineered MSCs. Further, both Tregs and MSCs were able to locate to the brain. The engineered suppressor cells hampered CNS inflammation and cured mice with EAE.

In conclusion, genetically engineered suppressor cells are able to localize to the CNS and cure EAE mice.

Mátrai J Dr 1 Cantore A Mr 2 Bartholomae CC Dr 3 Annoni A Mrs 4 Wang W Dr 3 Acosta-Sanchez A Mr 1 Samara-Kuko E Mrs 1 De Waele L Dr 1 Ma L Dr 1 Genovese P Mr 2 Damo M Mrs 2 Arens A Mrs 3 Nichols TC Professor 5 Von Kalle C Professor 3 Chuah MKL Professor 1 Roncarolo MG Professor 2 Schmidt M Professor 3 VandenDriessche T Professor 1 Naldini L Professor 2

1 Vesalius Research Center, Flanders Institute of Biotechnology (VIB), University of Leuven and Free University of Brussels (VUB), Belgium 2 San Raffaele Telethon Institute for Gene Therapy (TIGET), San Raffaele Scientific Institute, Milan; “Vita Salute San Raffaele” University, Milan, Italy 3 Nationales Centrum für Tumorerkrankungen (NCT) Heidelberg, Abteilung für Translationale Onkologie, Deutsches Krebsforschungszentrum (DKFZ) Heidelberg, Germany 4 San Raffaele Telethon Institute for Gene Therapy (TIGET), San Raffaele Scientific Institute, Milan 5 University of North Carolina, Chapel Hill, USA Or 72 Integrase-Defective Lentiviral Vectors Enable Tolerogenic Expression of Bioactive Molecules in the Liver With Minimal Genotoxic Risk

Lentiviral vectors are attractive tools for liver-directed gene therapy by virtue of their ability for stable gene expression and lack of pre-existing immunity in most human subjects. However, the use of integrating vectors may raise some concerns regarding the potential risk of insertional mutagenesis. Here we investigated integrase-defective lentiviral vectors (IDLV) containing an inactivating mutation in the integrase (D64V) and show that hepatocyte-targeted IDLV support a prolonged window of transgene expression from the liver upon intravenous injection in mice. Whereas this expression attains lower levels as compared to that obtained with the integrase-competent vector counterparts, it can result in relevant biological effects such as sustained therapeutic coagulation factor IX expression levels in hemophilia B mice and induction of transgene-specific regulatory T cells and immunological tolerance to foreign antigens. Deep sequencing of transduced livers showed that the majority of IDLV genomes remain episomal. Rare genomic integrations show no preference for gene coding regions and occur mostly by a mechanism inconsistent with residual integrase activity.Thus, IDLV provide an attractive platform for tolerogenic expression of bioactive molecules with minimal genotoxic risk.

Annoni A Dr * 1 Cantore A Mr * 2 Damo M Ms 2 Genovese P Mr 2 Sergi L Sergi Ms 1 Roncarolo MG Professor 2 Naldini L Professor 2

1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy 2 San Raffaele Telethon Institute for Gene Therapy and “Vita Salute San Raffaele” University, Milan, Italy Or 73 Liver gene transfer by integrase-defective vectors induces antigen-specific regulatory T cells

Integrase-Defective Lentiviral Vectors (IDLV) are emerging as an attractive gene delivery system. These vectors harness the pantropism and proficiency of lentiviral vector transduction without relying on integration and permanent modification of the cellular genome. The extent and duration of IDLV expression, however, can vary substantially with the target tissue and it is not known whether IDLV can achieve relevant transgene expression levels following hepatic gene delivery. Here we report a detailed characterization of IDLV performance in primary human hepatocytes and in the mouse liver. We show that IDLV have the same infectivity, efficiently transduce vector genomes, but express the transgene at lower levels on a per copy basis, as compared to their integration-competent counterparts. Using different experimental approaches, including TCR transgenic and Foxp3 reporter mice, we show that a pulse of IDLV-driven hepatocyte-targeted expression can trigger the conversion of naive CD4⁺ T cells to induced antigen-specific Tregs that mediate active tolerance to strong neoantigens (GFP, ovalbumin). We previously reported the tolerogenic outcome of LV gene transfer stringently targeted to hepatocytes. We did not know, however, whether this outcome depends on high levels of vector integration and transgene expression within hepatocytes, which would limit application of this finding outside of gene replacement strategies for the correction of monogenic diseases. Our new findings demonstrate that Treg induction in vivo can now be obtained in a safer way opening up new therapeutic opportunities for immune modulation.

Cohen-Haguenauer Odile On behalf of the GeneCellPro consortium (see below), CliniGene and LBPA at ENSC, F-94235 Cachan Cedex and Hospital St-Louis F-75475 Paris

Or 74 Relevance of an Academic GMP Pan-European Vector Infra-structure (PEVI)

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led “First-in-man” gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into “First-in-man” trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Members of the GeneCellPro consortium (by order of co-author if so required)

Odile Cohen-Haguenauer

Nicolas Creff

Pedro Cruz

Celia Tunc

Alessandro Aïuti

Christopher Baum

Fatima Bosch

Pontus Blomberg

Klaus Cichutek

Mary Collins

Olivier Danos

Frédéric Dehaut

Marc Federspiel

Eithan Galun

Henk Garritsen

Hansjörg Hauser

Martin Hildebrandt

David Klatzmann

Otto Merten

Eugenio Montini

Tim O'Brien

Amos Panet

Rasooly_Linda

Daniel Scherman

Manfred Schmidt

Pierre TIBERGHIEN

Thierry Vandendriessche

Holger Ziehr

Seppo Ylä-Herttuala

Christof von Kalle

Gösta Gahrton

Manuel Carrondo

Vallanti G Dr 1 Gatti A Dr 1 Spoldi E Dr 1 Marano G Dr 1 Rossetti F Dr 1 Massariello P Dr 1 Salomoni M Dr 1 Biffi A Dr 2 Aiuti A Professor 2 Naldini L Professor 3 Bordignon C Professor 4 Radrizzani M Dr 1

1 MolMed S.p.A., Milano, Italy 2 San Raffaele Telethon Institute for Gene Therapy 3 San Raffaele Telethon Institute for Gene Therapy and Vita-Salute San Raffaele University, San Raffaele Scientific Institute, Milano, Italy 4 MolMed S.p.A., Milano, Italy, Vita-Salute San Raffaele Scientific Institute, Milano, Italy Or 75 Large scale manufacturing of transduced CD34+ cells using lentiviral vectors for Metachromatic Leukodystrophy and Wiskott Aldrich Syndrome clinical trials

MolMed SpA is a biotechnology company focused – inter alia - on cellular and gene therapy products. It is formally authorised for the production and release of medicinal products for human use and it is acting as GMP manufacturing site for two innovative gene therapy clinical trials (Metachromatic Leukodistrophy, MLD and Wiskott Aldrich Syndrome, WAS) sponsored by Telethon. The large scale transduction process has been designed in collaboration with Tiget-HSR: CD34+ cells are transduced by 2 hits of ARSA or WASP GMP- grade LV at MOI 100 respectively, in RetroNectin^® coated bags in presence of cytokines. For GMP validation, three transduction runs for each type of vector were performed using CD34+ cells from healthy donor bone marrow. Validation results indicate that the GMP transduction process guarantees the quality of the product in terms of identity/potency, purity and safety. Recently, the GMP process has been used to prepare cells for the first two patients, one for each trial. CD34+ purification yield was 49% and 41% and 12 x 10⁶ and 9 x 10⁶ of transduced CD34+ /kg were obtained for MLD and WAS patient, respectively. High transduction efficiencies, in the range of the data obtained in validation phase, were observed. Moreover, ARSA activity and WAS protein were present in transduced cells. Clinical lots were sterile, free from mycoplasma and RCL; purity analyses demonstrated the absence of host cell DNA sequences integrated in cell genome.

Rauschhuber C Ms 1 Hausl M Mr 1 Ehrhardt A Dr 2

1 Max von Pettenkofer-Insitute, Munich 2 Max von Pettenkofer-Institute, Munich Or 76 Micro RNA knockdown significantly enhances adenovirus replication and vector production

The use of micro RNAs (miRNAs) fundamentally improved the generation of viral vectors for gene therapy. In this study we used our recently established RNAi knockdown cell line B6 based on the RNAi suppressor protein P19 (Rauschhuber and Ehrhardt, submitted) to analyze the influence of the RNAi pathway on adenoviral vectors. We investigated replication of wildtype adenovirus 5 (wtAd5) and first generation adenovirus (FgAd) on a genome level. An up to 10-fold increase in viral genome copy numbers could demonstrate that there is significantly enhanced viral DNA replication under RNAi knockdown conditions. To analyze whether we can also achieve higher viral titers in the B6 cell line, we infected with a common FgAd expressing firefly luciferase (FgAdluc). After re-infection of HEK293 cells we measured up to 5-fold increased luciferase activity indicating a higher titer of FgAdluc derived from B6 cells.

To reach even stronger effects we hypothesized that the amount of the RNAi suppressor protein P19 may be the limiting factor. Thus, we generated a recombinant adenovirus expressing p19 under the fiber promoter. We detected up to 100-fold increased genome copy numbers compared to wtAd5 and the corresponding control virus. Additionally, we developed a helper-virus expressing p19 for helper-dependent adenovirus (HC-AdV) production. In contrast to a conventionally used helper-virus we detected up to 12-fold increased HC-AdV titers.

In conclusion, we provide new insights into regulation of adenoviral vectors by RNAi and our RNAi knockdown cell line B6 could be used as a novel producer cell line for adenoviral vectors.

Koefoed M Mrs 1 Pedersen A.G Miss 1 Soballe K Professor 2 Jensen TG Professor 1 Ulrich-Vinther M Dr 2

1 Department of Human Genetics. Aarhus University, Denmark 2 Department of Orthopedic Surgery, Aarhus University Hospital, Denmark Or 77 Gene Transfer for Bone Healing Using Immobilized Freeze-dried Adeno-associated Viral Vectors

Bone allografts are widely used clinically although they often heal insufficiently leading to risk of fracture. These problems may be alleviated using allografts coated with freeze-dried AAV vectors carrying genes for osteogenic stimuli. We have evaluated the kinetics of the gene delivery from bone allografts and potential effects of stimulation of new bone formation.

For the in vitro studies AAV-GFP coated allografts were placed onto confluent layers of HEK 293 cells. Subsequently the grafts were moved to a new cell layer every minute, and GFP positive cells measured 2 days later. We show that within the first minute most of the viral vectors (88%) from the bone grafts were released.

A murine critical size fracture model was used for in vivo studies. Allografts coated with AAV-luciferase were implanted and bioimaging was used to assess luciferase activity. New bone formation was stimulated by VEGF and bFGF2 and evaluated after 10 weeks using micro-CT and histomorphometry.

Bioluminescence imaging showed localized gene expression reaching a plateau from 51 to 70 days. Interestingly VEGF coated allografts lead to an approx. 5 fold increase in total bone volume compared to bFGF2, a combination of VEGF and bFGF2 and controls, although with large variation within each group.

In conclusion allografts with freeze-dried AAV vectors on the surface is a promising new tool for enhancement of the formation of new bone. The positive effect of VEGF on bone formation points to new blood vessels as a critical step in the healing process.

Or 78 Insect cell-based AAV manufacturing—robust technology for clinical and commercial supply

Mavilio F Dr 1

1 San Raffaele Scientific Institute; San Raffaele – Telethon Institute for Gene Therapy (HSR-TIGET); Milan Or 79 Lentiviral Vector Common Integration Sites in Preclinical Models and a Clinical Trial Reflect a Benign Integration Bias and not Oncogenic Selection

HIV-derived lentiviral vectors (LV) have shown good efficacy and safety data in preclinical models and in a recent Hematopoietic Stem Cell (HSC)-based clinical trial for X-linked adrenoleukodystrophy (ALD). However, a careful analysis of LV integration sites in ALD patients' derived cells showed that a relevant number of Common Insertion Sites (CIS) were present. This observation raises concerns because the detection of CIS is a well established hallmark of insertional mutagenesis in mice and clinical trials. Indeed, in clinical trials for X-linked severe combined immunodeficiency and chronic granulomatous disease, several patients developed malignancies triggered by γretroviral vector (γRV) integrations at CIS recurrently targeting the same proto-oncogenes. Thus, it is possible that the occurrence of CIS in the ALD clinical trial is a still silent effect of genotoxicity caused by LV integrations that confer a selective advantage. To understand if CIS generated by LV integrations are the product of genotoxicity we generated our own dataset of LV integrations in human HSC and their progeny after engraftment in immunodeficient mice and studied the integration pattern and the clonal repertoire of vector marked cells in in vitro culture and in vivo. Our integration profile was extensively compared to LV integrations found in the ALD clinical trial, in other gene therapy trials that reported insertional leukemogenesis, as well as in retroviral and transposon-mediated oncogene tagging studies in mice. Interestingly, we found that our integration profile had the same CIS reported in ALD patients. More strikingly however, most CIS in our experimental model and in ALD patients cluster in megabase-wide chromosomal regions of high LV integration density. Conversely, cancer-triggering integrations at CIS found in tumor cells from clinical trials and oncogenes tagging studies in mice, do not form clusters, target always a single gene and are contained in narrow genomic intervals. These findings imply that LV CIS are produced by an integration bias towards specific genomic regions rather than by oncogenic selection.

Montini Eugenio San Raffaele Scientific Institute; San Raffaele – Telethon Institute for Gene Therapy (HSR-TIGET); Milan. Italy

Or 80 Lentiviral Vector Common Integration Sites in Preclinical Models and a Clinical Trial Reflect a Benign Integration Bias and not Oncogenic Selection

HIV-derived lentiviral vectors (LV) have shown good efficacy and safety data in preclinical models and in a recent Hematopoietic Stem Cell (HSC)-based clinical trial for X-linked adrenoleukodystrophy (ALD). However, a careful analysis of LV integration sites in ALD patients' derived cells showed that a relevant number of Common Insertion Sites (CIS) were present. This observation raises concerns because the detection of CIS is a well established hallmark of insertional mutagenesis in mice and clinical trials. Indeed, in clinical trials for X-linked severe combined immunodeficiency and chronic granulomatous disease, several patients developed malignancies triggered by γretroviral vector (γRV) integrations at CIS recurrently targeting the same proto-oncogenes. Thus, it is possible that the occurrence of CIS in the ALD clinical trial is a still silent effect of genotoxicity caused by LV integrations that confer a selective advantage. To understand if CIS generated by LV integrations are the product of genotoxicity, we generated our own dataset of LV integrations in human HSC and their progeny after engraftment in immunodeficient mice and studied the integration pattern and the clonal repertoire of vector marked cells in in vitro culture and in vivo. Our integration profile was extensively compared to LV integrations found in the ALD clinical trial, in other gene therapy trials that reported insertional leukemogenesis, as well as in retroviral and transposon-mediated oncogene tagging studies in mice. Interestingly, we found that our integration profile had the same CIS reported in ALD patients. More strikingly however, most CIS in our experimental model and in ALD patients cluster in megabase-wide chromosomal regions of high LV integration density. Conversely, cancer-triggering integrations at CIS found in tumor cells from clinical trials and oncogenes tagging studies in mice, do not form clusters, target always a single gene and are contained in narrow genomic intervals. These findings imply that LV CIS are produced by an integration bias towards specific genomic regions rather than by oncogenic selection.

Reichenbach J Dr 1 Siler U Dr 1 Paruzynski A Dr 2 Schmid M Dr 2 von Kalle CH Professor 2 Grez M Dr 3 Seger R.A Professor 1

1 University Children's Hospital Zürich, Steinwiesstrass 75, CH-8032 Zürich 2 German Cancer Research Institute, Heidelberg 3 Georg-Speyer Research Institute, Frankfurt Or 81 Gamma-Retroviral Salvage Gene Therapy for Two Children with Chronic Granulomatous Disease

Two children with X-linked CGD and therapy-refractory aspergillosis, age 5 (P1) and 8 (P2) years, received gammaretroviral salvage gene therapy after submyeloablative chemotherapy with Busulphan IV. Invasive Aspergillosis was eradicated within 8 weeks after expressing therapeutic gp91phox and reactive oxygen metabolite levels (15% and 30% oxidase positive cells, respectively). The children recovered from life-threatening paraparesis (P1) and respiratory insufficiency (P2). Subsequent myeloid expansion of transduced cells resulted from insertional mutagenesis by the vector and upregulation of Evi-1 expression. Expansion of gene transduced cells was seen in P1 after 1 year and up to 40% by one dominant MDS clone and in P2 after 3 months and up to 90% by two dominant clones (MDS1/CAMTA1 and MDS1/Stat3). While P2 showed beginning myeloid dysplasia (MDS) after 2.4 yr f/up, P1 remains clinically well (no MDS after 5yr f/up). Allogeneic haematopoietic stem cell transplantation has recently been performed for P2 and is planned for P1. A new SIN gamma-retroviral vector with an internal c-fes myeloid enhancer/promoter has been developed to avoid genotoxicity in the next CGD gene therapy study.

Recchia A. Dr Cattoglio C. Dr 2 Maruggi G. Dr Bartholomae C. Dr Malani N. Dr 4 Pellin D. Dr 5 Cocchiarella F. Dr Magnani Z. Dr 6 Ciceri F. Dr 7 Di Serio C. Dr 5 von Kalle C. Professor Bushman F.D Professor 4 Bonini C. Dr 6 Schmidt M. Dr Mavilio F. Professor

2 IIT Unit of Molecular Neuroscience, Istituto Scientifico H. San Raffaele, Milan, Italy 4 Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA19104, USA 5 Center for Statistics in Biomedical Sciences, Università Vita-Salute San Raffaele, Milan, Italy 6 Experimental Hematology Unit, PIBIC, Division of Regenerative Medicine, Gene Therapy and Stem Cells, Istituto Scientifico H. San Raffaele, Milan, Italy 7 Hematology Unit, Istituto Scientifico H. San Raffaele, Milan, Italy Or 82 High-definition mapping of retroviral integration sites defines the fate of allogeneic T cells after donor lymphocyte infusion

The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors, and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34+ hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. This study shows that retroviral transduction has a very low genotoxic potential for adult T cells.

von Kalle C Professor 1 von Kalle Christof 2

1 NCT Heidelberg, INF 460, 69120 Heidelberg 2 National Center for Tumor Diseases (NCT), Im Neuenheimer Feld 460, and German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg Or 83 Exploiting viral integration to analyse stem cell dynamics

Understanding the regulation of self-renewal and differentiation of stem cells in hematopoiesis as well as in normal and cancerous solid tissues is pivotally important to develop safe and efficient genetically modified stem cell therapeutics and to eradicate malignant cells in human cancer. Hematopoietic stem cells are an easily accessible source of adult stem cells for therapeutic interventions including their genetic correction.

Increasingly effective modalities have been developed to genetically modify human cells ex vivo. Clinical gene therapy studies demonstrated that insertional activation of regulatory genes may lead to a selective advantage in hematopoiesis. We hypothesize that preferred integration loci mark candidate genes and regulatory genomic elements of hematopoietic stem cells whose deregulation could represent an early event in leukemogenesis. Additional identification of candidate stem cell regulatory genes can be accomplished by analyzing the complete integrations site repertoire in a clinical gene therapy study. The impact of our pharmacokinetic analyses will shed new light on stem cell dynamic and biology.

Jacobsen Sten Eirik Haematopoietic Stem Cell Laboratory, Weatherall Institute of Molecular Medicine, Oxford University, United Kingdom

Or 84 Delineating the cellular pathways and molecular determinants of normal and leukemic hematopoiesis

The classical model for hematopoiesis predicts that the commitment to a single hematopoietic lineage requires that the first and obligatory lineage commitment step of hematopoietic stem cells (HSCs) results in a strict separation between the lymphoid and myelo-erythroid lineages. Although this model has been strongly supported by the identification of common lymphoid progenitors (CLPs) and common myeloid progenitor (CMPs), more recent data from us and others support the existence of alternative commitment pathways. The current status as for our understanding of the lineage commitment pathways in normal hematopoiesis in mouse and man will be reviewed.

As for malignant hematopoiesis, the existence of leukemic stem cells (LSCs) as distinct and rare populations of cells capable of reconstituting leukemia has recently been questioned. A related but distinct question, is whether there exists rare populations of LSCs which are uniquely resistant to conventional therapeutic targeting, and therefore also the likely source of relapses following such treatments. In a subgroup of myelodysplastic syndromes (MDS), the del(5q) syndrome, we have pinpointed the primary cellular target for the 5q deletion to a rare population (typically less than 0.1% of all BM cells) of multipotent (lympho-myeloid) stem or progenitor cells, which displays phenotypic and functional stem cell properties, and efficiently displaces the normal HSC compartment. Notably, a high fraction of otherwise transfusion-dependent del(5q) MDS patients go into complete clinical, morphological and molecular remission in response to Lenalidomide treatment. Whereas our investigation of the bone marrow of these 5q- patients before and after treatment with Lenalidomide, confirm that Lenalidomide efficiently eliminates the 5q- clone at the progenitor level, we can in all patients demonstrate that the 5q- MDS stem cell compartment is selectively resistant to Lenalidomide. Over time, lenalidomide resistance develops in most of the patients in partial and complete remission, with recurrence or expansion of the del(5q) clone and clinical and cytogenetic progression. Thus, these studies establish the existence of rare and phenotypically distinct malignant stem cells that are selectively resistant to therapeutic targeting at the time of complete clinical and cytogenetic remission.

Chien Kenneth R. MD, PhD Cardiovascular Research Center, Massachusetts General Hospital, Charles River Plaza/CPZN 3200, 185 Cambridge Street, Boston, MA 02114-2790, USA and the Harvard Stem Cell Institute, Cambridge, MA 02138, USA

Or 85 Regeneration Next: Towards Heart Stem Cell Therapeutics

Recent advances in stem cell biology hold great promise for a new era of cell-based therapy, sparking considerable interest amongst scientists, clinicians, and their patients. However, although our understanding of the biology of stem cells has expanded exponentially in the past few years, the translational arm of stem cell science is in a relatively primitive state. While a number of clinical studies have been initiated with autologous cells with a variety of delivery systems, the early returns point to several inherent problems. In this regard, the clinical potential of stem cells can only be fully realized by the identification of the key barriers to clinical implementation, including scalability, delivery, grafting, survival, and safety issues. In this discussion, we examine experimental paradigms to address the critical steps in the transition from stem cell biology to regenerative medicine, utilizing cardiovascular disease as a case study. Towards this goal, we will review a new promising system for cardiovascular regenerative medicine developed in the Chien lab. Recent studies in the mouse embryo have identified a multipotent, ”master” cardiac progenitor, which contributes to all the major cell types in the murine heart. In contrast to murine cardiogenesis, human heart development is associated with a longer onset of heart cell lineage diversification and expansion, suggesting the possibility of potentially divergent pathways. We have recently identified a diverse set of human fetal ISL1 + cardiovascular progenitor populations that give rise to the cardiomyocyte, smooth muscle, and endothelial cell lineages. A subset of these progenitor populations expressing ISL1 but not NKX2-5 or cardiac differentiation markers is localized to the fetal atria and proximal/medial OFT, and a diverse set of lineage-restricted progenitors and intermediates is marked by ISL1 in combination with NKX2-5 or cardiac differentiation markers. Using two independent transgenic and gene-targeting approaches in human embryonic stem (hES) cell lines, we demonstrate that purified populations of ISL1 + primordial progenitors are capable of self renewal and expansion prior to differentiation into the three major cell types in the heart: cardiomyocyte, smooth muscle, and endothelial lineages. Recent studies have documented the abilitiy to purify a rare, novel ventricular progentior in the islet lineage that can self expand and that can then spontanieous assemble into a mature strip of ventricular muscle, representing a convergence of tissue engineering and stem cell technology. Recent studies in the Chien lab have established a novel approach for the generation of vasculature and muscle cells in vivo form human islet-1 multipotent progenitors initially purified from human ES cells. These results lay the ground work for the generation of human model systems for cardiovascular disease and open novel approaches for human regenerative cardiovascular medicine. A new direction for heart stem cell therapeutics is on the horizon and joint ventures with the private sector are in a position to accelerate this transition from stem cell biology towards true regenerative medicine.

Biasco L Mr 1 Baricordi C Miss 1 Merella S Miss 2 Bartholomae C Dr 3 Ambrosi A Professor 4 Pellin D Mr 4 Di Serio C Professor 4 Von Kalle C Professor 3 Schmidt M Dr 3 Aiuti A Professor 5

1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET); Vita Salute University, Milano, Italy 2 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milano, Italy 3 National Center for Tumor Diseases (NCT), German Cancer Research Center (DKFZ), Heidelberg, Germany 4 CUSSB, Vita Salute University, Milano, Italy 5 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milano, Italy; University of Rome “Tor Vergata”, Rome, Italy Or 86 Uncovering haematopoietic system dynamics in gene therapy treated patients by retroviral tagging

Upon retroviral gene transfer, transduced cells are univocally tagged by vector insertions. We previously showed that ADA-SCID gene therapy (GT) with CD34+ cells and reduced intensity conditioning resulted in, multilineage engraftment, in the absence of aberrant expansion. Long-term studies in these patients provide a unique model to study in depth haemopoietic clonal dynamics by retroviral tagging. We performed a comprehensive multilineage longitudinal insertion profile of bone marrow (BM) (CD34+ , CD15+, CD19+, Glycophorin+) and peripheral blood (PB) (CD15+, CD19+, CD4+, CD8+cells, naïve and memory T cell subpopulations) cells in 4 patients 3-6 years after GT, retrieving to date 1055 and 1999 insertions from BM and PB cell lineages respectively. We could shape the insertional landscape of each lineage through a tri-factorial analysis uncovering the effects of selective advantages in periphery and the frequency of identical integrants in different haematopoietic compartments. BM cells displayed the highest proportion of shared integrants (up to 58.3%), reflecting the real-time repopulating activity of gene-corrected progenitors. Strikingly, we found “core integrants”, shared between CD34+ cells and both lymphoid and myeloid lineages, stably tagging active long-term multipotent progenitors overtime. Tracking two of these integrants (inside a fragile site of MLLT3 gene and downstream the LRRC30 gene) by specific PCRs we confirmed the multilineage contribution to haematopoiesis of these progenitor clones, showing fluctuating lineage outputs over 4 years. The application of mathematical models to our datasets is allowing to uncover new information on the fate and activity of gene corrected haematopoietic progenitors years after transplantation in humans.

Giustacchini A Miss 1 Schira G Miss Saini M Mr 1 Gentner B Dr 1 Naldini L Professor 1

1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Vita Salute San Raffaele University, Milano, Italy Or 87 Function of microRNA-126 in hematopoietic stem cells-implications for gene therapy

Little is known about miRNA function in hematopoietic stem and progenitor cells (HSPC). Using a functional reporter assay, we identified miR-126 as an HSPC-specific miRNA which is rapidly down-regulated upon differentiation, offering significant potential to engineer stem-cell protective, regulated expression cassettes for HSC gene therapy. To shed light on the biological function of miR-126, we generated a stable miR-126 knock-down (kd) or knock-in (ki) in mouse HSPC using lentiviral vectors. Kd or ki cells were competitively transplanted along with congenic, control vector-transduced cells, and hematopoietic chimerism was followed for >1 year in both primary and secondary recipients. No hematopoietic tumors were observed in >30 mice with long-term follow-up. miR-126 kd HSPC displayed enhanced myeloid and B cell contribution, particularly during the early phases of reconstitution, while subsequently reaching steady state. This enhanced contribution could be reproduced after secondary transplantation, again reaching a steady state, with some mice showing signs of exhaustion. On the other hand, miR-126 overexpression resulted in a competitive disadvantage in vivo involving all hematopoietic lineages, paralleled by a complete depletion of miR-126 ki HSC in the BM. While showing normal clonogenic activity in vitro, miR-126 ki cells were decreased as early as 3 days after bone marrow transplantation. Interestingly, KSL ki cells had an increased proliferative index as judged by EdU incorporation, and hematopoietic contribution temporarily recovered before exhaustion. These data suggest that miR-126 ki favors HSC commitment at the cost of self-renewal. We conclude that a precise level of miR-126 is required for robust HSPC function.

Kneissl S Dr 1 Abel T Mr 1 Verhoeyen E Dr 2 Koehl U Professor 3 Johnston I Dr 4 Cosset FL Professor 2 Cichutek K Professor 1 Buchholz CJ Professor 1

1 Division of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany 2 Human Virology Department, Inserm, Lyon, France 3 Paediatric Haematology and Oncology, University Hospital, Frankfurt, Germany 4 Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany Or 88 Specific gene transfer to CD133-positive hematopoietic Progenitor cells

We developed a flexible and highly specific targeting method for lentiviral vectors (LVs) relying on the specificity of single-chain antibodies (scFv) recognizing cell surface antigens. For the generation of such LVs the modified measles virus (MV) glycoproteins hemagglutinin (H), responsible for receptor recognition and fusion (F) protein were incorporated into LVs. The H protein was blinded for the MV receptors and displayed a scFv. Using an anti-CD20 scFv, we could demonstrate targeted gene transfer into primary human CD20-positive lymphocytes. Now, we show the versatility of this technology by targeting CD133-positive human hematopoietic stem cells (HSCs). This population is of extraordinary interest for gene therapy as correction of genetic defects in these cells will be implemented in the whole hematopoietic system.

αCD133-LV selectively transduced human G-CSF mobilized or cord blood CD133/CD34-positive cells and stably transferred the reporter gene to all hematopoietic lineages assessable by a colony forming assay, whereas VSV-G pseudotyped vectors also transduced CD34-negative cells. To determine the targeting capacity of αCD133-LV for strongly underrepresented target cells in presence of other blood cells such as granulocytes, monocytes and lymphocytes, primary human CD133-positive cells were mixed with peripheral blood. αCD133-LV transduced the CD133-positive cells with unimpaired efficiency even when only 5% target cells were present, while the surplus of non-target cells remained largely untransduced. These results are promising for targeted gene transfer into HSCs that might become possible by simply adding αCD133-LV to bone marrow harvests or mobilized peripheral blood samples making the purification of HSCs prior to gene transfer obsolete.

Ruiz Carmen

Or 89 European databases: what can we learn from the “promise” EBMT database?

The European Group for Blood and Marrow Transplantation (EBMT) is a non-profit organisation established in 1974 in order to allow scientists and physicians involved in clinical bone marrow transplantation to share their experience and develop co-operative studies. From the 70's onwards, clinical data started being collected into what is now known as the EBMT Registry.

When the EBMT started collecting data, transplantation was not a main stream procedure. Today it is for some indications and we believe that the continuous collection of Registry data has widened the experience and improved the outcome of transplanted patients. The Registry currently contains clinical data on more than 315,000 patients, submitted from more than 50 countries.

There is a growing desire on the part of donor registries, health departments, cord blood banks, study groups and other outcome registries to be able to access the content of the EBMT Registry and we developeding closer collaborations.

The EBMT overlaps with the community involved in cellular therapies. A Cellular Therapy Med-A (“minimum essential data”) was designed by the Cellular Therapy committee and has been in use for the last 2 years.

We believe that the EBMT could offer its expertise on data collection and part of the Registry infrastructure to kick start a similar data collection exercise in the Cellular Gene Therapy community. We are looking to further enhance our data collection system, to better serve our centres, improve collaboration with existing partners and create new partnerships. An invitation to do just that is offered to the Cellular Gene Therapy community.

Galli Christina

Or 90 Long-term follow-up of patients treated with Gene Therapy Medicinal Products

See supplement

Monaco Lucia Telethon

Or 91 From patient association sponsored research to pharmaceutical development

See supplement

Buchholz C Dr 1

1 Division of Medical Biotechnology, Paul-Ehrlich-Institut, 63225 Langen, Germany Or 92 The Hospital Exemption Regulation – New Options for Gene Therapy?

Until recently, there was only a single option available to bring gene therapy medicinal products to the market, i.e. run through clinical trials to demonstrate safety and efficacy for your product, then apply for marketing authorisation in the centralised procedure at the European Medicines Agency (EMA). With Regulation (EC) No 1394/2007 which amends Directive 2001/83/EC coming into force, Advanced Therapy Medicinal Products (ATMPs) including gene therapy products will be excepted from the obligation of obtaining marketing authorisation via the centralised procedure under certain conditions (so called “Hospital Exemption”). While by this regulation cell and tissue products can be excepted from the centralised procedure that were already on the market before the ATMP Directive came into force and are produced in small scale for individual patients, it may well apply also to certain types of gene therapy products currently under development. Such products could then be put onto the market on the basis of a national authorisation, but without centralised marketing authorisation. This presentation will discuss the implementation of this regulation into national law, the requirements to be fulfilled to fall under this exception as well as its impact on future gene therapy product development.

Bankiewicz Krystof S. MD, PhD 1 Richardson R. Mark MD, PhD 1 Kells Adrian PhD 1 Rosenbluth Kathryn. H. Ph.D 1 Martin Alastair J. PhD 2 Larson Paul MD 1 Starr Philip MD, PhD 1 Piferi Peter G. MBA 3 Bringas John BS 1

1 Department of Neurological Surgery and 2 Department of Radiology, University of California San Francisco, San Francisco, California; 3 SurgiVision Inc., Irvine, California Or 93 Intra-Cerebral Administration of Viral Vectors via MR Guided Convection Enhanced Delivery; Translational Studies

Optimal viral vector delivery into the brain is challenging and brain distribution of viral vectors is uncertain. To address this issue we developed viral vector delivery system that permits direct MRI monitoring of vector distribution within the brain in real-time. This significant advance allows for the first time to adjust parameters of vector infusion while delivering gene therapy, giving surgeon full control over gene transfer technology.

To allow for precise intracerebral delivery of biologics for therapy of neurological disease we developed a skull-mounted aiming device (SmartFrame) and integrated software platform (ClearPoint) for interventional MRI guided placement of deep brain stimulators. In anticipation of upcoming gene therapy clinical trials, we adapted this device for real-time convection enhanced delivery (RCD) of therapeutics via a custom designed infusion cannula. Based on real-time MRI data, this system allows selection of brain targets, provides instructions for cannula insertion along a chosen trajectory, and permits visual monitoring of infusions. The targeting accuracy of this delivery system and infusion cannula performance were validated in nonhuman primates (NHPs). Infusions of an MRI tracer (Gd-DPTA) were delivered to the thalamus (n = 4), subthalamus (4), substantia nigra (n = 1) hippocampus (n = 2) and enthorinal cortex (n = 4), and the targeting error was determined for each cannula placement.

We found that the average absolute targeting error for all targets (n = 15) was 0.8mm (95% CI = 0.14mm). No infusions in any target produced cannula reflux or leakage from the targeted structure, even when infusions occurred in close proximity to a previous needle tract. No hemorrhages occurred during cannula placement and no signs of unexpected tissue damage were seen on histology.

The ClearPoint system allows RCD to be performed with a high level of precision, predictability, and safety. This method may improve the success rate for clinical trials involving intracerebral drug delivery by direct injection.

Our advanced gene delivery system is currently tested for delivery of therapeutic genes in Parkinson's (PD), Huntington's (HD) and Nieman-Pick, AADC deficiency in children and brain tumors. Data will be provided to demonstrate promises and challenges in successful clinical translation of gene transfer technology for CNS disorders.

Azzouz M Professor 1 Valori C Dr 1 Ning K Dr 1 Wyles M Mr 1

1 The University of Sheffield Or 94 Current Advances in Gene Therapy for Spinal Muscular Atrophy

SMA, a recessive autosomal disorder, is one of the most common genetic causes of death in childhood. It is caused by mutations of the survival motor neuron (SMN) gene. We previously reported that Lentiviral vector expressing SMN was successfully used to increase the life expectancy by 5 days ¹ . The marginal efficacy of this therapeutic approach, however, prompted us to explore different strategies for gene delivery to motor neurons to achieve a more clinically relevant result.

The first part of this presentation will describe the efficiency of scAAV9 mediated SMN gene replacement in SMA mouse model. We report that a single systemic injection of SMN-expressing scAAV9 vector reversed the phenotype of SMNΔ7mice ² , a well established animal model of SMA. Most notably, SMN replacement led to substantial increase in the life expectancy, thereby achieving one of the highest therapeutic effects reported in the field to date ³ . The second part will discuss the role of PTEN, a negative regulator of the target of rapamycin (mTOR) pathway, in SMA. AAV6 expressing siPTEN into hind limb muscles at postnatal day 1 in SMNΔ7 mice leads to significant PTEN depletion and robust improvement in motor neuron survival. These studies report promising advances in AAV-based therapy for SMA. We are currently planning to initiate clinical trials using scAAV-based SMN replacement in the very near future.

References

1 Azzouz et al. J. Clin. Inv., 114:1726–1731. 2004. a-567 2 Le et al. Hum Mol Genet, 14:845–857. 2005. a-568 3 Valori et al. Sci. Transl. Med., 235ra422010. a-569 Capotondo A Dr 1 Milazzo R Dr 1 Politi L.S Dr 2 Nonis A Dr 3 Di Serio C Professor 3 Scotti G Professor 2 Naldini L Professor 4 Biffi A Dr 5 1 HSR-TIGET,San Raffaele Scientific Institute, Milan, Italy 2 Neuroradiology Research Group, Imaging Core, San Raffaele Scientific Institute, Milan, Italy 3 University Centre of Statistics for Biomedical Sciences,Vita-Salute University, San Raffaele Scientific Institute, Milan, Italy 4 HSR-TIGET; Vita-Salute University, San Raffaele Scientific Institute, Milan,Italy 5 HSR-TIGET, San Raffaele Scientific Institute, Milan,Italy Or 95 Critical factors affecting microglia reconstitution after hematopoietic stem cell transplantation

The occurrence of progressive myeloid infiltration and microglia reconstitution by donor cells in the brain following transplantation of hematopoietic stem progenitor/cells (HSPC) was demonstrated under specific conditions in several reports. However, the mechanisms of microglia reconstitution after the transplant are not well understood. We investigated the modalities, the kinetic and the factors that could affect this phenomenon in mice. In particular, we performed this study in the mouse model of Metachromatic Leukodystrophy (MLD), a lysosomal storage disorder characterized by the accumulation of the un-degraded substrates in glial cells and neuron, leading to microglia activation and neuroinflammation. After the transplantation of HSPC labeled with magnetic resonance contrast media and upon transduction with lentiviral vectors encoding GFP, we tracked the kinetic of the myeloid infiltration into the brain, in basal conditions or after the administration of different preparatory regimens, using magnetic resonance imaging, cytofluorimetry, tissue pathology and immunostaining. Independently from the conditioning regimen applied, we observed a short-term infiltration of the brain by labeled cells, with a preferential location within areas of neurogenesis or at disease sites. However, in order to obtain a long-term persistence of the transplanted cells into the brain and mature microglia reconstitution the use of conditioning regimens capable of exerting a direct CNS “myeloablative” effect was required. Interestingly, the extent of the microglia reconstitution was proportional to the extent of removal of the endogenous microglia by the conditioning regimen and to the disease burden which renders these cells more susceptible to the regimen-related toxicity.

Ruzo A Dr 1 García M Dr 1 Marcó S Miss 1 Villacampa P Miss 1 Ribera A Mr 1 Ayuso E Dr 1 Haurigot V Dr 1 Ruberte J Dr 1 Bosch F Dr 1

1 Center of Animal Biotechnology and Gene Therapy, Universitat Autònoma de Barcelona Or 96 AAV9-Based sulfamidase gene transfer reverses somatic and neurological pathology in mpsiiia mice

Mucopolysaccharidosis type IIIA (MPSIIIA) is a inherited lysosomal storage disease caused by the deficiency of the sulfamidase enzyme, resulting in intralysosomal accumulation of the glycosaminoglycan (GAG) heparan sulfate. Severe neurodegeneration and other non-neurological alterations lead to death of the affected subjects during adolescence. No effective treatment for MPSIIIA is currently available. Here we used AAV serotype 9 to deliver the sulfamidase gene to somatic tissues as well as the central nervous system of MPSIIIA mice at 2 months of age, when high levels of GAG accumulation are already present. Intravenous administration of sulfamidase-expressing AAV9 vectors resulted in a widespread reversal of lysosomal accumulation in all brain regions, which was parallel to a reduction of neuroinflammatory markers and an improvement of neuromotor alterations of MPSIIIA mice. Intravenous AAV9 treatment also corrected GAG accumulation in all somatic tissues. We also locally delivered AAV9 vectors carrying the sulfamidase gene to the cerebrospinal fluid of MPSIIIA mice, by direct injection into the cisterna magna. This treatment mediated normalization of GAG storage in all brain areas, concomitant with a complete reversal of the lysosomal accumulation in neurons, astrocytes and microglia, and the absence of neuroinflammatory process. Intracisternally treated animals showed also significant correction of GAG accumulation in the somatic tissues, due to leakage of the vector to systemic circulation. In summary, these results may constitute the basis for the development of a novel non-invasive therapeutic alternative for MPSIIIA patients.

Ungari S Dr 1 Visigalli I Dr 2 Gentner B Dr 3 Martino S Dr 4 Ungaro D Dr Sergi L Sergi Dr 2 Cerri F Dr 6 Del Carro U Dr Quattrini A Dr 6 Naldini L Professor 2 Biffi A Dr 2

1 San Raffaele Telethon Institute for Gene Therapy; Vita-Salute San Raffaele University, San Raffaele Scientific Institute, Milan, Italy; 2 San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy; 3 San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy 4 Department of Experimental Medicine and Biochemical Science, Perugia University, Italy; 6 Experimental Neurology, San Raffaele Scientific Institute, Milan, Italy Or 97 Towards a Safe and Effective Gene Therapy Approach for Globoid Cell Leukodystrophy

Globoid cell leukodystrophy (GLD) is a lysosomal storage disorder due to the deficiency of the enzyme Galactocerebrosidase (GALC). The enzymatic deficiency results in the storage of undegraded metabolites in the nervous systems, leading to progressive dysmyelination. We are developing a gene therapy strategy based on hematopoietic stem/progenitor cells (HSPC) and lentiviral vectors (LV) in the murine models of GLD. Our initial efforts were affected by the finding that GALC supra-normal expression in HSPC is responsible for apoptosis and functional impairment of transduced cells. Importantly, mature hematopoietic cells, including the myeloid HSPC progeny infiltrating the nervous system, are not affected by GALC supra-normal expression following gene transfer, suggesting a unique sensitivity of HSPC to enzyme toxicity. According to this evidence, we exploited miRNA-regulated LV for GALC gene transfer: the use of a LV containing the target sequence of an miRNA exclusively expressed in HSPC (miR126), allowed safe transduction of HSPC and GALC expression only in their differentiated progeny. Importantly, this approach led to a significant amelioration of the survival and overall phenotype of the transplanted GLD mice, also as compared to affected animals transplanted with wild type HSPC. Of notice, gene therapy reconstituted the enzymatic activity in the brain of the affected mice and led to correction of the metabolic defect in resident neuronal and glial cells. Overall, our findings provide the proof-of-principle of a novel gene therapy approach, which might provide GLD patients with a new treatment option more efficacious than transplantation of normal donor's HSPC.

Russo Vincenzo

Or 98 Genetically Modified Lymphocytes for Active Immunotherapy

See supplement

Squadrito ML Mr 1 Naldini L Professor 1 De Palma M Dr 1

1 San Raffaele Scientific Institute Or 99 Modulating microRNAs in tumor macrophages to inhibit tumor angiogenesis

TIE2-expressing macrophages (TEMs), an antigenically and functionally defined subpopulation of tumor-associated macrophages (TAMs), have been implicated in the promotion of tumor angiogenesis. In order to identify microRNAs (miRNAs) that could regulate the proangiogenic and protumoral activity of TEMs, we searched for miRNAs differentially expressed in TEMs vs. TIE2^– (or inflammatory) TAMs. Because miRNAs are sometimes lodged within intronic gene regions, we searched for intronic miRNA consensus sequences within genes that we previously found to be upregulated in TEMs vs. inflammatory TAMs. We found one gene highly expressed by TEMs but not by inflammatory TAMs that contains a previously undescribed intronic miRNA (int-miRNA). We validated the expression of the int-miRNA by qPCR and found it to be significantly upregulated in tumor-derived TEMs vs. inflammatory TAMs. By using multiple strategies, including intronic microRNA overexpression, we identified the active strand of this miRNA and measured its activity in vivo by using a lentiviral vector-based reporter system. Our data indicate that the int-miRNA is specifically active in TEMs among the circulating and tumor-infiltrating myeloid cells. Finally, in vitro data as well as bioinformatic analyses suggest the int-miRNA may be involved in regulating TEM motility in tumors. We are currently analyzing predicted target genes that could be regulated by the int-miRNA at the post-transcriptional level in TEMs that over-express the miRNA sequence. These data identify a novel miRNA, which is characterized by a remarkably specific expression pattern among TAMs and that might be targeted to improve anticancer therapies.

Van de Velde A Dr 1 Van Driessche A Dr 1 Sugiyama H Professor 2 Price DA Professor 3 Anguille S Dr 1 Cools N Dr 1 Berneman ZN Professor 1 Van Tendeloo VFI Professor 1

1 Vaccine & Infectious Disease Institute (Vaxinfectio), University of Antwerp University & CCRG, Antwerp University Hospital, Antwerp, Belgium 2 Osaka University Graduate School of Medicine, Osaka, Japan 3 Cardiff University School of Medicine, Cardiff, Wales, UK Or 100 WT1-targeted dendritic cell vaccine elicits clinical and immunological responses in AML patients

Background: Active immunization using tumor antigen-loaded dendritic cells holds promise for the adjuvant treatment of cancer to eradicate or control residual disease, but so far most dendritic cell trials have been performed in end-stage cancer patients with high tumor loads.

Methods: in a phase I/II trial, we investigated the effect of autologous dendritic cell vaccination in 10 patients with acute myeloid leukemia (AML). The Wilms' tumor 1 protein (WT1), a nearly universal tumor antigen, was chosen as an immunotherapeutic target because of its established role in leukemogenesis and superior immunogenic characteristics.

Results: Two patients in partial remission after chemotherapy were brought into complete remission following intradermal administration of full-length WT1 mRNA-electroporated dendritic cells. In these 2 patients, and in 3 other patients who were in complete remission, the AML-associated tumor marker returned to normal following dendritic cell vaccination, compatible with the induction of molecular remission. Clinical responses were correlated with vaccine-associated increases in WT1-specific CD8⁺ T-cell frequencies, as detected by peptide/HLA-A*0201 tetramer staining, and elevated levels of activated natural killer (NK) cells post-vaccination. Furthermore, vaccinated patients showed increased levels of WT1-specific IFN-γ-producing CD8⁺ T-cells and features of general immune activation.

Conclusions: These data support the further development of vaccination with WT1 mRNA-loaded dendritic cells as a post-remission treatment to prevent full relapse in AML patients.

Manzo T Mrs 1 Michelini R Hess Dr 1 Basso V Mrs 1 Freschi M Dr 1 Schumacher T Dr 2 Bellone M Dr 3

1 San Raffaele Scientific Institute, Milan 20132, Italy; 2 The Netherlands Cancer Institute, Amsterdam, Netherlands; 3 an Raffaele Scientific Institute, Milan 20132, Italy Or 101 Immunotherapy for prostate cancer with tumor and minor histocompatibility antigen-specific TCR-transferred T cells

We have demonstrated that the concomitance of minor histocompatibility (H) antigen- and high-affinity tumor-specific T cell responses found in allogeneic hematopoietic transplant recipients cause the eradicating of autochthonous prostate mouse adenocarcinoma (Hess Michelini, 2010). As in most cases donors might lack high affinity T cells specific for tumor-associated antigens, we have investigated the possibility to supplement donor T cells with sizeable frequencies of high-affinity tumor-specific TCR-transferred lymphocytes. Furthermore, as graft-versus-host disease (GVHD) is the major complication of allotransplantation, we also investigated whether combining tumor- and minor H antigen-restricted TCR-transferred T cells might confer graft versus tumor without evoking GVHD. We report that while allogeneic donor T cells or high affinity tumor-specific TCR-redirected CD8⁺ T cells per se are insufficient in causing disease eradication, their concomitant infusion promotes tumor regression. TCR-transferred cells promptly respond to tumor-specific vaccines and, although showing limited persistence in peripheral lymphoid organs, promote tumor infiltration and eradication. Likewise, the infusion of tumor-specific TCR-transferred T cells together with a limited number of minor H antigen-specific TCR-engineered T cells reveal therapeutically effective, in the absence of signs of GVHD. Thus, together these data support the value of combining TCR-transferred T cells specific for tumor and minor H antigens for the immunotherapy of prostate cancer.

Kaneko S Dr 1 Nishimura T Mr 1 Goto H Mr 1 Takayama N Dr 1 Tajima Y Ms 1 Shimizu T Dr 1 Iriguchi S Mr 1 Otsu M Dr 1 Eto K Dr 1 Nakauchi H Professor 1

1 Div Stem Cell Therapy, The Institute of Medical Science, The University of Tokyo, 4-6-1, shriokanedai, minatoku, Tokyo, 108-8639, Japan Or 102 Generation of monoclonal TCR-expressing human T-lineage cells from induced pluripotent stem cells of single peripheral T-lymphocyte origin

Adoptive immunotherapy is a promising approach to cancer immunotherapy that circumvents some of the limitations of active immunotherapy. However, its effectiveness is often hampered by exhaustion of antigen-specific T cells. To address this issue, we utilized induced pluripotent stem cells (iPSCs) generated from human peripheral T cells (T-iPSCs). Rearrangement of T-cell receptor (TCR) genes was maintained in these T-iPSCs which, because T-iPSCs have unlimited self-renewal capacity, could be expanded ex vivo. These T-iPSCs were then re-differentiated into CD3+TCRαβ + functional T-lineage cells in vitro with higher efficiency than was possible for embryonic stem cells, iPSCs derived from fibroblasts, or cord blood cells. Complete monoclonality of productive TCR transcripts, transcribed from the genome inherited from the original progenitor T cell, was observed in re-differentiated T-lineage cells: No signature of de novo TCR gene rearrangement could be identified. The technology described opens a way for effective T-cell therapy with antigen-specific monoclonal TCR.

Giacca Mauro Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy

Or 103 Searching for genes that induce myocardial protection, myocardial repair or neoangiogenesis by in vivo gene transfer using AAV vectors

The identification of novel genes and pathways controlling prenatal cardiomyocyte proliferation, regulating cardiomyocyte survival during the adult life or promoting new blood vessel formation in ischemic conditions holds paramount interest in view of developing new therapeutic approaches for patients with ischemic cardiomyopathy and heart failure. Over the last several years, we have exploited the potential of viral vectors based on the Adeno-Associated Virus (AAV) to explore the therapeutic potential of several genes in small and large animal models of cardiovascular disorders. AAV vectors have the capacity to transduce myocardial cells in vitro and in vivo with high efficiency and to promote the expression of their transgenes for indefinite periods of time.

These studies have led to the identification of a few novel regulators of cardiomyocyte function (among these, VEGFB, a selective activator of cardiomyocyte-expressed VEGFR1, which protects against pacing-induced heart failure in dogs) and cardiomyocyte proliferation (e.g. Jagged1, which induces neonatal cardiomyocyte expansion through the Notch1 pathway), as well as disclosing the role of defined mononuclear cell populations in the regulation of blood vessel formation.

More recently, we have undertaken an exhaustive approach in the identification of factors with beneficial effect upon myocardial damage by the construction of library of AAV vectors delivering the whole mouse secretome (1500 + mouse secreted proteins). Preliminary screening of a subset of this library has led to the identification of peptide hormone ghrelin as a powerful cardioprotective agent.

Reginato Miss S. 1 Gianni-Barrera R Mr 1 Heberer M. Professor 2 Banfi A. Dr 1

1 Cell and Gene Therapy, Department of Biomedicine, Basel University Hospital, Switzerland 2 Department of Surgery, Basel University Hospital, Switzerland Or 104 Coordinated co-expression of PDGF-BB accelerates stabilization of VEGF164-induced angiogenesis

Background: VEGF induces normal or aberrant angiogenesis depending on its expression level in the microenvironment around each transduced cell. Therefore even rare hotspots of high expression should be avoided, making it difficult to achieve safety and efficacy with direct gene delivery. Further, 4 weeks of sustained expression are required to achieve vascular stabilization. We found that co-expression of PDGF-BB from a single bicistronic construct induced only normal angiogenesis despite high and heterogeneous VEGF levels. Here we tested its effects on vascular stabilization.

Methods: We expressed mVEGF₁₆₄, hPDGF-BB or both in muscle implanting retrovirally transduced myoblasts. VEGF signaling was abrogated at defined time-points by systemic VEGF-Trap treatment.

Results: Two weeks after implantation, VEGF-induced vessels all regressed, whereas 50% of induced vessels already stabilized with PDGF-BB co-expression.

To investigate the role of factor dose, we implanted clonal populations homogeneously producing specific levels of VEGF, with or without PDGF-BB.

At low VEGF levels, 2% and 30% of new vessels were stable after 2 and 3 weeks respectively. PDGF-BB moderately improved these stabilization rates to 10% and 50%. However, at high levels, VEGF alone caused aberrant vessels that never stabilized, whereas PDGF-BB induced normal capillaries of which 50% and 80% were already VEGF-independent after 2 and 3 weeks.

Conclusions: PDGF-BB co-expression both prevented aberrant angiogenesis by heterogeneous and high VEGF levels and accelerated vascular stabilization in a time-frame compatible with short-term expressing viral vectors. Therefore, PDGF-BB co-expression could overcome the intrinsic limitations of direct VEGF gene delivery.

Zentilin LZ Dr 1 Lionetti VL Dr 2 Puligadda UP Dr 1 Moims SM Dr 1 Zacchigna SZ Dr 1 Pepe MP Dr 3 Mamdani MM Dr 3 Recchia FR Professor 4 Giacca MG Professor 1

1 ICGEB, Trieste, Italy 2 Scuola Superiore Sant'Anna, Pisa, Italy 3 New York Medical College, Valhalla, NY, United States 4 Scuola Superiore Sant'Anna, Pisa, Italy and New York Medical College, Valhalla, NY, United States Or 105 Potent cardioprotective effect exerted by AAV-VEGF-B in small and large animal models of myocardial

VEGF-B, a member of the vascular endothelial growth factor family, is emerging as a major cardiovascular protecting factor.

We have recently observed, in a rat model of acute myocardial infarction, that the intramyocardial injection of AAV2-VEGF-B167, which selectively binds VEGFR-1, significantly improves both regional and global cardiac contractility and maintains favorable tissue remodeling in the absence of a significant angiogenic response. We found that cardiomyocytes expressed physiologically active VEGFR-1, VEGFR-2 and Neuropilin-1.

Histological and molecular analysis revealed that VEGF-B is capable to elicite a compensatory, hypertrophic response and a marked antiapoptotic effect both in vitro on isolated neonatal cardiomyocytes and in vivo after induction of ischemia.

Consistent with this conclusion, we found that VEGF-B exerts a similarly striking, angiogenesis-unrelated cardioprotective effect in a pacing-induced model of non-ischemic heart failure in chronically instrumented dogs. Control animals subjected to 4 weeks of pacing suffered from an overt congestive heart failure; on the contrary, at the same time point, intramyocardial AAV9-mediated VEGF-B expression maintained heart contractility and prevented LV wall thinning.

Also in this model, apoptosis was significantly reduced in myocardium expressing VEGF-B. Consistently, protecting signaling through Akt was superphysiological in AAV-VEGF-B treated tissue, while there was a reduction or normalization of the activity of the pro-apoptotic intracellular mediators GSK-3β and FoxO3a. Cardiac VEGFR-1 expression was reduced four-fold in paced dogs, suggesting that exogenous VEGF-B exerted a compensatory receptor stimulation.

Together, these results point toward VEGFR-1 signaling in the heart as an important mediator of cardiomyocyte function, with clear therapeutic implications.

Abel T. Mr 1 Kneissl S. Dr 1 Kontermann R. Professor 2 Cichutek K. Professor 1 Buchholz CJ. Professor 1

1 Division of Medical Biotechnology, Paul-Ehrlich-Institut, 63225 Langen 2 Institut für Zellbiologie und Immunologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart Or 106 Selective gene transfer to endothelial cells using lentiviral vectors

Endothelial cells lining the inner side of blood vessels form an attractive target cell population for a number of different gene therapy concepts. To generate endothelial cell specific vectors we applied a re-targeting approach recently demonstrated for B lymphocytes via CD20 (Funke et al., 2008). This technology relies on the pseudotyping of lentiviral vectors with engineered measles virus glycoproteins displaying a single-chain antibody (scAb) for target recognition. For the targeting of endothelial cells a human endoglin (CD105) and a murine CD105 specific scAb were applied in the system.

The human CD105-targeting vector efficiently transduced CD105-positive HT1080cells and, interestingly, was almost 10-fold more efficient on different types of primary human endothelial cells. Gene transfer was stable in HUVEC cells and did not alter their phenotype as demonstrated by long-term cultivation and matrigel tube formation assays. Selectivity was demonstrated in cocultures of endothelial cells and lymphocytes. While VSV-G pseudotyped vectors did not discriminate between both cell types, the endoglin targeting vector selectively transduced HUVEC cells cocultivated with primary human lymphocytes (PBMC). The murine CD105-targeting vector was similarly specific and efficiently transduced primary CD105+murine cells isolated from heart tissue. In vivo applications of this targeting vector transferring a luciferase reporter gene are currently ongoing.

The data suggest that lentiviral endoglin-targeting vectors allow the specific genetic modification of endothelial cells and may thus improve the specificity of in vivo gene transfer applications.

Koornneef A Dr 1 Maczuga P Mr 1 van Logtenstein R Mr 1 Borel F Ms 1 Blits B Dr 1 Ritsema T Dr 1 van Deventer S Professor 2 Petry H Dr 1 Konstantinova P Dr 1

1 AMT, Meibergdreef 61, 1105 BA Amsterdam, the Netherlands 2 LUMC, Albinusdreef 2, 2333 ZA Leiden, the Netherlands Or 107 Apolipoprotein B100 knock-down by AAV-delivered shRNA lowers cholesterol in mice

Serum low-density lipoprotein cholesterol (LDL-C) levels are proportionate to the risk of atherosclerotic cardiovascular disease. In order to reduce serum total cholesterol and LDL-C levels, we used RNA interference to inhibit expression of the structural protein of LDL-C, apolipoprotein B100 (ApoB). We developed and screened 19 short hairpin RNAs targeting conserved sequences in human, mouse, and macaque ApoB mRNAs (shApoBs) and subsequently narrowed our focus to one candidate for in vivo testing in wild-type mice. Recombinant adeno-associated virus (AAV) was used for long-term transduction of murine liver with shApoB. We observed a strong dose-dependent knock-down of ApoB mRNA and protein levels, which correlated with a reduction in total cholesterol levels, without obvious signs of toxicity. Furthermore, shApoB was found to specifically reduce LDL-C in diet-induced dyslipidemic mice, while high-density lipoprotein cholesterol (HDL-C) remained unaffected. Finally, we demonstrated elevated lipid accumulation in murine liver transduced with shApoB, a known phenotypic side-effect of lowering ApoB levels. These results demonstrate a robust dose-dependent knock-down of ApoB by AAV-delivered shRNA in murine liver, thus providing an excellent candidate for development of RNAi-based gene therapy.

Auricchio Alberto Telethon Institute of Genetics and Medicine (TIGEM); Medical Genetics, Dept. of Pediatrics, Federico II University, Napoli, Italy

Or 108 AAV vectors for gene therapy of retinal degeneration

AAV vectors are safe, efficient and versatile tools for gene transfer to the retina. The availability of dozens of different serotypes with distinct transduction characteristics, appropriate regulatory elements and strategies to overcome the vector limited cargo capacity allow to design efficient protocols for retinal gene transfer in small and large animal models.

Recently, the safety and efficacy of AAV-mediated gene transfer to the retina of patients with a severe form of inherited childhood blindness has been shown, paving the way for additional trials for other blinding conditions.

Ali RR Professor 1 Bainbridge JW Dr 1 Smith AJ Dr 1 Robbie S Dr 1 Fitzke FW Professor 1 Holder GE Professor 1 Stockman A Professor 1 Yzer Z Dr 2 Van den Born I Professor 2 Rubin GS Professor 3

1 UCL Institute of Ophthalmology and Moorfields Eye Hospital Biomedical Research Centre for Ophthalmology 2 Rotterdam Eye Hospital 3 AUCL Institute of Ophthalmology and Moorfields Eye Hospital Biomedical Research Centre for Ophthalmology Or 109 Clinical trial of gene therapy for early onset severe retinal dystrophy resulting from defects in RPE65

Early-onset severe retinal dystrophy caused by defects in the gene encoding the retinal isomerase RPE65 is associated with poor vision at birth and complete loss of vision in early adulthood. In a phase I/II dose-escalation trial, we have delivered subretinally recombinant adeno-associated virus (rAAV) vector expressing RPE65 under the control of an RPE65 promoter in 9 human subjects with early onset severe retinal dystrophy associated with mutations in RPE65. We have examined systemic vector dissemination and immune responses following vector delivery, assessed visual function pre- and post- vector delivery using a range of psychophysical techniques, and performed detailed electrophysiology and retinal imaging studies.

There have been no serious adverse effects of surgical delivery of vector in the subjects enrolled to date. We have detected no systemic dissemination of vector genome. Although we have detected an increase in systemic neutralising antibodies to AAV capsid in two subjects but there have been no evidence of immune responses to RPE65 protein. We have measured significant improvements in retinal sensitivity by microperimetry and dark-adapted perimetry, and improved performance in a test of visually-guided mobility. The outcomes in the first 9 subjects to date suggest that subretinal delivery of rAAV vector can be safe in humans in the short term and can improve retinal sensitivity. These findings support further clinical studies in subjects with RPE65 deficiency and the development of gene therapy for other inherited retinal disorders.

Rama P Dr 1 Matuska S Dr 2 Paganoni G Dr 2 Spinelli A Dr 2 De Luca M Professor 1 Pellegrini G Professor 1

1 Centre for Regenerative Medicine “Stefano Ferrari”, University of Modena and Reggio Emilia, Modena, Italy 2 San Raffaele Scientific Institute, Ophthalmology Unit, Milan, Italy Or 110 Limbal Stem-Cell Therapy and Long-Term Corneal Regeneration

Background: Corneal renewal and repair are mediated by stem cells of the limbus. Ocular burns may destroy the limbus, causing limbal stem-cell deficiency. We investigated the long-term clinical results of cell therapy in patients with burn-related corneal destruction associated with limbal stem-cell deficiency, a highly disabling ocular disease.

Methods: We used autologous limbal stem cells cultivated on fibrin to treat 112 patients with corneal damage, most of whom had burn-dependent limbal stem-cell deficiency. Clinical results were assessed by means of Kaplan–Meier, Kruskal–Wallis, and univariate and multivariate logistic-regression analyses. We also assessed the clinical outcome according to the percentage of holoclone-forming stem cells, detected as cells that stain intensely (p63-bright cells) in the cultures.

Results: Permanent restoration of a transparent, renewing corneal epithelium was attained in 76.6% of eyes. The failures occurred within the first year. Restored eyes remained stable over time, with up to 10 years of follow-up. In post hoc analyses, success — that is, the generation of normal epithelium on donor stroma — was associated with the percentage of p63-bright holoclone-forming stem cells in culture. Cultures in which p63-bright cells constituted more than 3% of the total number of clonogenic cells were associated with successful transplantation in 78% of patients. In contrast, cultures in which such cells made up 3% or less of the total number of cells were associated with successful transplantation in only 11% of patients. Graft failure was also associated with the type of initial ocular damage and postoperative complications.

Conclusions: Cultures of limbal stem cells represent a source of cells for transplantation in the treatment of destruction of the human cornea due to burns.

Colella P Dr 1 Iodice C Dr 1 Di Vicino U Mr 1 Auricchio A Professor 2

1 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy 2 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy; Medical Genetics, Dept. of Pediatrics, Federico II University, Naples, Italy Or 111 Gene Transfer of Erythropoietin Derivatives protects from photoreceptor degeneration

Gene therapy based on delivery of neurotrophic factors provides a mutation-independent approach for inherited retinal degenerations (IRDs) that is desirable given their high genetic heterogeneity. The cytokine Erythropoietin (EPO) exerts protective functions in several tissues. We have previously reported that systemic adeno-associated viral (AAV) vector-mediated EPO delivery sustains retinal function and photoreceptors survival in IRDs models. However the EPO erythrodifferentiating function is associated with undesirable systemic side effects. Recently EPO derivatives that are tissue-protective but non-erythropoietic have been identified. To test their therapeutic efficacy in IRDs animal models and to understand EPO neuroprotective mechanisms in the retina, we have delivered AAV2/1 vectors expressing either EPO, the mutant S100E (EpoS100E) or two EPO-derived peptides to either muscle or retina of: light-damaged albino rats, the Aipl1^-/- and rds models of IRDs. We observed efficient EPO or S100E production from transduced tissues in the absence of significant hematocrit increase when expressing EPO derivatives. Systemic as well as intraocular gene delivery of EPO derivatives protected both retinal function and morphology from light-damage similarly to EPO. However systemic delivery was most effective. The effect of EPO and EPO derivatives gene delivery to the retina of Aipl1-/- and rds mice is being evaluated. Our data suggest that EPO derivatives protects from photoreceptor degeneration without erythropoietic activity.

Millington-Ward S Dr 1 O'Reilly M Dr 1 Chadderton N Dr 1 Palfi A Dr 1 Wolfrum U Professor 2 Goldmann T Dr 2 Kilty C Ms 1 Humphries P Professor 1 Kenna PF Dr 1 Farrar GJ Professor 1

1 Department of Genetics, Trinity College Dublin, Ireland 2 Johannes Gutenberg Universitaet, Mainz, Germany Or 112 Functional benefit in a mouse model for dominant retinitis pigmentosa following gene therapy

The development of gene therapies, targeting the primary genetic lesion, for many dominantly inherited diseases has been severely impeded by their inherent mutational heterogeneity. This is clearly demonstrated by the blinding disorder rhodopsin-linked autosomal dominant retinitis pigmentosa (RHO-adRP) in which well over 100 different mutations in the rhodopsin gene (RHO) have been characterised. In this study we have developed a mutation-independent AAV therapy for RHO-adRP administered to a murine disease model, Pro347Ser.

AAV2/5 vectors express a short hairpin RNA (shRNA), which targets both human and murine rhodopsin transcript. In addition, vectors express a replacement RHO gene, which is resistant to suppression due to five nucleotide alterations at the shRNA target site, all of which are at degenerate positions of the RHO coding sequence. Hence replacement genes encode wild type protein. Suppression and Replacement AAVs were subretinally injected into 5-day old Pro347Ser mice. Therapeutic benefit was determined by histology and electrophysiology.

The approach outlined in this study is potentially applicable to many dominantly inherited disorders where mutational heterogeneity demands a mutation-independent therapy.

Wadsworth S Dr 1

1 49 New York Ave, Framingham MA 01701-9322, USA Or 113 Gene Therapy for the Treatment of wet-AMD

VEGF plays a critical role in neovascular age-related macular degeneration and proliferative diabetic retinopathy. VEGF antagonists are useful for treating such disorders; however current treatments require monthly intravitreal injections. We have designed a soluble anti-VEGF molecule (sFLT01) and delivered it by intravitreal injection of an AAV vector. We have shown that AAV2-sFLT01 inhibits neovascularization in murine disease models. In the eyes of cynomolgus monkeys, AAV2-sFLT01 gives expression levels persistent for at least one year. We also performed laser-CNV experiments 5 months after vector administration in non-human primates and show that sFLT01 is effective at inhibiting neovascularization. We have now completed a 12 month safety study of AAV2-sFLT01 administered intravitreally in cynomolgus monkeys. Animals were dosed with vehicle, 2x10⁹or 2x10¹⁰ vector particles/eye. Electroretinograms, fluorescein angiograms and tonometry were assessed, and no test article-related findings were observed in any group. Indirect ophthalmoscopy and slit lamp exams revealed a mild to moderate vitreal haze and cells in the high dose group only, beginning at 1 month with most animals in this group affected by 3 months. This response resolved over time in the majority of animals. Histological evaluation of the eyes in this study found no structural changes in any part of the eye including the retina. In conclusion we have demonstrated long term efficacy with minimal side effects following intravitreal delivery of AAV-sFLT01 in rodents and non-human primate models. These results suggest an alternate method of long term treatment for diseases of ocular neovascularization without the need for repeated intraocular injections.

Piccolo P Dr 1 Dindot S Dr 2 Grove N Mr 3 Palmer D Dr 3 Brunetti-Pierri N Dr 1

1 Telethon Institute of Genetics and Medicine, Naples, Italy 2 College of Veterinary Medicine and Biomedical Sciences, Veterinary Pathobiology, Texas A&M University, College Station, TX, USA 3 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA Or 114 Intrathecal injection of HDAd vectors results in long-term transgene expression in neuroepithelial cells and neurons

Helper-dependent adenoviral (HDAd) vectors are devoid of all viral genes and result in long term transgene expression in the absence of chronic toxicity. Because of their ability to infect post-mitotic cells, including cells of the central nervous system (CNS), they are particularly attractive for brain-directed gene therapy. However, the blood-brain-barrier limits transduction of brain cells by HDAd vectors injected intravenously. Administration of viral vectors into the cerebrospinal fluid (CSF) through injection in the cisterna magna or lumbar puncture is an attractive approach to achieve widespread transgene expression in the CNS. Moreover, intrathecal injection is far less invasive than intracerebral injection. We show that intrathecal injection of HDAd results in mice in extensive transduction of neuroependymal cells, minimal toxicity, and sustained transgene expression for up to one year post-injection. We also demonstrate for the first time that HDAd delivered by intrathecal injections results in transduction of neuronal cells. These findings are significant because neuronal cells are important targets for correction of neurodegenerative diseases and long term expression is crucial for the treatment of genetic diseases. HDAd vector-mediated transduction of neuroepithelial cells and secretion of the therapeutic gene into the CSF may allow delivery of the transgene product to the whole CNS through the ventricular circulation and phenotypic correction of brain diseases with diffuse involvement, such as lysosomal storage disorders.

Carrer A. Dr Zacchigna S. Dr Moimas S. Dr Zentilin L. Dr Giacca M. Professor

Or 115 Recruitment of Neuropilin-1-expressing Mononuclear Cells (NEMs) by AAV2-Sema3A contributes to vessel stabilization and inhibits tumor growth

Initially discovered for their role in axon guidance, class 3 semaphorins, along with their neuropilin receptors (Nrp1 and Nrp2) have recently been implicated in angiogenesis. We have recently reported the ability of AAV-Sema3A to recruit a population of mononuclear cells expressing Nrp1 (CD11b+/Nrp1+/Gr1-), which are crucial for vessel maturation (Nrp1-Expressing Mononuclear cells, NEMs; J. Clin. Invest. 2008, 118, 2062). In addition, AAV-Sema3A exerted marked antitumoral activity by improving pericyte coverage of tumor blood vessels and restoring tissue normoxia (J. Clin. Invest. 2009, 119, 3356).

In order to characterize the anti-tumor role of NEMs, CD11b+/Nrp1+/Gr1- cells were isolated either from the bone marrow or from AAV-Sema3A-injected muscles, and administered to tumor-bearing animals. In both models, NEMs counteracted tumor growth. Consistent with the above findings, NEM-treated tumors exhibited a more mature vascular network, associated to a higher degree of a-SMA + /NG2 + mural cell coverage (3-fold) and decreased vascular leakiness (30% reduction), suggesting a role for NEM in triggering vessel normalization.

In addition, Sema3A expression inhibited the recruitment of pro-angiogenic tumor-associated macrophages (TAM), as unveiled by F4/80 staining of tumor sections. Phenotypic characterization of NEMs, purified by cell sorting, magnetic beads or laser microdissection, revealed that these are a unique population of bone marrow-derived cells, characterized by an expression profile reminiscent of M1 polarized macrophages. NEMs were in no case found incorporated in the vasculature, nor they displayed angiogenic properties, however, they contributed to vessel maturation through a paracrine effect, ensuing in the activation and proliferation of tissue-resident mural cells.

Casucci M Dr 1 Falcone L Ms 1 Camisa B Mrs 1 Magnani Z Dr 1 Bordignon C Professor 2 Dotti G Professor 3 Bonini C Dr 1 Bondanza A Dr 1

1 Experimental Hematology Unit, S Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, ITALY 2 Vita-Salute University, S Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, ITALY 3 Center for Advanced Cell and Gene Therapy, Baylor College of Medicine, Houston TX Or 116 Targeting of myelogenous leukemia stem cells through genetic redirection of T cells against CD44v6

Myelogenous leukemia is organized as a hierarchy dominated by leukemia stem cells (LSC). LSC are resistant to radiochemotherapy and ultimately responsible for relapse. The results of allogeneic hemopoietic cell transplantation suggest that LSC are sensitive to adoptive T-cell therapy. Nevertheless, the frequent occurrence of HLA-loss variants after transplantation recommends the targeting of antigens crucial to LSC biology. LSC self-renewal depends on signals provided within the bone marrow (BM) stem-cell niche, among which those mediated by CD44 molecules. With the aim of targeting LSC, we constructed a second-generation chimeric antigen receptor (CAR) based on the fusion of the single-chain fragment of a CD44v6-specific mAb with the signaling domains of the TCR z chain and CD28 (CD44v6CAR.28z). Retroviral-mediated gene transfer after stimulation with beads coupled to anti-CD3 and anti-CD28 mAb allowed robust and stable transgene expression and selective elimination of CD44v6-expressing cells, including primary leukemias, but not of healthy hemopoietic stem cells. Redirected T cells were T central memory cells and expressed the BM “addressin” CXCR4. During culture with IL-7 and IL-15, we observed the selective enrichment of CD44v6-redirected T cells, possibly through cross-stimulation by CD44v6 low-expressing activated T cells. When cultured with autologous monocytes, which also express CD44v6, redirected T cells expanded significantly. Co-transfer of the suicide gene HSV thymidine kinase enabled the elimination of redirected T cells, providing a safety switch in case of unexpected toxicity. Altogether these results warrant the clinical implementation of this novel strategy of T-cell gene therapy for the safe and effective eradication of LSC.

ESGCT 2010 Poster Presentations

Lovric JL Dr 1 Mano MM Dr 1 Zentilin LZ Dr 1 Giacca MG Professor 1

1 ICGEB, Padriciano 99, Trieste - Italy P 1 Increased AAV transduction in postmitotic tissues correlates with the downregulation of DNA-damage response machinery

Despite the broad success of rAAV vectors, several aspects of their biology still remain obscure. In particular, limited information is available on molecular determinants of AAV tissue permissivity. Previously, we have demonstrated that the host cell DNA damage response machinery and, in particular, the proteins involved in double-stranded DNA break repair (DSB) (Mre11-Rad50-Nbs1 (MRN) complex), interact and negatively regulate incoming rAAV genome processing.

Since in vivo AAV transduction is particularly efficient in postmitotic tissues such as heart, muscle, brain and retina, we envisioned elucidating the molecular mechanisms underlying this high rAAV permissivity and to specifically understand whether it could be mediated by the diminished expression of proteins involved in DSB repair in terminally differentiated cells.

In a comprehensive analysis using tissue extracts from adult mice, we observed decreased expression levels of MRN complex in postmitotic tissues in comparison with tissues with high proliferative activity known to be poorly permissive of AAV transduction.

Accordingly, in vitro, in a cell model of myoblast differentiation and in primary cultures of neonatal murine cardiomyocytes, we also demonstrated that permissivity to rAAV transduction increased with the cell differentiation status. Consistently, we observed a gradual decrease in the expression of the MRN complex along the process of myoblast differentiation.

In vivo, the increased cardiac transduction upon systemic delivery of AAV correlated with abrupt decrease of MRN expression from day 7 after birth.

These results indicate that the downregulation of the DNA damage response machinery in postmitotic cells results in the stabilization of rAAV genomes leading to increased functional transduction.

Nicolas A Mr 1 Alazard-Dany N Mrs 1 Biollay C Ms 2 Arata L Miss 2 Jolinon N Miss 1 Kuhn L Mrs 3 Ferro M Mrs 3 Weller SK Mrs 4 Epstein AL Mr 5 Greco A Mrs 5 Salvetti A Mrs 1

1 INSERM U758, ENS, Lyon F-69007, France 2 CNRS UMR5534, Villeurbanne, F-69622, France 3 INSERM, U880 CEA, Grenoble, F-38054, France 3 INSERM, U880 CEA, Grenoble, F-38054, France 4 University of Connecticut Health center, Farmington, Connecticut 06030-3205, USA 5 CNRS UMR5534, Villeurbanne, F-69622, France P 2 Deciphering AAV replication strategy in the presence of HSV-1

Recombinant AAV vectors are currently produced using either adenovirus (Ad), baculovirus, or herpes virus (HSV) helper activities. Understanding how AAV replicates under these different conditions is critical to further optimize vector production. This study is focused on the analysis of AAV replication in the presence of HSV-1, a powerful helper virus for vector production. We previously showed that nine HSV-1 factors were able to support AAV rep gene expression and genome replication (Alazard-Dany et al. PLoS Pathogens 2009). To further elucidate the strategy of AAV replication in the presence of HSV-1, we have recently undertaken the proteomic analysis of cellular and HSV-1 factors associated with Rep proteins during AAV replication. This study resulted in the identification of approximately 60 cellular proteins among which factors involved in DNA and RNA metabolism represented the largest functional categories. Finally, several HSV-1 factors were also found associated to Rep among which UL12. We demonstrated here for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms. Altogether these results indicate that, in the presence of HSV-1, AAV may replicate using a basal set of cellular DNA replication enzymes but also extensively relies on HSV-1-derived proteins for its replication, among which UL12 a newly discovered helper factors. They also suggest that AAV may be able to differentially adapt its replication strategy to the nuclear environment induced by the helper virus.

Alba R Dr 1 Bradshaw AC Dr 1 Mestre-Frances N Dr 2 Verdier JM Dr 2 Henaff D Dr 3 Baker AH Professor 1

1 British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow G12 8TA, UK 2 Inserm U710, EPHE, University of Montpellier 2, Montpellier, F-34095 France 3 Institut Génétique Moléculaire de Montpellier, Montpellier, France P 3 Transduction profiles of FX-binding ablated Ad5 vectors in non-human primates (Microcebus murinus)

The mechanism underlying Adenovirus type 5 (Ad5) entry into hepatocytes has been recently described. We demonstrated that coagulation factor X (FX) binds to Ad5-hexon protein at high affinity and this interaction mediates hepatocyte transduction following intravenous (i.v.) delivery into rodent models. FX-binding ablated Ad5 vectors have been genetically engineered to ablate the interaction with FX resulting in substantially reduced hepatocyte transduction following i.v. administration in mice and rats. However, many other interactions and anatomical features, including fenestrae size in liver sinusoids and interactions with key tropism-determining capsid proteins can differ between different species. The effect of FX-ablation on liver gene transfer in non-rodent models has not been reported. Here, we document the transduction profiles and viral genome accumulation of Ad5 and FX-binding ablated Ad5 vector in non-human primates (Microcebus murinus) following the i.v. delivery of 1×10¹¹ viral particles/animal. Transduction profiles analyzed at 48 h showed FX-binding mutated Ad5 vectors precluded liver transduction while Ad5 showed efficient liver gene transfer compared to FX-binding ablated Ad5 vectors (p < 0.05). Viral genome accumulation at 48h showed FX-binding ablated Ad5 vectors presented lower accumulation of viral genomes in most organs analyzed compared to Ad5. Of note, higher amounts of FX-binding ablated Ad5 viral genomes were found in the spleen, as previously observed in rodents, but at considerably lower levels than those observed for Ad5. In summary, FX-binding ablated Ad5 vectors showed similar biodistribution profiles in non-human primates than in rodent models.

Sweeney K Miss 1 Cheong SC Dr 1 Lemoine N Professor 1 Hallden G Dr 2

1 Centre of Molecular Oncology and Imaging, Queen Mary's University of London, John Vane Science Centre, Charterhouse Square, London, EC1BM 6BQ 2 Centre, Charterhouse Square, London, EC1BM 6BQ P 4 A non-replicating adenovirus expressing Hey1 is cytotoxic to prostate cancer cells

Androgen receptor (AR) cell signalling is constitutively active in most hormone-refractory prostate cancer (PCa) tumours and suppression hypothesized to impede PCa cell proliferation. Hey1, a co-repressor of the AR is being investigated as a therapeutic transgene for treatment of late-stage PCa. A replication-defective recombinant adenovirus deleted for E1 and E3, expressing Hey1 was constructed (Ad5Hey1). A luciferase reporter system demonstrated it repressed AR activity in a dose dependent manner in AR-expressing PCa 22Rv1 cells. Knock down of AR verified cell death by AR silencing. AR-specific siRNA suppressed expression and produced 37 ± 2.2% cell death compared to 13 ± 1.9% for control siRNA. Ad5Hey1 cytotoxicity in PCa cells assessed by MTS viability assays found A5Hey1 alone in 22Rv1 cells produced EC50 values comparable to the cytotoxic E1A-12S gene expressed from an E1-E3 deleted virus. In combination with docetaxel and mitoxantrone, Ad5Hey1 sensitised the cells in a Hey1-dependent manner. In these cells, protein expression of p53 increased with increasing virus doses. p21 levels also increased over time at low doses of virus however disappeared at higher. This suggests cells are being driven towards cycle arrest or apoptosis. Verified further by cell cycle analysis, at low virus doses the number of cells in G2/M-phases increased compared to untreated cells and at higher doses a larger proportion of cells were in sub-G1 indicating cells are being pushed towards apoptosis. Data will be presented to suggest that Hey1 can be expressed from a potent replication selective adenovirus to further improve on oncolysis anti-tumour efficacy both in vitro and in prostate cancer xenograft models.

Mersmann N Ms 1 Roeth P Mr 1 Kraemer-Albers EM Dr 2 Klugmann M Professor 3

1 Institute of Physiological Chemistry, University Medical Center, Johannes Gutenberg University, Mainz, Germany 2 Molecular Cell Biology, Department of Biology, Johannes Gutenberg University, Mainz, Germany 3 Translational Neuroscience Facility, Department of Physiology, School of Medical Sciences, University of New South Wales, Sydney, Australia P 5 Promoter targeting is sufficient to cause oligodenroglial tropism of adeno-associated virus vectors

Background: Recombinant adeno-associated viral vectors (rAAV) have become a versatile tool for gene transfer to the central nervous system (CNS). However, established AAV-serotypes do only transduce neurons, while efficient and widespread transduction of oligodenroglia has not been observed. Retargeting the natural AAV tropism is a prerequisite for a gene therapy of leukodystrophies, a group of disorders caused by oligodendrocyte dysfunction. Canavan disease (CD), a leukodystrophy caused by mutations in the gene encoding the enzyme aspartoacylase (ASPA), is ideally suited for a gene-replacement therapy, because the genetic defect is a null mutation.

Objectives: Identification and characterization of AAV-based vectors with oligodendroglial tropism and the application of these vectors for CD gene therapy.

Methods: We packaged AAV2 pseudotyped AAV1, AAV1/2, AAV8 vectors for expression of genes encoding green fluorescent protein (EGFP) or ASPA under the control of the chicken-beta-actin (CBA) promoter or the myelin-basic protein (MBP) promoter. Transduction efficacy and tropism was assessed by double immunochemistry after transduction of enriched oligodendrocyte cultures and delivery to the mouse brain.

Results: All vectors carrying the CBA promoter transduced neurons in vitro and in vivo. The MBP promoter was sufficient to shift transgene expression exclusively to cultured oligodendrocytes for AAV1/2 and AAV8, while AAV1 showed some additional astroglial transduction. However, only AAV1/2-MBP-injection resulted in robust, yet not exclusive, transduction of oligodendroglia in vivo. Transduction efficacy was unaffected by the transgene or the purification method.

Conclusions: Our findings suggest that AAV1/2-MBP is a viral vector for CD gene therapy and for the treatment of other leukodystrophies.

Pagès G Ms 1 Homs J Ms 1 Ariza L Ms 1 Chillón M Dr 2 Bosch A Dr 1

1 1: Centre de Biotecnologia Animal i Teràpia Gènica, Spain. 2: Dept. Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Spain. 2 1: Centre de Biotecnologia Animal i Teràpia Gènica, Spain. 2: Dept. Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Spain. 3: Institució Catalana de Recerca i d'Estudis Avançats P 6 Intrathecal adminstration of AAVrh10 efficiently transduces motor and sensory neurons in mice

Background: Adeno-associated viral (AAV) vectors are one of the most promising gene therapy vectors for human clinical trials as they are able to drive long-term expression, mostly as an episomal form. No toxicity is associated to wild type virus, which increases safety issues, and they can easily be produced at pure high-titer. Different AAV serotypes can efficiently transduce neurons both in central and peripheral nervous systems using various administration routes. Until now the transduction of motoneurons in spinal cord has been poorly achieved by intramuscular injection of serotypes 1, 6 and 9. Intravenous injections of AAV9, though, obtained efficient motoneuron transduction but rised safety concerns due to spread biodistribution.

Results: In vitro infection of DRG organotypic cultures with AAV1 and AAVrh10 showed preferential infection of small neurons. Among them, AAV1 mainly infected non-peptidergic small neurons and AAVrh10 transduced peptidergic small neurons. After intrathecal injection of AAV vectors in mice, GFP positive neurons were counted. In DRG slices 15% for AAV1 and around 50% of neurons for AAVrh10 were infected with similar neuronal pattern as observed in vitro. In spinal cord slices, AAVrh10 infected around 30% of motoneurons all along the spinal cord, while no infection was observed with AAV1.

Conclusion: Intrathecal injection of AAVrh10 is able to infect motoneurons in spinal cord, which hadn't been reported until now. Thus, this AAV serotype can be a promising tool to design gene therapy approaches for diseases affecting motor and sensory neurons.

Acknowledgements: ISCIII PS09/00720, SGR 2009/1300

Müther N. Dr 1 Zhang W. 1 Hausl M. 1 Harms N. 1 Wolf N.M 1 Mates L. 2 Izsvak Z. 2 Ivics Z. 2

1 Max von Pettenkofer-Institute, Department of Virology, Ludwig-Maximilians-University of Munich, Pettenkofer Str. 9A, 80336 Munich, Germany 2 Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany P 7 Evaluation of a novel AAV/transposase hybrid-vector system for somatic integration in vitro and in vivo

Recombinant adeno-associated viral (rAAV) vectors predominantly persist as extra-chromosomal genomes. However, in dividing cells, rAAV vector genome copy numbers and transgene expression levels decline rapidly. Herein, we inserted the hyperactive Sleeping Beauty (SB) transposase variants HSB5 and the novel SB100 displaying 10- and 100-fold increased integration efficiencies compared to wild-type SB into our AAV-based two-viral-vector system.

Previous work showed that transposition only works sufficiently from circular substrates. However, once inside the cell rAAV genomes form various episomal DNA forms including circular and linear DNA molecules. However, our initial experiments suggested that Flp-mediated circularization may not be required for AAV-delivered transposase activity significantly reducing the complexity of our system.

In order to avoid co-transduction of two AAV-vectors we generated a stably SB100 expressing cell line. Integration efficiencies were analyzed and we found that after infection with a rAAV encoding a neomycin transposon, the integration efficiencies in SB100- cells was 5-fold increased compared to cells with inactive SB (mSB) indicating for the first time that transposition works from rAAV Analysis of insertion sites after SB100-mediated integration into the host genome is ongoing.

To address whether the new version of the rAAV hybrid-vector-system also results into stable transgene expression levels in vivo, we co-injected C57Bl/6 mice with a rAAV transposon donor vector expressing the human coagulation factor IX and an adenoviral-vector encoding HSB5. After induction of rapid cell cycling in mouse liver, transgene expression levels were stabilized in mice which received HSB5 compared to the control mSB group indicating that transposition occured.

Mingozzi F Dr 1 Finn J Dr 1 Basner-Tschakarjan E Dr 1 Chen Y 1 Haurigot V 1 Maiden M 1 Hensley M 1 Herzog R 2 High K 1

1 Children's Hospital of Philadelphia, Philadelphia, PA USA 2 University of Florida at Gainesville, Gainesville, FL USA P 8 Influence of surface-exposed tyrosine phosphorylation on AAV2 capsid processing and presentation

Reducing the levels of capsid antigen presentation on MHC class I (MHCI) may represent a successful approach to reducing immunogenicity of adeno-associated viral (AAV) vectors. Upon entering the cell, AAV capsid gets processed and presented on MHCI molecules in amounts sufficient to trigger cytotoxic T lymphocyte (CTL) killing of transduced cells. Here we compared levels of antigen presentation and CTL killing of the recently described tyrosine-mutant capsids (PNAS 2008;105:7827) with unmodified AAV2. Mutation of tyrosine residues on the AAV capsid that are target for ubiquitination could potentially result in decreased MHCI antigen presentation. To test this hypothesis, a human hepatocyte cell line was transduced with an unmodified AAV2 vector, a vector carrying the Y730F mutation (SM), or a vector with the Y444F, Y500F, and Y730F mutations (TM). Antigen presentation was assayed using a Jurma/VPQ T cell line recognizing a conserved epitope within the AAV capsid and expressing luciferase upon T cell receptor engagement. No difference in antigen presentation over a wide range of MOI was noted between the unmodified or mutant vectors in this assay. This correlated with equivalent levels of CTL-mediated killing. Treatment with proteasome inhibitors reduced antigen presentation in vitro and enhanced transgene expression in vivo of unmodified and mutant vectors. While these results suggest that both unmodified and tyrosine mutant AAV vectors follow similar antigen processing and presentation pathways, the use of mutant capsids with higher transduction efficiency may allow to reach therapeutic efficacy at lower vector doses, thus decreasing the total vector antigen load.

Leggiero E. Dr 1 Astone D. Dr 1 Wonganan P. Dr 2 Mazzaccara C. Dr 1 Labruna G. Dr 1 Sacchetti L. Professor 1 Salvatore F. Professor 1 Croyle M. Dr 2 Pastore L. Professor 1

1 CEINGE-Biotecnologie Avanzate, Napoli, Italia 2 Division of Pharmaceutics, College of Pharmacy, The University of Texas at Austin, Austin, USA P 9 PEGylated helper-dependent adenoviral vector for gene therapy of Hyperlipidemias

Helper-dependent adenoviral (HD-Ad) vectors are very promising for gene therapy application; however, their administration induces an acute toxicity that impairs the possibility of a safe clinical administration. We previously observed that PEGylation of HD-Ad vectors strongly reduces acute inflammation both in murine and in primate models. In order to evaluate if PEGylated HD-Ad vectors (PEG-HD-Ad) can combine safety with the correction of the pathological phenotype, we treated a mouse model of hypercholesterolemia (LDLR-deficient mice) with a vector expressing apolipoprotein A-I (HD-Ad-ApoA-I), already proven successful in a previous study. In this study, we treated LDLR-deficient mice with high doses of HD-Ad-ApoA-I, the PEGylated version of the same vector (PEG-HD-Ad-ApoA-I) and PBS as control. After 12 weeks, mice treated with both HD-Ad-ApoA-I and PEG-HD-Ad-ApoA-I showed reduction in triglycerides and LDL-C, and elevation of HDL-C. Moreover, they showed significantly smaller areas of atherosclerotic plaques compared to untreated mice. We confirmed the increased safety of PEGylated vectors, analyzing cytokine response after treatment: PEGylated vector induced a lower increase in IL-6, IL-12, KC and TNF-alpha compared to the naïve vector. This data indicates, for the first time, that the reduction of toxicity due to the PEGylation of HD-Ad vectors is independent from the transgene expressed and that treatment with PEGylated vectors can correct pathological phenotypes. In conclusion, these data further support the possibility the clinical application of PEGylated HD-Ad vectors for correction of genetic diseases.

Jakobsen M Mrs 1 Stenderup K Mrs 2 Rosada C Mrs 2 Dam T.N Mr 3 Corydon T.J Mr 1 Mikkelsen J.G Mr 1 Jensen T.G Professor 1

1 Department of Human Genetics, Aarhus University, Aarhus, Denmark 2 Department of Dermatology, Aarhus University Hospital, Aarhus, Denmark 3 Department of Dermatology, Roskilde Hospital, Roskilde, Denmark P 10 AAV mediated shRNA expression in human skin

shRNA delivery by recombinant adeno-associated viral (rAAV) vectors has succesfully been applied to several tissues in vivo. However, RNAi in human skin by rAAV-delivered shRNAs has never been reported. In a previous study using lentiviral vector delivery, we demonstrated that TNF-a can be downregulated in inflammatory psoriatic skin in the xenograft transplantation model by RNA interference with resulting clinically and histologically improvements (Mol Ther 2009;17:1743–1753).

We are now using the same model for comparisons between rAAV vectors and lentiviral vectors for shRNA delivery into human skin in vivo. This is done using a highly potent TNF-a-directed shRNA (shTNFa3) which we designed and used in our previous study.

Currently, we are testing the efficiency of TNF-a knockdown in vitro by shTNFa3 when delivered by rAAV vectors with different designs and comparing these results to results obtained with shTNFa3 delivered by lentiviral vectors. Additionally, we are comparing different AAV serotypes in human skin grafted onto mice to determine the optimal serotype. This is done using luciferase as an in vivo marker.

Voigtlander R Mr Hausl M Mr Haase R Mr 2 Zhang W Mrs Baiker A Dr Ehrhardt A Dr

2 Department of Pharmacy, Munich P 11 A novel adenoviral hybrid-vector system carrying a plasmid replicon for gene therapy

In dividing cells the two aims a gene therapy should accomplish are defined as the nuclear distribution and retention of therapeutic DNA. Because monosystems fail to fulfil both tasks with equal efficiency, hybrid-vector systems are promising in facing the challenges of molecular medicine. Our hybrid-vector system HDAdV-pEPito synergizes the helper-dependent adenoviral vector(HDAdV) with the plasmid pEPito containing the therapeutic gene and special DNA sequence SMAR(Scaffold/Matrix-Attachment-Region) for episomal retention and replication. Our technique provides a powerful tool for stable maintenance of therapeutic DNA reducing the risk of insertional mutagenesis.

HDAdV-pEPito and the SMAR deleted control (HDAdV-pEPito-ゔSMAR) were generated with our novel BAC-technology. Both contain promotor hCMV/EF1 upstream of an eGFP-IRES-BSD-cassette. 83% of A549-cells infected with MOI of 50 were positive for eGFP-expression. pEPito has to be released from linear adenoviral genomes with Flpe-recombinase to function as a plasmid. PCR proved this recombination for both constructs. Episomal status of recombined plasmids was verified with rescue experiments at day 4 after coinfecting A549-cells with our constructs and Flpe-expressing virus HDAdV-mSB-Flpe. More bacterial colonies grew after transformation of genomic DNA containing HDAdV-pEPito with Flpe (n = 281) than of DNA containing HDAdV-pEPito-ゔSMAR with Flpe (n = 25), suggesting replication of excised plasmids.

Ongoing experiments compare HDAdV-pEPito and HDAdV-pEPito-ゔSMAR concerning their long-term peristence in mice and their in-vitro establishment in a colony forming assay (CFA). Previous CFAs with the original plasmids inserted into our viruses seemed to show a tendency towards SMAR-positive pEPito (48 surviving colonies) compared to SMAR deleted pEPito-ゔSMAR (27 surviving colonies) after 4 weeks of selection.

Loiler SA Dr 1 Vervoordeldonk MJ Dr 1 Tak PP Professor 1

1 Arthrogen BV, and Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands P 12 Induction of MX1 Gene Expression across Species by Mouse and Human Interferon Beta

Towards our development of gene therapies to treat rheumatoid arthritis an assay was needed for the measurement of interferon beta (IFNβ) activity after rAAV5 mediated gene delivery. Our pre-clinical vector is a rAAV5 that will deliver a human IFNβ gene under the control of a nuclear factor kappa B (NF-κB) promoter. The myxovirus resistance protein 1 (MX1) is a key mediator of innate immunity against pathogens. MX1 gene expression is induced by type 1 interferon and is currently used to evaluate IFNβ activity and anti-IFNβ neutralizing antibodies in multiple sclerosis patients. These tests determine the cross-species ability of mouse and human IFNβ to induce MX1 gene expression.

Mouse fibroblast like synoviocytes (mFLS) and human 2V6.11 cells were infected with AAV5 expressing either mouse or human IFNβ (moi 10⁵). After 72 hours IFNβ expression was measured by ELISA and MX1 gene expression was determined by real-time PCR.

Mouse FLS infections produced more than 10 ng/ml of both mouse and human IFNβ whereas human 2V6.11 infections produced more than 100 ng/ml of both mouse and human IFNβ. In mFLS, human IFNβ induced 21% of the level of MX1 gene expression compared to mouse IFNβ. In human 2V6.11 cells, mouse IFNβ induced 49% of the level of MX1 gene expression compared to human IFNβ.

Infection of mouse FLS with AAV5 expressing human IFNβ induced a modest level of MX1 gene expression. Additionally, mouse IFNβ induced a moderate level of MX1 gene in human cells. Induction of MX1 gene expression across species may be a useful tool for the evaluation of IFNβ activity during pre-clinical testing.

Mathis JM Professor 1 Terry T Ms 1 Odaka Y Mr 1 Curiel DT Professor 2

1 LSU Health Sciences Center, Gene Therapy Program, 1501 Kings Hwy, Shreveport, LA, USA 2 University of Alabama at Birmingham, Division of Human Gene Therapy, 901 19th Street South, Birmingham, AL, USA P 13 Incorporation of a Metallothionein Fusion into the Adenovirus Protein IX for Non-invasive Imaging

Introduction: Adenovirus (Ad) vectors have been exploited for a wide range of gene therapy applications. However tracking Ad biodistribution in vivo is limited to invasive procedures such as biopsies, which are error prone, non-quantitative, and do give a full representation of the pharmacokinetics involved. Also, the major drawback to approaches using reporter genes is that these systems require initial viral infection and subsequent cellular expression of reporter gene expression to allow quantification. To overcome these limitations, we incorporated the human metallothionein (MT) protein as a fusion to the Ad minor capsid protein pIX. The pIX protein is a locus capable of presenting incorporated ligands on the virus capsid surface, and MT has high affinity for heavy metals, including radiometals such as ^99mTc.

Methods: After incorporating the MT coding sequence as a C-terminus fusion to pIX, we used the E. coli recombination system to generate a non-replicative vector containing an E1A expression cassette with GFP under control of the CMV promoter (Ad5-CMV5-tGFP-pIX-MT). We tested whether Ad5-CMV5-GFP-pIX-MT could be radiolabeled by ^99mTc binding. After radiolabeling the Ad5-CMV-GFP-pIX-MT, we determined whether SPECT imaging could be used to monitor Ad biodistribution and uptake in vivo.

Results: Our results strongly demonstrate imaging capability in vivo using pIX-MT, visualizing the biodistribution of Ads.

Conclusions: These results demonstrate the feasibility of ^99mTc binding to the pIX-MT fusion on the Ad capsid using a simple transchelation reaction. The genetic Ad capsid labeling system offers noninvasive dynamic imaging of biodistribution that can be monitored by existing nuclear medicine imaging.

Freidig A Dr 1 Salmon F Dr 1 Jenal U Dr 2 Petry H Dr 1

1 Amsterdam Molecular Therapeutics 2 Jenal Partners Biosafety Consulting P 14 Use of non-clinical and clinical data for the environmental risk assessment of Glybera (alipogene tiparvovec)

Purpose: Glybera (alipogene tiparvovec) is an AAV-1 based gene therapy medicinal product developed for long term correction of lipoprotein lipase deficiency (LPLD), a seriously debilitating inherited lipid metabolism disorder. The environmental risk assessment (ERA) forms an important part of the submission in the European Union but gene therapy specific experimental assessment protocols are not available. Glybera is the first AAV-based gene therapy medicinal product filed in Europe for Market Authorization.

Methods: We screened the non-clinical and clinical studies to select data that should be included in the ERA. This data was supplemented with use scenarios from clinical trials and literature on the biology of wild-type AAV and modified AAV-based gene therapy vectors, to assess magnitude and likelihood of potential adverse events.

Results: All essential experimental data for the ERA were generated as part of the non-clinical and clinical development. The clinical vector shedding from different excreta including urine, saliva, faces and seminal fluid is the most critical element in the assessment. This data allowed estimating the fraction of the dose excreted (as low as 0.000013%/d on day 1; lower thereafter) after Glybera administration. A thorough description of the characteristics of the recombinant AAV-vector, such as replication deficiency, low infectivity and non-pathogenicity were pivotal in assessing the hazard of the shed vector in absence of specific environmental data.

Conclusion: Based on the available data the overall assessed risk of Glybera for people and the environment was concluded to be negligible. Hence, specific risk management measures can be kept to a minimum.

Martinez-f F Dr 1 Sandoval-zamora HE Dr 2 Lomeli-rodriguez CY Mr 2 Villegas-castrejon H Dr 2

1 National Institute of Rehabilitation, Mexico Xochimilco 283, 14389 Mexico D.F. 2 National Institute of Rehabilitation, Mexico P 15 Betamethasone delays the Egr-1 promoter response to UV light of Ad-Egr1-Luc-7 on human and mouse fibroblasts

Background: Egr1 is a finger zinc transcriptional factor involved closely with transcriptional activation of collagen type1 on skin. Transcriptional activation is induced by UV light, serum, hormones and drugs. Hereby, we describe the effect of Betamethsone on human primary fibroblast (HPF) transduced with Ad-Egr1-Luc-7, exposed to UV, in order to propose as betamethasone like repressor of Ad-Egr1-Luc-7.

Methods: 5×10⁵ 3T3 mouse fibroblasts and HPF cells were seeded and infected at 75 MOI with Ad-Egr1-Luc, and AD-CMV-Luc as reference control. Cells were deprived of serum (FBS) 24 hours before induction assay. The Induction assay was performed in 1).SFB(+); 2).FSB(−). 3).FBS(−)/betamethasone(+). All conditions were grouped in A).UV(−) and, B) UV(+). Reporter activity was measured by luciferase activity assay at 3 and 6 hours.

Results: Transcriptional activity of Egr-1, was induced by UV light en both cell lines at 3 hours, however, the maximum activity was observed at 6 hours (10 Folds of luminescence). Activity of Egr-1 not shows significant change in presence of serum. However, Egr-1 activity increases at ∼7 fold times (46.2/10³LC) compared to (5.2/10³LC) of CMV in presence of UV. When cells are exposed to UV and Betamethasone, luciferase activity are in 3T3 cells but in PHF the egr-1 level is lower than CMV, this correlation is inverted at 6 hours 3 fold times.

Conclusion: In this model of UV induction, the egr1 activity in PHF is 10 times over 3T3 and early delayed by betamethasone. Betamethasone could be proposed as temporal repressor of Ad-Egr1-Luc-7.

Research granted by ICYT-PIFUTP-169

Martinez-f F Dr 1 Sakamoto K Dr 2 Sandoval L Dr 1 Machuca C Dr 3 Villegas H Dr 1

1 National Institue of Rehabilitation, Mexico-Xochimilco 289, 14389 Mexico D.F. 2 Childrens Hospital of UCLA, USA 3 Fes-Zaragoza National University of Mexico P 16 AdEgr-1-Luc7 Adenoviral vector is functionally activated by UV light

Background: the Early grow response (EGR-1) protein is finger zinc transcriptional factor with transcriptional activation of collagen type1 on skin and TGF-B. The promoter activity is induced by UV, serum, hormones and drugs. Hereby, we describe the functional activity of Ad-Egr1-Luc-7 of luciferase activity under transcriptional control Egr-1 promoter inducible for UV light.

Methods: The fragment of − 615 pb of human Egr1-promoter was subcloned in p-Shutle-Luc-poly-A vector derived by pShuttle of the adeasy1 system (Kindly donated by B. Vogelstein) to obtain pEgr-1-Luc7-poly A construct. Homologous recombination was performed in bacterial competent cells. The recombinant adenoviral plasmid obtained was purified and packaged in HEK-293 cells, according standard conditions of our lab. Adenovirus large scale production was performed from lytic plaques and purified using protocol for animal grade applications. Adenovirus stock was quantified by OD and titled by Plaque Assay in HEK-93 Cells. Transduction efficiency of Ad-Egr-1- Luc7 was performed in Human fibroblast (HF) using 25, 50 75 100, and 150 MOI´s. The functional induction by UV, was performed in 3×10⁵infected HF starved of serum (FBS), infected and exposed to UV (120″/254 nm). Luciferase reporter activity was evaluated 6 Hours after exposition to UV, using Ad-CMV-Luc Control vector

Results: Optimal MOI of Ad-Egr-1-Luc7 in HF was 50. 120″ of UV exposition induce transcriptional activity AdEgr-1-Luc7 (4.1×10⁶LC) is 10 fold times more than Ad-CMV-luc. (4.5x10⁵LC). Interestingly, the CMV activity is 2 fold times more over than egr-1 in presence of SFB.

Conclusion: Hereby we demonstrate the functional activation AdEgr-1-Luc7 induced by UV light.

Ni W Mr 1 Le Guiner C Dr 2 Penaud-Budloo M Mrs 3 Moullier P Dr 4 Snyder RO Dr 5

1 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville - USA 2 INSERM UMR 649, Nantes & GENETHON, Evry - France 3 INSERM UMR 649, Nantes - France 4 INSERM UMR 649, Nantes & GENETHON, Evry - France & Department of Molecular Genetics and Microbiology, University of Florida, Gainesville - USA 5 Department of Molecular Genetics and Microbiology & Departement of Pediatrics, University of Florida, Gainesville - USA & INSERM UMR 649, Nantes - France P 17 Development of a reliable blood test for the detection of rAAV-mediated gene doping

The goal of our project is to develop a test for the detection of the presence of a vector delivered transgene of interest encoding a protein capable of enhancing athletic performance. Several in vivo studies have shown that vectors derived from Adeno-Associated Virus (AAV) are able to provide long-term expression of a transgene after a single administration. It has been demonstrated especially after one intramuscular injection of a recombinant AAV (rAAV) vector that vector genomes are detectable in blood cells several months or years after injection. These results suggested that a sensitive-PCR technique can be used to detect rAAV-mediated gene doping from a simple blood sample. Our project consisted of a single intramuscular injection of macaques with two different rAAV serotypes known for their efficiency to transduce skeletal muscle cells: the rAAV1 and rAAV8. Various clinically relevant doses were tested. Each vector carried the expression cassette CMV-cmEpo-SV40polyA that codes the cynomologous Erythropoietin gene. Our initial goal was to establish the minimal dose of vector that can cause an increase of ≈ 15% of the hematocrit compared to the normal level. Once the minimum dose was determined, an optimized PCR method was used to highlight the presence of the transgene in blood. We validated Taqman real-time PCR sensitive conditions that allow us to detect at least 5 copies of the rAAV Epo transgene in the background of endogenous genomic DNA.

Data generated in our nonhuman primates will be the basis for developing a legally defensible commercial real-time PCR assay.

Guadarrama-Pérez V Miss 1 Peralta-Zaragoza O Dr 2 Merchant-Larios H Dr 3 Meneses-Acosta A Dr 1

1 Faculty of Pharmacy, Autonomus University of the State of Morelos 2 National Institute of Public Health, Health National Institutes of México 3 Institute of Biomedics Research of the National Autonomous University of Mexico P 18 Different types of cell death in HEK293 in response to Ad5 infection and environmental factors

Background: HEK293 cell line and its derivatives are widely used in the production of viral vectors and recombinant proteins. Nevertheless, information of mechanisms of cell death are limited and controversial. Studies related to cell death are important in the design and optimization of strategies to improve the production process. This study aims to identify the type of cell death developed by HEK293 in a static culture in response to different stress conditions.

Methods: HEK293 cell line were maintained in DMEM-F12 media supplemented with 10% FBS. There were 3 trials of cell death induction: Temperature (47°C), lack of growth factors and infection with Adenovirus 5 (Ad5). Analysis of cell growth, morphology by epifluorescence microscopy and transmission electron microscopy, determination of cell cycle by flow cytometry and the presence of p53 protein by Western blot were performed.

Results: Cell death related to autophagic features were observed during the experiments at high temperature (42 °C) and in those related to the lack of growth factors and control cultures. Meanwhile, HEK293 cells presented apoptotic characteristics in response to adenoviral infection with Ad5 at MOI of 50. An independent pathway of p53 was developed by this cell death because it did not show a specific pattern of DNA fragmentation and changes in the expression of it.

Conclusion: HEK293 culture has a mixed pattern of programmed cell death depending the type of stimulus presented in the culture. This knowledge helps to improve the infection process and the strategies of viral vector production.

Cots D Mr 1 Chillon M Dr 1 Alba R Dr 1 Segura MM Dr 2 Kremer EJ Dr 3 Hearing P Dr 4 Chillon M Dr 1

1 Departament de Bioquimica i Biologia Molecular, Centre de Biotecnologia Animal i Teràpia Gènica. Bellaterra, Catalunya. 2 Departament de Bioquimica i Biologia Molecular, Centre de Biotecnologia Animal i Teràpia Gènica. Bellaterra, Catalunya. 3 Institut Génétique Moléculaire de Montpellier, Montpellier, Languedoc-Roussillon, France 4 Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, Long island, United States P 19 Robust recombinase-free system Production of HD adenoviral vectors with negligible contamination

Helper dependent adenoviral vectors (HDAd) are devoid of all viral coding sequences and retain only minimal viral cis-acting sequences required for vector propagation. The Cre/loxP system is the classical and most widely method for producing HDAds. In this system, the helper adenoviral packaging signal is excised rendering the helper virus unpackageable but able to trans-complement HDAd propagation. However high activity of the Cre recombinase needed for low Ad helper contamination is associated to high toxicity. To better address this issue, we have cloned attB/attP-C31 sequences flanking the packaging signal of both, human and canine adenovirus (Ad-5 and CAV-2) vectors to develop a new system based on delayed packaging of Helper-Ad respect to HDAds. Surprisingly, the mechanisms causing this delay were not related to the presence of the recombinase. Subsequent band-shift studies suggested that an unknown cell factor (in both, permissive human 293 and canine DK cells) interacts with the attB sequence and affects the correct functioning of the packaging complex. Viral cycle studies indicated a clear delay in both canine and human attB-Ad, showing its potential in different adenoviral serotypes.

In addition, production assays in 293 suspension cells showed that human HDAd vectors can be produced at high titer with negligible helper contamination levels.

Future experiments will be performed to identify the unknown factor and to optimize large-scale production/purification of HD-Ad vectors using both human and canine adenovirus for long-term gene therapy strategies.

Hsu P Mr 1 Yang YW Dr 1

1 1, Jen-Ai Road, Section 1, Room 1214, Jen-AI Road,Section 1, Taipei 10051, TAIWAN P 20 Complexation of recombinant adeno-associated virus with polyelectrolytes for gene delivery

Background: This study attempts to study the effect of complexation of recombinant adeno-associated virus (rAAV) with polyelectrolytes on gene delivery.

Method: A previously constructed recombinant adeno-associated virus vector, containing the furin-mutated human insulin gene downstream of rat insulin I promoter and the enhanced green fluorescent protein (EGFP) gene driven by the cytomegalovirus immediate early (CMV_IE) promoter, was employed to investigate the roles of reactive oxygen species (ROS) in rAAV-mediated gene transfer. Huh7 hepatoma cells were transduced with the virus vector complexed with polyethyleimine (PEI), a cationic polyelectrolyte, followed by flow cytometric analysis, and the generation of reactive oxygen species (ROS) was determined with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFH-DA) or dihydroethidine (DHE).

Results: Flow cytometric analysis showed that complexation of rAAV with polyethyleimine (PEI), a cationic polyelectrolyte, resulted in an enhanced uptake of rAAV. To examine the virus uptake by the cells, rAAV was labeled with the carbocyanine dye Cy3, and the enhancement of rAAV binding by PEI was confirmed by confocal fluorescence microscopy. Treatment of Huh 7 cells with NaClO₃, on the other hand, reduced the transduction efficiency as compared to the cells treated with rAAV alone, suggesting the roles of proteoglycans in gene transfer. Complexation of rAAV with PEI increased the generation of ROS, including superoxide and hydrogen peroxide, in the rAAV-treated cells, and pretreatment of the cells with the anti-oxidants reduced the transduction efficiency of the rAAV-PEI complexes.

Conclusion: Results obtained in this study demonstrated the roles of ROS generation in gene delivery mediated by the rAAV-PEI complexes.

Schäfer A Ms 1 Pahnke A Ms 1 Schaffert D Mr 1 van Weerden W M Dr 2 Rödl W Mr 1 Vetter A Ms 1 Spitzweg C Professor 3 Kraaij R Dr 2 Wagner E Professor 1 Ogris M Dr 1

1 Center for System based Drug Research, Department of Pharmacy, Pharmaceutical Biotechnology, Ludwig-Maximilians-University, Butenandstr. 5-13, D-81377 Munich, Germany 2 Dept of Urology, Josephine Nefkens Institute, BE 362, Erasmus MC Rotterdam 3 Dept. of Internal Medicine II, Klinikum der Universitaet Muenchen – Campus Grosshadern, Ludwig-Maximilians-University, Munich, Germany P 21 EGF-receptor targeted nonviral gene delivery in vitro and in vivo avoiding receptor activation

Background: The epidermal growth factor receptor (EGFR) is upregulated in many solid tumors and an attractive target for tumor targeted gene therapies. Epidermal growth factor (EGF) is used for this purpose, although the risk of mitogenic effects due to EGFR activation exists. We have developed a synthetic, EGFR targeted gene delivery system, enabling to evaluate different EGFR binding peptides in terms of transfection efficiency and EGFR activation.

Method: Thiolated, recombinant murine EGF (mEGF), peptides CMY, MYI (derived from human EGF) or C-GE11 (recently identified by phage display) containing a terminal cysteine, were coupled to LPEI via a heterobifunctional polyethylene glycol linker. Cellular binding and internalization was monitored by flow cytometry and laser scanning microscopy. In vitro transfections and intratumoral injections in a clinically relevant orthotopic prostate cancer model were performed with luciferase encoding plasmid.

Results: EGFR targeted polyplexes specifically enhanced transfection on EGFR overexpressing glioblastoma and hepatoma tumor cells. mEGF, CMY and MYI polyplexes induced EGFR activation leading to phosphorylation of Erk and Akt. In contrast, C-GE11 polyplexes led to highest transfection levels without activation of EGFR. mEGF polyplexes diminished cell surface expressed EGFR for up to four hours, while this effect was absent with C-GE11 polyplexes. Intratumorally injected C-GE11 polyplexes with a plasmid optimized for in vivo transfections were superior in inducing luciferase transgene expression as compared to untargeted ones.

Discussion: With this fully synthetic system, targeted, safe and efficient transgene expression is achieved in vitro and in vivo paving the way for further therapeutic studies.

Jose A Mrs 1 Huch M Dr 1 Andreu N Dr 1 Sobrevals L Dr 1 Ayuso E Dr 2 Navarro P Dr 3 Fillat C Dr 4

1 Programa Gens i Malaltia. Centre de Regulació Genòmica, Centro de Investigación Biomédica en Red CIBERER 2 Centre de Biotecnologia Animal i Teràpia Gènica, Centro de Investigación Biomédica en Red CIBERDEM 3 Institut Municipal d'Investigació Mèdica, IMIM 4 Programa Gens i Malaltia. Centre de Regulació Genòmica, Centro de Investigación Biomédica en Red CIBERER. Barcelona, Spain P 22 Cytotoxic adenoviruses delivered into the common bile duct induce tumor regression in Ela1-myc mice

Gene-based anti-cancer therapies delivered by adenoviral vectors or oncolytic adenoviruses are commonly applied in vivo through intratumoral injections or by systemic delivery. However, intratumoral administration results in a limited spread of the viruses and systemic injections are often accompanied by toxic effects. In the current work we have explored the feasibility of targeting pancreatic tumors by adenoviruses through a loco-regional route. We have retrogradelly injected adenoviruses through the common bile duct of wild type (wt) and Ela1-myc mice, a transgenic model of pancreatic cancer, by a technique very similar to the established endoscopic retrograde cholangiopancreatography procedure applied in humans and studied tumor targeting and anti-cancer effects. Administration of AdCMVLuc or AduPARLuc in wt mice revealed a peak of luciferase activity at 4 days post-injection that localized in the pancreas with no expression in other abdominal tissues such as liver or intestine. Viral administration into Ela1-myc mice resulted in lower luciferase activity when injected with AdCMVLuc but not after AduPARLuc administration, suggesting increased activity of the uPAR promoter in the tumors. Furthermore we evaluated the impact of AduPARTat8TK/GCV therapy in Ela1-myc mice. Primary carcinoma cells from Ela1-myc pancreatic tumours displaying a ductal-like phenotype showed relevant cytotoxicity when exposed to AduPARTat8TK/GCV viral therapy. Importantly the in vivo application of AduPARTat8TK through the common bile duct followed by GCV treatment significantly delayed tumor growth in Ela1-myc mice. In summary our data shows that uPAR designed adenovirus retrogradelly injected into the common bile duct can be a feasible approach for pancreatic cancer treatment.

Yun Chae-Ok Professor 1 Kim Pyung-Hwan Mr 2 Tae-il Kim Mr 3 Yockman >James W. Mr 3 Kim Sung Wan Professor 3

1 Institute for Cancer Research, Yonsei Uni. College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul, Republic of Korea 2 Institute for Cancer Research, Yonsei University College of Medicine 3 Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah P 23 Bioreducible polymer-conjugated hepatoma-specific oncolytic adenovirus for systemic cancer gene therapy

Systemic administration of adenovirus (Ad) is hampered by a multitude problems including immediate accumulation in the liver, resulting in a very short circulatory half-life and induction of immune responses. An alternative to overcome the limitations of Ad in vivo is to engineer a hybrid gene transfer vector system combining viral and non-viral elements. In this study, we have generated a hybrid polymer-virus vector through chemical conjugation. The surface of Ad was chemically conjugated to cationic polymer ABP (arginine-grafted bioreducible polymer) via the cross-linker, DTSSP (3,3´-Dithiobis[sulfosuccinimidylpropionate]). The size distribution and zeta potential of Ad-linker-ABP complex were increased depending on polymer ratio compared with those of naked Ad. The transduction efficiency of Ad-linker-ABP complex was enhanced compared with that of naked Ad in various cancer cell lines. In particular, a higher efficacy was observed in coxsackie and adenovirus receptor (CAR)-deficient cancer cells showing 4-fold increase of transduction efficiency. Earlier accumulation in endosomes was identified by Lysotracker staining when Ad was conjugated with ABP. When hepatoma-specific oncolytic Ad was conjugated with ABP, the Ad-linker-ABP complex elicited an enhanced cell killing effect towards liver cancer cells (Huh7 and HepG2) expressing α-fetoprotein (AFP) while sparing non-liver cancer cells. Moreover, the level of interleukin-6 (IL-6) cytokine released from macrophage cells was significantly reduced when Ad was conjugated with ABP compared with that of naked Ad, suggesting that Ad-linker-ABP complex can elude the innate immune response. Taken together, these hybrid Ad vectors may provide an interesting option for the delivery of therapeutic viruses to disseminated tumor masses by systemic administration.

Li L Dr 1 Sarver A Dr 2 Subramanian S Dr 3

1 14-217A Moos Tower, 515 Delaware Street SE, Minneapolis, MN 55455, USA 2 5-134 MCB, 420 Washington Ave SE, Minneapolis, MN 55455, USA 3 11-212 Moos Tower, 515 Delaware St, SE, Minneapolis, MN 55455, USA P 24 miR-183 functions as a potential oncogene by targeting EGR1 and promoting tumor cell migration

Background: MicroRNAs (miRNAs) are ∼22nt non-coding RNAs that post-transcriptionally regulate gene expression. Decreased levels of many common tumor suppressors, for example, Early growth response 1 (EGR1) as well as PTEN, are implicated in a wide range of tumor types. We hypothesize that transcriptional perturbations in specific miRNA(s) observed across multiple tumor types may regulate the level of important tumor suppressors like EGR1 and PTEN in these tumor types.

Method: An integrative network analysis was used to identify potential microRNA candidates shared in synovial sarcoma(SS), rhabdomyosarcoma(RMS) and colon cancer. In vitro target validation was conducted using Luciferase reporter systems. Western blotting was used to verify protein level. Further analyses including cell migration/invasion, proliferation, cell cycle, apoptosis and live/dead assay were performed to reveal function effects of antimiR-183.

Results: We identified that miR-183 is significantly over-expressed in all tumor types being tested (SS, RMS, and colon cancer) and their corresponding cell lines. We proved that EGR1 is a bona fide target of miR-183, at least in the 3 tumors we tested. miR-183 knockdown with antimiR rescued the protein level of EGR1 and PTEN. Functional analyses showed that miR-183 is an important contributor to cell migration/invasion in all the cell lines tested. Interestingly, different cell lines showed different response in cell cycle shift after antimiR-183 treatment.

Conclusion: miR-183 is a potential oncomir through regulating two tumor suppressor genes EGR1 and PTEN. The deregulation of miR-183-EGR1-PTEN network may be central to many tumor types. miR-183 may have a therapeutic role to regulate cancer metastasis.

Shimizu K Professor 1 Ishida E Ms 2 Kitahara M Mr 3 Yawata T Dr 4

1 Kohasu, Okh-cho, Nankoku, Kochi, Japan P 25 Development of a suicide gene therapy for the treatment of glioblastoma using tumor-specific retroviral vector

Suicide gene therapy has been shown to be effective in inducing tumor regression. The expression of suicide genes is best restricted to tumor cells but is ubiquitous in most brain tumor tissues. In this study, a human brain tumor–specific promoter was identified and used to develop transcriptionally targeted gene therapy. We identified SSX4 and MAGEA3 as genes expressing in glioma cell lines at high frequency among CTA and MAGE gene family by in silico and RT-PCR screening. SSX4 gene promoter activity was high in human brain tumor cells but not in normal human astrocyte cells, whereas the MAGE-A3 promoter showed activity in both tumor and normal cells. In order to define a useful tumor-specific promoter for targeting in context of retroviral-mediated gene therapy, the SSX4 promoter were used to restrict the expression of suicide gene HSVtk in retroviral vector. Glioma and human telomerase catalytic subunit (hTERT)–immortalized fibroblast BJ-5ta cell lines transduced with retrovirus vectors were assayed for killing activity by ganciclovir. Glioma cell lines were effectively killed by ganciclovir in a concentration-dependent manner, whereas BJ-5ta cells were not. In addition, mouse glioma RSV-M cells transduced with retrovirus vector also showed suppressed tumor formation activity in syngeneic mice in response to ganciclovir administration. These results suggest that the promoter of SSX4 is useful for tumor-specific gene therapy, and promising the safe and effective retroviral-mediated gene therapy. Efficacy of the gene therapy against mouse glioma model and tumor stem cells is under investigation.

Lévy C Mrs 1 Frecha C Dr 1 Costa C Mrs 1 Rachinel N Dr 2 Salles G Professor 2 Cosset FL Dr 1 Verhoeyen E Miss 1

1 INSERM U758, Human Virology Department; ENS de Lyon, Université Lyon 1, F-69007, Lyon, France 2 Hospices Civils de Lyon, Université Lyon, UMR CNRS 5239, Lyon, France P 26 Efficient gene transfer in human malignant B cells by a Measles virus gp pseudotyped lentiviral vector

B-lymphocytes are attractive targets for gene therapy of diseases associated with B-cell dysfunction and also for immunotherapy by their potential to induce specific immune activation or tolerance. Moreover, malignant B-cell transduction would allow the design of gene transfer based therapies as well as biological studies. However, up to now efficient lentiviral transduction of resting healthy or malignant B-cells had not been reported.

Recently, we generated lentiviral vectors pseudotyped with measles virus glycoproteins (MV-LVs) that allowed efficient transduction of quiescent human T-cells and B-cells after a single exposure. Indeed, MV-LVs can efficiently transduce either BCR-activated as well as quiescent human B-cells. Additionally, MV-LVs are the first vector tools that allow efficient stable gene transfer into leukemic cancer B-CLL cells, one of the most prominent adult blood cancers that are blocked in G0/G1 phase of the cell cycle. MV-LVs proved also to be the candidate of choice for stable gene transfer into marginal zone lymphoma (MZL) B-cells, another B-cell malignancy and were highly superior to VSV-G-LVs. Interestingly, efficient transduction by MV-LVs co-incided with signaling-activating-molecule (SLAM) expression, one of the measles virus receptors. A poor gene transfer profile for VSV-G-LVs was also evident when the cells were pre-stimulated through the B-cell receptor while MV-LV transduction of B-cells augmented significantly for the 12 B-CLL and 6 MZL donors evaluated. Summarizing, MV-LVs overcome the limitations of current LVs in B-cells by making B-cell-based gene therapy and immunotherapy applications feasible as well as the study of gene functions in healthy and malignant B-cells.

Hiraoka K Dr 1 Inagaki A Dr 1 Logg C Dr 1 Kamijima S Dr 1 Ostertag D Dr 2 Perez O Dr 2 Gruber H Dr 2 Robbins J Dr 2 Jolly D Dr 2 Kasahara N Professor 3

1 Department of Medicine, UCLA 2 Tocagen, Inc. 3 Departments of Medicine and Molecular & Medical Pharmacology, UCLA P 27 Preclinical Studies Support the Clinical Utility of RCR Vectors for Cancer Gene Therapy

Unique among replicating viruses being developed as oncolytic agents, replication-competent retroviruses (RCR) replicate without immediate lysis of host cells and maintain viral persistence through stable integration. Due to its intrinsic inability to infect quiescent post-mitotic cells and susceptibility to innate antiviral defenses that exist in normal cells but are inactivated in many cancers, MLV-based RCR vector replication has proven to be highly tumor-selective. We have previously demonstrated that RCR vectors are capable of highly efficient replication in tumor cells, and we have found significantly enhanced survival benefit in a variety of cancer models in vivo when RCR vectors are employed for delivery of prodrug activator genes. In the present study, we tested a newly developed RCR vector with modified virus backbone sequences and a codon-optimized cytosine deaminase prodrug activator gene (Toca511), which has now entered Phase I/II clinical trials for glioblastoma. We examined the transduction efficiency of this newly developed RCR vector in U87 human glioblastoma models and confirmed that >99% tumor transduction could be achieved over time. Next, therapeutic efficacy was evaluated, and the minimum effective doses of 5FC prodrug and Toca511 vector determined. Significant survival benefit was seen after 5FC treatment compared to control groups, even after injecting only 1x10e4 total units of RCR vector (p = 0.0058). These data support the use of Toca511 to treat highly aggressive solid tumors such as glioblastoma. Now, with the use of RCR vectors, the original promise of retrovirus-mediated gene therapy strategies for cancer may finally be fulfilled.

Fonseca L Miss 1 Martins L Miss 2 da Silva F Aires Dr 2 Barata J Dr 2 Barbas C Dr III 3 Gonçalves J Professor 1

1 URIA-CPM, Faculdade de Farmácia de Lisboa, Portugal 2 Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Portugal 3 The Scripps Research Institute, La Jolla, CA, USA P 28 Cell-specific targeting using a Sindbis-pseudotyped lentiviral vector expressing anti-FITC scFv

A crucial factor for successful gene therapy is the efficacy of specific gene transfer, which is usually done by lentiviral vectors. Fusion and specificity of lentiviral vectors must be provided by envelope glycoprotein domains. Sindbis envelope can pseudotype lentiviral particles and display exogenous protein domains. Previous results demonstrated that Sindbis envelope can accommodate anti-receptor single-chain antibodies (scFv) and target cell-specific viral infection. Additionally, it was shown that protein A-chimeric Sindbis envelope can specifically target cells immunolabelled with anti-receptor IgG via Fc recognition. However, these strategies might present some problems for in vivo applications, since there may be non-specific reactions with plasma antibodies and the need for cloning a receptor specific antibody each time a new molecule is to be targeted. To overcome these problems we developed a new lentiviral vector capable of transducing several cell types in a specific manner without the above constraints, that consists of a chimeric scFv-Sindbis virus envelope that binds fluorescein (FITC) with high affinity and consequently recognize FITC-conjugated proteins. Using this targeting strategy we were able, in vitro, to target efficiently and specifically cells expressing a receptor labelled by a FITC-conjugated antibody. Moreover, we could specifically kill those transduced cells using an HSVtk/GCV suicide gene strategy. We are currently testing the in vivo efficiency of this gene therapy proposal in a mouse model of T-cell acute lymphoblastic leukaemia, although the strategy herein proposed has the potential to be applied to a broad range of diseases.

Rivera Gonzalez G Mr 1 Georgopoulos L Mrs 1 Maitland N Professor 1

1 University of York P 29 Human prostate transglutaminase, a prostate specific gene with potential applications for gene therapy

Background: Targeted gene therapy offers an alternative means to kill prostate cancer cells. One method to achieve efficient targeting is the use of tissue specific promoters that drive expression of therapeutic genes only in the desired tissue. To make use of such regulatory sequences more knowledge about their control is needed. Human prostate transglutaminase (hTGp) is a highly prostate specific gene, but the mechanisms that control its expression are not well understood.

Results: Androgen had previously been shown to have a positive effect on hTGp expression, but the hTGp promoter contains not only multiple androgen receptor binding sites but also retinoic acid responsive elements (RAREs). atRA exerted a major effect on hTGp expression while androgen played a minor and indeed negative role, in contrast to previous reports. The RAREs that activate hTGp transcription were located within the promoter sequence in a region of 4.5kb. Cells were treated with TTNPB, a synthetic retinoid that activates RAR's, showing increased transcription afterwards. siRNAs against the different transcripts encoding RAR's were used showing a major role for RAR gamma in hTGp transcription after atRA treatment.

Conclusion: hTGp transcriptional regulation is dependent primarily on atRA but not on androgen. Control is exerted by the RAR's, mainly by RAR gamma. This study shows that even when most prostate specific genes are androgen regulated, there are more elements capable of controlling their regulation. By investigating the whole set of factors that control prostate specificity, we will be able to make use of these regulatory elements to express therapeutic genes in the prostate.

Zhang Guo ZH Professor 1

1 No.42 Xiao-He-Yan Road, Da-Dong District, Shen yang, 110042, Liao-ning Province, China P 30 Clinical Effect of rAd-p53 Combined with FOLFOX4 on the Prognosis of Advanced Colorectal Cancer

Background: This research was to determine the safety and prognosis of recombinant adenovirus p53(Gendicine®) injection combined with FOLFOX₄ on the patients with advanced colorectal cancer.

Methods: Twenty-two patients were recruited and analyzed in the study from July 2008 and April 2010. Gendicine® (1 × 10¹²VP) was injected introtumorally, intravenously or intraperitoneally, to all the patients once a week for two weeks, and then followed by systemic chemortherapy (FOLFOX₄ regimen) as one cycle. 12 cases with local advanced disease then received operation.

Results: All patients were alive during the follow-up (68∼766 days). KPS Score were evaluated in the patients after treatment as 68.2%(15) in 90-100, 22.7% (5 cases) in 90-70, and 9.1% (2 cases) in 70-50, compared with that of 59.1% (13 cases) in 90-70, 18.2% (4 cases) in 70-50, 18.2% (4 cases) in 50-30, and 4.5% (1 case) in 30-10 before the treatment. Self-limiting fever was reported in 10 patients (45.5%) and nausea and diarrhea in 1 (4.5%) after Gendicine® administration. 1 patient was assessed as complete respone and 4 as partial response in the 5 patients with recurrent disease after the treatment, while 1 was assessed as partial response and 2 as ste disease in the 3 patients with liver metastasis. No recurrent symptom were dignosed in the palliative operation group.

Conclusion: Recombinant adenovirus-p53 combined with FOLFOX₄ provides a safe and effective choice of treatment for prolonging progression-free survival time and improves the quality of life of patients with local recurrent and/or metastatic colorectal cancer.

Tagawa M Dr 1 Li Q Dr 1 Kawamura K Mrs 1 Okamoto S Mr 2 Yang S Miss 1 Yamauchi S Mr 1 Kobayashi H Professor 2 Numasaki M Professor 3 Tada Y Dr 4 Hiroshima K Professor 5 Shimada H Professor 6

1 Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba, Japan 2 Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan 3 Department of Nutritional Physiology, Faculty of Pharmaceutical Science, Josai University, Sakado, Japan 4 Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan 5 Department of Pathology, Tokyo Women's Medical University Yachiyo Medical Center, Yachiyo, Japan 6 Department of Surgery, School of Medicine, Toho University, Tokyo, Japan P 31 Adenoviruses expressing interferon-lambda induced apoptosis to the infected human tumors and produce anti-tumor effects in vivo

Recently identified cytokines, IL-28A, IL-28B and IL-29, belong to type III interferon (IFN), IFN-lambda, but the precise biological properties remain uncharacterized. IFN-lambda could have a distinct activity other than type I IFN, IFN-alpha and IFN-beta despite the similar signal cascades. We found that human esophageal carcinoma cells, which expressed the receptor complex consisting of IL-10Rbeta and IL-28Ralpha, were susceptible for IFN-lambda due to either cell cycle arrest or apoptosis. The cell cycle arrest was accompanied by up-regulated p21 expression and dephosphorylated pRb protein. Apoptosis was evidenced by increased sub-G1 populations and annexin-V-positive cells. We constructed adenoviruses bearing the IFN-lambda gene with a fiber-knob modification (Ad/IFN-lambda) and investigated the anti-tumor effects. Transduction of the carcinoma cells with Ad/IFN-lambda increased expressions of cleaved caspase-9, Bax and cytoplasmic cytochrome C, suggesting that IFN-lambda activated the mitochondria-mediated apoptosis pathway. We observed loss of tumorigenicity of esophageal carcinoma YES-2 cells in nude mice when they were infected with Ad/IFN-lambda. Human fibroblasts, negative for the IL-28Ralpha expression, were resistant to IFN-lambda but Ad/IFN-lambda-infected fibroblasts induced apoptosis of co-cultured YES-2 cells and retarded the tumor growth in vivo. Further analyses showed that increased NK cells activity and anti-angiogenesis were not involved in the anti-tumor effects. These data collectively suggest that Ad/IFN-lambda induce apoptosis of the infected receptor-positive tumor cells and fibroblasts-mediated delivery of IFN-lambda is a possible strategy for cancer therapy by inducing direct cell death of the target carcinoma.

Walther W Dr 1 Kobelt D Mr 2 Aumann J Mrs 1 Fichtner I Dr 2 Schmidt M Dr 3 Schlag PM Professor 4

1 Experimental and Clinical Research Center, Charitè at the Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125 Berlin 2 Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125 Berlin 3 Mologen AG, Berlin, Germany 4 Charité Comprehensive Cancer Center, Charité University Medicine, Berlin, Germany P 32 Use of the minimalistic nonviral MIDGE expression system for improved in vitro and in vivo transfer

Great efforts are made for improvement of nonviral vectors to reduce size, proportion of unnecessary sequences to increase transfer- and expression efficiencies. The minimalistic immunologically defined gene expression (Mologen, Berlin, Germany) vector system provides a nonviral linear DNA molecule that is depleted of bacterial origin, the replication backbone sequences, CpG motifs and the antibiotic resistance sequences. Therefore, MIDGE vectors are significantly reduced in size. In our in vitro experiments using equimolar amounts of luciferase expressing plasmid or MIDGE vector we observed 3 to 13-fold increased expression by the MIDGE vector in transfected human colon carcinoma cell lines HCT116 and SW480, and melanoma cell lines A375, MeWo and SKMEL-28. FACScan analysis demonstrated improved transfection efficiency of MIDGE vectors in these tumor cell lines. To analyze applicability of MIDGE-based vector for therapeutic gene expression, we analyzed the tumor necrosis factor alpha (TNF) expressing MIDGE in human HCT116 and A375 cells in vitro. The MIDGE vector generated significantly higher TNF-levels in comparison to plasmid-mediated expression. The use of the TNF-expressing MIDGE vector for nonviral in vivo gene therapy approved its effectiveness. The intratumoral nonviral jet-injection in vivo transfer of the TNF-expressing MIDGE vector into A375 melanomas resulted in efficient high level cytokine expression. We show, that this high level TNF-expression leads to chemosensitization of melanoma in vivo for improved chemotherapy with vindesine. This is reflected by significant tumor growth inhibition. The study demonstrates applicability of MIDGE vector based gene therapy for local gene therapy approaches.

Tyciakova S Dr 1 Hlubinova K Dr 1 Kucerova L Dr 1 Markus JAN 1

1 Laboratory of Molecular Oncology, Cancer Research Institute of Slovak Academy of Sciences, Vlarska 7, bratislava, Slovak Republic P 33 Self-inactivating retroviral vectors expressing human anti-angiogenic genes for cancer gene therapy

Most of the solid tumors growth and metastasis critically depends on angiogenesis. Endostatin and angiostatin are potent angiogenesis inhibitors, which suppress endothelial cell proliferation and migration, tumor vascularization and slow down the growth of tumors. Previously we have successfully used mesenchymal stromal cells isolated from human adipose tissue (AT-MSCs) as delivery vehicles for tumor-targeted suicide gene therapy of mouse xenograft. Now the anti-angiogenic genes were used as therapeutic genes.

In order to improve and prolong the expression of angiostatic genes in tumor area, new safer self-inactivating bicistronic retroviral vectors were constructed. The human endostatinXVIII and the synthetic fusion endostatin-angiostatin genes were cloned into these vectors by standard cloning techniques. Replication-deficient retroviral particles prepared by transient transfection in appropriate packaging cell line were used for infection of target cells, human HT-29 colon carcinoma cell line and AT-MSCs. HT-29-Endo, HT-29-Endo-Angio, AT-MSCs-Endo and AT-MSCs-Endo-Angio cell cultures were prepared. Integration of provirus containing therapeutic genes into the cell genome and gene expression was confirmed by real-time PCR. Biological effect of secreted antiangiogenic proteins was confirmed against the endothelial cell proliferation. Therapeutic cells – genetically engineered AT-MSCs-Endo and AT-MSCs-Endo-Angio were tested for inhibitory effect on tumor cells in vivo on mouse xenograft tumors. Endostatin secretion was proved by ELISA and tumor growth inhibition was achieved.

Yue Wei ZH Professor

P 34 rAd-p53 combining transcatheter arterial embolization with gelatin sponge particles in HCC

Background: Hepatocellular carcinoma(HCC) is one of the most common cancer worldwide. This study aims to evaluate the efficacy of rAd-p53 gene therapy combining transcatheter arterial embolization using gelatin sponge (GS)particles in treatment of HCC.

Methods: Forty patients with HCC were randomly divided into 2 groups : only GS embolization therapy (n = 20) + GS embolization combining rAd-p53 group (n = 20). GS was used in the form of 350–560μm cubes. Tumor was embolized completely. Changes of liver function，responses of tumor and complications related to embolization and the change of digital substract angiogram were analyzed before and after TACE.

Results: In 7 days,values of the serum bilirubin, GPT of two groups patients decreased to normal level. CT showed deceased tumor density, part of these tumors became gas formation in one month, digital substract angiogram was completed in all patients,tumor supplying artery were obstructed completely in 36% patients in GS embolization combining rAd-p53 group, whereas there was only 15% patients in only GS embolization therapy.

Conclusion: Embolization by GS combined with rAd-p53 gene is an effective treatment for HCC. Wide-type p53 gene probably play an important role in inactivation of vascular growth.

Kiselev A Dr 1 Egorova A Dr 1 Bogacheva M Miss 2 Baranov V Professor 1

1 Ott's Institute of Obstetrics & Gynecology 2 Saint-Petersburg State University P 35 Development of non-viral gene delivery system using CXCR4 ligand-conjugated cross-linking peptides

Application DNA as therapeutics requires cell-specific targeting which can be achieved by modification of vehicles with a ligand for certain receptor. CXCR4 is a receptor of chemokine SDF-1 and is expressed on some types of cancer and stem cells. Cystein-flanked peptides which are capable to form DNA condensates because of cross-linking are considered to be a perspective group of non-viral vehicles. The aim of this project is to characterize a CXCR4 ligand-conjugated cross-linking peptides as a receptor-mediated gene delivery system.

We studied four types of DNA/peptide complexes with different ratio between cystein-flanked arginine-rich peptide modified with N-terminal sequence of the chemokine SDF-1 (residues 1–17) and peptide (CHRRRRRRHC) – 100%, 50%, 10% and 0% (ligand-free control). The peptides modification with histidine residues facilitates the escape of DNA from endosomes. Template polymerization of cross-linking peptides was used to form DNA/peptide complexes. EtBr exclusion assay proved peptides ability to condense DNA. Transfection activity was studied in two CXCR4(+) cell lines (A172 and HeLa) and CXCR4(-) cell line (CHO) with lacZ as a reporter gene. Transfectional efficacy of ligand-conjugated vehicles in CXCR4(+) HeLa and A172 cells was 10-times higher compared to control peptide. The level of transgene expression with ligand-conjugated peptides in low N/P ratios was comparable with the efficacy of PEI. Otherwise transfection efficacy of ligand-conjugated peptides on CXCR4(-) CHO cells was lower than in control PEI. Thus these results demonstrate that ligand-conjugated vehicles reported can be a perspective approach for effective gene delivery to CXCR4 expressing cells.

This work was supported by Carl Zeiss fellowship and RFBR grant 10-04-01236-a.

Lavilla-Alonso S Mr 1 Ylösmäki E Mr 1 Saksela K Professor 1

1 PO Box 21 (Haartmaninkatu 3, room E359). 00014-University of Helsinki. Finland P 36 Inclusion of miR122 targets in wild-type serotype 5 adenovirus to ablate liver replication

Adenovirus -based oncolytic therapies present important limitations regarding tissue selectivity and toxicity, an issue well-documented for the commonly used adenovirus serotype 5 (Ad5).

Basic research has shown a specific micro-RNA (miR) pattern for many tissues, like the virtually exclusive expression of miR122 by liver. We have shown that this feature can be utilized to selectively ablate adenovirus replication in cells of hepatic origin by genomic insertion of miR122 targets to reduce E1A gene expression.

In this study we have examined in more detail the optimal number of miR122 targets, studied the cell type-specific inhibitory potential of miR122 via careful cell viability analyses, and extended these studies to more relevant experimental systems.

These results will provide a rational basis for proceeding towards ex vivo and in vivo studies to test the applicability of this system for clinical gene therapy purposes.

Günzburg W Dr 1 Hlavaty J 2 Jandl G 1 Petznek H 1 Liszt M 1 König-Schuster M 1 Sedlak J 1 Egerbacher M 3 Weissenberger J 4 Salmons B 5 Renner M 6

1 Institute of Virology, Department of Pathobiology, University of Veterinary Medicine, A-1210 Vienna, Austria 2 Christian Doppler Laboratory for Therapeutic Vector Development, University of Veterinary Medicine, A-1210 Vienna, Austria 3 Institute of Histology & Embryology, Department of Pathobiology, University of Veterinary Medicine, A-1210 Vienna, Austria 4 Experimental Neurosurgery, Goethe University Hospital, Neuroscience Center, D-60590 Frankfurt, Germany 5 Austrianova Singapore Pte Ltd, Singapore 138668 6 Department of Medical Biotechnology, Paul-Ehrlich-Institute, D-63225 Langen, Germany P 37 Comparative evaluation of replicating retroviral vectors carrying a suicide gene in multiple preclinical in vivo models of glioblastoma

Recently, efficacy of suicide gene therapy based on replication-competent retroviral (RCR) vectors as delivery vehicles for the therapeutic gene has been described in the treatment of experimental cancer, including malignant gliomas. One such promising suicide gene is cytosine deaminase (CD) since is converts 5-flurocytosine (5-FC) to 5-flurouracil (5-FU), which is freely diffusible and tumour toxic. We have critically evaluated a panel of human and rodent glioma/glioblastoma cell lines (U-87MG, U-118MG, LN-18, LN-229, 8-MG-BA, 42-MG-BA, A-172, T-98G, UVW, C6, 9L, G-26, GL-261, Tu-2449, Tu-9648) with respect to spread of an RCR virus vector carrying the CD gene, sensitivity towards the CD mediated conversion of 5-FC to 5-FU suicide system, and orthotopic growth characteristics in mice to identify suitable preclinical animal models for the development of a glioblastoma gene therapy. Rapid virus spread was observed in eight out of nine human cell lines tested in vitro. As expected, only CD-expressing cells became sensitive to 5-FC, due to their ability to convert the prodrug in its toxic form, 5-FU. All LD₅₀ values were within the range of concentrations obtained in human body fluids after conventional antifungal 5-FC administration. In addition, a significant bystander effect was observed in all human glioma cell lines tested. Injection of the RCR vector into pre-established orthotopic mouse tumor xenografts revealed substantial infection and virus spread of tumor tissue from most cell types. This preclinical data forms part of the requirement for a recently approved human phase I/II clinical trial.

Lee HJ Ms 1 Jeong YY Professor 2 Lee IK Professor 3 Namgung R Ms 4 Kim WJ Professor 4 Yu MK Ms 5 Jon S Professor 5 Lee SJ Mr 6 Park IK Professor 1

1 Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju 501-746, South Korea 2 Department of Diagnostic Radiology, Chonnam National University Medical School, Gwangju 501-746, South Korea 3 Department of Genome Research Center for Hematopoietic Disease, Hwasun Chonnam National University Hospital, Hwasun 519-809, South Korea 4 Department of Chemistry, BK21 Program, Polymer Research Institute, Pohang University of Science and Technology, San 31, Hyoja-dong, Pohang 790-784, South Korea 5 Department of Life Science, GIST, 1 Oryong-dong, Buk-gu, Gwangju 500-712, South Korea 6 BioImaging Research Center, GIST, Gwangju 500-712, South Korea P 38 Tumor Suppressor p53 Gene Therapy mediated by PEI-coated, thermally crosslinked Superparamagnetic iron oxide nanoparticles

Targeting therapeutic drug using Superparamagnetic iron oxide nanoparticles (SPION) as carrier systems gained increased attention in the biomedical field. Generally, particles carrying different core sizes and various coatings were used in in vitro and in vivo. According to their desired purpose, they were also additionally functionalized with drugs, plasmids, peptides or proteins of interest. The p53 gene is a tumor suppressor and genome defense. It plays important role in various activities like cell cycle regulation, apoptosis, and DNA repair. In this study, we proposed gene carrying SPION for anti-cancer gene therapy. thermally crosslinked SPION was conjugated with branched PEI (bPEI) 1800Da by EDC-NHS chemistry for DNA delivery. The presence of bPEI 1800Da was functionalized to bind with DNA due to its electrostatic interaction. The synthesized bPEI 1800Da-conjugated TCL-SPION (bPEI-SPION) was investigated for its potential as an imaging-guided gene carrier. For the first work, bPEI-SPION achieved higher cell viability compared to bPEI 25kDa and 1800Da. And internalized SPIONs were tracked by Magnetic resonance imaging (MRI). MR contrasts were strong and became more intense with increasing amounts of the bPEI-SPION. The shortening of the MR T2 relaxation time was also correlated to increasing amounts of the bPEI-SPION. Finally, delivery of p53 tumor suppressor gene by bPEI-SPION obtained the suppressive effect on CT26, 4T1, Ca46 and Daudi cells. Protein expressions of p53 gene after transfection into the cell lines (CT26, CA46) were measured by RT-PCR. In conclusion, bPEI-SPION could be used for an efficient gene delivery carrier tracked by MR imaging.

Hasenpusch GH Mr 1 Geiger JP Mr 1 Mykhaylyk O Miss 2 Wagner K Dr 3 Wiekhorst F Dr 4 Rudolph C Dr 1

1 Department of Pediatrics, Ludwig-Maximilians-University Munich, 80337 Munich, Germany 2 3Institute of Experimental Oncology, Klinikum Rechts der Isar, Technical University of Munich, 81675 Munich, Germany 3 Institute of Pathology, Ludwig-Maximilians-University Munich, 80337 Munich, Germany 4 Physikalisch-Technische Bundesanstalt, 10587 Berlin, Germany P 39 Magnetic field derived aerosol delivery (Magnetosol) facilitates targeted gene delivery to distinct lung regions

Severe lung diseases, such as lung cancer, are usually treated by intravenous administration of drugs. This application route however requires relatively high drug amounts in order to maintain a therapeutically effective drug level in the lungs. This circumstance leads to severe side effects, especially when cytotoxic drugs are applied to the patient. Aerosol application however has been shown to increase drug levels in the lungs, while reducing systemic side effects, because less of the therapeutic agent is needed. Nevertheless the lungs represent a delicate organ, predisposed for inflammation and irritation. In order to reduce side effects and coincidentally increase drug concentrations in distinct regions of the lungs we applied Superparamagnetic Iron Nanoparticles (SPIONs) and Fluorescein-Natrium as an aerosol to conscious mice, which were exposed to a magnetic field during inhalation. This experiment resulted in a 3-fold increase of Fluoresceine-Natrium in the lungs in total and to a 3-fold increase of Iron in the right lung lobe, when the mice were exposed a magnet field on the right chest during inhalation. Moreover we demonstrated magnet field gene expression in one distinct lung lobe under the influence of a magnetic field during the inhalation procedure. Summarizing we postulate that Magnetosols could be an effective tool for the gene therapeutic treatment of lung diseases, such as lung cancer or tuberculosis.

Jankowski M Dr 1 Wojciechowska M Mrs 1 Wandtke T Mr 1 Lenartowski R Dr 2 Tyrakowski T Professor 3 Kopinski P Dr 1

1 Dept. of Gene Therapy, Nicolaus Copernicus University, Torun, Poland 2 Dept. of Genetics, Institute of General & Molecular Biology, Nicolaus Copernicus University, Torun, Poland 3 Dept. of Pathobiochemistry & Clinical Chemistry, Nicolaus Copernicus University, Torun, Poland P 40 Plasmid delivered S.typhi flagellin induces inflammation and cell death in a model of non-small cell lung cancer

Tumor cells, including those of non-small cell lung cancer, developed numerous mechanism impairing maturation of dendritic cells (DCs). The delivery of stimulatory signals to dendritic cells in the tumor microenvironment is believed to be an effective means to break tumor-induced tolerance. The work presented here evaluates the immunostimulatory properties of a TLR5 ligand Salmonella typhi flagellin (FliC). We developed a novel plasmid vector containing a cell membrane-displayed humanized FliC ORF under control of tumor-specific two-step transcriptional activation system. In vitro, transduction of A549 tumor cells with plasmid coding for humanized Salmonella flagellin induced the maturation of human monocyte-derived DCs in co-cultures.

Two groups of C57BL6 mice bearing a subcutaneous syngenic Lewis lung cancer (LLC) tumor were intratumoraly injected with in vivo-JetPEI and FliC plasmid or as a control with in-vivo JetPEI and phosphate-buffered saline. Four administrations were performed within 1 week. Tumor volumes and survival were monitored for 6 weeks. Plasmid injections delayed the growth of implanted LLC tumors and significantly improved survival rates. Histological examination of plasmid-injected tumors revealed lymphocyte infiltration and significant necrosis. These results suggest that plasmid-delivered FliC could be effective immunoadjuvant for cancer immunotherapy.

Jing Qiang ZH Professor 1

1 37#, Guoxuexiang,Renmin South Road, Wuhou District,Chengdu,610041,China P 41 Randomized and controlled clinical trial for thyroid cancer treated by rAd-p53

Background: TCs are insensitive to radiotherapy and chemotherapy. Surgery is the main options for TC. Studies show there are p53 genetic mutations in TC with various manifestations.This study aims to discuss the safety and efficacy of rAd-p53 gene therapy to treat TC in combination with surgery and other traditional methods.

Method: This is a radomized and controlled clinical trial. 22 patients were enrolled into the Trial Group and received rAd-p53 plus surgery, or surgery and ¹³¹I,or surgery and radiation, or surgery, radiation and ¹³¹I. 18 patients received p53 local injections, 2 received intervenous infusion, and 2 received administration by the both methods. Another 22 patients were enrolled into the Control Group.

Results: All patients in the Trial Group got fever and muscular soreness after the 1st injection of rAd-p53, which were all tolerable and disappeared after 2-3 times injections without special treatment. Other side effects related to rAd-p53 injection were arthralgia in 4,nausea in 2, and numbness of tongue tip in 1,which all dissapeared without special treatment. ECOG Scores decreased in different degree in all the patients in the Trial Group,which were better than that of the control group. 2 patients in the study were sensitive to radiotherapy, which might be sensitized by rAd-p53. In some patients,tumors were softened after 2 times injections.

Conclusion: The study shows that it is safe to use rAd-p53 in TC patients with main adverse reaction of flu symptoms like fever and muscular soreness. It suggests that rAd-p53 may change the insensitity of TC cell to external radiotherapy.

Şalva E. Mrs 1 Kabasakal L. Dr 2 Özkan N. Ms 3 Eren F. Mr 4 Çakalağaoğlu F. Professor 5 Akbuğa J. Professor 6

1 1 Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, İstanbul, Turkey. 2 Marmara University, Vocational School of Health Related Professions, Pathology Laboratory 2 3Marmara University, Faculty of Pharmacy, Department of Pharmacology, İstanbul, Turkey. 3 2 Marmara University, Vocational School of Health Related Professions, Pathology Laboratory Department, İstanbul, Turkey. 4 Marmara University, Institute of Gastroenterology, İstanbul, Turkey. 5 Izmir Ataturk Education and Research Hospital, Pathology Department, İzmir, Turkey 6 1 Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, İstanbul, Turkey P 42 Chitosan Mediated VEGF shRNA Transfer for the Treatment of Breast Cancer

Background: Angiogenesis is an exciting and promising area of anti-cancer drug research. VEGF and its receptors is the most extensively investigated system in tumor angiogenesis. RNA interference includes strategies that result in the down-regulation of the expression of a target gene at the post-transcriptional level using RNAi agents. The aim of this study was to investigate whether intratumoral injection of chitosan/shVEGF complexes could suppress VEGF expression in the breast cancer model of rats.

Method: Chitosan/shVEGF complexes were prepared in 2/1 ratio. For tumor formation, Sprague Dawley rats in the experiment were injected intraperitonally 50 mg/kg NMU (Sigma) on the 45-50 day of age. After tumor formation, chitosan/shVEGF complexes were injected intratumorally to rats. Tumor progression was reported. VEGF expression were examined by immunohistochemistry, RT-PCR and western blot.

Results: Tumor volume decreased at the end of experiments after chitosan/shVEGF complexes treatment (96%). VEGF immunexpression was reduced approximately 67.4% by i.t applied complexes. VEGF-A mRNA expression was also reduced approximately 84.60%. Free shRNA injection indicated lower tumor suppression. The western blotting results correlated with the RT-PCR and tumor volume measurements.

Conclusion: The data suggest that chitosan/shVEGF complexes can be used to inhibit tumor growth and VEGF expression in breast carcinoma model of rats. Chitosan is very suitable and effective gene delivery system for VEGF shRNA in breast cancer models.

Acknowledgements: This study was supported by Marmara University Scientific Research Projects Association, Turkish Association for Cancer Research and Control (Terry Fox Cancer Foundation).

e-mail: jakbuga@marmara.edu.tr

Cronin M Ms 1 Collins S Ms 2 Inagaki A Dr 3 Hiraoka K Dr 3 Kasahara N Professor 3 Gahan CGM Dr 4 O'Sullivan GC Professor 5 Tangney M Dr 2

1 Cork Cancer Research Centre, University College Cork, Ireland 2 Cork Cancer Research Centre, University College Cork, Ireland & UCLA School of Medicine, USA 3 UCLA School of Medicine, USA 4 Dept. of Microbiology, University College Cork, Ireland 5 University College Cork P 43 Non-pathogenic bacteria as cell therapy vectors for cancer treatment

The ideal cancer treatment would eradicate tumour cells selectively with minimum side effects on normal tissue. Bacteria have emerged as gene vectors with natural tumour specificity, capable of homing to tumours and replicating locally to high levels when systemically administered. Pathogenic, invasive species have been utilized to deliver plasmid intracellularly to tumour cells, and some genera possess oncolytic capability, such as Clostridium and Salmonella. However, their inherent toxicity has outweighed therapeutic responses in patients, despite efforts to reduce toxicity through genetic modification.

A promising alternative exploits non-pathogenic bacteria. Non-invasive, apathogenic species expressing heterologous genes can secrete therapeutic proteins locally within the tumour (analagous to Mesenchymal Stem Cell therapeutic strategies.) We are investigating a range of commensal bacteria (natural inhabitants of the human GIT, and often employed as probiotics). Our group has developed imaging systems using luminescent reporter gene (lux) tagging of various bacteria permitting real-time visualization of vector, in subcutaneous and orthotopic murine models (melanoma, breast, lung, glioma).

We have also demonstrated that certain species (Bifidobacterium breve) are capable of trafficking to systemic tumours following oral administration, with equal efficiency to intravenous injection. Our studies indicate a low level, delayed anti-vector immune response within tumours, with no bacterial clearance from tumours for up to 1 month post administration. Through engineering of secreting constructs, these replication competent bacteria can mediate high-level production of soluble agents within tumour masses, presenting a powerful and safe approach to specific gene/cell therapy of primary tumours and secondary metastases.

Longjiang LI Professor Zhuang ZH Dr Yi LI Dr Yang D Dr Ning GAO Dr

P 44 p53 Gene Have a Role in Phenotype Reversion of Melanoma in vitro

Background: When accompanied with abnormal p53 expression, Melanoma suffered a higher risk of recurrence and metastasis. We evaluate the optimum multiplicity of infection (MOI) and transfection efficiency of recombinant Ad-p53 gene to malignant melanoma cell line（A375）in vitro, and observe the effect of exogenous p53 gene on biologic phenotype of A375 cells.

Methods: A375 cells in logarithmic growth phase were seeded onto six-well plate. After 24h, the cells attached and rAd-GFP solution were added into the medium with MOI 0 to 500.The transfection efficiency of rAd-p53, expression of exogenous p53 gene, proliferation inhibition rate of each group, cell cycle and apoptosis, as well as the expression of P21CIP/WAF, bcl-2, MMP-9 before and after the transfection, were all detected or determined.

Results: Exogenous p53 gene was successfully transfected into A375 cells with the transfection efficiency more than 95% and inhibited A375 cell proliferation significantly with MOI ranged from 100 to 500. G1 stage increasing and S stage decreasing were detected after transfection. The percentage of apoptosis increased 24h after transfection and apoptotic peak was obvious (P < 0.01). The expression of p53 mRNA, P21CIP/WAF mRNA andP21CIP/WAF protein increased obviously, while bcl-2 mRNA and protein decreased 72h after transfection when compared with the control group.

Conclusions: Exogenous wild type p53 gene could be effectively transfected into malignant melanoma cell line and strongly inhibited cell proliferation, altered the cell cycle and induced cell apoptosis. p21CIP/WAFand bcl-2 were partly responsible for the inhibition of A375 cells growth.

Şalva E Mrs Özbaş-Turan S. Mrs Akbuğa J. Professor 3

3 1 Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, İstanbul, Turkey P 45 Chitosan nanoparticles containing shRNA targeting VEGF as cancer therapeutic

Background: RNAi is a highly promising technology for gene therapy application in treatment of different cancers and preliminary results of trials with siRNAs targeted against VEGF for treatment of breast cancer are encouraging. However, RNAi is challenged by the method of delivery and stability in vivo. Therefore, the efficiency of carrier system or delivery is an important subject in this technology. The aims of this study are to evaluate shRNA-loaded (shRNA-expressing pDNA targeting vascular endothelial growth factor-A (VEGF-A)) chitosan nanoparticles for cellular uptake and to investigate the silencing effect of siRNA using human breast cancer cells in vitro.

Method: Chitosan nanoparticles were prepared using ionotropic gelation method. Physicochemical and morphological characterization of nanoparticles were studied. Cell uptake of nanoparticles were investigated using fluorescence microscope. The VEGF silencing activity of shRNA-loaded chitosan nanoparticles were measured by ELISA method in different cell lines; MCF-7, MDA-MB-435, HeLa, HEK293.

Results: Nanoparticles protected the shRNA against the degradative effects of serum and enzyme. As a result of in vitro transfection studies made in different cell lines with nanoparticles containing shRNA in different amounts, the highest gene inhibition (56%) was measured in MCF-7 after transfection while the lowest gene inhibition (48%) was observed in MDA-MB435.

Conclusion: Chitosan nanoparticles show high potential as carrier for safer and cost effective shRNA delivery in breast cancer.

Acknowledgements: This study was supported by Marmara University Scientific Research Projects Association, Turkish Association for Cancer Research and Control (Terry Fox Cancer Foundation).

e-mail: jakbuga@marmara.edu.tr

Matuskova M Dr 1 Baranovicova E Dr 2 Kucerova L Dr 1

1 Cancer Research Institute SAS, Vlarska 7, 83391 Bratislava, Slovakia 2 Faculty of Natural Sciences, Mlynska dolina, 84215, Bratislava, Slovakia P 46 Comparison of Bystander Effect Mediated by MSC Expressing Cytosine Deaminase or HSV Thymidine Kinase towards Tumor Cells

Background: Adipose tissue-derived mesenchymal stromal cells (AT-MSC) retroviraly transduced with either cytosine deaminase::phoshoribosyl transferase (CD-MSC) or Herpes simplex virus thymidine kinase (TK-MSC) in combination with appropriate prodrug can be used for cancer gene therapy. We aimed to compare the efficiency of the above mentioned systems and to mechanisms responsible for sensitivity of different tumor cells to cell-mediated prodrug therapy. We used a fluorimetric assay in order to determine the extent of bystander effect mediated by engineered AT-MSC toward various tumor cells.

Method: Tumor cells stably expressing the green fluorescent protein were cocultured with therapeutic AT-MSC in the presence of ganciclovir (GCV) or 5-fluorocytosine (5-FC). Gap junctional intercellular communication (GJIC) was examined using flow cytometry and expression of genes responsible for drug metabolism and efflux was detected by RT-PCR.

Results: We observed significant differences in sensitivity of tested tumor cell lines. Both therapeutic systems were effective on glioblastoma cells 8-MG-BA. CD-MSC exhibited strong bystander effect on melanoma A375 cells. GJIC defective HeLa cells responded neither TK-MSC/GCV nor CD-MSC/5-FC therapy. In spite of functional GJIC between A375 cells and AT-MSC, this cell line was resistant to TK-MSC/GCV treatment. Cell line MDA-MB-231, resistant to CD-MSC/5-FC system, was very sensitive to TK-MSC/GCV treatment.

Conclusion: Taken together, neither CD-MSC/5-FC nor TK-MSC/GCV approach should be considered a universal tool for cancer therapy. Although additional studies are necessary, it is obvious that intercellular communication, multidrug resistance proteins and enzymes included in drug metabolism contribute to bystander effect efficiency of cancer gene therapy mediated by AT-MSC.

Kichler A Dr 1 Mason AJ Dr 2 Leborgne C Mr 3 Bechinger B Professor 4 Scherman D Dr 1

1 Laboratoire de Pharmacologie Chimique et Génétique UMR 8151 CNRS-U1022 INSERM, Université René Descartes, Chimie Paristech, Paris, France 2 Pharmaceutical Science Division, King's College London, 150 Stamford Street, London, UK 3 Genethon, 1 rue de l'Internationale, 91000 Evry, France 4 Faculté de Chimie, Institut Le Bel, 4 rue Blaise Pascal, F-67000 Strasbourg, France P 47 Histidine-rich amphipathic peptides promote efficient delivery of nucleic acids into mammalian cells

Besides being a useful tool in research, gene transfer has a high potential as treatment for a variety of genetic and acquired diseases. However, in order to enable a gene to become a pharmaceutical, efficient and safe methods of delivery have to be developed. We found that cationic amphipathic histidine-rich peptide antibiotics can efficiently deliver DNA into mammalian cells. Our lead compound, LAH4 (KKALLALALHHLAHLALHLALALKKA), demonstrated in vitro transfection efficiencies comparable to those of commercially available reagents. Synthesis and evaluation of LAH mutants provided evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Our results also suggest a mechanism of selective destabilization by LAH4 of anionic lipids in the membranes of cells during transfection. Further results indicate that acidification of the endosome results in high local concentrations of free peptide in this organelle. These peptides become then available to interact with the endosomal membranes and thereby are responsible for the delivery of the plasmid DNA complex to the cytoplasm. When these data are taken together, they indicate a dual role of the peptide during the transfection process, namely DNA complexation and membrane permeabilization. Finally, we will report that peptides of the LAH family are efficient siRNA delivery vehicles.

Zhang Guo ZH Professor

P 48 A Case Report of Recurrent and Metastatic Rectal Cancer Responding to rAd-p53 and FOLFOX4

Background: Locally recurrent and metastatic disease carries a poor prognosis and remains the most frequent cause of death due to colorectal adenocarcinoma. Although aggressive operation is the only curative operation, it is only feasible in a few cases. Patients with nonresectable recurrent and metastatic tumors are always treated with palliative modalities, and long-term survival is limited. Gene therapy has been used as a possible treatment modality for verious malignent diseases.

Methods: The patients presented here is a 57-year-old man who was operated with APR for rectal cancer 3 years ago, and was diagnosed locoregional recurrence and multiple lung metastases, accompanied with ureteric dilatation, pelvis effusion, acute renal failure, hyperkalaemia, oedema of lower extremity (KPS < 20). The patient was treated with p53 gene (totally 20 × 10¹²VP) (Gendicine^@, Shenzhen Sibino Gentech, China) combined with FOLFOX for ten cycles.

Results: During a 13 months follow-up perio, the patient was alive in good condition (KPS >90). Obstruction of urinary passage was relieved and a steady improvement in plasma biochemistry toward normal was observed. We also found that the locally recurrent disease was necrosis and marked lung metastatic tumor shrinkage or disappeared, and sustained normal CEA and liver function. In the patient treated with Gendicine + FOLFOX, there was no severe side effect.

Conclusion: It can be concluded that Gendicine^@ combined with FOLFOX chemotherapy is an effective and safe strategy for the treatment of patient with advanced rectal cancer.

Ahmadi M Dr 1 King J Dr 1 Nicholson E Dr 1 Morris E Dr 1 Stauss H Professor 1

1 Department of Immunology, University College London, Royal Free Hospital, Rowland Hill Street, London NW3 2PF, United Kingdom P 49 Enhancing the Efficacy of TCR Gene Therapy by Co-Transfer of CD3 Molecules

T cell receptor (TCR) gene transfer is an efficient strategy to redirect the specificity of T cells. Efficient cell surface TCR expression requires the formation of a stable TCR-CD3 complex. CD3 may be rate-limiting for the expression of TCR, which may result in reduced cell surface expression of the introduced TCR. The increased expression of TCR may improve the functional avidity of the genetically modified T cells. Co-transfer of CD3 and TCR into a murine T cell line enhanced expression of an Influenza-specific TCR (NP-specific), resulting in increased NP tetramer binding compared to cells transduced with TCR only. We found that transduction of both CD8⁺ and CD4⁺ T cells with MHC-class I restricted TCR results in antigen-specific function of these cells in vitro. The co-transfer of CD3 into these cells increases expression of the introduced TCR and the functional avidity of these cells to their specific peptide. Data suggest that TCR-CD3 co-transduced T cells are preferably expanded compared to the TCR-only transduced T cells in response to an NP-expressing tumour, and are capable of memory response formation upon secondary challenge with tumour in vivo. Furthermore, TCR-CD3 T cells eradicate tumour faster than TCR-only transduced cells. Using in vivo imaging, we found that increased efficiency in tumour eradication may be linked to faster tumour infiltration by TCR-CD3 co-transduced T cells compared to TCR-only transduced T cells. The data suggest that co-transduction of CD8⁺ with TCR and CD3 enhances functional avidity and anti-tumour immunity of cells without resulting in toxicity in vivo.

Pincha MP Miss 1 Salguero GS Dr 2 Sundarasetty BS Mr 3 Wedekind DW Dr 3 Schambach AS Dr 4 Baum CB Professor 4 Ganser AG Professor 2 Stripecke RS Professor 2

1 Department of Hematology, Hemostasis, Oncology and Stem cell Transplantation 2 Department of Hematology, Hemostasiology, Oncology and Bone Marrow Transplantation 3 Department of Hematology, Hemostasiology, Oncology and Bone Marrow Transplantation 4 Department of Experimental Medicine P 50 Integration-Deficient tetracistronic Lentiviral vector engineered SMART-DC vaccine against Melanoma

Background: Novel approaches facilitating consistent and potent Dendritic cell (DC) production for large scale vaccination trials are warranted. We previously demonstrated, human and mouse DC precursors programmed with lentiviral vectors (LV) encoding growth factors (GM-CSF, IL-4) and tumor antigen (TRP-2) led to induction of highly viable and therapeutically potent “SMART-DCs” (Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors) in a B-16 melanoma mouse model (Koya et al, 2007). Their biosafety was enhanced 2-fold by introducing a suicide gene (HSV-TK) and a cell-specific MHCII promoter (Pincha et al, submitted). In this study, we tested, integration-deficient (ID) packaging system to program even safer SMART-DCs with state of art tetracistronic LVs (HSV-TK, GMCSF, IL-4, TRP2) under both CMV and MHCII promoters. Additionally, we explored preconditioning with Flt3L to expand and direct hematopoietic stem cell precursors toward DC differentiation.

Methods and Results: Mouse bone marrow cells were preconditioned (8 hours) + /− Flt3L and transduced with tetracistronic ID-LVs (CMV and MHCII), lead to autonomous self differentiation into DCs. Cytospin and FACS analysis of surface markers: CD11c, CD11b and MHCII were performed every 7 days to confirm DC phenotype over a period of 3 weeks. Highest purity was obtained at day 14 (upto 65%). C57BL6 mice vaccinated with all SMART-DC groups protected (upto 40%) mice against melanoma. Flt3L (+) groups doubled SMART-DC yield in vitro and prolonged survival in vivo.

Future: In summary, we demonstrate rationale programming of SMART-DCs with tetracistronic ID-LVs capable of protecting mice against melanoma. We are currently exploring intravenous administration of tetracistronic ID-LVs as direct vaccines.

Meyerhuber P Mr 1 Conrad H 2 Stärck L 1 Leisegang M 1 Bernhard H 3 Uckert W 1

1 Department of Molecular Cell Biology and Gene Therapy, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany 2 Department of Hematology/Oncology, Technical University of Munich, Klinikum rechts der Isar, Munich, Germany 3 Department of Hematology/Oncology, Klinikum Darmstadt, Darmstadt, Germany P 51 Targeting the epidermal growth factor receptor (HER) family by T cell receptor gene-modified T lymphocytes

HER2 is over-expressed in 25–40% of all breast cancers and in a variety of other tumors. Due to the selective over-expression in malignant tissue, HER2 is considered one of the most attractive targets for therapeutic interventions.

In this study, we isolated the TCR genes of a HER2-reactive, allo-HLA-A2-restricted CTL clone and introduced the genes into a retroviral vector. Efficient cell surface expression of the TCR and improved functional avidity of the gene-modified T cells were achieved after murinization (replacement of the human TCR constant regions by mouse counterparts), codon-optimization and application of the P2A gene linker (HER2-TCR-opt). Thus, the TCR expression in transduced T cells increased from 1.5% to 41% measured by A2/HER2-multimer staining. The ability to secrete IFN-g of HER2-TCR-opt-transduced T cells was comparable to that of the CTL clone, which showed a half maximum IFN-g secretion at 10⁻⁷ M towards HER2 peptide-loaded T2 cells. Furthermore, HER2-TCR-opt-transduced T cells lysed HER2-expressing tumor cell lines as efficient as the parental clone (lysis of 58% at a E:T of 30:1). The TCR showed a cross-reactivity to HER3 and HER4 that was similar to the parental CTL clone. Extensive testing with HER2/3/4 negative cell lines did not reveal any further epitopes that were recognized by the TCR.

Our results contribute to the development of a TCR-based approach for the treatment of HER2-positive breast cancer, as well as of other malignancies expressing HER2, HER3 and/or HER4.

Schillinger U. Dr 1 Rutz M. Mrs 2 Hüttinger C. Dr 2 Jahnke A. Dr 2 Walsch F. Dr 2 Ferizi M. Mrs 1 Benda V. Mrs 1 Hirschberger J. Professor 2 Köstlin R. Professor 3 Gänsbacher B. Professor 1 Brill T. Dr 1 Plank C. Dr 1

1 Institute of Experimental Oncology, Klinikum rechts der Isar der TU Munich, Germany 2 Department of Small Animal Medicine, Ludwig-Maximilians-Universität Munich, Germany 3 Department of Small Animal Surgery and Reproduction, Ludwig-Maximilians-Universität Munich, Germany P 52 Adjuvant immunomagnetofection prolongs relapse-free survival of fibrosarcoma bearing cats

Feline fibrosarcoma is an everyday challenge in veterinary practice. Despite aggressive pre-or post-operative treatment it has a high relapse rate of aprox. 75 % within 6 months after surgical resection. To obtain a better therapeutic outcome, novel strategies are necessary. Hence, we established immunostimulatory therapy by magnetofection. Here we report preliminary results from a comparative clinical study where the genes for feline GM-CSF, IFN-g and IL-2, feline GM-CSF alone or human GM-CSF were administered. The study design is prospective, randomized, placebo-controlled ( = standard therapy) and includes four arms: (1) standard therapy, i.e. surgery alone; (2) nonviral magnetofection of the triple-combination of feline GM-CSF, IFN-g and IL-2 genes into the tumor before surgery or nonviral administration by magnetofection of feline (3) or human GM-CSF (4) gene alone. Preliminary clinical endpoints of the studies are relapse-free survival. Nonviral magnetofection, a procedure developed in our laboratory, is gene therapy by plasmids associated with magnetic nanoparticles under the influence of a magnetic field. The magnetic field was applied to achieve improved retention of the injected vector dose in the tumor. All gene-therapeutic treatments were well tolerated and led to significantly prolonged relapse-free survival. Recent results from FACS-analysis of the primary tumor cells from the treated groups showed increased MHC-II expression compared to those of control cats. This is encouraging concerning future use in veterinary practice as this treatment can be easily administered. The results are promising with respect to their potential in human medicine, as they have been obtained in real patients instead of experimental tumor models.

Kottke T Mr 1 Pulido J Dr 1 Thompson J Ms 1 Harrington K Dr 2 Selby P Professor 3 Melcher A Dr 3 Vile R Dr 1

1 Mayo Clinic 2 The Institute of Cancer Research 3 University of Leeds P 53 Tumour Immune Evasion Enforced by Viral cDNA Library Vaccination Can Be Exploited for Ambush of Emerging Tumour Cell Variants

The tumor associated antigens (TAA) most relevant for tumor rejection remain largely unidentified in most tumor types. We have shown previously that autoimmune T cell reactivity, which also mediates tumor rejection, can be activated in vivo by inflammatory killing of normal cells. In addition, expression of a TAA in an immunogenic virus generates potent T cell responses against the TAA. Therefore, we hypothesized that it would be possible to use a cDNA library from a normal tissue, expressed from an immunogenic viral platform, to vaccinate tumor bearing hosts against a wide range of TAA expressed on tumors of the same histological type. We show here that a cDNA library from normal human prostate expressed by Vesicular Stomatitis Virus cured established murine prostate tumors. However, under suboptimal vaccinating conditions, tumor escape was possible, but only by the acquisition of a radically new tumor cell phenotype. Recurrent tumors could then be treated by sequential vaccination with a cDNA library from the recurrent tumors or by exploiting a novel acquired sensitivity to chemotherapy. This two-step ‘trap-and-ambush’ strategy uses initial vaccination to trap tumor cells into a very restricted evolution to ensure escape from the selective pressure imposed against the very broad antigenic repertoire encoded by the cDNA library. Thereafter, a highly targeted second line treatment against the new, enforced tumor phenotype can ambush these emerging escape variants. This combination represents a novel way to exploit the ability of tumor cells to escape powerful selective pressures in vivo for therapeutic advantage.

Cieri N Dr 1 Provasi E Dr 1 Camisa B Dr 1 Magnani Z Dr 1 Bondanza A Dr 1 Bonini C Dr 1

1 San Raffaele Scientific Institute, Milan, IT P 54 Memory T cells masquerading as naïve cells: Implications on adoptive T cell immunotherapy

Ex vivo T cell manipulation often induces their terminal differentiation, resulting in poor persistence and activity of transferred cells. We previously showed that costimulation and culture with gchain cytokines generates gene modified T cells with a functional central memory (T_CM) phenotype superior to effector/effector memory (T_EM) counterparts for expansion potential and antitumor activity. Here we investigated the consequence of initial targeting of selected T cell subpopulations. We activated and efficiently transduced FACS-sorted T Naïve (T_N), T_CM and T_EM cells by viral vectors. In contrast to T_CM and T_EM, T_N had a greater expansion potential and more sustained expression of IL7-Ra. Strikingly, manipulation of T_N, resulted in a predominant population of post-mitotic lymphocytes expressing the CD45RA + CD62L + CCR7 + naïve phenotype. These cells expressed markers common to early differentiated cells (CD27 + CD28 + CD127 + Bcl2+) and markers proper of memory lymphocytes (CD45RO + CD122 + CXCR3+). Post-mitotic T_N produced lower levels of IFNg and Granzyme A, expressed higher levels of c-Kit and CXCR4, and lower levels of HLA-DR, CCR5 and PD1 than memory counterparts. To verify their self-renewal and differentiation potential upon antigen stimulations, T_N, T_CM and T_EM were transduced to express a WT1-specific TCR. Upon multiple stimulation T_N expanded at higher numbers and were unique in the ability to generate a mixed population of CD127 + /CD127- lymphocytes. When infused in immunodeficient mice, transduced T_N proved higher engraftment and persistence potential than memory counterparts, reconstituted a mixed CD45RA ± CD62L ± phenotype and highly xenoreactive. These results suggest that gene transfer into T_N lymphocytes might increase the efficacy of cancer immunotherapy.

Stripecke R. Professor 1 Kuhs S. Dr 1 Pincha M. Mrs 1 Gutzmer R. Professor 1 Salguero G. Dr 1 Sundarasetty B. Mr 1 Garritsen H. Dr 2 Farzaneh F. Professor 3 Chan L. Dr 3 Woelfel T. Professor 4 Schendel D. Professor 5 Schmiedeknecht G. 6 Ganser A. 1

1 Hannover Medical School, Germany 2 Institute for Clinical Transfusion Medicine, Städtisches Klinikum Braunschweig 3 Dept. of Hematological Medicine, Kings College, London, UK 4 Dept. Hematology and Oncology, University of Mainz, Germany 5 Institute for Molecular Immunology, Helmholtz Center Munich, Germany 6 Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany P 55 Monocytes transduced with tricistronic lentiviral vectors in a closed bag system induce differentiation of loaded “SMART-DCs” for melanoma immunotherapy

The large-scale use of Dendritic Cells (DCs) to boost anti-tumor responses in the clinics require methods easy to standardize and consistent cell viablity. A novel concept for DC production consists of overnight lentiviral vector (LV) transduction of growth factors and full-length antigens into monocytes that results into induction of “SMART-DCs” (Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors). This concept has been validated in the pre-clinical B16 melanoma mouse model (Koya et al, 2007). Third generation self-inactivating tricistronic lentiviral vectors containing interspacing 2A elements co-expressing human GM-CSF, IL-4 and MART-1 or TRP-2 were constructed. Cytokine preconditioning and 16h transduction of CD14 + monocytes with high titer LVs resulted in persistent (3 weeks) viability, DC imunophenotype (CD209 + , MHCII + , CD80 + CD86+), secretion of GM-CSF and IL-4 and expression of MART-1 or TRP2. Monocytes that were transduced with LVs overnight in a closed GMP-grade bag system, washed, frozen and thawed also resulted in effective SMART-DC recovery and differentiation. PBMCs primed/boosted in vitro with autologous SMART-DCs/ MART-1 demonstrated the induction of MART-1-specific T cell responses assayed by IFN-γ-ELISPOT-Assay. A consortium for clinical development of SMART-DCs has been formed for additional preclinical testing (optimal vector dose, toxicity studies), development of Standart Operating Procedures (for vector production and cell transduction) and establishment of identity and potency markers for a phase I immunotherapy clinical trial.

Sutlu T Mr 1 Gahrton G Professor 1 Alici E Dr 1

1 Cell and Gene Therapy Center, Division of Hematology, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden P 56 Expansion of NK cells for cancer immunotherapy: from process optimization to clinical evaluation

Natural killer (NK) cell based immunotherapy is a prominent approach for the treatment of cancer. We have previously reported a GMP-compliant method for the expansion of NK cells from healthy donors or cancer patients. These cells showed cytotoxic activity against primary tumor cells in vitro and in experimental models, a response that stimulated evaluation in clinical settings.

Infusion of ex vivo expanded NK cells in five cancer patients following allogeneic transplantation has been evaluated. The treatment was safe whether administered alone or with subcutaneous interleukin-2. No signs of acute GvHD developed. Although tumor response was not a primary goal, the decreasing alpha-fetoprotein levels and autopsy results in a patient with hepatocellular carcinoma indicated possible anti-tumor effect.

As the ex vivo expansion of NK cells under GMP conditions are crucial for facilitating large clinical trials, we also aimed to optimize a large-scale, feeder-free, closed system. In this study, the cells were cultured for 3 weeks in bags as well as an automated bioreactor and compared to flask-based cultures. Significant expansion of NK cells was obtained in all systems. The bioreactor yielded a final product rich in NK cells ensuring that a clinically relevant cell dose was reached. Moreover, we have observed that NK cells expanded in the bioreactor displayed significantly higher cytotoxic capacity. It was possible to partially attribute this to higher expression level of NKp44.

The large-scale expansion process has been optimized and validated under GMP conditions and further clinical trials for the use of these cells are being initiated.

Oliveira G Mr 1 Vago L Dr 1 Noviello M Dr 1 Soldati C Dr 2 Ghio D Dr 2 Nicoletti R Dr 2 Brigida I Dr 3 Aiuti A Dr 3 Stanghellini MT Lupo Dr 4 Peccatori J Dr 4 Bondanza A Dr 1 Fleischhauer K Dr 5 Bordignon C Professor 6 Ciceri F Dr 4 Bonini C Dr 1

1 Experimental Hematology Unit, San Raffaele Scientific Institute, Milan, Italy 2 Radiology Unit, San Raffaele Scientific Institute, Milan, Italy 3 TIGET, San Raffaele Scientific Institute, Milan, Italy 4 Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy 5 Immunogenetics, San Raffaele Scientific Institute, Milan, ITALY 6 Vita-Salute University, San Raffaele Scientific Institute, Milan, Italy P 57 Thymic renewal in adults after haploidentical hematopoietic stem cell transplantation and suicide gene therapy

Background: In haploidentical Hematopoietic Stem Cell Transplantation (HSCT), the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine kinase (HSV-Tk) suicide gene allows to control GvHD, to mediate GvL, and to rapidly provide an effective and polyclonal anti-infective T cell repertoire (Ciceri and Bonini et al., Lancet Oncology, 2009). Even though their engraftment is necessary to achieve these effects, HSV-Tk⁺ cells represent the minority of lymphocytes circulating in treated patients. Therefore, we investigated the putative role of HSV-Tk⁺ cells in promoting thymic activity and T cell development from graft progenitors.

Methods: Twenty-eight adult patients received genetically modified donor T cells in the TK007 study. In selected patients, post-transplantation thymic function was assessed by qPCR amplification of single joint T cell Receptor Excision Circles (sjTREC) and immunophenotype analysis of CD31⁺ recent thymic emigrants (RTEs) in CD4⁺ naïve T cells. Thymic volume was assessed by CT scans.

Results: Post-transplant recovery of naïve T cells not carrying the HSV-Tk transgene occurred. The newly reconstituted CD4⁺ naïve T cells were almost entirely comprised by CD31⁺ RTEs. Comparative analysis with a cohort of patients undergoing haplo-SCT in the absence of TK⁺ cells, suggested a direct role of Tk⁺ cells in promoting thymopoiesis. Accordingly, CT scans documented an increase in thymic volume and TREC counts rose following Tk⁺ cell add-backs.

Conclusions: These data show that the infusion of suicide gene-modified T cells prompts the renewal of thymic activity and the recovery of a polyclonal T cell repertoire.

Uckert W Professor 1 Leisegang M 2 Wilde S 3 Spranger S 3 Milosevic S 3 Frankenberger B 3 Schendel DJ 3

1 MDC, Berlin, Germany 2 MDC, Berlin, Germany 3 Helmholtz Zentrum München, Munich, Germany P 58 MHC-restricted fratricide of human lymphocytes expressing survivin-specific transgenic T cell receptors

The apoptosis inhibitor protein survivin is overexpressed in many tumors making it a candidate target molecule for various forms of immunotherapy. To explore survivin as a target antigen for adoptive T cell therapy using lymphocytes expressing transgenic T cell receptors (tg-TCR), we isolated HLA-A2 allo-restricted survivin-specific T cells with high functional avidity. Recipient lymphocytes expressing tg-TCR derived from these T cells specifically recognized HLA-A2⁺ survivin⁺ tumor cells. Surprisingly, HLA-A2⁺ but not HLA-A2^- lymphocytes expressing tg-TCR underwent extensive apoptosis over time. This demise was caused by HLA-A2-restricted fratricide that occurred due to survivin expression in lymphocytes, which created ligands for tg-TCR recognition. Therefore, survivin-specific TCR gene therapy would be limited to application in HLA-A2-mismatched stem cell transplantation. We also noted that recipient lymphocytes that expressed survivin-specific tg-TCR killed T cell clones of various specificities derived from HLA-A2⁺ but not HLA-A2^- donors. These results raise a general question regarding development of cancer vaccines that target proteins which are also expressed in activated lymphocytes, since induction of high avidity T cells that expand in lymph nodes following vaccination or later accumulate at tumor sites might limit themselves by self-MHC-restricted fratricide while at the same time inadvertently eliminating neighboring T cells of other specificities.

Marin V Dr 1 Pizzitola I Dr 1 Agostoni V Dr 2 Attianese GMP Giordano Dr 1 Finney H Dr 3 Lawson A Dr 3 Pule M Dr 4 Rousseau R Dr 5 Biondi A Professor 1 Biagi E Dr 1

1 M. Tettamanti Research Center, Monza, Italy 2 Centro di Ricerca Matilde Tettamanti, Department of Pediatrics, University of Milano-Bicocca, San Gerardo Hospital, Monza, Milan, Italy 3 UCB CellTech, Slough, UK 4 Department of Hematology, University College London, London, United Kingdom 5 Centre Leon Berard, Lyon, France P 59 Improvement of anti-leukemic activity of CIK cells through CD33-specific chimeric receptors (CARs)

CIK cells are ex-vivo expanded effector cells with potent antitumoral activity.We showed that CIK cell infusion in AML patients is well tolerated, but with limited clinical responses.To improve their functions,CIK cells were tranduced with anti-CD33-z or anti-CD28-OX40-z CAR (mean CAR expression,65%). Transduced CIK cells acquired potent cytotoxicity against AML targets:after 4-h we observed,at E:T ratio 5:1,a mean lysis of HL-60 cells of 79% and 75%,for anti-CD33-z and anti-CD33-CD28-OX40-z cells (p < 0.005 vs untransduced CIK cells). Analogous lytic efficiency was observed against the KG-1 cell line and primary AML blasts and in long-term killing experiments. In these assays, anti-CD33-CD28-OX40-z-CIK cells eliminated almost all leukemic cells after 6 days co-culture on human stromal layer w/o IL2 at E:T ratio 1:200, with 16% mean residual primary AML cells compared to 31% (p ≤ 0.05) of anti-CD33-z CIK cells and 91% (p ≤ 0.005) of untransduced CIK cells. Moreover, a prominent CD33-specific proliferative activity was observed,with a mean proliferation index of 3.7 (p ≤ 0.005) in anti-CD33-CD28-OX40-z and of 2.4 (p ≤ 0.05)in anti-CD33-z-CIK cells after primary AML cell-mediated stimulation compared to 1.4 for untransduced CIK cells. In addition, when stimulated with irradiated HL-60, anti-CD33-CD28OX40-z and anti-CD33-z. CAR-transduced CIK cells secreted 10-fold and 11-fold higher amount of IFN-g, 180-fold and 120-fold more TNF-a, 600-fold and 250-fold more TNF-b, 3800-fold and 1400-fold more IL2 (p ≤ 0.05 for anti-CD33-CD28-OX40-z and for anti-CD33-z) compared to unmanipulated cells. Importantly, anti-CD33-CAR-expressing CIK cells showed toxicity against normal hematopoietic progenitors, but a consistent number of clonogenic progenitors could be recovered in colony forming-assays. Our results indicate that anti-CD33 CAR strongly enhances anti-leukemic CIK functions, suggesting that CD33.CAR-expressing CIK cells might represent a promising tool for AML immunotherapy.

Chicaybam L Mr 1 Sodre A Ms 2 Bonamino M Dr 3

1 lcpeixoto@inca.gov.br 2 deda.sodre@gmail.com 3 mbonamino@inca.gov.br P 60 A conditional system for the activation of lymphocytes expressing activating and inhibitory CARs

The use of Chimeric Antigen Receptors (CAR) represents a new strategy to redirect cytotoxic T lymphocytes specificity toward tumor cells. The data generated by the clinical utilization of CARs evidenced their potential for cancer treatment and to cause off-target effects. In an attempt to circumvent the off-target effects, we tested a system based on conditional responses to two CARs: the anti CD19scFv-4-1BB-zeta activating CAR and an inhibitory antiCD20scFvCAR bearing the intracytoplasmic tail of an inhibitory receptor (CTLA4, BTLA or PD1). To check if the engagement of both CARs results in inhibition of the activation promoted by the antiCD19CAR, Jurkat cells bearing the pGL4.30 plasmid (NFAT responsive element driving luciferase (Luc) expression), were transduced with both CARs and stimulated with PMA + ionomycin (PMA + I) or K562 cells transduced to express CD19, CD20 or both antigens. Stimulation with PMA + I or K562/CD19 + showed at least 25 and 5 fold increase in Luc activity respectively. The co-incubation with K562/CD19 + /CD20 + reduced Luc signal by at least 3 fold compared to incubation with K562/CD19 + alone, independent of the inhibitory CAR used. We also evaluated the expression of the early activation marker CD69. While antiCD19CAR activation increased CD69 expression from 65% (background) to above 84% in Jurkat cells,the co-stimulation of antiCD19CAR and a CARs expressing CTLA4, BTLA or PD1 reduced this expression to 60, 47 and 53% respectively. These preliminary data indicate that inhibitory CARs can counterbalance the activation promoted by antiCD19CAR engagement. We are currently evaluating this approach in cytotoxicity assays against cells expressing target antigens.

Uckert W Professor Leisegang M Turqueti-Neves A Blankenstein T Engels B Schendel DJ Noessner E

P 61 T cell-based immunotherapy for renal cell carcinoma

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Adoptive therapy with genetically engineered T cells carrying redirected antigen specificity (TCR gene therapy) is a new option to treat cancer, especially melanoma. However, this treatment is not yet available for metastatic renal cell carcinoma (RCC), due to the scarcity of therapeutically useful reagents. We analyzed tumor-infiltrating lymphocytes (TIL) from RCC to identify T cells with shared tumor-specific recognition for the generation of T cell receptor (TCR)-engineered T lymphocytes for TCR gene therapy of RCC.

We established a T cell clone from TIL that recognized an HLA-A2-restricted tumor antigen. The TCRalpha- and beta-chain genes (TCR53) were isolated, modified by codon optimization and murinization, and transduced into peripheral blood lymphocytes (PBL). TCR53-engineered PBL recapitulated the specificity of the TIL and demonstrated tumor-specific, HLA-A2-restricted effector activites (e.g. IFN-gamma, cytotoxicity). TCR53-engineered PBL of healthy donors and RCC-patients exhibited similar TCR expression levels, expansion, and polyfunctional profile.

A TCR53-expressing indicator line (B3Z-TCR53) was established for the screening of the antigen prevalence in RCC, in other malignancies, and in normal cell counterparts. Using B3Z-TCR53 cells, 130 tumor and normal cells were analyzed and shared TCR53 peptide : MHC expression was found in more than 60% of RCC and 25% of tumor cell lines of other histology, while normal tissue cells were not recognized.

To date, TCR53 is the only TCR with shared HLA-A2-restricted recognition of RCC. It fulfills important criteria for utilization in TCR gene therapy and could advances T cell-based immunotherapy to patients with RCC and other malignancies expressing the TCR-ligand.

Marin V Dr 1 Pizzitola I Dr Cribioli E Biondi A Professor Biagi E Dr Pule M Dr

P 62 Comparison of Different suicide genes for the safety improvement of genetically manipulated T Cells

Immunotherapy with T cells transduced with retroviral vectors carrying TCRs or CARs may be toxic due to direct target effects, off-target effects or insertional mutagenesis. Therefore a suicide gene-approach has to be considered. We compared different suicide genes in vitro in EBV-CTLs. Herpes Simplex Virus Thymidine Kinase (HSV-TK), human inducible Caspase 9 (iCasp9), human CD20 and mutant human thymidylate kinase (mTMPK) genes were cloned in frame with 2A-truncated CD34 (dCD34)in a SFG-vector. EBV-CTLs could be similarly and efficiently transduced with iCasp9-2A-dCD34, HSV-TK-2A-dCD34, mTMPK-2A-dCD34 and CD20-2A-dCD34 (mean % CD34⁺, 80%, n = 5), similarly to the control vector containing dCD34 alone.Expression of the suicide genes was not associated with alterations in the expansion rate,immunophenotype and capacity to kill autologous LCLs. Transduced and CD34-selected EBV-CTLs have been tested for their sensibility to the corresponding activator in vitro by evaluating residual CD34⁺ cells. iCasp9- transduced cells were rapidly killed with high efficiency by CID,(mean survival, 11% after 24 hours, and 5% after 7 days; n = 7). Gancyclovir treated HSV-TK-expressing cells showed similar levels of efficacy only after 3 days and CD20 and mTMPK-transduced cells showed only minimal killing at all time points (mean survival after 7 days, 84% and 32%). The same results were obtained by analyzing apoptosis induction through Annexin-7AAD staining. In fact, after 24 hours of incubation with CID, nearly 100% iCasp9⁺ cells were apoptotic, whereas a significant lower % of apoptotic cells was observed with the other suicide genes. Altogether our results suggest that the faster activity of iCasp9 might be advantageous in case of occurring toxicity, and, together with its lack of immunogenicity and the absence of side-effects of CID, support the clinical applicability of iCap9-based suicide strategy.

Mineno J Dr 1 Okamoto S Dr 1 Ikeda H Dr 2 Fujiwara H Dr 3 Nukaya I Dr 1 Yoshioka H Mr 1 Enoki T Mr 1 Ideno M Ms 1 Yasukawa M Professor 3 Shiku H Professor 2 Takesako K Dr 1

1 Center for Cell and Gene Therapy, Takara Bio Inc., Shiga, Japan 2 Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan 3 Department of Bioregulatory Medicine, Ehime University Graduate School of Medicine, Ehime, Japan P 63 The next generation TCR gene therapy using siTCR vector and RetroNectin expansion method

Tumor antigen-specific T-cell receptor (TCR) gene therapy has been shown as an attractive strategy to treat cancer patients. However, the introduced TCR α and β chains have been reported to mispair with endogenous TCR subunits, resulting in insufficient formation of heterodimers of therapeutic TCR. Moreover, a serious safety concern on the generation of T cells with unexpected specificities, including self-reactive T cells caused by TCR mispairing has been postulated.

In this study, we developed novel “siTCR” retroviral vectors encoding both siRNA constructs that knockdown endogenous TCRs and codon-optimized, siRNA-resistant TCR αβ chains specific for tumor antigens. Human lymphocytes transduced with these vectors exhibited high expression of the introduced tumor-specific TCR on the cell surface accompanied with reduced endogenous TCR at low proviral copy numbers, resulting in enhanced cytotoxic activity against antigen-expressing tumor cells. Because the target of this novel strategy is endogenous TCR, siTCR vectors may work as a powerful tool for any TCR gene therapy without dependency of TCR variation.

We have also developed an efficient method to generate large numbers (over 10⁹ cells) of genetically modified T cells from small amount of whole blood (50-100 ml) without apheresis. “RetroNectin”, a recombinant human fibronectin fragment, co-stimulated with anti-CD3 mAb enhanced cell proliferation while conserving the CCR7 + CD45RA + naive phenotype of T cells.

We are in process to start clinical trials using siTCR vectors expressing HLA-A*2402 restricted MAGE-A4 or WT1 specific TCRs, combined with RetroNectin expansion method.

Almåsbak H Ms 1 Rian E Dr 2 Hoel J Ms 1 Pule M Dr 3 Wälchli S Dr 1 Gaudernack G Professor 1 Rasmussen AM Dr 1

1 Section for Immunology, Radiumhospitalet, Oslo University Hospital (OUS), Norway 2 DiaGenic ASA, Oslo, Norway 3 Department of Haematology, Cancer Institute, University College London, UK P 64 Transiently redirected T cells for adoptive transfer; co-electroporation of chimeric antigen receptors

Background: Clinical trials are underway investigating the safety and efficacy of adoptively transferred T-cells expressing Chimeric Antigen Receptors (CAR). Recent experiences with on-target/off-organ toxicity call for caution. Transient rather than permanent expression might represent a safer alternative in first-in-man studies with novel CARs and additionally in applications where long-lasting transgene activity is undesired.

Methods: We describe the preclinical evaluation of a method based on transient modification of bioreactor expanded T-cells with a CD19-CAR directed against B-cell malignancies. CAR mRNA is generated under cell-free conditions in a scalable process using recombinant RNA polymerase. Efficient and non-toxic square-wave electroporation is used to load the mRNA into the cytoplasm of T-cells with no risk of insertional mutagenesis.

Results: After transfection >80% of T-cells are viable with 94% CAR-expression. Transfected cells are cytolytic to CD19 + targets and produce IFN-g, even at day 8 post-electroporation with undetectable CAR-expression. Increased mRNA concentration results in higher CAR-expression, improved killing and more IFN-g release but at the expense of increased activation-induced cell death. Finally, we demonstrate that a second transgene can be introduced by co-electroporation of CXCR4 or CCR7 with the CAR to facilitate migration.

Conclusions: We advocate that the transient redirection approach is well suited to meet safety aspects for early-phase studies, prior to trials using stably transduced cells once the CAR has been proven safe. The simplicity of this methodology also facilitates rapid screening of candidate targets and novel receptors in preclinical studies.

Attianese GMP Giordano Dr Hoyos V Dr 2 Marin V Dr Savoldo B Professor 2 Pizzitola I Dr Agostoni V Dr Parma M Dr 3 Bertilaccio MTS Dr 4 Ghia P Professor 4 Biondi A Professor Biagi E Dr Dotti G Professor 2

2 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA 3 Department of Adult Hematology, San Gerardo Hospital, Monza, Italy 4 University Vita-Salute San Raffaele, Milan, Italy P 65 A New Chimeric Antigen Receptor (CAR) Targeting the CD23 Antigen

Background: B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a progressive accumulation of mature CD19⁺CD5⁺CD20^dim B-lymphocytes that over-express the B-cell activation marker CD23. Here we cloned and expressed in T-lymphocytes a CAR targeting the CD23 antigen (CD23.CAR) on B-CLL cells.

Methods: Cytotoxic activity was measured using a ⁵¹Chromium release assay. Co-culture experiments of non-transduced (NT) and CAR⁺T-cells with viable LCLs have been perform to assess expansion capability and soluble CD23(sCD23)-mediated inhibition of the CD23.CAR⁺T-lymphocytes. IFN-g, TNF-a, TNF-b and IL2 release were measured by Flow Cytomix Assay. In vivo experiments: eight-week-old-rag2^-/-g_c ^-/-male mice were injected subcutaneously with 10x10⁶MEC1-cells. 21-days later, mice were injected with 2x10⁶NT or CD23.CAR⁺T-cells intravenously: animals were monitored twice a week for tumor growth.

Results: CD23.CAR⁺T-cells showed specific cytotoxic activity against CD23⁺tumor cell lines(average lysis 54%) and primary CD23⁺B-CLL cells(average lysis 58%). This effect was obtained without relevant toxicity against normal B-lymphocytes. Moreover, CD23.CAR⁺T cells released 4-fold more IFN-gamma, 157-fold more TNF-alpha and 1445-fold more TNF-beta in response to CD23⁺target cells. IL-2 was also released(average release 2681 pg/mL) and sustained the antigen-dependent proliferation of CD23.CAR⁺T-cells without the addition of any exogenous cytokines. Preliminary in vivo data showed CD23.CAR⁺T-cells can exert their cytotoxic activity toward the B-CLL derived MEC1-cell line, reducing tumor diameter and prolonging mice survival as compared to NT-cells-treated mice.

Conclusions: Altogether these data suggest gene modification of T-cells to express the CD23.CAR represents a selective immunotherapy approach to eliminate CD23⁺leukemic cells, while sparing normal B lymphocytes, in patients with B-CLL.

Tsukahara T Dr 1 Ohmine K Dr 2 Uchibori R Mr 1 Urabe M Dr 1 Mizukami H Dr 1 Kume A Dr 1 Riviere I Dr Sadelain M Dr Brentjens R Dr Ozawa K Professor 1

1 Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University, Tochigi, 329-0498, Japan 2 Division of Hematology, Department of Medicine, Tochigi, 329-0498, Jichi Medical University, Japan P 66 Validation of anti-tumor effects mediated by anti-CD19CAR T-cells for B cell lymphoma

In order to develop a new therapeutic strategy for refractory B-cell non-Hodgkin lymphoma, we evaluate the efficacy of an adoptive immunotherapy using genetically engineered autologous T lymphocytes that express a CD19-antibody fragment fused to the TCRz/CD28 receptor (19-28z) in model experiments. Here we examined whether 19-28z-transduced T lymphocytes could eradicate CD19 + tumors in vitro. Human peripheral blood T lymphocytes from healthy volunteers were activated by immobilized anti-CD3 antibody/Retronectin, and transduced with retrovirus vectors encoding 19-28z prepared from producer cells, established by stable transduction of the 19-28z plasmid into the PG13 packaging cells. Transduced T lymphocytes were expanded ex vivo in the presence of NIH3T3 fibroblasts expressing human CD19. Following antigen stimulation, 19-28z-transduced cell numbers increased about 1000-fold, and the surface expression of 19-28z positive CD3 + T lymphocytes was approximately 95%, as assessed by flow cytometry. Expanded 19-28z + T lymphocytes efficiently lysed CD19 + tumor cell lines (Raji and Daudi burkitt lymphoma cell lines) in ⁵¹Cr release assays. These results indicate that functional 19-28z + T lymphocytes would be effective for the treatment of refractory B-cell non-Hodgkin lymphoma.

Pizzitola I Dr Agostoni V Dr Finney H Dr Lawson A Dr Pule M Dr Rousseau R Dr Biondi A Professor Biagi E Dr Marin V Dr

3 UCL, London, UK P 67 Identification of the best car-modified T-cell population: Is it possible without Human studies?

A crucial issue in adoptive cell therapy is the identification of the optimal T-cell effectors to be used in vivo. The ideal T-cell population must be able to home toward the tumor site and exert prolonged anti-tumoral activity. We compared EBV-CTLs, Cytokine Induced Killer (CIK) cells and g₉d₂ T (GDT) cells after transduction with a chimeric receptor (CAR) specific for CD33 (anti-CD33-z.CAR). EBV-CTLs, CIK and GDT cells were generated and efficiently transduced (mean % CAR expression, 71% for EBV-CTL, 65% for CIK cells and 67% for GDT cells). Anti-CD33-z.CAR-redirected effector cells showed potent and similar cytotoxicity against several leukemic targets both in 4-h ⁵¹Chr-release assays (mean killing vs primary AML at E:T ratio of 5:1, 50%, 61% and 50%, n = 3, for EBV-CTL, CIK and GDT cells) and in long-term killing assays (6 days) on a human stromal layer w/o IL2 (mean survival of primary AML at E:T ratio 1:100, 18%, 16% and 29% for EBV-CTL, CIK and GDT cells,n = 8). Moreover, all effector cells released high and comparable levels of immunostimulatory cytokines (IFN-g, IL2, TNF-a and TNF-b). Our results indicate that anti-CD33-z CAR expression potently and similarly increases the in vitro anti-leukemic activity of various T cell types and underlines the weakness of the in vitro model for this kind of analysis.These results need to be supported not as much by murine studies -where the microenvironment might not be permissive for the maintenance of the cells-but more by analysis in the human setting.This is consistent with recent evidences from a human clinical trial with CAR-modified T cells (Pule et al. Nat Med 2008), suggesting that T-cell in vivo persistence rather than cytotoxic/immune activity potential is crucial to demonstrate an anti-tumoral clinical relevant effect.

Perna S Dr 1 De Angelis B Dr 1 Pagliara D Dr 1 Zhang L 1 Rooney C Professor 1 Heslop H Professor 1 Brenner M Professor 1 Dotti G Dr 1 Savoldo B Dr 1

1 CAGT, Baylor College of Medicine, Houston, Texas, United States, 77030 P 68 IL-15, un-like IL-2, supports T cells proliferation and function even in the presence of Tregs

Adoptive T-cell immunotherapies are safe and clinically effective in cancer patients. Clinical responses strongly correlate with in-vivo T-cell survival. Systemic administration of recombinant IL-2 is used to sustain T-cell persistence. However, although effective, IL-2 induces side effects and regulatory T-cells (Tregs) expansion. We have now explored whether IL-15, able to sustain T-cell expansion and function, shares with IL-2 the unwanted effect on Tregs. We used Epstein-Barr-Virus (EBV)-specific cytotoxic T-lymphocytes (CTLs) as a model. Treg inhibitory activity was assessed using CSFE-based assays (to evaluate inhibition of proliferation), and co-culture experiments with lymphoblastoid-cell-lines (LCLs) (to assess inhibition of effector function), with or without IL-2 (25UI/mL) or IL-15 (2.5ng/mL).

T-cell proliferation was inhibited by Tregs alone (from 56% ± 6% to 20% ± 5%, p < 0.05) or IL-2 + Tregs (from 76% ± 3% to 53% ± 3%, p < 0.05), but not by IL-15 + Tregs (from 81% ± 3% to 74% ± 3%, p = 0.1). Similarly, the anti-tumor activity of EBV-CTLs was significantly impaired by Tregs alone (residual LCLs increased from 31% ± 13% to 55% ± 13%, p < 0.05) or IL-2 + Tregs (from 16% ± 8% to 36% ± 14%, p < 0.05) but not by IL-15 + Tregs (from 7% ± 6% to 11% ± 7%, p = 0.2).

Our study suggests that IL-15, unlike IL-2, can relieve effector cells from Tregs inhibition. We are currently exploring potential mechanisms of this observation by studying the effects of IL-2 and IL-15 on each component (Tregs vs effector cells). Preliminary data suggest that IL-15 does not directly revert Tregs inhibitory properties, but preferentially enhance T-cell proliferation and anti-tumor activity. Transgenic production of IL-15 by tumor-specific CTLs could help in overcoming Tregs effects while maintaining their ability to kill tumor cells.

Yun Chae-Ok Professor 1 Choi Kyung-Ju Mrs 1 Zhang Song-Nan Mr 1 Kim Ji-Seong Mrs 1

1 Yonsei University College of Medicine P 69 New insight into the antitumor efficacy of oncolytic adenovirus co-expressing IL-12 and GM-CSF

IL-12 and GM-CSF have recently been used as immunotherapeutic agents in cancer gene therapy. IL-12 and GM-CSF have differential roles in the antitumor immune response, as IL-12 targets T, NK, and NKT cells, and GM-CSF principally targets APCs. To strengthen the therapeutic efficacy of these two cytokines, we generated an oncolytic adenovirus (Ad), Ad-ΔB7/IL12/GMCSF, coexpressing IL-12 and GM-CSF. Using a murine B16-F10 syngeneic tumor model, we show that Ad-ΔB7/IL12/GMCSF promoted antitumor responses and increased survival compared to an oncolytic Ad expressing IL-12 or GM-CSF alone (Ad-ΔB7/IL12 or Ad-ΔB7/GMCSF, respectively). By measuring cytotoxic T lymphocyte (CTL) activity and interferon-γ (IFN-γ) production, we show that the enhanced therapeutic effect was mediated by the induction of immune cell cytotoxicity. In situ delivery of Ad-ΔB7/IL12/GMCSF resulted in massive infiltration of CD4⁺ and CD8⁺ T cells and CD86⁺ APCs into the tissue surrounding the necrotic area of the tumor. Moreover, GM-CSF effectively promoted anti-tumor immune memory, which was significantly augmented by IL-12. Lastly, IL12-expressing oncolytic Ads prevented tumor-induced thymic atrophy and was associated with reduced apoptosis and increased proliferation in the thymus. Taken together, these data demonstrate that an oncolytic adenovirus coexpressing IL-12 and GM-CSF is a potential therapeutic tool for the treatment of cancer.

Rosas-Taraco A Dr 1 Villanueva-Olivo A Dr 1 Saucedo-Cárdenas O Dr 1 Esparza-Gonzalez SC Dr 1 Villatoro-Hernandez J Dr 1 Loera-Arias MJ Dr 1 González-Saldívar G Miss 1 Barrera-Hernández A Mr 1 Martínez-Ortega JI Mr 1 Montes-deOca R Dr 1

1 Universidad Autónoma de Nuevo León P 70 Targeting and retention of antigen to the endoplasmic reticulum enhances immune tumor protection

Background: Endoplasmic reticulum (ER) is the main place where the MHC I are loaded with epitopes to elicit an immune cellular response. Most of the protein antigens are degraded in the cytoplasm to aminoacids and very few epitopes reach the ER. Antigen targeting to this organelle by means of fusion to Calreticulin, an ER living protein, avoid this degradation problem enhancing the immune response. In order to simplify this strategy and to determine if most of the antitumor properties conferred by Calreticulin are dependent on its ability to be retained in the ER, we engineered a new version of E7 gene bearing the signals required for ER targeting (SP) and ER retention (KDEL: Lys-Asp-Gln-Leu).

Method: We constructed recombinant adenovirus to express the E7 antigen with the SP and KDEL signals. E7 targeting was demonstrated by immunofluorescence on cell cultures transfected with these Sp-E7-KDEL and Calreticulin-E7 adenovirus. IFN-g induction and tumor protection assay were performed on C57B6 mice.

Results: The SP-E7-KDEL protein showed a similar pattern distribution as the control calnexin, an endogenous protein localized in the ER. Mice infected with the SP-E7-KDEL adenovirus showed an interferon induction and tumor protection response, similar to that provided by the adenovirus expressing the fusion protein Calreticulin-E7.

Conclusion: This work demonstrate that just by adding a signal peptide and KDEL sequence, the antigens can be targeted and retained in the ER with a consequent enhancement of immune response and tumor protection.

Schmidt P Dr 1 Abken H Professor 1

1 CMMC and Department I of Internal Medicine, University of Cologne 1 CMMC and Department I of Internal Medicine, University of Cologne P 71 The proteasome inhibitor Bortezomib sensitizes melanoma cells towards adoptive T cell attack

Adoptive transfer of tumor-specific cytotoxic T lymphocytes (CTLs) results in target cells lysis by activating the intrinsic apoptotic cell death program. Not surprisingly, deregulation of the apoptotic machinery is one of the central mechanisms by which tumor cells escape immune destruction despite specific CTL recognition. We show that treatment with the proteasome inhibitor bortezomib sensitizes previously resistant tumor cells for cytolytic T cell attack. Human T cells were redirected to target melanoma cells by engineered expression of an immunoreceptor with binding specificity for high molecular weight-melanoma associated antigen (HMW-MAA). Established melanoma cell lines as well as primary melanoma cells from tumor biopsies, which are notoriously resistant towards T cell lysis, became sensitive upon bortezomib treatment. Detailed analysis of the underlying molecular mechanism revealed that bortezomib-treatment induced the mitochondrial accumulation of NOXA which potentiated the release of mitochondrial SMAC in response to CTL effector functions including caspase-8 and granzyme B. The results of our study support the concept that proteasome inhibition increases the sensitivity of tumor cells towards a cytolytic T cell attack by NOXA-mediated enhancement of mitochondrial release of SMAC.

Rashkova V Miss 1 Essand M Professor 1

1 Uppsala University, Clinical Immunology, SE-75185 Uppsala, Sweden P 72 Generation of antigen–specific cytolytic T cells for prostate cancer therapy

Adoptive transfer of T cells specific for cancer antigens is a novel approach to treat cancer. Such immunotherapy has been successful in a setting where tumor infiltrating lymphocytes are isolated from patients, expanded ex vivo and given back to the patients in large numbers. However, in some patients and some malignancies such cells are not found or difficult to detect. An alternative approach would be to genetically modify T cells with a specific T cell receptor (TCR) to a given antigen. This approach has been used in the past for melanoma patients with encouraging results.

Using cytomegalovirus (CMV)–specific T cells against the pp65 antigen as a model we show that tetramer-specific T cells can be obtained, T cell clones can be generated and specific TCR sequence can be amplified. Our experiments confirm sequences that have already been published for the same peptide from pp65.

Using this validated protocol, we aim to develop cytolytic T cells against peptides derived from prostate antigens TARP, STEAP and PSA, which are only expressed in the prostate. With the help of HLA -A2 tetramers for TARP, STEAP and PSA peptides we can detect specific T cells and fluorophore directed beads can be used for the isolation of those cells. Our results indicate that such cells can be found in blood from prostate cancer patients. To deliver antigen specific TCR to T-cells we use a lentivirus vector with a self-cleaving peptide between the alpha and beta chain of the TCR to assure their equal expression.

Schiffer-Mannioui SMC Dr 1

1 102 Av. Gaston Roussel 93230 Romainville P 73 Meganucleases : a novel anti-viral approach

Julianne Smith, Roman Galetto, Stephanie Grosse, Cécile Schiffer Mannioui, Sylvain Arnould, Marine Gailledrat, Philippe Duchateau, Frédéric Pâques & Carole Desseaux.

The majority of current anti-viral treatments are based on the prevention of productive viral replication through the utilization of agents that inhibit essential virally encoded proteins. These anti-viral treatments, however, do not eliminate the viral genome from cells and thus removal of treatment or the presence of drug resistant mutations result in viral proliferation. Many chronic viral infections are due to double-stranded DNA viruses or viruses that involve a double-stranded DNA intermediate during their replicative cycle. Thus, an attractive alternative anti-viral strategy is to specifically cleave and either partially excise or eliminate viral DNA from infected cells and thus render them virus free.

Meganucleases are endonucleases that recognize large cleavage sites (>12bp) with a high specificity. We have shown that the expression of the meganuclease I-SceI, either before or after infection with a modified Herpes Simplex Virus (HSV) containing a meganuclease recognition site, results in a dramatic reduction of viral DNA. Meganucleases specific for viral DNA could thus represent a novel class of agents for the treatment of viral infections, targeting latent viral DNA in infected cells and rendering them virus free. Using a semi-rational approach, we have used a two step strategy to produce meganucleases cleaving several different viral genomes, including HIV, HSV and HBV. We will present data concerning the use of virus specific meganucleases for the development of a new anti-viral approach and therapeutic strategies for certain persistent infections.

Xue S.A Dr 1 Ghorashian S Dr 1 Pospori C Dr 1 Ahmadi M Dr 1 Holler A Dr 1 Thomas S Dr 1 Stauss HJ Professor 1

1 Department of Immunology, Royal Free Hospital, Rowland Hill Street, London NW3 2PF P 74 Development of CMV-specific T cells for the management of transplantation patients

Development of CMV-specific T cells forthe management of transplantation patients

Background: Bone marrow transplantation is a curative approach for the management of leukaemia patients. However, the requisite patient immunosuppression can lead to reactivation of viruses such as CMV or EBV, causing significant morbidity and mortality. Adoptive transfer of donor-derived CMV-specific CD8⁺ CTL clones has proven effective in the prevention and treatment of viral infections that are unresponsive to antiretroviral therapy. But, this procedure has not been widely adopted due to the technical and financial demands of extensive T cell isolation and expansion ex vivo, and moreover is impossible when the donor is seronegative for the virus. Thus, new strategies are needed. We report here the development of CMV-specific CTL via T cell receptor (TCR) gene transfer.

Methods: CMV-specific TCR genes were cloned and transduced into human T cells. The antigen-specific activity of these engineered T cells was tested in vitro by CTL killing and cytokine secretion assays, and in vivo using a NOD/SCID mouse tumour model.

Results: We show that CMV-TCR can be expressed on the surface of human T cells, and these TCR engineered T cells both recognize and kill CMV viral antigen-expressing target cells. Using a K562 leukaemia cell line expressing HLA-A2 and the CMV antigen pp65, we demonstrate that the engineered human T cells can inhibit tumour growth in NOD/SCID mice.

Conclusion: This is the first report that CMV-TCR engineered human T cells can inhibit tumour development in vivo, and paves the way for using CMV-TCR engineered T cells for the management of transplant patients.

Stripecke R. Professor Salguero G. Dr Sundarasetty B. Mr Borchers S. Dr Wedekind D. Dr Velaga S. Dr Warnecke G. Dr Knöfel A Dr Blasczyk R. Professor Eiz-Vesper B. Professor Mischak-Weissinger E. Professor Ganser A. Professor

P 75 Conditioning therapy with lentivirally reprogrammed dendritic cells accelerate the expansion and bio-distribution of CMVpp65-reactive human T cells

We evaluated the impact of conditioning therapy with genetically programmed dendritic cells (DCs) in order to enhance the engraftment and stimulation of donor T cells reactive against human cytomegalovirus (CMV). Ex vivo transduction of human monocytes with lentiviral vectors (LV) expressing GM-CSF and IL-4 induced their self-differentiation into “SMART-DCs” that were stable antigen-presenting cells viable for three weeks in vitro and in immunodeficient NOD.Rag1^−/−.IL2rγ^−/− (NRG) mice. SMART-DCs co-expressing the full length pp65 CMV antigen also maintained high viability and a stable DC immunophenotype. We analyzed the in vivo effects of SMART-DC-pp65 pre-administration on the engraftment and stimulation of anti-pp65 autologous T cells obtained from sero-positive CMV reactive donors using optical imaging, flow cytometry, immunofluorescence microscopy and immune analyses. “Conventional” DCs pulsed with a pp65 overlapping peptide pool were used as controls. NRG mice pre-conditioned s.c. with SMART-DC-pp65 showed: 1. Superior expansion of CD4⁺ and CD8⁺ human T cells in the spleen and peripheral blood; 2. Massive recruitment of lymphocytes to the DC injection site; 3. Stimulation of human CD8⁺ T cells against different pp65 epitopes. Thus, conditioning the host with engineered DCs prior to transfusion with donor T cells is a potential protective approach for CMV immunotherapy.

Loader J Mrs 1 Ferrige G Mrs 1 Angell-Manning D Mrs 1 Carlucci M Mrs 1 Miskin J Dr 1

1 Oxford Biomedica, Oxford Science Park, Oxford, OX4 4GA P 76 Nonclinical studies for the treatment of wet age-related macular degeneration for a first in man clinical trial using direct administration of an EIAV lentiviral vector (Retino Stat^®)

RetinoStat^® is currently being developed at Oxford BioMedica (OBM) for the initiation of a first in man (FIM) clinical study for the treatment of wet age-related macular degeneration (AMD). AMD is the leading cause of blindness in patients over 60 years in the developed world, resulting from neovascularisation in the retina. RetinoStat, expressing endostatin and angiostatin, will be administered by subretinal administration, which should result in the long-term expression of these angiostatic factors and suppression of neovascularisation. RetinoStat is an equine infectious anaemia virus (EIAV)-based Lentiviral vector, utilising the same platform technology that is currently being used in a Phase I/II clinical trial for the treatment of Parkinson's disease (ProSavin^®).

GLP non-clinical combined toxicology, shedding and biodistribution studies have been conducted in two species to support the regulatory submission of RetinoStat, using subretinal administration of RetinoStat. A wide range of biological samples were taken both in-life and at necropsy to evaluate the bio-distribution of vector, vector shedding and vector persistence and included a variety of target and non-target sample types. Samples were assessed for the presence of vector by quantitative real-time reverse transcriptase PCR (qRT-PCR) for vector genomic RNA. Persistence of vector was assessed by quantitative real-time PCR (qPCR) for vector associated DNA.

The most striking general observation from these studies was the fact that vector and vector associated sequences were highly restricted to target tissue (eye). An overview of the data from these studies will be presented.

Pandor A Ms 1 Ramesar R Professor 1 Prince S Dr 2

1 Division of Human Genetics, Department of Clinical and Laboratory Sciences, Faculty of Health Sciences, University Of Cape Town, South Africa 2 Department of Cell Biology, Faculty of Health Sciences, University Of Cape Town, South Africa P 77 Investigation of novel gene-based therapies for retinitis pigmentosa 17

Retinitis pigmentosa is a highly heterogeneous form of inherited blindness. The RP17 form of the disease is caused by an arginine to tryptophan (R14W) mutation in the signal sequence of carbonic anhydrase IV (CAIV). While CAIV is expressed in the choriocapillaris of the eye and renal epithelium, the R14W mutation results in an exclusively ocular phenotype in affected individuals. We show using immunocytochemistry, western blot analysis and flow cytometry experiments that the R14W mutant form of CAIV is misfolded and mis-trafficked in COS-7 and HT-1080 cells. In HEK-293 cells, which are of kidney origin, we have shown that the R14W mutant form of CAIV is correctly folded and targeted, perhaps explaining the lack of kidney phenotype in RP17 patients, despite high expression of the mutant protein in these cells. We have investigated the expression of ER-specific chaperones in HEK-293 cells expressing R14W mutant CAIV, in order to determine whether high expression of these proteins induces correct folding of the mutant protein in these cells. We believe that over-expression of these chaperones in COS-7 and HT-1080 cells expressing R14W mutant CAIV will result in correct folding of the protein, as is seen in HEK-293 cells. In addition, we have also designed allele-specific shRNA to silence R14W mutant CAIV, but not the wildtype form of the protein. We believe that by using these approaches, we have explored two possible means of providing therapy for RP17.

Cambon K Dr 1 Ruiz M Dr 1 Drouet V Dr 1 Zala D Dr 2 Feyeux M Dr 3 Auregan G Ms 1 Hassig R Ms 1 Dufour N Ms 1 Perrier A Dr 3 Saudou F Dr 2 Hantraye P Dr 1 Déglon N Dr 1

1 1CEA, Institute of Biomedical Imaging (I2BM) and Molecular Imaging Research Center (MIRCen), Fontenay-aux-Roses, France 2 3Institut Curie, Orsay, France 3 5Institut National de la Santé et de la Recherche Médicale/Université d'Evry-Val-d'Essonne Unité Mixte de Recherche 861, Institute for Stem Cell Therapy and Exploration of Monogenic Diseases, Associat P 78 Allele-specific silencing of mutant Huntingtin in HD neural stem cells and in vivo

Huntington's disease is a progressive and fatal neurodegenerative disorder, characterized by disturbances in mood and deterioration in cognitive and motor functions. This dominant and inherited disease is caused by the expression of mutant Huntingtin (Htt) protein with a CAG repeat expansion. Suppression of Htt expression, using RNA interference represents an interesting potential therapeutic strategy for this untreatable pathology. Nevertheless, the loss of normal Htt function might also contribute to the pathogenesis of HD. The reduced physiological activity of Htt in HD might also affect the development of therapeutic strategies with RNAi. Allele-specific silencing of the mutant allele using siRNAs targeting heterozygous single nucleotide polymorphisms SNPs that distinguish between the two alleles represents a very promising therapeutic strategy for HD.

In this study, we show the efficacy of shRNA targeting three SNPs covering the majority of HD patients. Lentiviral-mediated delivery resulted in efficient and selective in vitro silencing of a chimeric mutant Htt reporter system consisting of the sequence of the first 171 amino acids of the mutant human Htt, fused to the Htt exons containing the SNP. Furthermore, the defect in the vesicular transport of BDNF along microtubules was corrected in HD neural stem cells.

In vivo, siSNP efficiently degraded the mRNA of fully matched chimeric mutant Htt expressing the various SNP and prevented the apparition of neuropathology. On the contrary, the presence of one mismatch in the targeted mRNA prevented its degradation in almost all cases, leading to the accumulation of Htt aggregates and the appearance of striatal pathology.

Neri M Dr 1 Ricca A Miss 2 Di Girolamo I Dr 3 Martino S Dr 3 Sergi Setgi L Dr 1 Naldini L Professor 1 Gritti A Dr 1

1 San Raffaele Scientific Institute, Telethon Inst. for Gene Therapy (HSR-TIGET), Via Olgettina 58, 20132 Milano, Italy 2 Vita-Salute San Raffaele University Medical School, Via Olgettina 58, 20132 Milano, Italy. 3 Department of Experimental Medicine and Biochemical Sciences, Section of Biochemistry and Molecular Biology, University of Perugia, via del Giochetto, 06126 Perugia, Italy P 79 Therapeutic potential of genetically modified neural stem cells (NSCs) in a mouse model of Globoid Cell Leukodystrophy (GLD)

GLD is a rare genetic disorder caused by the deficiency of the lysosomal enzyme β-galactocerebrosidase (GALC), characterized by progressive central and peripheral demyelination. We evaluated the potential of Neural Stem Cell (NSC)-based approach to correct the metabolic defect and to ameliorate CNS pathology in Twitcher (Twi) mice, a true model of GLD. NSC lines derived from neonatal Twi and WT mice were efficiently transduced with bidirectional lentiviral vectors encoding for GALC and GFP, achieving supraphysiological GALC activity. In order to obtain a stable source of GALC-secreting cells in the brain we transplanted 5×10⁵ WT or GALC over-expressing NSCs into the telencephalic lateral ventricles of neonatal Twi mice. Forty days after transplant we found that 1-3% of the total injected NSCs were engrafted and distributed into the brain, some of them expressing neural lineage markers. Engrafted NSCs robustly produced and secreted the GALC protein, as assessed by immunofluorescence and western blot analysis. Circulation of the enzyme in the cerebrospinal fluid flow coupled to active cross-correction resulted into restoration of GALC activity up to 50% of WT levels in the brain and spinal cord tissues of NSC-transplanted Twi mice. Metabolic correction resulted in partial clearance of glycosphingolipid storage in CNS tissues and improvement of walking ability in NSC-treated mice as compared to untreated controls.

These results warrant further consideration of NSC gene therapy for the treatment of GLD, likely in combination with other approaches ensuring enzymatic reconstitution in visceral organs and in the PNS.

Valori CF Dr 1 Ning K Dr 1 Wyles M Mr 1 Mead RJ Dr 1 Grierson AJ Dr 1 Shaw PJ Professor 1 Azzouz M Professor 1

1 University of sheffield beech hill road Sheffield S10 2RX P 80 Systemic delivery of scAAV9 expressing SMN prolongs survival in a mouse model of SMA

Background: Spinal Muscular Atrophy (SMA) is one of the most common genetic causes of death in childhood. It is caused by mutations of the survival motor neuron (SMN) gene, leading to depletion in SMN protein levels. To date there are no effective drug treatments for this disease. SMN replacement by gene therapy seems therefore a promising strategy for successful treatment of the disease. Consistently, we previously reported that intramuscular administration of a lentiviral vector expressing SMN lead to a small but significant increase in the lifespan of SMNΔ7 mice. However, the marginal efficacy of this original therapeutic approach prompted us to explore different strategies for gene therapy delivery to motor neurons to achieve a more clinically relevant result. Self complementary adeno-associated virus 9 (scAAV9) mediates efficient and sustained transgene expression in cells of the nervous system following systemic administration. Therefore, we evaluated the efficiency of scAAV9-mediated SMN gene replacement in a mouse model of SMA.

Method: A single injection of SMN-expressing scAAV9 vector was performed into the facial vein of neonate SMNΔ7 mice. The pups were evaluated on a daily basis for survival and their motor function was assessed by behavioural tests.

Results: scAAV9-SMN gene therapy restored SMN protein expression and mediated correction of the motor deficits in these animals resulting in a substantial extension in their lifespan.

Conclusion: These data demonstrate a clinically relevant extension of survival in SMNΔ7 model and provide evidence for the most efficacious therapy observed in this field to date.

Anliker B Dr Brynza J Ms Münch R Mr Caputi A Dr 2 Hlavaty J Dr 3 Petznek H Ms 3 Müller UC Professor 4 Keinänen K Professor 5 Hohenadl C Professor 3 Monyer H Professor 2 Cichutek K Professor Buchholz CJ Professor

2 Institute of Clinical Neurobiology, University Hospital of Neurology, Heidelberg, Germany 3 University of Veterinary Medicine, Department of Pathobiology, Institute of Virology, Vienna 4 Institute for Pharmacy and Molecular Biotechnology, University of Heidelberg, Germany 5 Department of Biological and Environmental Sciences, University of Helsinki, Finland P 81 Re-targeted lentiviral vectors for specific neuronal gene transfer in vivo

Lentiviral vectors (LVs) equipped with the glycoprotein of vesicular stomatitis virus (VSV-G) allow transduction of basically all types of cells including neurons. However, such LVs do not discriminate between neuronal and non-neuronal cells if administered to the brain. To achieve exclusive gene transfer to neurons or even subpopulations of neurons, we took advantage of our recently established re-targeting system that relies on the pseudotyping of LVs with engineered measles virus glycoproteins. Specificity for cell entry is provided by displaying a single-chain antibody (scAb) recognizing a unique cell surface protein of the target cell population.

For neuronal targeting we displayed a scAb specific for the murine glutamate receptor subtype GluA2 and/or GluA4. In vitro, such GluA-LVs transduced 293T cells only if the cells expressed recombinant GluA-2 or GluA-4 receptor proteins. In primary cerebellar cultures containing neurons, astrocytes, and fibroblasts, the re-targeted vector was selective for GluA-positive neurons. When injected into the striatum or the hippocampus of adult mice, transduced neurons were detectable 3-6 weeks after injection, while astrocytes remained untransduced. In contrast, VSV-G pseudotyped LVs mediated gene transfer to both neurons and astrocytes. Efficient and specific gene transfer was also observed if a transfer vector encoding the Cre recombinase was packaged into GluA-LV. Injection of such GluA-LV^Cre particles led to successful DNA recombination within neuronal subpopulations of Cre reporter mice.

In future, these new LVs could be used for exclusive transfer of a therapeutic gene to neurons or for functional genomics studies allowing conditional gene ablation in a defined neuronal subpopulation.

Donsante A Dr 1 Yi L 1 Zerfas P 2 Brinster L 3 Sullivan P 4 Goldstein D 4 Prohaska J 5 Centeno J 6 Kaler SG 1

1 Molecular Medicine Program, NICHD, NIH 2 Division of Veterinary Resources, Office of Research Services, NIH 3 Division of Veterinary Resources, Office of Research Services, OD, NIH 4 Clinical Neurocardiology Section, NINDS, NIH 5 Department of Biochemistry and Molecular Biology, University of Minnesota, Duluth, MN 6 Division of Biophysical Toxicology, U.S. Armed Forces Institute of Pathology, Washington, DC P 82 Transduction of choroid plexus epithelia is crucial for gene therapy rescue in a murine model of Menkes disease

The mottled-brindled (mo-br) mouse manifests a lethal (dies by 14d) abnormality in copper transport to the brain caused by mutation of atp7a, a P-type ATPase, and is a model for Menkes disease. The mutant allele, a small in-frame deletion, retains a low level of residual copper transport, and rescue is possible by intraperitoneal copper administration depending on genetic background. We documented that mo-br mice on C57BL/6J were not rescued with intraperitoneal or intravenous copper, and exploited this model to gain insight concerning normal copper delivery to the CNS, using a brain-directed gene therapy approach.

We treated neonatal mo-br C57BL/6J mice with either intracerebroventricular recombinant adeno-associated virus serotype 5 (AAV5) harboring a human atp7a homolog, intracerebroventricular copper, or both. Only combination (AAV5 plus copper) treatment rescued mo-br, with tripling of mean survival and 31% living beyond 110d. As expected from prior characterization, AAV5 transduction occurred primarily in choroid plexus epithelia, without detectable transgene expression in neurons or brain capillary endothelial cells. Survival was associated with higher brain copper and increased activity of dopamine-beta-hydroxylase, a copper enzyme metallated in the trans-Golgi compartment. In contrast, activities of cytosolic and mitochondrial copper enzymes did not differ across treatment groups. At 300d, electron microscopy showed no overt ultrastructural brain abnormalities in combination treated mutants.

Our findings suggest that 1) transduction of choroid plexus epithelia in mo-br enhances brain copper retention and utilization, 2) copper enzymes processed in the trans-Golgi contribute to the rescue, and 3) ATP7A gene therapy may have future clinical applications.

Piersanti S. Dr 1 Astrologo L. Dr 2 Schwarz S.C Dr 3 Schwarz J. Professor 4 Chillon M. Dr 5 Kremer E. 6 Saggio I. Professor 7

1 Department of Biology and Biotechnology “C.Darwin”, University of Rome “La Sapienza”, and Biomedical Park of Rome “S. Raffaele”, Rome, Italy. stefania.piersanti@uniroma1.it 2 Department of Biology and Biotechnology “C.Darwin”, University of Rome “La Sapienza”, and Biomedical Park of Rome “S. Raffaele”, Rome, Italy 3 Department of Neurology, University of Leipzig, Leipzig, Germany. 4 Department of Neurology, University of Leipzig, Leipzig, Germany 5 Department of Biochemistry and Molecular Biology, Center of Animal Biotechnology and Gene Therapy (CBATEG), Universitat Autònoma de Barcelona, Barcelona 08193, Spain 6 Institut Génétique Moléculaire de Montpellier, Montpellier, France and Université Montpellier I & II, Montpellier, France 7 Department of Biology and Biotechnology “C.Darwin”, University of Rome “La Sapienza”, and Biomedical Park of Rome “S. Raffaele”, and Institute of Molecular Biology and Pathology, CNR, Rome, Italy P 83 High-throughput transcriptional analysis of gene therapy viral vectors effects on brain cells

The gene therapy approach using viral vectors currently represents one of the best hopes for treating numerous genetic and acquired brain disorders. Different viral vector platforms have been extensively studied and utilised in clinical trials on the Central Nervous System (CNS), by taking advantage of the specific viral features. However, the improvement of viral systems to mediate safe and long-lasting expression of therapeutic transgenes in brain is particularly challenging due to the post-mitotic nature of nervous cells, the high level of compartmentalisation of the CNS, the potential toxicity and the alteration of the neuronal physiology triggered by the virus. Although many studies have proved the efficacy of the viral sources in transducing the brain in vivo, little is known on neuronal cells perturbations following the vector interaction. To address this issue, we have analysed the global transcriptome of differentiated midbrain-derived human neuronal progenitor cells transduced in vitro with HIV-1-, AAV9-, Helper Dependent human adenoviral (HD hAd)- and Helper Dependent canine adenoviral (HD CAV-2)- vectors, at early and late time points. In particular, canine adenovectors have proved to be an interesting alternative to the human Ad, because of their efficiency in transducing human cells and the absence of CAV-specific neutralizing antibodies in human serum, that inhibit the vector effect. This study intends to provide insights in vector development for CNS, consisting in the ability to predict the neuronal functions altered by the vectors and the possibility to act on these with tools aimed at improving the efficacy and reducing the toxicity.

Nascimento-Ferreira I Miss 1 Nóbrega C Dr 2 Onofre I Miss 1 Déglon N Dr 3 Pereira de Almeida L Professor 1

1 Center for Neurosciences & Cell Biology, University of Coimbra, 3004- 517 Coimbra, Portugal and Faculty of Pharmacy, University of Coimbra, Portugal 2 Center for Neurosciences & Cell Biology, University of Coimbra, 3004- 517 Coimbra, Portugal 3 CEA, Institute of Molecular Imaging (I2BM) and Molecular Imaging Research Center (MIRCen), Fontenay-aux-Roses, France and CNRS/CEA URA2210, Fontenay-aux-Roses, France P 84 Activation of autophagy rescues behavioral and neuropathological cerebellar deficits in a lentiviral mouse model of Machado-Joseph disease

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3) is a fatal, dominant neurodegenerative disorder. MJD results from an abnormal increased repetition of the trinucleotide CAG in the MJD-1 gene, translating into an expanded polyglutamine tract that confers a toxic gain of function to the ataxin-3 protein. Clinical manifestations include cerebellar ataxia and pyramidal signs culminating in severe neurodegeneration.

Even though we previously reported a lentiviral (LV)-based model for MJD (Alves et al., 2008), a LV-model displaying the neuropathological features in the cerebellum, one of most severely attained brain regions in the disorder, had not yet been produced.

In this study, we generated a new mouse model of Machado-Joseph disease by injection of lentiviral vectors encoding human mutant ataxin-3 to the mouse cerebellum. Expression of the mutant protein with 72 glutamines in this region induced a severe behavioral phenotype starting at 4 weeks and progressing until 10 weeks post-injection. At this time-point animals were sacrificed and analyzed at by immunohistochemistry. Accumulation of ubiquitinated inclusions, neuropathological abnormalities and neuronal death were observed in animals expressing mutant ataxin-3 but not in animals expressing GFP (control). Importantly, overexpression of the autophagy-related protein beclin-1 in this model mitigated behavioral deficits and pathological hallmarks.

These data suggest that: a) lentiviral-mediated expression of mutant ataxin-3 in the mouse cerebellum provides a quick and cost-effective model of MJD and b) that stimulation of autophagy is a promising therapeutic strategy for this disease.

Support: Portuguese Foundation for Science and Technology: SAU-FCF/70384/2006; INF- SFRH/BD/29479/2006; CN- Pos Doc SFRH/BPD/62945/2009; IO- SFRH/BD/61461/2009; National Ataxia Foundation, USA (2010).

Ariza L Ms 1 Homs J Ms 1 Pages G Ms 1 Noguera E Mr 1 Udina E Dr 2 Chillon M Dr 3 Corfas G Dr 4 Navarro X Dr 2 Bosch A Dr 5

1 1Centre de Biotecnologia Animal i Teràpia Gènica, 2Dept. Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Barcelona, Spain 2 4Dept. Biologia Cel · lular, Fisiologia i Immunologia. Universitat Autònoma de Barcelona, Barcelona, Spain 3 1Centre de Biotecnologia Animal i Teràpia Gènica, 2Dept. Bioquímica i Biologia Molecular, 3Institució Catalana de Recerca i d'Estudis Avançats (ICREA) 4 5Children's Hospital. Dept of Neurology and Otolaryngology. Harvard Medical School of Boston, Boston, MA, USA 5 1Centre de Biotecnologia Animal i Teràpia Gènica, 2Dept. Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Barcelona, Spain P 85 Gene therapy for diabetic neuropathy by intrathecal NRG1typeIII administration

Background: Diabetic neuropathy is one of the most common long-term complications of diabetes mellitus. The discovery of an effective treatment requires a greater understanding of the causes leading to the development of diabetic neuropathy. Among several proposed pathogenic mechanisms, the process of myelination might be affected since patients have significant deterioration of peripheral nerve function. In this context, NRG1typeIII has been described as an important factor involved in myelination and is vital for this process in the peripheral nervous system.

Results: We analyzed the effect of high concentration of glucose in vitro and in vivo in various tissue cultures and mice models of type I diabetes. The results showed Schwann cell alteration in both, myelin proteins and transcription factors involved in myelination, already at prediabetic stage. Moreover, neurons showed a significant decrease in NRG1typeIII expression in vitro and in vivo already at prediabetes.

Sensory and motor neuron transduction was achieved in diabetic mice by delivering intrathecally AAVrh10 coding for NRG1typeIII. Electrophysiological studies showed an improvement in motor and sensory conduction tests, compared to untreated or mock-treated diabetic animals. Electrophysiological data correlated with a decrease in the number of degenerated axons, with an increase in the diameter of myelinating fibres and with the recovery of myelin-related protein levels.

Conclusions: Our results suggest that AAV vectors coding for NRG1typeIII might be good candidates for gene therapy of diabetic neuropathy and other de-myelinating neuropathies but treatment may be necessary since the very first days of hyperglycemia.

Acknowledgements

ISCIII PS09-00720

SGR 2009-1300

Pardo L Asin Miss 1 Anton M Dr 2 Thalhammer S Dr 3 Adigüzel D Mr 3 Döblinger M Dr 4 Goya G.F Dr 5 Goya R Professor 6 Plank CH Dr 2 Mykhaylyk O Dr 2

1 Klinikum rechts der Isar der TUM, Institute of Experimental Oncology and Therapy Research, München, Germany and Nanoscience Institute of Aragon, University of Zaragoza, Spain 2 Klinikum rechts der Isar der TUM, Institute of Experimental Oncology and Therapy Research, München, Germany 3 Helmholtz Zentrum München, AG NanoAnalytics, Neuherberg, Germany 4 Department of Chemistry and Biochemistry, LMU, Munich, Germany 5 Nanoscience Institute of Aragon, University of Zaragoza, Spain 6 Institute for Biochemical Research, National University of La Plata, Argentina P 86 Magnetic field-assisted transduction of neuronal and glial cells with magnetic virus vectors

We aim at using magnetic viral vectors and a magnetic field-assisted transduction to enhance the expression of neurotrophic factors that prevent the degeneration and enhance recovery of the neurons at the target site in the brain. We used RAd-(TK/GFP)fus, LVSFeGFP and LVSFds-Red2 vectors coding for reporter genes and in house synthesized magnetic nanoparticles (MNPs) with the an average core size of 10 and 80 nm. We quantified the association of the vectors with both MNPs and internalization of the vector using radioactively labelled adenovirus. We evaluated the magnetophoretic mobility of the complexes from the time course of the turbidity of the virus-MNP complex suspensions in defined magnetic fields. The optimal MNPs-to-virus particle ratio ensured almost complete vector association with MNPs into the complexes stable enough in CSF. TEM and AFM data showed structurally intact viruses “decorated” by multiple MNPs. In B92 glial and Neuro2A neuroblastoma cells magnetotransduction with optimized formulations of the magnetic viral complexes resulted in considerably improved efficiency compared to standard infection. The complexes possess magnetic responsiveness high enough to allow trapping of the vectors at specific target sites of the rat brain after intracerebroventricular (ICV) injection, thus achieving therapeutic effect at relatively low vector and MNP dosage and decreasing virus associated toxicity and immune response. Additionally, the ICV route should provide a minimally invasive approach for gene therapy in the brain. In further experiments we will use optimized magnetic vectors in order to implement neuroprotective gene delivery by magnetotransduction in the brain of aging rats with dopaminergic neurodegeneration.

Sugano H. Dr 1 Miyake N. Dr 1 Endo A. Ms 1 Miyake K. Dr 1 Shimada T. Professor 1

1 Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan P 87 Systemic injection of AAV type 9 vector in utero facilitates global gene expression in the CNS

Background: Since both the immune system and the blood brain barrier (BBB) are developmentally immature, systemic delivery of viral vector during the perinatal period may be a potential strategy to treat genetic neurological diseases. In this work, we examined biodistribution of adeno-associated viral vector serotype 9 (AAV9) following intraperitoneal injection into prenatal and postnatal mice.

Method: AAV9 vector encoding GFP (5 × 10⁴ viral genomes/g) was injected into the peritoneal cavity of fetal (embryonic day 15; n = 10) or neonatal (postnatal day 1; n = 14) C57BL/6 mice. The mice were sacrificed at 8 weeks of age and the tissues were analyzed by real-time PCR for biodistribution of vector and by immunohistochemistry for GFP expression.

Results: GFP expression was detected in all organs including the brain. The copy number of the AAV genomes in fetal injected mice were significantly lower than those of neonatal injected mice in all visceral organs. In the brain of neonatal injected mice, GFP expression was detected mainly in the olfactory bulbs. In contrast, global gene delivery occurred in the whole brain after fetal injection. The higher copy number of AAV was observed in the areas of the cerebrum, hypothalamus and hypocampus, compared to neonatal injected mice.

Conclusion: Our data suggest that systemic injection of AAV9 in utero is an effective strategy to cross the BBB for gene transfer into the CNS, which is tightly protected from viral infection in adult mice, and an important option for treatment of early onset genetic neurological diseases.

Tsalenchuck Y Miss 1 Cohen M Miss 1 Braun E Dr 1 Steiner I Professor 2 Panet A Professor 3

1 Laboratory of Neurovirology and Department of Biochemistry, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem, Israel 2 Laboratory of Neurovirology, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem, Israel and Department of Neurology, Rabin Medical Center Campus Beilinson, Petach Tikva, Israel 3 Department of Biochemistry, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem, Israel P 88 Neurotropic viruses tropism and spread in the CNS

Background: Neurotropic viruses, such as HSV-1 and Rabies virus (RV), may infect the CNS causing severe diseases, while carry the potential to serve as vectors in gene therapy to the nervous system.

Method: In order to study neurotropic virus tropism and spread in the brain, an ex vivo system of organ culture was established.

Results: i) HSV-1 infection was confined to leptomeningeal, periventricular and cortical brain regions.

ii) HSV-1 did not preferentially infect proliferating cells, although it had a predilection to ventricular zones and the Infection was localized to early progenitor cells.

iii) The infection pattern of lentivirus pseudotyped by VSV-G and RV-G was compared to that of HSV-1. While the tropism of the VSV-G pseudotyped vector had a striking resemblance to HSV-1, the RV-G pseudotype infection was weak and more diffuse in the entire parenchyma. VSV-G pseudotype widely infected neurons, sparing astrocytes, while the RV-G pseudotype infected astrocytes, but not neurons.

iv) The infection and tissue spread of HSV-1, expressing the GFP protein, was evaluated after injection into the brain ventricular zone. Within a day, infection was observed as a focal site and subsequently the virus spread along distinct anatomical structures. Neutralizing antibodies failed to inhibit viral spread, suggesting that the mechanism of spread is not mediated by extracellular re-infection.

Conclusions: Taken together these results indicate unique patterns of lentiviral and HSV-1 infections in the brain and furthermore, HSV-1 spread in the brain is determined both by anatomical neuronal networks as well as by intracellular factors.

Ahmed SO Mr 1 Peluffo H Dr 1 Foster E Dr 2 Lago N Dr 2 Hutson TH Dr 2 Moon L Dr 2 Yip P Dr 2 Caraballo-Miralles V Mr 3 Lladó J Dr 3 McMahon SB Professor 2 Yáñez-Muñoz RJ Dr 1

1 School of Biological Sciences, Royal Holloway-University of London, UK 2 Neurorestoration Group, Wolfson Centre for Age-Related Diseases, King's College London, UK 3 Department of Biology/IUNICS, Universitat de les Illes Balears, Spain P 89 Efficient gene expression in the spinal cord from integrase-deficient lentiviral Vectors

Gene transfer to spinal cord cells may be crucial for therapy in diseases including Spinal muscular atrophy and Amyotrophic lateral sclerosis, as well as in spinal cord injury. Methods are required that combine high gene transfer efficiency with enhanced bio-safety. Lentiviral vectors are efficient for transduction of a variety of cell populations, but like all integrating vectors they pose a risk of causing insertional mutagenesis. We have recently developed integrase-deficient lentiviral vectors (IDLVs) that remain episomal and yet retain the transduction efficiency of standard integrating lentivectors. IDLVs are particularly adequate for applications in non-dividing cells, where the episomes, which lack replication sequences, are not diluted out through repeated cell division. Following work in the eye and brain, we have now applied IDLVs for transduction of spinal cord in vitro, in explants and in vivo. Our results demonstrate similar efficiency of eGFP expression from integrating lentivectors and IDLVs in most cell types tested, including motor neurons, interneurons, dorsal root ganglia neurons and astroglia. Microglia is less efficiently transduced by IDLVs in pure cell cultures but not in explants. After intra-parenchimal injection in vivo, transduction is mainly neuronal, with both motor and interneurons being efficiently targeted. We have also demonstrated that IDLV-mediated expression of a growth factor rescues motor neuron cultures from death caused by removal of exogenous trophic support, and efficient IDLV-mediated RNA interference and SMN expression. These results suggest that IDLVs could be efficient and safer tools for cord transduction in therapeutic strategies, particularly for Spinal muscular atrophy.

Lindenmaier W Dr 1 Pohl S Ms 1 Zghoul N Dr 1 Schön O Dr 1 Lachmann K Dr 2 Dohse A Ms 3 Thomas M Dr 2 Meyring W Dr 4 Garritsen H Dr 4 Dittmar KEJ Dr 1

1 Helmholtz Zentrum für Infektionsforschung, Inhoffenstr. 7, D-38124 Braunschweig. Germany 2 Fraunhofer IST, Bienroder Weg 54 E, D-38108 Braunschweig, Germany 3 Fraunhofer IST, Bienroder Weg 54 E, D- 38108 Braunschweig, Germany 4 Klinikum Braunschweig, Celler Straße, D-38114 Braunschweig, Germany P 90 Cultivation of human MSC in a closed bag cultivation system modified by dielectric barrier discharge

For safe, GMP compliant production of therapeutic cells a closed disposable cell culture bag system has many advantages. We have previously shown that the closed cell culture bag is suitable for the cultivation of suspension cells but did not support growth of adherent cells. However, when the inner bag surface is functionalized by an atmospheric-pressure plasma process in the presence of suitable film forming agents to create an amino group or silane coating, adherently growing cells could be expanded, whereas the unmodified cell culture bag had cell repellent properties. Biocompatibility of the surface treated cell culture bag was assessed with adherently growing cell lines and primary human bone derived mesenchymal cells (MSC). Cellular properties of MSC cultivated in bags were compared to standard culture conditions. We evaluated cell morphology, expression of cell surface markers, attachment, cell proliferation and osteogenic and adipogenic differentiation. Both osteogenesis and adipogenesis of the MSC could be induced in the modified bags. Gene transfer by adenoviral vectors allowed efficient modification of bag cultivated cells. Also in FCS free cultivation conditions expansion was effective. Further, we successfully cryopreserved adherent cell layers in situ on the modified culture bag surface. The influence of bag cultivation on the cells was analyzed by expression profiling in comparison to classic cultivation in polystyrene flasks. In conclusion we have shown that adherent cell expansion, differentiation and cryopreservation is possible on surface adapted cell culture bags with potential application as a disposable closed bag system for GMP compliant cell cultivation.

Abdullah S Dr 1 Wendy-Yeo WY Miss 1 Rosli R Dr 1 Rahman SA Dr 1 Domb AJ Professor 2

1 UPM-MAKNA Cancer Research Laboratory, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 2 Department of Medicinal Chemistry and Natural Products, School of Pharmacy, the Hebrew University-Hadassah Medical School, Jerusalem, Israel P 91 Transfection Efficiency and Safety Profiles of Dextran-Spermine/Plasmid DNA in Mouse Lungs

Generation of an efficient gene delivery vector with low toxicity profile is required to achieve successful gene delivery for pulmonary diseases. Dextran-spermine (D-SPM), a cationic-based gene delivery vector, has been shown to be capable of transfecting cells and tissues in vitro and in vivo. However, no study has been performed to determine the efficiency and safety of this delivery agent exclusively in the mouse lungs via intranasal delivery to date. In this study, we determined the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA) in the lungs of BALB/c mice by varying the weight-mixing ratio of D-SPM to pDNA. The levels of transgene expression were also measured at different time points. Reporter gene expression levels were observed highest at weight-mixing ratio of 16 with 13.5μg pDNA and at day 1 post-administration. Re-administration with similar conditions of D-SPM/pDNA was performed at 24 hr post initial administration, but it did not augment the first administration to enhance or even to restore the transgene expression. Quantifications of cytokines and neutrophil inductions in bronchial alveolar lavage fluids were performed to assess the safety profile of the complex in the lungs. Administration of D-SPM/pDNA resulted in modestly elevated level of IL-12 with no difference in the levels of IFN-γ when compared to the untreated mice, although an increased in the neutrophil count was observed. These results demonstrate that D-SPM has the potential to be further developed into an efficient and safe gene delivery vector to the lungs.

Rodrigues AF Miss 1 Guerreiro M Mr 1 Santiago V Miss 1 Dalba C Dr 2 Klatzmann D Professor 3 Alves PM Dr 1 Carrondo MJT Professor 1 Coroadinha AS Dr 1

1 IBET/ITQB-UNL, Apartado 12, 2781-901 Oeiras, Portugal 2 Epixis SA, 75013 Paris, France 3 AP-HP, 75013 Paris, France P 92 Silencing host cell proteins associated with retroviral particles

Background: Retrovirus and Lentivirus derived vectors have already demonstrated their potential as gene transfer tools in gene therapy protocols, in vaccinology and as oncolytic agents for cancer treatment. HEK 293 cell lines have showed to be an excellent platform for the production of these viruses. Retroviral particles incorporate several host cell proteins namely membrane proteins that retain function and may participate in the infection process, but on the other hand can be also immunogenic reducing the efficiency of the viral particles.

Methodology: In this work we assess the feasibility of reducing host cell protein incorporation in retroviral particles by knockdown RNA interference. The CD81 was selected since it is a membrane protein virtually present in all cells and known to be incorporated on retroviral and lentiviral particles surface and, being highly immunogenic can lead to vector inactivation in vivo, reducing its therapeutic potential. A 293 derived cell line producing retrovirus like particles from MLV (RVLPs) was stably silenced using short hairpin RNAi.

Results: CD81 was successfully silenced up to 90-95%, clonal variability was found regarding to cell growth and metabolism but clones with normal patterns of growth without impaired RVLP production were successfully obtained. The analyses of RLPs showed low or no detection of CD81 protein.

Conclusion: The work herein presented constitutes a proof-of-concept, showing that it is possible to manipulate the incorporation of host cell proteins in the produced viral particles either for studying the effect of their incorporation on vector efficiency and safety or to improve the producer cell lines.

Vlaskou D. Dr 1 Mykhaylyk O. Dr 1 Pradhan P. Dr 1 Bergemann C. Mr 2 Klibanov A.L Professor 3 Hensel K. Mrs 4 Schmitz G. Professor 4 Plank C. Professor 1

1 Institute of Experimental Oncology, Technical University of Munich, Germany 2 Chemicell GmbH, Berlin, Germany 3 Department of Biomedical Engineering, Division of Cardiovascular Medicine, University of Virginia, Box 800158, Charlottesville, VA 22908, USA 4 Institute of Medical Engineering, Dept. for Electrical Engineering and Information Sciences, Ruhr-University Bochum, Germany P 93 Magnetic microbubbles as mediators of gene delivery

In recent years microbubbles technology has gained lot of interest in the field of gene and drug delivery. Based on the “Magnetofection” concept, besides the development of magnetic acoustically active lipospheres [1], we have been able to prepare lipid monolayer shelled microbubbles loaded with highly positively charged naked magnetic nanoparticles (composed of iron oxide) through electrostatic and matrix affinity interactions. These magnetic microbubbles show strong ultrasound contrast.

Treatment of cancer cells with these microbubbles using ultrasound exhibited strong dose-dependent cytotoxic effects, although ultrasound alone, lipid microbubbles alone, magnetic nanoparticles or magnetic microbubbles alone at the corresponding concentrations did not affect the cell viability. On the other hand, when these magnetic microbubbles were mixed with plasmid DNA encoding a reporter gene, we achieved gene delivery to cultured adherent cells only when ultrasound was applied. Gene transfer efficiency was strongly dependent on the application of a gradient magnetic field to sediment the microbubbles on the target cell membranes.

From the preliminary experiments we conclude that magnetic microbubbles could be used as magnetically targeted diagnostic agents for real-time ultrasound as well as magnetic resonance imaging. At the same time, such magnetic microbubbles may be useful for therapeutic purposes such as in cancer therapy, vascular thrombolysis and gene therapy. However, further improvements are required to control their cytotoxicity.

References

1 Vlaskou D. , Mykhaylyk O. , Krötz F. , Hellwig N. , Renner R. , Schillinger U. , Gleich B. , Rüger A. , Schmitz G. , Hensel K. , Plank C. Adv Funct Mat., 2010[accepted] a-1234 Silva AC Miss 1 Simão D Mr 2 Sousa MFQ Mr 2 Cruz PE Dr 2 Carrondo MJT Professor 2 Alves PM Dr 1 1 IBET/ITQB-UNL, Apartado 12, P-2781-901 Oeiras, Portugal 2 IBET, Apartado 12, P-2781-901 Oeiras, Portugal P 94 pO2 and pH profile evolution in shake flasks during 293 cell growth and infection with adenovirus

The metabolic state of the culture is essential for quality assurance at the early stages of a process. Key parameters in cell culture are the partial oxygen pressure (pO₂) and the pH, both of which decisively affect the culture quality. Continuous monitoring of oxygen consumption and pH is advantageous for optimization of cell culture processes.

Shake flasks are used in the biotechnology industry for a variety of tasks however, the data obtained from the cultures, have not been so far translated to scale-up due to the lack of knowledge of the conditions under which they are performed. The new tendency to use modern optical sensors and bioprocess monitoring devices will increase the relevance of shake flasks in bioprocess development.

Adenoviruses have increasingly been used as vectors in strategies for gene therapy and vaccination. Since the process analytical technologies initiative, where it is encouraged to design, analyze, and control manufacturing as early as possible in the development phase, it become important for all proceedings, even for well characterized processes as producing adenovirus, to increase the information at the early stages for quality assurance.

In this work, the new Shake Flask Reader from PreSens, which measure the pO₂ content and pH of the medium accurately and non-invasively, was used to monitor the production of adenoviruses in 293 cells. Data on the metabolic characterization during cell growth and infection at different cell concentrations will be presented. These results should aid the scaling-up process to bioreactors providing more information to develop process control strategies.

Bagnis C Dr 1 Chapel S Dr 1 Chiaroni J Dr 1 Bailly P Dr 1

1 EFS Alpes Méditerranée, 149 Bd Baille, 13005, Marseille, France P 95 Lentiviral vectors to generate blood samples expressing selected blood group antigens: an innovation

To avoid Haemolytic Transfusion Reaction (HTR), blood transfusion safety and efficiency relies on analysis of donor blood and patient sera, with some very rare blood group phenotype being difficult to obtain from donor population. An in vitro process to generate blood samples expressing preselected rare blood group phenotypes as control for diagnosis purposes remains an important issue to address. Our projects focuses on genetic manipulation to mediate silencing or expression of blood group system antigens. CD34+ hematopoietic stem cells were transduced with a lentiviral vector expressing a short hairpin RNA directed against UT-B mRNA, or the cDNA coding for the UT-B protein (JK/Kidd blood group system). Cells were then cultivated to promote terminal erythroid differentiation prior to be analyzed for Kidd expression. Silencing experiments performed with CD34+ cells from donor with Jk(a + b+) phenotype showed transduced cell populations exhibiting a Jknull phenotype when subjected to affinity column analysis. Conversely, erythroid cell populations derived from a Jk(a + b-) donor and transduced with the lentiviral vector carrying the UT-B cDNA coding for Jkb showed a de novo expression of the Jkb specific antigen. In parallel, as a prerequisite to explore the proof of concept to generate an “universal” blood, lentiviral vectors were designed to silence the expression of the gene coding for the fucosyltransferase that generates the ABO group, and the gene coding for the RhD antigen that generates the Rhesus group. Whether this strategy can be considered as a tool to generate rare blood group control samples for diagnosis remains to be discussed.

Marie C Dr 1 Quiviger M Mr 1 Vandermeulen G Dr 2 Richard M Dr 1 Préat V Professor 2 Scherman D Professor 1

1 INSERM U1022, CNRS UMR8151, Université Paris Descartes, ENSCP, Paris, France 2 Université Catholique de Louvain, Louvain Drug Research Institute, Unité de Pharmacie Galénique, Brussels, Belgium 2 Université Catholique de Louvain, Louvain Drug Research Institute, Unité de Pharmacie Galénique, Brussels, Belgium 1 INSERM U1022, CNRS UMR8151, Université Paris Descartes, ENSCP, Paris, France P 96 The biosafe pFAR vectors display superior expression efficiency in skin, liver and tumours

Nonviral gene therapy requires the production of plasmid DNA that meets defined criteria such as biosafety and high quality.

To produce biosafe plasmids, we have designed a novel antibiotic-free selection system that is based on the suppression of an amber mutation by a plasmid-borne function. The nonsense mutation was introduced into the essential chromosomal thyA gene of Escherichia coli, resulting in thymidine auxotrophy. In parallel, pFAR4, a small plasmid vector carrying a suppressor t-RNA gene, was entirely synthesised. The introduction of pFAR4 into the thyA mutant restored normal growth to the auxotrophic strain and allowed, after optimisation of the bacterial producer strain, an efficient production of predominantly monomeric supercoiled plasmids.

The potentiality of pFAR4 as an eukaryotic expression vector was first assessed by monitoring luciferase activities after electrotransfer of LUC-encoding plasmids into various tissues. In muscle, high luciferase expression levels were monitored, thus demonstrating an efficient gene delivery. In mouse skin, whereas luciferase activities decreased within three weeks after intradermal electrotransfer of a conventional expression vector, a sustained luciferase expression was observed with the pFAR4 derivative. Interestingly, tumour cells transplanted into mice and subsequently transfected by LUC-encoded pFAR4 display a higher luciferase expression level than those electrotransferred with a control vector. Similarly, higher expression levels were obtained after plasmid hydrodynamic injection of a mSEAP-encoded pFAR4 into liver.

Thus, we have designed a novel strategy for an efficient production of biosafe plasmids and demonstrated their potentiality as nonviral eukaryotic expression vectors in several organs and tissues.

Schleef M. Dr 1 Schmeer M. Dr 1 Blaesen M. Dr 1 Baier R. Dr 1

1 PlasmidFactory GmbH & Co. KG, Bielefeld, Germany P 97 Minicircle – an overview

For future gene therapy and genetic vaccination approaches it is crucial to develop safe and efficient vector systems. Currently, viral and non-viral vectors are used, having their advantages and limitations. The dissemination of antibiotic resistance genes, as well as the uncontrolled expression of backbone sequences present in plasmid DNA may have profound detrimental effects. Additionally, CpG motifs have been shown to contribute to silencing of episomal transgene expression. Hence, removal of bacterial backbone DNA can greatly improve safety and efficiency of DNA used in gene therapy and vaccination.

Here, we give an overview on earlier and recent approaches for the production, purification, and application of such minimal constructs. Different approaches have been described so far, from plasmids where the antibiotics resistance gene has been replaced by another marker to minicircle DNA consisting almost only of the gene of interest. Minicircles can be produced by in vivo site-specific recombination of a so-called parental plasmid resulting in a miniplasmid and the minicircle. This recombination can be achieved using different enzymes which need specific target sequences as recombination sites. The most important difference between these approaches is the efficiency of the recombination step as well as the purification procedure used in order to remove the miniplasmid (with the bacterial sequence motifs) and residual amounts of the parental plasmid if necessary.

In addition to their improved safety profile, minicircles have been shown to increase the efficiency of transgene expression in in vitro and in vivo studies.

Scale-up was recently possible in fermentation and results in an extremely pure DNA product.

Frecha C Dr Costa C Mrs Lévy C Mrs 1 Nègre D Dr Cosset FL Dr Verhoeyen E Miss

P 98 Unraveling the transduction mechanism used by MV-LVs for quiescent lymphocyte gene transfer

Gene transfer into quiescent T and B cells is of importance for gene therapy and immunotherapy approaches to correct multiple hematopoietic disorders. We generated LVs pseudotyped with measles virus glycoproteins (MV-HEd-LVs), which represent the first tool allowing efficiently transduction of quiescent human T and B cells. Indeed, MV-HEd-LVs allow an efficient transduction of primary target cells since they express CD46 and SLAM, the natural receptors of the vicinal MV-Edmonston strain. Interestingly, LVs pseudotyped with MV H gps, blind for the CD46 binding site, were completely inefficient for resting lymphocyte transduction. Similarly, SLAM-blind H mutants give rise to particles that recognize only CD46 as entry receptor, were also inefficient for transduction. We revealed that SLAM-blind LVs accomplish cell entry and reverse transcription, at similar levels as MV-HEd-LVs, but unlike for MV-Ed-LVs, this proviral DNA was not translated into efficient proviral integration. In contrast, only background levels of proviral DNA were found with CD46-blind-LVs. Moreover, our results indicate that both CD46- and SLAM-binding sites need to be present in cis in the H gp to allow successful engagement of both receptors resulting in a stable transduction of quiescent lymphocytes. The mechanism of entry seems to be crucial to determine the fate of these LVs in quiescent lymphocytes and our results point out that macropinocytosis may be involved. Altogether, our data suggest that even if vector entry can occur through CD46 receptor, SLAM-triggered signaling may be needed for an efficient transduction.

Kennedy CK Mr Havelin RH Mr O Flaherta COF Mr Barry FB Professor

P 99 A non-invasive animal in vivo imaging system to investigate MSC homing in rheumatoid arthritis

Introduction: Despite the common use of Collagen Induced Arthritis (CIA) as a model for rheumatoid arthritis (RA) in mice, homing of mesenchymal stem cells (MSCs) to diseased joints has not being demonstrated definitively to date. The Bazooka-SPECT, a low cost small animal in-vivo imaging system, allows spatial resolution of radioactively labeled cells administered to live animals. In this study, MSCs were engineered to express human sodium iodide symporter (NIS) which allows uptake of the gamma ray emitter technetium (^99mTc) in these cells. . Localisation of NIS expressing MSCs was determined using the Bazooks-SPECT imaging platform.

Materials & Methods: CIA was induced in ten male DBA/1 mice aged 8-10 weeks . MSCs from a GFP transgenic mouse (FVB strain) were transduced with adenovirus expressing NIS. AdNIS MSCs, MSCs transduced with empty adenovirus (AdNull) and untransduced MSCs were administered via IV and IP routes. All MSC injections were performed 42 days post immunization and 3- 5 days post MSC injection ^99mTc was administered by IP followed by in vivo imaging . As a backup to the imaging data, all joints were harvested for DNA extraction and detection of GFP genomic DNA by PCR.

Results: ^99mTc uptake was detected in mice with high clinical RA scores which received IV adNIS MSC and IP ^99mTc injections, while mice receiving adNull and untransduced MSCs did not show signal following in-vivo imaging. PCR for GFP genomic DNA, confirmed the in-vivo imaging platform results.

Conclusion: Using the Bazooka SPECT we determined intravenous administration of MSC to be the optimal route of delivery in the CIA model.

Farley DC Dr Bannister R Dr Carlucci M Mrs Miskin J Dr Mitrophanous K Dr

P 100 Characterisation of the replication kinetics of infectious EIAV in permissive cell lines: Application

We have developed a minimal gene therapy lentiviral vector system based on Equine Infectious Anemia Virus [EIAV], which is being utilised in a number of indications, including Parkinson's disease. Safety testing for RCL in batches of clinical product and end-of-production cells is a prerequisite for drug release. Since wild type EIAV dose not replicate in human cells, we previously developed an assay using murine leukaemia virus (MLV) as a surrogate RCL positive control and HEK293 cells (human) as an indicator cell line to amplify RCL/MLV from a minimal infectious dose (M.I.D.) [Miskin et al., 2006]. Detection of reverse transcriptase (RT) within culture supernatants by qRT-PCR provides the assay end-point, with a read-out differential of 4-5 logs between RT-positive and RT-negative samples.

As part of the ongoing characterisation of EIAV vectors we evaluated the replication kinetics of the wild type virus on a number of cell lines reported to be permissive for infection. EIAV grew maximally in 92BR (donkey) cultures. Accessory gene knock-out strains were generated to assess growth of attenuated EIAV. No amplification of EIAV lacking either Tat or Rev was observed in 92BR cultures over a period of at least 40 days, but EIAV lacking an intact S2 open reading frame replicated with similar kinetics as EIAV. In addition we will present empirical data supporting the use of Poisson distribution equations to determine the theoretical minimal infectious dose required to set confidence levels for detection of an RCL in a cell based amplification assay.

Benati C Dr Marano G Dr Salvatori F Dr Marzocca M Miss Martelli L Dr Catamancio S La Seta Dr Frizzale G Dr Imro MA Dr Silvani A Dr Tonlorenzi R Dr 2 Radrizzani M Dr Ziliotto R Dr Cossu G Professor 2 Bordignon C Professor 3

2 Division of Regenerative Medicine, San Raffaele Scientific Institute, Milano 3 MolMed S.p.A., Milano, Italy and Vita-Salute San Raffaele University, San Raffaele Scientific Institute, Milano, Italy P 101 Cell therapy of DMD: development of GMP manufacturing and testing methods for clinical application

MolMed S.p.A. has extensive expertise on cellular and gene therapy product development. Our GMP facility is formally authorized for the production and release of medicinal products for human use. MolMed will act as GMP manufacturing site for an upcoming clinical trial for Duchenne Muscular Dystrophy (DMD), conducted at HSR, based on the use of human mesoangioblasts (hMAB), cells showing myogenic potential and ability to negotiate vascular walls.

Methods for hMAB isolation, expansion and characterization were scaled-up, optimized and adapted to GMP.

A good cell expansion (range 2.2- 3.6) was obtained in flasks and cell factories up to p20. As expected, cultured hMAB expressed the mesenchimal stem cell/hMAB markers CD44 and CD13, while they did not express the endothelial/hematopoietic cell markers CD31, CD34, CD45; satellite cells (CD56+) were < 5%.

The senescent cells were < 6% up to p29, no chromosome abnormalities were detected.

Spontaneous differentiation into myotubes was observed up to p16.

Thawed cultured cells were maintained for 4 days, and then formulated as required for patient treatment. Viability was >90% and cell expansion was ∼3.

The following GMP process was designed: hMAB from dedicated donors will be expanded in cell factories (∼1 ± 0.3x10⁹ total cells at p5-p11), then frozen constituting the Intermediate Product (IP); IP cells will be thawed, briefly cultured, then formulated in saline solution/heparin, constituting the fresh Medicinal Product (MP).

IP and MP will be characterized for Identity/Potency, Safety, and Purity.

Bellintani F Dr Rossetti F Dr Martelli L Dr Vallanti G Dr Addamo R Raffaele Dr Marano G Dr Piacenza L Mr Marzocca M Miss Ziliotto R Dr Cantore A Dr 2 Bordignon C Professor Naldini L Professor 4 Radrizzani M Dr

2 San Raffaele Telethon Institute for Gene Therapy, Milano, Italy 4 San Raffaele Telethon Institute for Gene Therapy, Milano, Italy and Vita-Salute San Raffaele University, San Raffaele Scientific Institute, Milano, Italy P 102 Large scale LV production and purification: process optimization for in vivo gene therapy studies

Molmed S.p.A. is a biotech company focused – inter alia – on development of cell/gene therapy products. A GMP process for LV production and purification was developed in collaboration with HSR-TIGET and Généthon and is currently used for LV manufacturing for ex-vivo gene therapy clinical trials sponsored by Telethon.

Based on such process, the production of high quality purified LV for in-vivo haemophilia B gene therapy in large animal models was designed.

Vector production is based on quadri-transfection of 293T cells with a third generation LV system in 12 ten-trays cell factories (CF10), followed by purification through endonuclease treatment, anion exchange chromatography, concentration, gel filtration and final sterilizing filtration.

The current process has an overall yield around 30% in terms of total viral particles and results in 99% reduction of key contaminants such as plasmid DNA, host cell DNA and proteins.

To obtain a product suitable for in-vivo application in dogs, the following parameters have been considered:

Selection of protein-free formulation buffer (PBS) and evaluation of vector stability in storage conditions (-80°C)

Increase of LV production efficiency using strategies improving transcription and translation of packaging proteins and viral RNA

Improvement of vector purity by further reducing DNA contaminants

Results indicate that PBS as protein-free formulation buffer is suitable and guarantees vector stability for at least 12 months, without significant variations in infectious titer and infectivity.

Up to 5x increase in LV production has been obtained at 1 CF10 scale.

The addition of a second endonuclease treatment is under evaluation.

Kichler A Dr 1 Mason AJ Dr 2 Leborgne C Dr 3 Bechinger B Dr Scherman D Dr 1

1 Laboratoire de Pharmacologie Chimique et Génétique UMR 8151 CNRS-U1022 INSERM, Université René Descartes, Chimie Paristech, Paris, France 2 Faculté de Chimie, Institut Le Bel, 4 rue Blaise Pascal, F-67000 Strasbourg, France & Pharmaceutical Science Division, King's College London, 150 Stamford Street, London, UK 3 Genethon, BP60, 91002 Evry cedex, France P 103 Histidine-rich amphipathic peptides promote efficient delivery of nucleic acids into mammalian cells

Besides being a useful tool in research, gene transfer has a high potential as treatment for a variety of genetic and acquired diseases. However, in order to enable a gene to become a pharmaceutical, efficient and safe methods of delivery have to be developed. We found that cationic amphipathic histidine-rich peptide antibiotics can efficiently deliver DNA into mammalian cells. Our lead compound, LAH4 (KKALLALALHHLAHLALHLALALKKA), demonstrated in vitro transfection efficiencies comparable to those of commercially available reagents. Synthesis and evaluation of LAH mutants provided evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Our results also suggest a mechanism of selective destabilization by LAH4 of anionic lipids in the membranes of cells during transfection. Further results indicate that acidification of the endosome results in high local concentrations of free peptide in this organelle. These peptides become then available to interact with the endosomal membranes and thereby are responsible for the delivery of the plasmid DNA complex to the cytoplasm. When these data are taken together, they indicate a dual role of the peptide during the transfection process, namely DNA complexation and membrane permeabilization. Finally, we will report that peptides of the LAH family are efficient siRNA delivery vehicles.

Ryu DW Mr 1 Kim HA Miss 1 Lee M Professor 1

1 Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, Republic of Korea P 104 VEGF receptor binding peptide linked amphiphilic peptide micelles for targeting delivery of plasmid

Angiogenic endothelial cells are important target for cancer gene therapy. In this research, an angiogenic endothelial cell-targeting carrier was developed with VEGF receptor binding peptide (VRBP) and amphiphilic peptide micelles. The amphiphilic peptide was RRRVVVVVV (R3V6), which formed peptide micelles in aqueous solution. In the previous study, it was proved that V3R6 delivered plasmid DNA (pDNA) to various types of cells and did not have any toxicity. VRBP was a previously identified short peptide, whose sequence was ATWLPPR. VRBP-linked R3V6 was synthesized chemically for targeting gene delivery. In gel retardation assay, pDNA was completely retarded at a 2:1 weight ratio (peptide:pDNA). Heparin competition assay suggests that VRBP-R3V6/pDNA complex was dissociated more easily than poly-L-lysine (PLL)/pDNA complex. In transfection assay to calf pulmonary aortic endothelial (CPAE) cells, VRBP-R3V6/pDNA had the highest transfection efficiency at a 30:1 weight ratio (peptide:pDNA). In addition, VRBP-R3V6 had higher transfection efficiency than PLL and R3V6. In cytotoxicity assay, VRBP-R3V6 did not show any detectable toxicity to cells. Considering higher transfection efficiency to CPAE and lower cytotoxicity than PLL, VRBP-R3V6 may be useful for targeting gene delivery to angiogenic endothelial cells.

Stevenson L Dr 1 Farley DC Dr 1 Rodrigues T Dr 1 Freail K Ms 1 Stewart HJ Dr 1 Mitrophanous KA Dr 1

1 Oxford BioMedica (UK) Limited, Medawar Centre, Robert Robinson Avenue, The Oxford Science Park, Oxford OX4 4GA P 105 Development of a High Throughput Immunoassay for Screening ProSavin® Producer Cell Line Clones

ProSavin^® is an Equine Infectious Anaemia Virus (EIAV) based lentiviral vector for the treatment of Parkinson's disease that encodes three enzymes required for dopamine synthesis; aromatic amino acid decarboxylase (AADC), tyrosine hydroxylase (TH) and GTP-cyclohydrolase 1 (CH-1). Producer cell lines (PCLs) are being developed for the large scale manufacture of ProSavin^®. Such cell lines contain a regulatory protein (Tet repressor) and the vector components (EIAV Gag/Pol, VSV-G envelope and the EIAV ProSavin^® vector genome) stably integrated into the cellular genomes. To identify cell lines that are capable of producing ProSavin^® at high titre a large number of clones have to be screened. This process is labour intensive, involving clonal cell expansion, preparation of a clone cryo-bank, manipulation of clones for screening, and assaying of vector-containing cell culture supernatants to assess titres. For this reason a high throughput titration assay would significantly expedite the selection of high vector producing clones.

Currently, ProSavin^® is quantified by transduction of HEK293T cells cultured in 12-well plates. Transduced cells are determined using a flow cytometry based method to quantify fluorescently labelled cells, or by DNA integration assay which uses a quantitative PCR approach. Both methods are time consuming and are not considered high throughput. Here we discuss the development of a number of 96-well plate based ProSavin^® titration assays which employ the use of immunodetection for TH. The applicability of these assays as a general screening strategy will be discussed in greater detail.

Schmeer M. Dr Blaesen M. Dr Baier R. Dr Schleef M. Dr

P 106 Progress in minicircle manufacturing and performance testing

The dissemination of antibiotic resistance genes, as well as the uncontrolled expression of backbone sequences present in plasmids may have profound detrimental effects. Additionally, unmethylated CpG motifs have been shown to silence episomal transgene expression. Therefore, an important goal in vector development is to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle DNA.

PlasmidFactory´s technology facilitates the production of highly pure minicircle DNA for applications in gene therapy and vaccination as well as virus production.

The production technology is based on two processes:

1) An inducible, sequence specific, and very efficient in vivo recombination process

2) A chromatographic purification technology for the isolation of the minicircle DNA.

The resulting minicircle DNA only consists of the gene of interest and a tiny residual sequence stretch including one of the two recombination sites. The chromatographic purification results in an exceptional purity, proven by various QC tests, which is extremely important since even small amounts of contaminants can produce dramatic experimental artefacts.

For first efficacy studies, reporter genes for different types of analyses within various tissues, cells, animals and for testing the mode of administration (e.g. electro gene transfer, sonoporation, lipofection, magnetofection etc.) have been used. Also biodistribution studies using these constructs have been performed. Additionally, minicircle constructs for vaccines, virus production etc. are currently investigated.

The results demonstrate a significant increase of gene expression of minicircle DNA in comparison to a standard plasmid.

As a result of the work presented here, PlasmidFactory has launched several commercially available minicircle DNA products.

Hansen T Ms 1 Nehlin J Dr 1 Barington T Professor 1

1 Department of Clinical Immunology, Institute of Clinical Research, University of southern Denmark, Sdr. Boulevard 29, DK-5000 Odense C, Denmark P 107 RAG1 and RAG2 as Potential Mediators of Targeted Gene Integration

Gene therapy is a promising method for treating a number of genetic and non-genetic diseases. The most straight forward strategy to treat individuals with deleterious mutations is by inserting a functional version of the gene in the genome. However, the occurrence of vector-related side effects has highlighted the requirement for improved vector designs.

The present project explores an up until now ignored possibility of using the naturally occurring recombinases, RAG1 and RAG2, to introduce the wanted transgene specifically into the human immunoglobulin (Ig) gene loci. These enzymes recognize unique Recombination Signalling Sequences (RSS) that could act as potential integration sites for the transgene.

Different B cell lines were tested for their RAG1 and RAG2 expression and their integration sites were mapped. Molecular cloning was used to generate an expression vector containing the integration mediating enzymes, Rag1 and Rag2. We are currently in the process of establishing an in vivo assay for testing the functionality of exogenously expressed Rag1 and Rag2's. We will observe their ability to execute recombination both on artificial substrate introduced into a B cell line but also directly within the Ig Loci.

After succeeding with the in vivo assay, it is our ambition to use the RAG1/RAG2 expression vector as mediator of targeted integration of a reporter gene.

Bartholomae C Dr 1 Cartier N Dr 2 Hacein-Bey-Abina S Dr 3 Veres G Dr 4 Arens A Mrs 1 Vidaud M Mr 2 Dal-Cortivo L Dr 3 Mahlaoui N Dr 5 Kiermer V Dr 6 Mittelstaedt D Mrs 7 Bellesme C Dr 8 Lahlou N Dr 9 Blanche S Dr 5 Audit M Dr 10 Bougneres P Dr 11 Fischer A Professor 12 Cavazzana-Calvo M Professor 3 Aubourg P Professor 2 Schmidt M Dr 1 von Kalle C Professor 1

1 Translational Oncology, German Cancer Research Center, Germany 2 Inserm UMR745, University Paris-Descartes, France 3 Department of Biotherapy, Hopital Necker-Enfants Malades, France 4 117 Sw 72nd Place, 117 Sw 72nd Place, United States of America 5 Department of Pediatric Immuno-Hematology, Hopital Necker-Enfants Malades, France 6 Nature Publishing Group, Nature Publishing Group, United States of America 7 13687 Quinton Road, 13687 Quinton Road, United States of America 8 Department of Pediatric Endocrinology Neurology, Hopital Saint-Vincent de Paul, France 9 Biochemistry Department, Hopital Saint-Vincent de Paul, France 10 Genosafe, Genosafe, France 11 Department of Pediatric Endocrinology Neurology, Hopital Saint-Vincent de Paul, France 12 Inserm UMR768, University Paris-Descartes, France P 108 Distribution of Lentiviral Vectors in Gene Therapy for X-Adrenoleukodystrophy

Gene therapy using retroviral vectors has been successful in treating monogenic diseases, but insertion of functional retrovirus LTRs can lead to severe side effects. Lentiviral vectors with self-inactivating (SIN) configuration are a promising alternative showing a reduced genotoxic risk. Here, we report on the first clinical trial using HIV-1 lentiviral SIN-vector to treat monogenetic diseases. The treatment of the genetic defect in cerebral adrenoleukodystrophy (X-ALD) showed stable and sustained restoration, and is so far not accompanied by signs of clonal dominance or even premalignant disproportional distribution of cellular contributions. We performed a large scale vector integration sites analysis on ex vivo transduced cells prior to reinfusion and on engrafted cells by LAM-PCR and subsequent pyrosequencing on samples from the first three patients treated by now. Our results revealed the excepted insertions profile for lentiviral vectors, showing gene coding regions as preferred targets for lentiviral vector integration. Interestingly, we observed insertion of the lentiviral vector as common integration sites in same genes or gene regions common in all three patients, likely reflecting a preference of lentiviral vector insertion for particular genes. Furthermore, the occurrence of identical IS identified in myeloid and lymphoid lineages indicated a successful ex vivo transduction of early hematopoietic progenitors. High throughput distribution analysis of the IS repertoire indicates that lentivirus vectors offer great promise for safe and effective correction of human stem and progenitor cells.

Fehse B Professor 1 Cornils K Dr 1 Heinzelmann M Ms 2 Wolschke C Dr 2 Ayuk F Dr 2 von Laer D Professor 3 Zoufaly A Dr 4 Kühlcke K Dr 5 Naundorf S Ms 5 Blau IW Dr 6 Thiel E Professor 6 Fätkenhauer G Professor 7 van Lunzen J Dr 4 Kröger N Professor 2 Zander AR Professor 2

1 Research Department Cell and Gene Therapy, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 3 Institute of Virology, Innsbruck Medical University, Innsbruck, Austria 4 I. Department of Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 5 EUFETS GmbH, Idar-Oberstein, Germany 6 Tumor Medicine, Charité - Universitätsmedizin Berlin, Berlin, Germany 7 Department I of Internal Medicine, Cologne Medical University, Cologne, Germany P 109 Transplantation of gene-modified HSCs for HIV patients with malignant diseases indicating HSCT

Highly active antiretroviral therapy (HAART) treatment has shown great efficacy in suppressing HIV replication and AIDS, but is still not available to all patients. Moreover, HAART does not lead to virus elimination and is often accompanied by severe side effects. Additional limiting factors include patient compliance and appearance of multiple-resistant HIV strains. On the other hand, transplantation of genetically protected haematopoietic stem cells (HSC) may lead to complete suppression of viral replication, as convincingly demonstrated by Hütter and colleagues who transplanted an HIV-positive leukaemia patient with HSC from a donor homozygous for the CCR5^Δ32/Δ32 mutation. However, due to severe side effects allogeneic HSCT would be an option for high-risk patients, and approx. 1% of Caucasians have a CCR5^Δ32/Δ32 donor, only. Therefore, gene therapy may represent a promising alternative. We have recently introduced a novel gene therapy principle based on expression of a membrane-anchored entry inhibitor (maC46) in HIV target cells transduced with the retroviral vector M87o. maC46 has successfully been tested in numerous preclinical systems and a first clinical study using autologous T cells from HIV patients (van Lunzen et al 2007). We have now developed a new clinical protocol involving the genetic modification of HSC with M87o. Based thereon, HIV target cells (T cells, monocytes) derived from gene-modified HSC are expected to be protected from HIV entry. The clinical protocol is open for patients in need of autologous or allogeneic HSCT, depending on the underlying disease. Study outline and preliminary patient data will be presented.

Paruzynski A Ms 1 Boztug K 2 Nowrouzi A 1 Ball C 1 Arens A 1 Lulay C 1 Böhm M 2 Naundorf S 3 Kühlke K 3 Blasczyk R 4 Kondratenko I 5 Maródi L 6 Glimm H 1 Klein C 2 von Kalle C 1 Schmidt M 1

1 Department of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany 2 Department of Pediatric Hematology/Oncology, Hannover Medical School, Germany 3 EUFETS AG, Idar-Oberstein, Germany 4 Institute for Transfusion Medicine, Hannover Medical School, Germany 5 Department of Clinical Immunology, Russian Clinical Children's Hospital, Moscow, Russia 6 Department of Infectious and Pediatric Immunology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary P 110 Two Patients of a Successful WAS Gene Therapy Trial Show a Polyclonal Integration Site Distribution

In a German clinical gene therapy trial two WAS patients were successfully treated by receiving autologous CD34+ cells that were transduced with a MLV based retroviral vector. LAM-PCR analysis combined with high throughput sequencing showed a polyclonal and lineage-specific integration site distribution with >5700 unique integration sites (IS) for patient 1 (P1) and >9500 unique IS for patient 2 (P2). The strongest integration clones show an IS upstream of CCND2 for P1 and an IS within the MDS1 locus for P2. Both clones contribute continuously to the gene-corrected hematopoiesis until the latest time point analyzed. We further detected a high clustering of up to >90 IS within the proto-oncogenes MDS1-EVI1 (P1: 81 IS; P2: 94 IS), PRDM16 (P1: 10 IS; P2: 28 IS), LMO2 (P1: 13 IS; P2: 29 IS) and CCND2 (P1: 11 IS; P2: 18 IS). The analysis of those IS in sorted cell fractions showed a distinct distribution. Thus, IS within or close to the gene loci MDS1-EVI1 and PRDM16 were predominantly detected in the myeloid fraction whereas IS within or close to LMO2 and CCND2 occurred preferentially in the lymphoid fraction. Despite the detection of IS within or close to already known proto-oncogenes, we found a highly polyclonal reconstitution of the hematopoietic system until 3 years after gene therapy (1108 and 1071 days after gene therapy, respectively). Further analysis of the clonal composition and a high throughput tracking analysis will help to further study the efficacy and safety of the used vector system.

Karussis D Professor 1 Slavin S Professor 2 Karageorgiou C Dr 3 Kassis I Mr 4 Vaknin-Dembinski A Dr 1 Petrou P Dr 4 Gomori J Dr 1 Gowda-Kurkalli B Dr 2 Ben-Hur T Professor 1 Bulte J Dr 5

1 Hadassah-Hebrew University Medical Center, Jerusalem, Israel 2 The International Center for Cell Therapy & Cancer Immunotherapy (CTCI), Tel Aviv, Israel 3 Gennimatas General Hospital, Athens, Greece 4 Hadassah-Hebrew University Medical Center, Jerusalem, Israel 4 Hadassah-Hebrew University Medical Center, Jerusalem, Israel 5 Russell H. Morgan Department of Radiology and Radiological Science, Department of Biomedical Engineering, and Department of Chemical & Biomolecular Engineering, The Johns Hopkins University School of P 111 Pilot phase I/II clinical trial with autologous mesenchymal stem cells in patients with multiple sclerosis

Mesenchymal stromal cells (MSC) have shown neuroprotective and immunomodulatory effects. A phase I/II clinical trial was designed to evaluate the feasibility and safety of intrathecal and intravenous administration of autologous MSCs in patients with multiple sclerosis(MS) and amyotrophic lateral sclerosis(ALS). Fifteen MS (mean EDSS = 6.7 ± 1.05) and 19 ALS patients (mean ALSFRS = 20.26 ± 8.56), were enrolled. Following culture, a mean number of 63.2 ± 2.5x10⁶ MSCs was injected intrathecally(n = 35) and intravenously(n = 14). In 9 cases, MSCs were magnetically labeled with the superparamagnetic iron oxide, FeridexÒ. In 21 patients there were injection-related side effects consisting of transient fever and headache. No major side effects were reported during a follow up period of up to 25 months. The mean ALSFRS remained stable during the 6 first months of observation, whereas the mean EDSS score improved from 6.7 ± 1.05 to 5.9 ± 1.6. MR imaging visualized the MSCs in the occipital horns of the ventricles, and indicated a possible migration of FeridexÒ-labeled cells in the meninges, the subarhachnoid space and the spinal cord. Immunological analysis, revealed an increase in the proportion of CD4 + CD25 + regulatory T-cells, and a decrease in the proliferative responses of lymphocytes, the IFNg and IL-17 production and of the expression of CD40, CD83, CD86, and HLA-DR on myeloid dendritic cells, at 24hrs following MSC transplantation. Our findings suggest that intrathecal and intravenous injection of MSCs in patients with MS and ALS, is a clinically feasible and relatively safe procedure. Controlled studies are needed to evaluate the long term safety and the potential clinical efficacy of MSC treatment

Delai S Dr

^*1

Visigalli I Dr *1 Politi LS Dr 2 Mrak E Dr 3 Di Domenico C Dr 4 Mariani E Dr 3 Stok M Dr 5 Del Carro U Dr 6 Rubinacci A Dr 3 Brambilla R Dr 7 Quattrini A Dr 8 Di Natale P Professor 4 Ponder K Professor 9 Naldini L Professor Biffi A Dr

2 Neuroradiology Group, Imaging Core, San Raffaele Scientific Institute, Milan, Italy 3 Bone Physiopathology Program (BoNetwork), San Raffaele Scientific Institute, Milan, Italy 4 Department of Biochemistry and Medical Biotechnology, University of Naples Federico II, Naples, Italy 5 Erasmus MC, Rotterdam, NL 6 Experimental Neurophysiology, San Raffaele Scientific Institute, Milan, Italy 7 Molecular Genetics of Behavior Unit, Neuroscience Dept, San Raffaele Scientific Institute, Milan, Italy 8 7Experimental Neuropathology Unit, Neuroscience Dept, San Raffaele Scientific Institute, Milan, Italy 9 Division of Hematology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA P 112 Therapeutic efficacy of hematopoietic stem cell gene therapy for Hurler Type 1 Mucopolysaccharidosis

Type 1 Mucopolysaccharidosis (MPS I) is an inherited lysosomal storage disorder caused by the deficiency of α-L-iduronidase (IDUA), which is responsible for systemic glycosamminoglycans (GAGs) accumulation, leading to skeletal and joint disease, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly and mental retardation. The available treatment options (enzyme replacement therapy and hematopoietic cell transplantation -HCT) are poorly effective on skeletal and brain disease manifestations. In order to improve the therapeutic efficacy of HCT on these skeletal and neurological manifestations, we are developing a gene therapy (GT) strategy based on IDUA supra-normal expression in hematopoietic stem cells (HSC) and their progeny by lentiviral vector (LV) gene transfer. Transduction of murine HSC with a LV encoding the human IDUA cDNA led to enzyme over-expression up to 150 fold above normal levels in their differentiated progeny, which could thus more abundantly correct affected tissues upon infiltration. Indeed, when these cells were transplanted into Idua-/- mice, supra-normal enzyme expression was observed in circulating hematopoietic cells and in all tested diseased tissues, including the brain, in which wild type HCT failed to deliver the functional enzyme. Importantly, GT corrected all major disease manifestations, decreasing GAGs storage and improving auditory impairment and retinal structure. Moreover, GT demonstrated a greater efficacy than wild type HCT in correcting the MPS I-related skeletal and behavioral defects. Thus, these results demonstrate that enzyme over-expression renders HSC GT effective in tissues refractory to correction following transplantation of normal donors' HSC, paving the way for future clinical testing.

Otsu M Dr 1 Onodera M Dr 2 Yamada M Dr 3 Kawamura N Dr 3 Kobayashi R Dr 3 Kobayashi E Mr 4 Kitagawa M Mr 4 Mineno J Mr 4 Bali P Dr 5 Hershfield M Dr 5 Candotti F Dr 6 Kawamura N Dr 3 Sakiyama Y Dr 3 Ariga T Professor 3

1 4-6-1 Shirokanedai, Minato-ku, Tokyo, Japan 2 2-10-1 Okura Setagaya-ku, Tokyo, Japan 3 N 15 W 7, Kita-ku, Sapporo, Japan 4 Seta 3-4-1, Otsu, Shiga, Japan 5 Durham, NC 27710, USA 6 Bethesda, MD 20892, USA P 113 Update on a Japanese clinical trial of stem cell gene therapy for ADA-deficiency

We report an update of ∼6 year follow-ups of our ADA-SCID gene therapy (GT) trial carried out without cytoreductive conditioning.
 Pt 1 received BM CD34⁺ cells transduced with the RV vector GCsapM-ADA in Dec 2003, and so did pt 2 in Feb 2004. At present, both patients remain off PEG-ADA with toxic metabolite levels in erythrocytes being kept low, indicating continuous production of substantial amounts of ADA from hematopoietic compartments reconstituted by gene-corrected cells. Immunity still stays protective in both patients, although their lymphocyte counts are currently low requiring replacement of IVIG. Hematopoietic gene marking has been evident with the highest levels in T cells. These cells showed supra-normal ADA activity, demonstrating functional correction in circulating T lymphocytes. Despite this, recent thymic emigration has not been demonstrable, indicating that full T cell reconstitution in ADA-GT likely necessitates measures to enhance thymic repopulation, such as mild preconditioning. Gene insertion analysis revealed long-term engraftment of gene-marked primitive hematopoietic cells sustaining lympho-myeloid reconstitution with common RV integration sites shared between lineages. No serious adverse events have been observed. Overall, GT for ADA-deficiency without cytoreductive conditioning may be considered to be a safe treatment capable of providing patients with sustained generalized detoxification. Immune reconstitution, however, may not be achieved with the current protocol at the levels comparable to those observed in pioneering GT trials utilizing mild preconditioning. Thorough improvement in strategies is necessary in a way that enables enhancement in reconstitution abilities of hematopoietic stem/progenitor cells and/or T cell precursors.

Scholz S Ms 1 Diaz M 2 Stein S 2 Schambach A 3 Glimm H 1 von Kalle C 1 Grez M 2 Schmidt M 1

1 German Cancer Research Center and National Center for Tumor Diseases, Heidelberg, Germany 2 Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt, Germany 3 Experimental Hematology, Hannover Medical School, Hannover, Germany P 114 Integration Site Analysis and Deep Sequencing for Vector Biosafety Assessment in CGD Gene Therapy

Insertional activation of MDS1/EVI1 has led to the clonal expansion of gene marked myeloid progenitor cells, triggering the development of monosomy 7 and a myelodysplastic syndrome in both patients enrolled in a clinical trial for the treatment of X-linked chronic granulomatous disease (X-CGD). To decrease vector-host interactions and associated side effects a new self-inactivating (SIN) gammaretroviral vector (SINfes.gp91s) was developed. The internal cellular promoter and a codon-optimized transgene aim to improve the safety and efficacy of gene delivery (Manuel Grez, Georg-Speyer-Haus, Frankfurt). We performed high-throughput integration site (IS) analysis in combination with deep sequencing (454/Roche) for vector biosafety assessment on mice transplanted with gene modified hematopoietic stem cells. The full LTR SFFV-promoter driven vector which was used in the previous trial was analyzed as a control (SF91eGFP).

We could detect a total of 1159 unique IS in SF91eGFP (n = 4) and 2261 in SINfes.gp91s mice (n = 5). In average, we retrieved 281 unique IS in SF91eGFP mice, showing a less polyclonal IS pattern than SINfes.gp91s transplanted mice (442 unique IS/mouse). Moreover, (pre)dominant clones were found more frequently in mice transplanted with the SF91eGFP vector than with SINfes.gp91s. Furthermore, in SINfes.gp91s mice no CIS formation occurred in/near EVI1/MDS1 while in SF91eGFP mice 5 IS formed 2 CIS upstream of MDS1 and EVI1, respectively. Finally, SF91eGFP secondary recipients revealed an increased in vivo skewing towards cancer related genes compared to SINfes.gp91s (p = 2.9*10⁻⁶). Overall, the SINfes.gp91s vector showed no obvious signs of vector-induced side-effects.

Scaramuzza S Dr Bosticardo M Dr Ripamonti A Dr Castiello MC Dr Biasco L Dr Vallanti G Dr 2 Radrizzani M Dr 2 Galy A Dr 3 Bredius R Dr 4 Biffi A Dr Roncarolo MG Professor Naldini L Professor Villa A Dr 5 Aiuti A Professor

2 MolMed S.p.A., Milan Italy 3 Genethon, Evry, France 4 Department of Pediatrics, University Medical Center, Leiden, The Netherl 5 ITB-CNR, Segrate, Milan, Italy P 115 LV mediated Gene therapy for Wiskott-Aldrich Syndrome: in vitro and in vivo preclinical studies

Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency characterized by thrombocytopenia, infections, autoimmunity and lymphomas. We previously demonstrated that a lentiviral vector (LVV) encoding for human WAS under the control of endogenous 1.6 kb promoter efficiently corrected human and mouse cells. We then set up a clinically applicable and efficient transduction protocol based on 60 hours of culture and 2 hits of gene transfer (MOI 100) on CD34⁺ cells from normal donors. The selected protocol was applied to WAS patients bone marrow CD34⁺ cells for the validation of the GMP grade LVV. Transduced cells showed a vector copy number per cell of 1.4 ± 0.3 and about 80% of transduced colonies. WAS cells proliferated less than normal donors (with or without LVV exposure) but there was no toxicity of LVV on clonogenic progenitors. Following gene transfer, WASp expression was restored in patients' differentiated cells, including megakaryocytes.Analyses of vector integrations on in vitro transduced CD34⁺ cells showed polyclonal integrations with the expected bias of LVV for transcriptional units. Pharmacokinetic of transduced CD34⁺ cells was studied by injection in sub-lethally irradiated neonate Rag2^-/-/γc^-/- mice. Transduced cells showed a normal biodistribution to hematopoietic organs and differentiation capacity in the absence of vector shedding and germline transmission.In conclusion, we demonstrate that our GMP grade LVV allows robust and reproducible transduction of CD34⁺ cells leading to restoration of WASp expression with no toxicity. A phase I/II gene therapy protocol based on infusion of transduced CD34⁺ cells combined with a reduced intensity conditioning has recently started.

Koch C Mr 1 Kolk A Dr 2 Probst F Dr 2 Bissinger O Dr 2 Weitz J Dr 2 Bauer F Dr 2 Plank C Dr 1

1 Institute of Experimental Oncology and Therapy Research, Technische Universität München, Ismaninger Strasse 22, 81675 Munich, Germany 2 Department of Oral and Maxillofacial Surgery, Technische Universität München, Ismaninger Strasse 22, 81675 Munich, Germany P 116 Acceleration of osseointegration by nanoparticle loaded dental implants: A study in mini pigs

The loss of dental implants is a major obstacle in reconstructive surgery and is often caused by an insufficient initial formation of a tight bone implant interface. Using a large animal model i.e. the Göttingen mini pig we examined whether nanoparticle loaded implants could be used to accelerate the contact between bone and the metallic surface of the implant. Nanoparticles were generated using plasmid DNA/ branched polyethylenimine complexes shielded with a protective polyethylene glycol polymer which were lyophilized and dispersed in an organic poly(D,L-lactic acid) solution to obtain stable biodegradable films of the implant surface. A new loading procedure “template guided coating” resulted in uniform films on the metallic surface as seen in raster electron microscopy with nanoparticle loading efficiencies of about 60 % as estimated by ¹²⁵I labeled DNA. Ex vivo studies were performed by drilling coated radioactive implants into pieces of bone which resulted in an abrasion of about 50 % of the nanoparticle layer. For the animal study each mini pig (n = 18) received six implants coated either with BMP-2 or Luciferase plasmid (used as control) into the maxilla in a split-mouth-design with a DNA concentration of 6 μg and 12 μg respectively. Additional implants were coated with 50 or 150 μg recombinant BMP-2 protein. Exact evaluation of the bone implant contact area performed by μCT analysis using a new algorithm demonstrated that plasmid BMP-2 nanoparticle loaded implants lead to a significant greater bone regeneration and osseointegration.

Anghel A Professor 1 Ionac M Professor 2 Seclaman E Dr 1 Taranu G Dr 2 Popovici E Dr 3 Anghel M Dr 3 Tamas L Dr 1

1 Biochemistry Department, University of Medicine and Pharmacy Victor Babes, Romania 2 Vascular Surgery Department, Clinical Emergency County Hospital Timisoara, Romania 3 Epidemiology Department, University of Medicine and Pharmacy Victor Babes, Romania P 117 Angiogenetic gene therapy with VEGF and HGF

Background: Critical limb ischemia (CLI) is an ideal research target, in that its treatment outcome is clear-cut, ending in either limb amputation or functional limb salvage. Surgical and endovascular revascularization is the treatment of first choice for CLI, but with current therapy, it can be expected that >25% of CLI patients will require major limb amputation within 12 months. Therapeutic angiogenesis is a new strategy that attempts to improve the perfusion of ischemic vascular beds by promoting the formation of new blood vessels using gene therapy.

Methods and Results: A double-blind, placebo-controlled, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) trial was conducted in patients with critical limb ischemia (with claudication, rest pain, ulcer lesions and an ankle/brachial index < 0.5). Patients were randomly assigned to placebo (n = 10) and to drug administration with 10¹³ copy of both VEGF and HGF pBLAST plasmids (n = 10). Study drug was injected directly into the muscles of the ischemic limb. Intramuscular administration was safe and well tolerated. The clinical evolution has been monitored by ankle-brachial index, walking distance and the relief of rest pain. Six months after therapy 7 patients show partial or complete relief of rest pain, improvement of ischemic ulcer lesions and increased walking distance on the rolling carpet. The improved perfusion to the distal ischemic limb was demonstrated by a mean increase of 0.1 in the ankle-brachial index.

Conclusions: Therapeutic angiogenesis with VEGF and HGF gene transfer is a safe and non-inflammatory procedure with a good effectiveness in the revascularisation of the ischemic limb.

Kim YY Ms 1 Lee SY Ms 1 Park SH Ms 1 Chae BC Ms 1 Kim SH Ms 2 Park SN Dr 2

1 Modern Cell & Tissue Techonologies Inc. Seoul TechnoPark, 172, Gongneung, Nowon, Seoul, 137-743, Korea 1 Modern Cell & Tissue Techonologies Inc. Seoul TechnoPark, 172, Gongneung, Nowon, Seoul, 137-743, Korea 2 Advanced Therapy Products Research Division, Pharmaceutical and Medical Device Research Department, P 118 A study of purity test forkeratinocyte therapy products

Many keratinocyte therapy products are in development or have recently been introduced onto the market. Recently new products are constantly produced and approved for clinical use in burn surgery and other chronic wounds. The purpose of this study is to suggest the purity test method for keratinocyte therapy products. In this study, we investigated the cellular impurity test and in-process impurity test for keratinocyte therapy products. For cellular impurity test, the ratio of inactive cell (fibroblast, endothelial cell and melanocyte) to against active cell was detected using real-time PCR and FACS (fluorescent activated cell sorter). Because of unavailability of specific surface marker, cellular impurity results by real-time PCR was more objective than FACS. For in-process impurity test, the amount of residue gentamicin and FBS were detected. A sensitive and robust high-performance liquid chromatographic method with fluorescence detection (HPLC-FLD) was developed for the determination of gentamicin residues in keratinocyte therapy product. Gentamicin is consists of four major components, C1, C1a, C2, and C2a. BSA (bovine serum albumin) residues of FBS were detected by commercial test kit based on ELISA (Enzyme-Linked Immunosorbent Assay).

Nguyen TM Ms 1

1 740 Dr. Penfield Ave., Room 5206 P 119 Overview of Regulations and Standards for Stem Cell-Based Therapies: An International Perspective

As stem cell research continues to move towards more clinical applications, it is important to question how these stem cell-based products will be regulated. Stem cell-based products are novel and have complex characteristics due to their different sources, potency or uses, and so may be defined and classified in different ways. Even then, these products may not fit perfectly within existing categories leaving them potentially unmonitored or unregulated. As a result, this exposes challenges with respect to ensuring safety and quality in the processing, distribution and international circulation of human cell and tissue products. Stem cell-based therapy regulation varies between jurisdictions according to product characterization. However, there is a trend towards ensuring proper oversight of these therapies through different regulatory requirements and product approval processes.

This study seeks to illuminate particular barriers to harmonizing international laws and policies related to clinical translation of stem cell and to investigate potential methods for overcoming these challenges. This study will provide a comparative analysis of the regulatory frameworks surrounding the clinical translation of stem cells in various jurisdictions.

It is concluded that while the challenges to harmonization are diverse and important, so too are the benefits of establishing uniformity in the regulation of stem cell-based therapies worldwide. There is still much to learn and discover about stem cell-based therapies but it is important that the infrastructure is in place to determine the safety and efficacy of these therapies and products.

Aiuti A Professor 1 Ferrua F Dr 2 Scaramuzza S Dr 3 Bosticardo M Dr 3 Castiello C Dr 3 Evangelio C Dr 2 Casiraghi M Dr 4 Ripamonti A Dr 3 Vallanti G Dr 5 Radrizzani M Dr 5 Salomoni M Dr 5 Galy A Dr 6 Finocchi A Dr 7 Cicalese MP Dr 4 Biffi A Dr 2 Ciceri F Dr 8 Naldini L Professor 9 Villa A Dr 10 Roncarolo MG Professor 11

1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; University of Rome “Tor Vergata”, Rome, Italy 2 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; Pediatric Immunohematology and Bone Marrow Transplant Unit, Scientific Institute HS Raffaele, Milan, Italy 3 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy 4 Pediatric Immunohematology and Bone Marrow Transplant Unit, Scientific Institute HS Raffaele, Milan, Italy 5 MolMed SpA, Milan, Italy 6 Genethon, Evry, France 7 University of Rome “Tor Vergata”, Rome, Italy 8 Division of Hematology, Scientific Institute HS Raffaele, Milan, Italy 9 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; Università Vita-Salute San Raffaele, Milan, Italy 10 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; ITB-CNR, Segrate, Milan, Italy. 11 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; Pediatric Immunohematology and Bone Marrow Transplant Unit, Scientific Institute HS Raffaele, Milan, Italy; Università Vita- P 120 Gene therapy trial with lentiviral vector transduced CD34+ cells for the treatment of Wiskott-Aldrich Syndrome

Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency characterized by thrombocytopenia, infections, autoimmunity and lymphomas. Gene therapy with autologous hematopoietic stem cells (HSC) could represent a valid alternative to allogeneic transplant for patients lacking an HLA-identical donor or who are at high risk of complications. We have developed a novel approach based on a self-inactivating lentiviral vector (LVV) encoding for human WAS under the control of the endogenous 1.6 kb promoter. We previously showed that this vector is safe and efficiently corrects the WAS defect in the murine model of the disease and in human cells. A highly purified, GMP grade, LVV transduced at high efficiency human CD34⁺ cells from healthy donors and patients, allowing restoration of WASp expression in multiple lineages without toxicity. A phase I/II protocol aimed at studying safety, biological activity, and efficacy of gene therapy in 6 WAS patients was opened in April 2010. Patients will receive preconditioning with anti-CD20 monoclonal antibody and reduced intensity busulfan and fludarabin; ATG will be included in case of autoimmune manifestations. The first patient was recently enrolled and treated with autologous CD34⁺ cells obtained from bone marrow (BM) and mobilized peripheral blood (MPB), achieving an optimal target HSC dose. Transduced cells met all quality specifications and showed a vector copy number per cell of 1.4 in the MPB and 1.9 in the BM, respectively, with high gene transfer efficiency in clonogenic progenitors (88-92%). The patient did not experience toxicity or adverse events, recovered well from transient neutropenia and is currently independent from platelet transfusions, 3 months after gene therapy. Initial engraftment analyses is showing the presence of vector transduced cells in multiple lineages of peripheral blood and BM, with evidence of WASp expression. Long-term assessment study will provide key information on the safety and efficacy of gene therapy for WAS patients using lentiviral-vector transduced HSC in combination with reduced intensity conditioning.

Passerini L Dr 1 Fousteri G Dr 2 Amendola M Dr 2 Jofra T Dr 2 Naldini L Professor 1 Roncarolo MG Professor 1 Bacchetta R Dr 2

1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Division of Regenerative Medicine, Stem Cell and Gene Therapy, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy 2 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Division of Regenerative Medicine, Stem Cell and Gene Therapy, Milan, Italy P 121 Lentiviral vector-mediated gene transfer of FOXP3 into CD4+T cells isolated from patients with IPEX Syndrome generates potent suppressor regulatory T cells

IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked) is a genetic disease caused by mutations of the transcription factor FOXP3, the master gene for naturally occurring (n) CD4⁺CD25⁺ regulatory T (Treg) cell fate and function. It is well established that nTreg cells play a central role in the control of immune responses to auto-antigens, allergens, and tumor antigens. In IPEX patients the lack of functional nTreg cells leads to the development of early onset life-threatening systemic autoimmunity. Current therapies for the cure of patients with IPEX are limited. The majority of patients are treated with immunosuppressive drugs, with only partial control of the clinical manifestations. At present, haematopoietic stem cell transplantation (HSCT) is the only definitive cure, but it is available for a limited number of patients.

An alternative strategy to restore tolerance could aim at generating high numbers of human Treg cells by lentiviral-vector- (LV-) mediated gene transfer of FOXP3 (LV-FOXP3). We previously showed that transduction of human CD4⁺ T cells generates a homogeneous population of Treg cells that can be propagated in vitro. Here we demonstrate that high an stable FOXP3 expression can be induced in CD4⁺ T cells isolated from patients carrying different FOXP3 mutations. FOXP3-mutated effector T (Teff) cells isolated from severely affected patients could be converted into functional Treg cells displaying potent in vitro suppressive activity. Preliminary results further suggest that LV-FOXP3 transduced cells can efficiently inhibit Teff cell responses in vivo in a xenogeneic graft versus host disease model (xeno-GvHD).

These results demonstrate that suppressive capacity is conferred to human T cells upon high and stable FOXP3 expression even in the presence of FOXP3 mutations, as in the case of IPEX patients. Overall, these findings pave the way for the development of a gene therapy approach using adoptive transfer of Treg-converted mature lymphocytes for the treatment of patients with IPEX Syndrome.

Rangel-Colmenero BR Dr 1 Gómez-Gutierrez JG Dr 2 Villatoro-Hernández J Dr 3 Villanueva-Olivo A Mr 4 Guzmán-López S Dr 5 Montes-de-Oca CR Dr 4 Elizondo-Omaña RE Dr 5 Montes-de-Oca-Luna R Dr 4 Saucedo-Cárdenas O Dr 6

1 Departamento de Histología, Fac. de Medicina, UANL Monterrey, NL, México 2 School of Medicine, University of Louisville, Louisville, KY, USA. 3 University of Groningen, Kerklaan 30, 9751 NN Haren, NED. 4 Departamento de Histología, Fac. de Medicina, UANL, Monterrey, NL, México 5 Departamento de Anatomía Humana, Fac. de Medicina, UANL, Monterrey, NL, México 6 Area de Genética, CIBIN, Instituto Mexicano del Seguro Social and Departamento de Histología, Fac. de Medicina, UANL, Monterrey, NL, México P 122 Enhancement of Ad-CRT/E7-mediated antitumor effect by preimmunization with L. lactis expressing HPV-16 E7

Background: Developing effective vaccines that target HPV-16 E6 and/or E7 has become a necessity, because current prophylactic vaccines against HPV infection do not protect against cervical cancer already established. We previously showed the effectiveness of adenovirus expressing CRT/E7 (Ad-CRT-E7) in a cervical cancer animal model. We also demonstrated that intranasal immunization of Lactococcus lactis encoding HPV-16 E7 (Ll-E7) anchored to its surface, induced significant HPV-16 E7-specific immune response. In this study we assessed the combination of both approaches in a cervical cancer animal model.

Method: Groups of mice were immunized intranasally with Ll-E7 on days 0, 14, and 28. One week later, the mice were challenged with HPV-16 E7-expressing murine tumour cells (TC-1) in the right leg. Ad-CRT-E7 vector was injected intratumorally once they reached a diameter of 6 mm. Tumor growth and survival were monitored. Infiltration of CD8 + cytotoxic T lymphocytes and the presence of apoptosis within the tumour were analyzed.

Results: A single dose of Ad-CRT/E7 was able to reduce the tumour size 60%, with a 20% of survival, compared with the controls. Interestingly, this antitumour and survival effect were increased to 80% and 70% respectively by the intranasal preimmunization with Ll-E7. Significant CD8 + cytotoxic T lymphocyte infiltration was detected in the tumours of mice treated with Ll-E7 + Ad-CRT/E7. In situ TUNEL apoptosis analysis showed a strong correlation between tumour regression and a higher number of apoptotic cells.

Conclusion: Preimmunization with Ll-E7 enhances the antitumour and survival effect of the Ad-CRT/E7 in a cervical cancer animal model.

Goyvaerts C Mrs 1 Robays L Dr 1 De Groeve K Dr 2 Raes G Dr 3 De Baetselier P Professor 3 Thielemans K Professor 1 Breckpot K Dr 1

1 Laboratory of Molecular and Cellular Therapy, Department of Physiology-Immunology, Vrije Universiteit Brussel, Brussels, Belgium 2 LaLabortory of Cellular and Molecular Immunology, Department of Molecular and Cellular Interactions, Vrije Universiteit Brussel, Brussels, Belgium 3 Labortory of Cellular and Molecular Immunology, Department of Molecular and Cellular Interactions, Vrije Universiteit Brussel, Brussels, Belgium P 123 Targeting lentiviral vectors to dendritic cells by the Nanobody display technology

Professional antigen-presenting cells (APC), such as dendritic cells (DCs) and macrophages, are targets for immunotherapy. Lentiviral vectors (LVs) have been successfully used to modify APC in vivo. However, their broad tropism limits their use in the clinic. As selective targeting will improve safety and efficacy, we developed the “Nanobody (Nb) display technology”. Herein, LVs are pseudotyped with a fusogenic, but binding-defective envelope together with a DC- and macrophage-specific Nanobody (DC2.1) or an irrelevant control (BCII10). We demonstrated the expression of Nbs on virus producing cells, their incorporation in the viral surface, and the production of high titer LVs. Flow cytometry revealed that BCII10 LVs didn't mediate infection in vitro, whereas DC2.1 LVs selectively transduce primary DCs and macrophages. Broad tropism LVs additionally transduced T- and B-cells. We further evaluated the biodistribution of the LVs by in vivo bioluminiscence imaging and flow cytometry demonstrating a similar transduction profile of the respective LVs in vivo as in vitro. In conclusion, we report for the first time on the “Nb display technology” for targeting of LVs to DCs and macrophages, a strategy that can be exploited in future applications, such as diagnostics and immunomodulatory therapies.

Sioud MS Professor 1

1 Department of Immunology, Institute for Cancer Research, The Norwegain Radium Hospital, Montebello, N-0310, Oslo, Norway P 124 Modulation of dendritic cell maturation and function with small interfering RNAs targeting indoleamine 2,3dioxygenase

Background: Among the described mechanisms involved in the failure of immunotherapy, is the expression of indoleamine 2,3-dioxygenase (IDO) by dendritic and tumour cells. Therefore, strategies to silence IDO gene expression may improve anti-tumour immunity.

Methods: Human monocytes or monocyte-derived DCs were transfected with anti-IDO siRNAs and the effects of IDO gene silencing on DC maturation and function were investigated using standard methods.

Results: The designed siRNA totally inhibited IDO gene expression without impairing DC maturation and function. Depending on the design and chemical modifications, it is possible to design either monofunctional siRNAs devoid of immunostimulation or bifunctional siRNAs with gene silencing and immunostimulatory functions. Inhibition of IDO expression with both classes of siRNAs inhibited DC immunosuppressive function on T cell proliferation. Immature monocyte-derived DCs that had been transfected with siRNA-bearing 5′-triphosphate activated T cells, indicating that, even in the absence of external factors such as TNF-a, those DCs were sufficiently mature to initiate T-cell activation.

Conclusions: The inhibition of IDO expression and deliberate induction of cytokine with a single siRNA in immune cells should provide an important new immunotherapeutic option. The activation of DC maturation with 5′-triphosphate-bearing siRNA opens the possibility of generating mature DCs without the addition of external cytokines. This strategy of DC maturation may be extended into a therapeutic vaccination setting.

Muehlebach M Dr 1 Heidmeier S Mrs 1 Frank C Mr 1 Cattaneo R Professor 2 Cichutek K Professor 1

1 Division of Medical Biotechnology, Paul-Ehrlich-Institut, 63225 Langen, Germany 2 Department of Molecular Medicine, Mayo Clinic, Rochester, 55905 MN, USA P 125 Bivalent Measles Virus Vaccines Presenting Different Forms of a Lentiviral Glycoprotein

Live attenuated vaccines against measles virus (MV) are among of the safest vaccines and recombinant MVs that express foreign proteins have been shown to elicit significant immune responses and protection against the respective pathogens in animal experiments. Interestingly, all but one candidate vaccine published elsewhere expressed a soluble form of the foreign antigen. We therefore ask here whether the soluble, membrane-bound or other forms of the foreign antigen presented by recombinant MVs is critical for the induced immune responses.

We report the construction of soluble or stabilized (i.e. non-shedding) forms of the envelope (Env) glycoprotein of simian immunodefiency virus SIVsmmPBj1.9, a model virus strain inducing acute viral pathogenesis. To this end, the SIV Env protein was genetically truncated to express only the ectodomain. Alternatively, the protease cleavage site was modified to prevent cleavage and allow expression of a stabilized gp160-Env. Bivalent recombinant MVs each encoding a different form of the SIVsmmPBj1.9 Env protein were rescued, successfully tested for Env expression, and shown to retain similar growth rates as parental MV with titers of up to 1x10⁸ TCID₅₀/ml. The bivalent vaccines were inoculated twice intraperitoneally into transgenic IFNARko-CD46Ge mice (1x10⁵ TCID₅₀/dose). Transgenic CD46 expression allows MV replication in these mice in vivo. We are measuring the quality of the cellular and humoral immune responses to different forms and amounts of the Env proteins presented by MV in mice. These studies will be used to select candidate MV-SIV vaccines for the analysis of the immune response and levels of protection in monkeys.

Krmenčíková M. Miss 1 Staněk L. Mr 1 Dundr P. Dr 2 Vonka V. Professor 1

1 Department of Experimental Virology, Institute of Hematology and Blood Transfusion, U Nemocnice 1, Prague, 128 20, Czech Republic 2 Institute of Pathology, General Faculty Hospital, Studnickova 2, Prague, 128 00, Czech Republic P 126 Altered properties of BCR-ABL-Transformed mouse cells expressing endostatin

Backround: Neoangiogenesis plays an important role in chronic myeloid leukemia. We investigated whether gene modification of bcr-abl – transformed mouse cells resulting in endostatin production changed their oncogenic potential.

Method: Mouse B210 cells expressing BCR-ABL fusion protein and inducing leukemia and very rarely intraabdominal solid tumors after intravenous administration, were used. The cells were transfected with a plasmid carrying genes for mouse endostatin and resistence to blasticidine. Transduced cell clones were isolated in the presence of blasticidine. Production of endostatin was determined by Western blotting. BALB/c mice were inoculated intravenously and were followed for at least 75 days. The materials from the autopsied animals were tested histologically, immunohistologically and by the FISH technique.

Results: When compared with the original B210 cells, the capability of the gene-modified cells to induce leukemia was somewhat lower. However, mice which did not succumb to leukemia, later on developed aggressively growing intraabodominal and intrathoracic solid tumors. In the autopsied animals hepatosplenomegaly was also observed. The tumors were difuse, penetrating into the neighbouring tissues. They were composed of lowly differentiated cells with irregular nuclei and roughly granular chromatin. FISH revealed the presence of bcr-abl fusion gene both in the tumours and spleens. In cultivated tumor-derived cells endostatin production was detected.

Conclusion: Surprisingly, B210 cells producing the angiogenesis inhibitor endostatin tended to produce solid tumors in intravenously inoculated mice much more frequently than the parental unmodified cells. This oncogenic activity was not associated with the loss of endostatin production.

Acknowledgement MZCR IGA NS 10634

Stanek L. Mr 1 Petrackova M. Mrs 1 Dundr P. Dr 2 Krmenciková M. Miss 3 Sidlova J. Mrs 2 Vonka V. Professor 1

1 Department of Experimental Virology, Institute of Hematology and Blood Transfusion, U Nemocnice 1, Prague, 128 20, Czech Republic 2 Institute of Pathology, General Faculty Hospital, Studničkova 2, Prague, 128 00, Czech Republic 3 Department of Experimental Virology, Institute of Hematology and Blood Tranfusion, U Nemocnice 1, Prague, 128 20, Czech Republic P 127 Toxicity of GM-CSF overproduced by gene-modified mouse cells

Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is strong stimulator of the immune system.

Methods: Mouse bcr-abl-transformed 12B1 cells which induce leukemia and also subcutaneous tumors in syngeneic animals were transfected with mouse GM-CSF gene. Groups of mice were inoculated subcutaneously with either 12B1-GM-CSF cells or parental 12B1 cells. At intervals two mice from each group were sacrificed. Blood sample was taken for cytological investigation and for determining the GM-CSF level in sera. At autopsy several organs were taken for histopathological, immunohistological, and cytogenetic investigation.

Results: After administration of 12B1 GM-CSF cells the first clinical symptoms were observed on day 6, i.e. about one week prior to appearance of subcutaneous tumors. Simultaneously a raise of GM-CSF level in serum was detected, which gradually increased until day 16. On day 6 the first signs of damage to lungs, heart, spleen, liver and kidneys were detected. These progressed in the subsequent days. Except of hepatosplenomegaly similar changes were not seen in mice inoculated with parental 12B1 cells Starting on day 8 a marked increase of myeloid- derived suppressor cells was seen in 12B1-GM-CSF inoculated mice. Their numbers steadily increased and on day 18 they surpassed more than 10 times their counts in 12B1-inoculated mice and control animals On the other hand, similar counts of T-reg cells were detected in both 12B1- and 12B1-GM-CSF inoculated animals.

Conclusion: The data strongly suggest that the extensive organ damage seen was caused by the overproduction of GM-CSF by the gene-modified cells.

Acknowledgement MZCR IGA: NS10634

Perche F Mr 1 Pichon C Professor 1 Berchel M Dr 2 Benvegnu T Professor 3 Jaffrès PA Professor 2 Midoux P Dr 4

1 Centre de Biophysique Moléculaire CNRS UPR 4301, 45071 Orléans Cedex 2, France 2 CEMCA, UMR CNRS 6521, Université de Brest-UBO, 29238 Brest cedex 3, France 3 UMR CNRS 6226, Ecole Nationale Supérieure de Chimie de Rennes, 35708 Rennes, France 4 Centre de Biophysique Moléculaire CNRS UPR 4301, 45071 Orléans Cedex 2, France P 128 mRNA-based vaccine: Dendritic cells targeting with Mannosylated and Histidylated lipopolyplexes and induction of antitumor vaccine

Background: The transfection of dendritic cells (DCs) with mRNA encoding tumour antigen is a promising anti-cancer vaccine strategy that offers advantages over DNA transfection in terms of efficiency and safety. We report the preparation of mannosylated and histidylated lipopolyplexes (Man₁₁-LPD100) as nanovectors that enhance mRNA transfection of DCs in vivo and induce anti-B16F10 melanoma vaccine effect in mice.

Method: Man₁₁-LPD100 was prepared by addition of mannosylated and histidylated liposomes containing β-D-mannopyranosyl-N-dodecylhexadecanamide (11 mol %) on PEGylated histidylated polylysine/mRNA polyplexes. Liposomes contained a cationic [O,O-dioleyl-N-(3N-(N-methylimidazolium iodide)propylene) phosphoramidate)] and a neutral [O,O-dioleyl-N-histamine Phosphoramidate] co-lipid. The imidazole groups of the co-lipid and polymer favour endosome destabilisation and nucleic acids delivery in the cytosol.

Results: By scintigraphy, we showed that upon IV injection of Man₁₁-LPD100 with ^99mTc-DNA, 9% of the dose was accumulated in the spleen, a representative lymphoid tissue. We demonstrated that spleen from mice injected with EGFP mRNA Man₁₁-LPD100 contained 4-fold more DCs expressing EGFP than the spleen from mice injected with LPD100. This better transfection of DCs was correlated with a better inhibition of B16F10 melanoma growth and an increased survival time when mice were immunized with Man11-LPD100 made with MART-1 mRNA.

Conclusion: These results indicate that Man₁₁-LPD100 is an efficient system to deliver tumour antigen mRNA in splenic DCs for inducing antitumor vaccine.

This project is developed in the frame of Ligue contre le Cancer (Nationale and Région Centre) and Cancéropole Grand Ouest.

Young Cho Sun Young Lee Bo Yang Jai Myung Shin Sungho Department of Life Science, Sogang University, Shinsu-Dong, Mapo, Seoul, 121-742, Republic of Korea

P 129 Codon-optimized HC gene of Clostridium botulinum neurotoxin serotype E is an effective botulism DNA Codon-optimized HC gene of Clostridium botulinum neurotoxin serotype E is an effective botulism DNA vaccine candidate

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most toxic substance known and act by preventing the release of the neurotransmitter acetylcholine at neuromuscular junction. BoNT consists of two polypeptide chains which are linked by a disulfide bond. The most commonly used human botulism vaccine is prepared from culture supernatant from C. botulinum. However, disadvantages associated with this vaccine, including simplicity of production and purity of product. Therefore, new generation botulism vaccines are required. In the present study, immunogenicity of plasmid DNA vaccine encoding the heavy chain (HC) domain of C. botulinum neurotoxin E was investigated. Using codon-optimized genes, DNA vaccines which expressed the carboxyl-terminal 50 kDa fragment of HC fused to IgM signal peptide were prepared. We transfected 293T cells with constructed plasmids and performed Western and ELISA assay. Transfection studies proved that fragment of HC protein was expressed from plasmid-transfected cells and was secreted into culture medium. By employing DNA vaccine in Balb/c mice, the immune response and corresponding neutralizing antiserum titers were markedly increased. Our results suggest that our plasmid DNA vaccine can elicit strong immunogenicity and may be a potential alternative strategy to conventional protein vaccines against C. botulinum neurotoxin serotype E.

Lin CC Professor 1 Chu CL Dr 2 Shi CC Miss 1 Yu YL Dr 3

1 Institute of Biomedical Sciences, National Chung Hsin University, Taichung, Taiwan 2 Immunology Research Center, and 5National Institute of Cancer Research, National Health Research Institutes, Miaoli, Taiwan 3 Immunology Research Center, and 5National Institute of Cancer Research, National Health Research Institutes, Miaoli, Taiwan P 130 A Novel Adjuvant Ling Zhi-8 Enhances the Efficacy of DNA Cancer Vaccine by Activating Dendritic Cell

DNA vaccine has been suggested to use in cancer therapy, but the efficacy remains to be improved. The immunostimulatory effect of a fungal immunomodulatory protein Ling Zhi-8 (LZ-8) isolated from Ganoderma lucidum has been reported. In this study, we tested the adjuvanticity of LZ-8 for HER-2/neu DNA vaccine against p185^neu expressing tumor MBT-2. We first examined the effect of LZ-8 on mouse dendritic cells (DCs) because DCs play a key role in initiating vaccine responses. Recombinant LZ-8 activated mouse bone marrow-derived DCs and the stimulatory effect of LZ-8 was not due to any microbe contaminant. As shown by the poor responses of TLR4 mutant DCs to LZ-8, TLR4 was involved in LZ-8-induced DC activation. In addition, LZ-8 enhanced the ability of DCs to induce antigen-specific T cell activation in vitro and in a subunit vaccine model in vivo. Surprisingly, co-delivery of LZ-8 strongly improved the therapeutic effect of DNA vaccine against MBT-2 tumor in mice. This increase of anti-tumor activity was attributed to the enhancement of vaccine-induced Th1 and CTL responses. Consistent with the results from DCs, the promoting effect of LZ-8 on DNA vaccine was diminished when the MBT-2 tumor cells were grown in TLR4 mutant mice. Thus, we concluded that LZ-8 may be a promising adjuvant to enhance the efficacy of DNA vaccine by activating DCs via TLR4.

Vahedian V Dr 1

1 Yerevan State Medical University P 131 Prevention of streptozocin-induced diabetes mellitus via embryonal cell therapy

In our research experimental plan we investigated the new property of glycoprotein of embryonal genesis (PEG) which is created by my scientific consoultant prof.l.n.mkrchyan and before it has been proved its anticancer effects as a new biocompound of embryonal cell origin that could prevent tumor progress and it is known as antitumor modulator (EATM).

Due to some observations in cancer patients who had simultaneously diabetes, this study was designed.

We have a very interesting result as other bioimmunological activity and preventive role in diabeteic models, and could show decrease of glucose and modulation in some clinical biochemistry factors in hyperglycemic and diabetic condition. By means of a new combination of embryonal proteins as one of the natural fetus cell resource, this can be considered as a new anti-diabetic biodrug or diabetes new vaccine in future after complementary study and clinical evaluations.

Koornneef A Dr 1 van Logtenstein R Mr 1 Timmermans E Mr 1 Pisas L Ms 2 Blits B Dr 1 Abad X Dr 3 Fortes P Dr 4 Petry H Dr 1 Konstantinova P Dr 1 Ritsema T Dr 1

1 AMT, Meibergdreef 61, 1105 BA, Amsterdam, the Netherlands 2 Meibergdreef 61, 1105 BA, Amsterdam, the Netherlands 3 Digna Biotech/Division of Gene Therapy and Hepatology, CIMA, Universityof Navarra, Pio XII 55. 31008, Pamplona, Spain 4 Division of Gene Therapy and Hepatology, CIMA, Universityof Navarra, Pio XII 55. 31008, Pamplona, Spain P 132 AAV-mediated in vivo knock-down of luciferase using RNAi and U1i

RNA interference (RNAi) has been successfully employed for specific inhibition of gene expression; however, safety and delivery of RNAi remain critical issues. We investigated the combinatorial use of RNAi and U1 interference (U1i), another method of gene silencing that acts on the pre-mRNA by preventing polyadenylation. RNAi and U1i have distinct mechanisms of action in different cellular compartments and their combined action may allow usage of minimal effector doses, while retaining high target inhibition. As a proof of concept, we investigated knock-down of the firefly luciferase reporter gene by combinatorial use of RNAi and U1i, and evaluated their inhibitory potential both in vitro and in vivo. Co-transfection of RNAi and U1i constructs in the HEK293T cell line reduced luciferase expression by 95%, which was more than observed for either inhibitor alone. Furthermore, we were able to attain similar knock-down when RNAi and U1i constructs were hydrodynamically transfected in mice, demonstrating for the first time the in vivo application of U1i. Moreover, we demonstrated long-term gene silencing by AAV-mediated transduction of murine muscle with RNAi/U1i constructs targeting firefly luciferase. In conclusion, these results provide a proof of principle for the combinatorial use of RNAi and U1i to enhance target gene knock-down in vivo.

Baranyi U Dr 1 Linhart B Dr 2 Pilat N Dr 1 Gattringer M Ms 1 Klaus C Mr 1 Muehlbacher F Professor 1 Iacomini J Professor 3 Valenta R Professor 2 Wekerle T Professor 1

1 Division of Transplantation, Department of Surgery, Medical University of Vienna, Austria 2 Div. of Immunopathology, Dept. of Pathophysiology, Center of Pathophysiology, Immunology and Infectiology, Medical University of Vienna, Austria 3 Transplantation Research Center, Renal Division, Brigham and Women´s Hospital and Children´s Hospital, Harvard Medical School, Boston, MA, USA P 133 Induction of long-term tolerance in IgE-mediated allergy by a gene therapy approach

Background: Robust strategies for the prevention of allergy are currently not available. In a preventive murine model employing myeloablative irradiation we showed that tolerance towards an allergen can be induced through engraftment of retrovirally transduced syngeneic bone marrow (BM) expressing a membrane-anchored allergen (i.e. molecular chimerism). In the present studies we investigated whether tolerance can be induced in a non-myeloablative protocol.

Methods: BALB/c BM-cells (BMC) were transduced (VSV-Phl p 5) in vitro to express Phl p 5 (a major grass pollen allergen) (transduction efficiency: 7%). Non-myeloablated (6 Gy TBI) BALB/c mice received 2-4x10⁶ Phl p 5-transduced BMC iv and were challenged with recombinant Phl p 5 and the major birch pollen allergen Bet v 1 (specificity control) at multiple time points post-BM transplantation (BMT).

Results: Non-myeloablated mice (n = 10) transplanted with Phl p 5-transduced BMC developed low levels of white blood cell molecular chimerism (10/10) (approx. 1%). Serum levels of Phl p 5-specific IgE and IgG1 remained undetectable in 8/10 chimeras throughout follow-up (44 weeks post-BMT). Phl p 5-specific IgG2a and IgG3 was undetectable in any of ten chimeras at late time points (week 39/44), while in contrast Bet v 1-specific antibodies were detectable in all mice. Basophil degranulation assays revealed the absence of Phl p 5-specific degranulation in all chimeras. In T-cell proliferation assays chimeras showed specific non-responsiveness to Phl p 5.

Conclusion: This gene therapy approach demonstrates that even low levels of molecular chimerism are sufficient to maintain tolerance in allergy at the B-cell, T-cell and effector-cell levels long-term.

Catucci M Dr 1 Prete F Dr 2 Bosticardo M Dr 3 Benvenuti F Dr 2 Aiuti A Professor 4 Roncarolo MG Professor 1 Villa A Dr 5

1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy. Vita-Salute San Raffaele University, Milan, Italy. 2 International Centre for Genetic Engineering and Biotechnology, Trieste, Italy. 3 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy. 4 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy. University of Rome Tor Vergata, Rome, Italy. 5 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy. Human Genome Department, Istituto di Tecnologie Biomediche, CNR ITB, Segrate, Milan, Italy P 134 Restoration of dendritic cell functionality in gene therapy treated was^−/− mice

Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by defective expression of the WAS protein (WASP) in haematopoietic cells. Several immune cell functions are altered in WAS patients, thus resulting in a complex clinical phenotype. Increasing evidences show the contribution of DC defects in WAS pathogenesis. Reduction of WASP expression impairs cytoskeleton reorganization and inhibits podosome formation in DCs, dramatically reducing their motility and functionality. Our group has previously demonstrated the efficacy of a gene therapy (GT) protocol in was murine model. Haematopoietic stem cells from was ^-/- mice can be efficiently transduced with a lentiviral vector carrying human WAS cDNA driven by the endogenous promoter and reinfused in nonlethally irradiated recipient was ^-/- mice. As we showed correction in T cell proliferation and cytokine production, we are now interested in evaluating DC functionality in GT treated mice. We detected normal DC count in secondary lymphoid organs and WASP positive DCs in spleen (∼35%), lymph nodes (∼22%) and thymus (∼29%) of GT treated mice. To test DC functionality we differentiated them from bone marrow (BMDCs) of GT treated mice. We found that although only 20% of BMDCs from gene therapy treated mice expressed WASP, in vitro phagocytosis, in vivo migratory capacity and T cell priming are ameliorated compared to was ^-/- BMDCs. These results suggest that GT can efficiently restore DC functionality despite a reduced frequency of WASP expressing DCs.

Hösel M Dr 1 Broxtermann M Mr 2 Arzberger S Dr 2 Gillen S Dr 3 Kleeff J Professor 3 Stabenow D Dr 4 Knolle P Professor 4 Hallek M Professor 1 Protzer U Professor 2 Büning H Dr 1

1 Department I of Internal Medicine, University of Cologne 2 Institute of Virology, Technische Universität München / Helmholtz Zentrum München 3 Department of Surgery, University Hospital Rechts der Isar, Technische Universität München 4 Institute of Molecular Medicine, University of Bonn P 135 Structural components of AAV capsids induce cell autonomous immune responses in human non-parenchymal liver cells

Clinical efficacy of adeno-associated viral vectors (rAAV) is limited by humoral and cytotoxic T cell responses towards the viral capsid. Since adaptive immune responses are a consequence of pathogen recognition by the innate immune system, we investigated cell autonomous immune responses towards rAAV2 and rAAV8, which are both used for hepatic gene transfer. The liver consists of hepatocytes and non-parenchymal cells. While the former were successfully transduced by rAAV2 and rAAV8 in the absence of innate immune activation, in primary human non-parenchymal liver cells an NFkB-mediated inflammatory response was induced. Interestingly, neither serotype elicited type I interferon, which was in contrast to anti-AAV immune responses in plasmacytoid dendritic cells (pDC). In line, pDC and human non-parenchymal liver cells differed in the mechanism of recognition since viral capsids, but not genomes, were identified as the main pathogen associated molecular pattern (PAMP). The pathogen receptor of this newly identified PAMP is likely located at the cell surface, but seems non-functional in murine LSEC as neither vectors nor empty capsids have mounted cytokine up-regulation in murine LSEC. Hence, mechanism and consequences of the innate immune response in human liver cells differ from known responses and are key to understand the limitations of gene therapy using rAAV.

van Til NP Dr 1 Sarwari R 1 Visser TP 1 de Boer H 1 Li Y 1 Hauer J 2 Lagresle-Peyrou C 2 Danos O 3 Jollet A 4 Zhang F 5 Thrasher A 5 Cassani B 6 Verstegen M 1 Villa A 7 Cavazzana-Calvo M 2 Wagemaker G 1

1 Department of Hematology, Erasmus University Medical Center, Rotterdam, the Netherlands 2 INSERM, U768, Université René Descartes, and Hôpital Necker–Enfants Malades, Paris, France 3 INSERM U781, Université René Descartes, and Hôpital Necker–Enfants Malades, Paris, Franc 4 INSERM U781, Université René Descartes, and Hôpital Necker–Enfants Malades, Paris, France 5 Molecular Immunology Unit, UCL Institute of Child Health, London, UK 6 Fondazione Humanitas per la Ricerca, Rozzano, Milano,Italy 7 CNR-ITB ; Telethon Institute for Gene Therapy-HSR, Milano, Italy P 136 Lentiviral gene therapy of RAG1 SCID: risks and implications for treatment

Recombination activating gene 1 (RAG1) deficiency results in severe combined immunodeficiency (SCID) due to complete lack of T and B lymphocytes. Consequently, these patients succumb from recurrent infections.

Aim of the study was to develop lentiviral gene therapy for RAG1 SCID. Constructs containing the viral SF or cellular PGK, EFS and UCOE or more pathway-restricted promoters of the RAG1, RAG2, IL2RG and TCRβV6.7 gene driving sequence optimized RAG1 were compared for efficacy and safety in Rag1^-/- mice. A standard 5x10⁵ transduced lineage negative bone marrow cells containing on average 1-3 integrations per cell were transplanted in mice subjected to 6 Gy (γ-rays) conditioning.

Peripheral blood (PB) CD3⁺ T cell reconstitution was achieved with all promoters (n = 54, 191 ± 199 cells/mL at month 5), but 8-fold lower than wildtype Rag1^-/- transplanted mice (Rag1-WT, n = 7; 1479 ± 251 cells/mL). However, IL-2 mediated T cell responses, T cell receptor diversity and T-cell dependent immune responses were restored. In contrast, reconstitution of mature PB B220⁺IgM^lowIgD^high B-cells was low for the SF vector (42 ± 47 cells/mL) and the other cassettes (6 ± 9 cells/mL) as compared to Rag1-WT (596 ± 233 cells/mL). Although PB B-cell numbers were low, plasma immunoglobulin isotype levels were detected in the majority of the mice.

Two months onward, GT treated mice developed rashes with cellular tissue infiltrates, activated PB CD44⁺CD69⁺ T-cells and high IgE plasma levels. Although immunological functions were restored in a proportion of these mice, these results underline that further development is required for successful RAG1 gene therapy with an inherent potential risk to develop autoimmune symptoms.

Arce F Dr 1 Breckpot K Dr 2 Stephenson H Miss 1 Karwacz K Miss 1 Ehrenstein MR Professor 1 Collins M Professor 1 Escors D Dr 1

1 University College London 2 Vrije Universiteit Brussel P 137 Therapeutic application of selective ERK activation for the treatment of inflammatory arthritis

Rheumatoid arthritis is an autoimmune disease characterized by chronic joint inflammation and destruction. However, arthritogenic antigens are currently unknown and most therapeutic treatments rely on immunosuppressive drugs which have side effects. We demonstrate that co-delivery of a specific ERK activator with a model antigen, using lentivectors, induces antigen-specific immune suppression by differentiation of antigen-specific regulatory T cells (Tregs) and inhibition of inflammatory T cell subsets. Differentiated Tregs strongly proliferate after antigen re-encounter in inflammatory conditions and exhibit antigen-dependent suppressive activities. ERK activation causes mouse and human dendritic cells (DCs) to secrete bioactive TGF-beta, which is required to suppress T cell responses and differentiate antigen-specific Tregs. In vivo administration of the ERK activator with antigen inhibits inflammatory arthritis partially through Treg activity if this antigen is administered during arthritis induction. Thus, antigen-specific tolerance was re-directed to circumvent the need for targeting an arthritogenic antigen. We also demonstrate equivalent mechanisms in human DCs, setting the scene for rapid translation to patients with rheumatoid arthritis. This strategy resulted in an effective therapeutic protocol with substantial advantages over DC or T cell vaccination.

Wally V Dr 1 Brunner M Ms 1 Lettner T Mr 1 Koller U Dr 1 Hintner H Professor 1 Bauer JW Professor 1

1 Division of Molecular Dermatology and EB-house Austria, Department of Dermatology,Paracelsus Medical University Salzburg, Salzburg, Austria P 138 Keratin 14 mRNA trans-splicing in dominant Epidermolysis bullosa

Mutations in the K14 gene underly Epidermolysis bullosa simplex (EBS), a hereditary skin disease characterized by erosions and blistering of the skin and mucous membranes after minor trauma. One subgroup of EBS (Dowling-Meara) is inherited in an autosomal dominant way, rendering it a challenge for conventional cDNA-based gene therapy.

In our model of EBS-DM, the heterozygous missense mutation R125P in the KRT14 gene leads to protein misfolding and subsequent self-aggregation.

We replaced exons 1 to 7 of the K14 gene using “Spliceosome Mediated RNA Trans-Splicing”, thereby reducing levels of the mutated allele and increasing the amount of functional protein. Essential trans-splicing components were brought in by an RNA-trans-splicing molecule (RTM). Besides the wildtype gene-portion to be replaced and important splicing features, the RTM incorporates a binding domain (BD), crucial for the trans-splicing specificity and efficiency. In a reporter based screen we identified best BDs for an RTM specifically replacing exons 1 to 7 of the K14 gene. Therefore, a library consisting of RTMs with random BDs was screened, resulting in the isolation of a highly functional RTM, which was adapted for endogenous trans-splicing. We showed specific trans-splicing into exon 8 of the endogenous K14 gene and a decrease of cis-spliced K14 transcripts by 40 to 60%. Scratch assays revealed a reversion of the migratory behaviour of RTM-transfected cells versus wildtype. Finally, we detected a 81kD protein band representing trans-spliced K14 by western blotting.

We conclude that successful trans-splicing in this model constitutes a novel approach to treat autosomal dominant diseases.

Ruggiero E Ms Bendle G 2 Linnemann C 2 Bies L 2 Schumacher T 2 Glimm H von Kalle C Schmidt M

2 The Netherlands Cancer Institute, Amsterdam, The Netherlands P 139 Vector Biosafety in a Mouse Model of TCR Gene Therapy Developing Graft-Versus-Host Disease

Engineering T cell specificity with the genetic introduction of antigen-specific T cell receptor (TCR) represents a promising tool for the treatment of tumors and viral infections.

We applied our developed strategies for comprehensive genome wide retrieval of retroviral integration sites (IS) to analyse IS distribution and the clonal dynamics of reprogrammed T cells in a mouse model of TCR gene therapy developing Graft-versus-Host Disease (GvHD).

Mouse T cells were transduced with a gammaretroviral vector encoding an ovalbumin-specific OT-I TCR and the cohort receveing a subsequent IL-2 administration developed GvHD.

By performing linear amplification-mediated PCR (LAM-PCR) combined with high-throughput sequencing on CD4 and CD8 T cells sorted from the different mice cohorts, we were able to retrieve 841 unique IS in OT-I transduced mice and 1063 unique IS in control GFP transduced mice. The analysis of the clonal composition revealed in OT-I transduced samples a high clustering of IS in genes involved in the immune response and an enrichment of IS in specific gene classes, Hematological Disease and Inflammatory Response, compared to the GFP transduced cells (p = 7.84*10⁻⁴ and p = 2.13*10⁻², respectively). Furthermore, the IS analysis of samples from mice developing GvHD revealed an increased distribution of IS located in genes and in the surrounding 10 kb (73,60% and 65.34%, respectively; p = 3*10⁻²) and an increased frequency of (pre)dominant clones compared to the control cohort.

Our results highlight the importance of high-throughput IS analyses for monitoring the efficacy and the safety of vectors used in TCR gene transfer.

Lin DS Dr 1 Lin SP Dr 2 Chuang CK Mr 3 Liui HL Professor 4

1 Dept. Pediatric, Mackay memorial Hospital, Taipei, Taiwan 2 Dept. Pediatrics, Mackay Memorial Hospital, Taipei, Taiwan 3 Dept. Medical Research, Mackay Memorial Hospital, Taipei, Taiwan 4 Dept. of Chemical engineering and Biotechnology, National Taipei university of technology, Taiwan P 140 Axonal Transport of AAV Vector Provides Widespread Correction in Murine Globoid Cell Leukodystrophy

Globoid cell leukodystrophy (GLD) is a devastating lysosomal storage disease caused by a deficiency of galactocerebrosidase (GALC). This leads to accumulation of toxic psychosine in the CNS, and progressive neurodegenerative demeylination. Given the global involvement of CNS and rapidity of neurological deterioration in the GLD, the treatment calls for a rapid and robust distribution of therapeutics throughout the CNS before the disease progresses. The high efficiency with which AAV vectors are transported antero-and-retrograde axonally opens the possibility of targeting a transgene to neurons population remote from the injection sites and difficult to access after a relative localized intracranial injection. We investigate the feasibility and efficacy of axonal transport of therapeutics to diseased CNS globally by inoculating AAV vector directly into brain structures with defined axonal connections with other structures. Broad distribution of AAV vector is seen in the entire brain, cerebellum, brain stem, and throughout spinal cord. Neuroanatomical mapping of transduced cells in CNS are consistent with the pattern of major axonal circuit in CNS. Widespread restoration of functional Galc activity in CNS leads to reduction of toxic substrate accumulation, correction of CNS inflammation, axonal damage, and demyelination, and amelioration of devastating phenotype. Our results show that targeting the brain structures with divergent axonal connection is an effective approach for achieving widespread therapeutics distribution throughout the CNS. This therapeutic strategy with axonal transportation might be promising as gene therapy for GLD, but also disorders indications with global neurodegeneration in future clinical trials.

P 141

Abstract Withdrawn

Andolfi G Mrs 1 Rossetti M Dr 1 Magnani CF Dr 1 Fousteri G Dr 1 Jofra T Mrs 2 Gregori S Dr 3 Roncarolo MG Professor 3 1 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Department of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy 2 San Raffaele Diabetes Research Institute (HSR-DRI), Department of Immunology, Transplantation and Infectious Diseases, Via Olgettina 58, 20132 Milan, Italy 3 an Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Department of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy P 142

Enforced IL-10 expression confers Tr1 cell phenotype and functions to human CD4+T cells

Background: Type 1 regulatory T (Tr1) cells are an inducible subset of regulatory T cells producing high levels of interleukin-10 (IL-10) and suppressing antigen-specific effector T cells via cytokine-dependent mechanisms. Several protocols have been developed to expand Tr1 cells in vitro. However, in most cases the resulting population still includes a consistent fraction of contaminating effector T cells that might represent an obstacle for the development of therapeutic adoptive transfer strategies.

Method: We constructed a bidirectional lentiviral vector (LV) encoding for human IL-10 and carrying GFP as marker gene (LV-IL-10/GFP). A detailed characterization of resulting LV-IL-10/GFP-transduced human CD4⁺ T cells in terms of phenotype and functions will be reported.

Results: LV-IL-10/GFP efficiently transduced activated human CD4⁺ T cells (CD4^IL-10/GFP). CD4^IL-10/GFP T cell lines express higher levels of ICOS, ICOS-L, IL-10R and HLA-G antigen compared to cells transduced with control LV-GFP (CD4^GFP). CD4^IL-10/GFP T cell lines secrete high amounts of IL-10 and low levels of IL-4, displaying a Tr1 cytokine production profile, are anergic, and suppress the proliferation of allogeneic CD4⁺ T cells in vitro and in vivo. CD4^IL-10/GFP T cell lines express high levels of granzyme B and lyse target cells of myeloid origin.

Conclusion: Constitutive over-expression of IL-10 in human CD4⁺ T cells results in a stable cell population which recapitulates the phenotype and function of Tr1 cells.

Kahraman S Mrs 1 Dirice E Dr 2 Elpek O Professor 3 Ertosun MG Dr 4 Balci MK Professor 5 Omer A Professor 6 Sanlioglu S Professor 7 Sanlioglu AD Professor 1

1 Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics, Department of Medical Biology and Genetics, Antalya, Turkiye 07058 2 Section of Islet Cell and Regenerative Medicine, Joslin Diabetes Center, Harvard Medical School, Boston MA, 02215, USA 3 Department of Pathology, Akdeniz University Faculty of Medicine, Antalya, Turkiye 07058 4 Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics, Antalya, Turkiye 07058 5 The Division of Endocrinology and Metabolism, Akdeniz University Faculty of Medicine, Antalya, Turkiye 07058 6 Sn.Vincent Hospital, Massachusetts, USA 7 Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics, Department of Medical Genetics, Antalya, Turkiye 07058 P 143 Spontaneous type 1 diabetes in NOD mice is related to an increase in DcR1 expression

Background: Type 1 Diabetes (T1D) results from selective autoimmune destruction of the pancreatic beta islets by the infiltrating inflammatory cells. There is evidence for both destructive and protective roles for TNF-Related Apoptosis-Inducing Ligand (TRAIL) in T1D [1],[2]. We aimed to detect the expression profiles of TRAIL ligand and receptors in spontaneous disease development in NOD mice for implications on the role of these molecules in the developmental course of T1D.

Materials and Methods: Pancreatic beta islets at tissue sections prepared from a total of 10 three days diabetic NOD mice were immunohistochemically analysed for TRAIL ligand and receptor expressions. The results were compared with those of pre-diabetic NOD mice and age-matched Non-Obese Diabetes Resistant (NOR) mice. TUNEL was used for detection of the related apoptotic profile.

Results: DcR1 expression levels displayed a significant increase in diabetic mice, while the expression levels for the other two membrane-bound receptors or the TRAIL ligand did not change. Apoptotic cell count was highest at day 7.

Conclusion: Increase in the DcR1 expression levels in the beta islets in three days diabetic mice may reflect a protective strategy against the infiltrating cells expressing TRAIL.

References

1 Sanlioglu A.D. et al. Molecular mechanisms of death ligand-mediated immune modulation: a gene therapy model to prolong islet survival in type 1 diabetes. J Cell Biochem, 2008; 104,3:710–20. a-1556 2 Dirice E. et al. Adenovirus-mediated TRAIL gene (Ad5hTRAIL) delivery into pancreatic islets prolongs normoglycemia in streptozotocin-induced diabetic rats. Hum Gene Ther, 2009; 20,10:1177–89. a-1557 Alonso-Ferrero ME Dr Valeri A Dr Yañez R Navarro S Garin MI Ramirez JC 2 Bueren JA Segovia JC 2 Viral Vector facility and Gene Targeting Unit. CNIC. Madrid. Spain P 144 Immunoresponses limit in utero hematopoietic engraftment of transduced fetal liver progenitors

In utero cell and gene therapies constitute alternative strategies to the postnatal treatment of inherited diseases. Fetal hematopoietic progenitors could be a potential source of donor cells for this strategy. In the present study, hematopoietic lineage-negative fetal liver cells from 14.5 days old fetuses were transduced using different cytokine and culture combinations with a lentiviral vector expressing the enhanced green fluorescent protein (EGFP). Up to 70% of granulo-macrophage colony forming cells expressed the EGFP reporter gene when cells were transduced for 6 hours in presence of mSCF, hTPO and FLT3-L in retronectin-coated dishes at an MOI 10 TUs/cell. In utero transplantation experiments showed that conditions leading to high transduction efficiencies did not result in high engraftment levels of EGFP-expressing cells in the syngeneic recipients. In these animals we detected the presence of antibodies and cellular responses against EGFP, indicating immunoreaction against the transduced cells. Analysis of mother' and pups' immunoresponses showed that the first events of the immunoreaction came from the operated pregnant females, as early as two days after in utero transplant. Our results show that fetal liver hematopoietic progenitors can be transduced very efficiently after short periods of incubation with low lentiviral vector doses and demonstrate for the first time that in utero transplanted animals can develop both humoral and cellular immunoresponses against exogenous transgenes. These observations should be considered in future protocols of prenatal gene therapy.

Bisgin A Dr Sanlioglu AD Dr Yoldas B Dr Aydin C Mrs Yazisiz V Dr 4 Tuzuner S Professor 5 Gorczynski RM Professor 6 Akdis CA Professor 7 Terzioglu E Professor 4 Sanlioglu S Professor

4 Department of Internal Medicine, Division of Rheumatology and Immunology, Akdeniz University Medical Faculty, Antalya, Turkiye 07058 5 Department of Orthopedics and Traumatology, Akdeniz University Medical Faculty, Antalya, Turkiye 07058 6 Division of Cellular & Molecular Biology, Toronto Hospital, University Health Network, Toronto, ON, Canada 7 Swiss Institute of Allergy and Asthma Research, Davos, Switzerland P 145 Trail profile of synoviocytes and peripheral blood lymphocytes of rheumatoid arthritis patients

Background: Rheumatoid arthritis (RA) is a common, progressive, destructive inflammatory disease modulated by death ligand/receptor expression both on T cells and RA synoviocytes. TNF Related Apoptosis Inducing Ligand (TRAIL) appears to play major roles during the development of autoimmune diseases such as diabetes [1]. However, its role in RA development remains unknown. For this reason, the primary objective of this study was to explore TRAIL and TRAIL receptor composition in peripheral blood lymphocytes of RA patients and compare it to that of synoviocytes [2].

Methods: Flow cytometry (FC) analyses were utilized to characterize the expression pattern of TRAIL and its receptors on synoviocytes and peripheral blood lymphocytes isolated from RA patients.

Results: Our results suggested that while DR5 was the predominant receptor type expressed in RA synoviocytes, other TRAIL receptors exhibited a heterogeneous expression profile. On the other hand, the expression of TRAIL and its receptors both death and decoy were increased on the peripheral lymphocytes of RA patients, compared to healthy controls.

Conclusion: Differential alteration of TRAIL and TRAIL receptor expression on T cells and synoviocytes indicates that the profile might be important in decision-making process of cell death or survival.

References

1 Sanlioglu A.D. et al. Molecular mechanisms of death ligand-mediated immune modulation: a gene therapy model to prolong islet survival in type 1 diabetes. J Cell Biochem, 2008; 104,3:710–20. a-1564 2 Terzioglu E. et al. Concurrent gene therapy strategies effectively destroy synoviocytes of patients with rheumatoid arthritis. Rheumatology (Oxford), 2007; 46,5:783–9. a-1565 Oviedo A Mr 1 Yañez R Dr 1 Aldea M Ms 1 Rubio A Mr 1 Bueren J Dr 1 Lamana M Dr 1 1 CIEMAT/CIBERER. Hematopoiesis and Gene Therapy Department P 146 Effect of mesenchymal stromal cells on GVHD and GVL reactions in a CML relapse mouse model

To study factors that affect Graft v. Host Disease (GVHD) and Graft vs.Leukemia effect (GVL) in haplo-BMTs, first, we generated a CML mouse model. For this, B6D2F1 (H2^b/d) bone marrow lin^- cells were transduced with p210-tNGFR retroviral vector that encodes BCR/ABL oncogene and tNGFR. Cells were infused into lethally irradiated syngeneic mice, generating a CML-like syndrome that killed mice around day + 18. To mimic CML-relapse after BMT, BM cells from haploidentical C57Bl/6 mice (H2^b/b) were infused, producing a slower development of CML with + 28-72 day deaths. In both models, necropsies detected splenomegaly, hepatomegaly, hemorrhagic lungs, all with NGFR⁺ leukemic infiltration, as well as in hypercellular bone marrows. To develop a GVL effect, haploidentical splenocytes from C57Bl/6 mice were additionally infused. The animals died of GVHD + 13-36 days after co-transplants, similarly to the GVHD control group. No organomegaly or NGFR⁺ cells were detected in the analyzed tissues. In a previous work we demonstrated that MSCs infusion on days + 0, + 7 and + 14 prevented GVHD in 60-80 % of the mice in haplo-BMT (Yañez, 2006). Now, MSCs were additionally infused to study MSCs' immunosuppressive effects on GVL. This infusion did not increase mice survival, although it prolonged survival until day + 55. Low values of NGFR⁺ cells were detected in bone marrow and spleen, and the death of the animals was attributed to a moderately delayed GVHD. Our results show that, in this CML-relapse/GVL mouse model, MSCs did not induce an evident increase in leukemic relapses but had a modest efficacy in preventing GVHD.

Yilmazer AY Ms 1 Al-Jamal WAJ Dr 1 Van den Bossche JVB Dr 1 Kostarelos KK Professor 1

1 Nanomedicine Laboratory, Centre for Drug Delivery Research, School of Pharmacy, University of London, London WC1N 1AX, United Kingdom P 147 Artificial Envelopment of Adenovirus Dramatically Alters Viral Liver Tropism and Tissue Distribution

Human adenovirus serotype 5 (Ad5) is the most frequently used gene delivery vector in clinical studies. Following intravenous administration, Ad is rapidly cleared from systemic blood circulation with a half life of 2 minutes, and more than 99 % of the injected dose is sequestered in liver resulting in significant hepatotoxicity. These issues currently restrict the use of Ad-based vectors, particularly for clinical gene therapy protocols that involve systemic administration. We propose that such limitations can be overcome by engineering artificial lipid envelopes around Ad. Liver tropism of artificially enveloped Ad vectors in cationic non-pH-sensitive DOTAP:Chol or anionic pH-sensitive DOPE:CHEMS lipid bilayers was studied after intravenous (tail vein) administration to Balb/C mice. Real-time PCR and transgene expression levels showed that the type of lipid envelope dramatically changed the uptake of viral particles in parenchymal cells (PC) and non-parenchymal cells (NPC) of the liver compared to naked Ad. Artificial lipid bilayers around Ad prevented PC transduction in the following order DOTAP:Chol >DOPE:CHEMS. Whole-body imaging of luciferase expression using an IVIS camera at 24 hr after administration of enveloped Ad showed an overall decrease in liver transduction similar to that observed for naked Ad injected in warfarin-treated mice. Furthermore, DOTAP:Chol envelopment significantly increased lung accumulation compared to DOPE:CHEMS enveloped or naked Ad. These data suggest that artificial lipid envelopes for Ad offer an approach to significantly divert viral particles from their natural liver tropism and may allow safe delivery to target organs in vivo.

Piguet F Ms 1 Sondhi D Dr 2 Colle M.A Dr 3 Raoul S Dr 4 Roujeau T Dr 5 Vanier M.T Dr 6 Bouquet C Dr 1 Moullier P Professor 7 Cherel Y Professor 3 Hackett N Dr 2 Zerah M Professor 5 Aubourg P Professor 1 Crystal R.G Professor 2 Cartier N Dr 1 Sevin C Dr 1

1 INSERM U745, Paris, France 2 Weill Medical College of Cornell University, New York, United States 3 UMR INRA 703, Nantes, France 4 Neurosurgery, CHU Nord, Nantes, France 5 INSERM U745 & Necker hospital, Paris, France 6 INSERM U820, Lyon, France 7 Inserm U649, Nantes, France P 148 Brain gene therapy for metachromatic leukodystrophy: towards clinical trial

Brain gene therapy seems to be a suitable option to treat patients affected with the most severe forms of Metachromatic leukodystrophy (MLD), a lethal lysosomal storage disorder due to a deficiency in arylsulfatase A (ARSA) enzyme, leading to sulfatide storage, demyelination and neuronal degeneration.

Proof of efficiency and safety of intracerebral delivery of adeno-associated-vector serotype 5 (AAV5) encoding human ARSA was established in MLD mice and non-human primates (NHP).

We demonstrated that, in comparison with AAV5/ARSA vector, intracerebral injection of AAVrh.10/ARSA resulted in a more rapid and complete correction of sulfatide storage and neuropathological lesions in symptomatic MLD mice. ARSA enzyme was detected in oligodendrocytes and oligodendrocyte-specific sulfatide isoforms accumulation was completely corrected.

Safety and efficiency study was performed in non-human primates. AAVrh.10/ARSA vector was injected in one or both hemispheres (1.1e11 PP/hemisphere) in white matter areas, targets selection based on brain MRI. In a clinical perspective, surgical device was optimized to allow simultaneous infusion of 12 deposits, thus reducing time of surgical procedure. Animals were analyzed 3 months post-injection. We documented diffusion of the vector in up to 73% and up to 31% increased activity of ARSA enzyme in the whole injected hemisphere. As compared to our previous results with AAV5/ARSA, the use of a 20-fold lower dose of AAVrh.10-derived vector led to increased amounts of vector in the non-injected hemisphere, and increased level of ARSA activity in the injected hemisphere. Toxicological evaluation is currently ongoing prior to a phase I/II clinical trial for rapidly evolutive forms of MLD.

Joubert V Dr 1 Falk H Dr 2 Panet A Professor 2 Mir LM Dr 1

1 CNRS UMR 8203, Institut Gustave Roussy, Villejuif, France 2 Department of Virology, The Hebrew University – Hadassah Medical School, Jerusalem, Israel P 149 Electrical parameters for ex vivo plasmid electrotransfer to dermis and epidermis

Previous studies have shown the ability to culture and to ex-vivo transducer organ fragments such as lungor skin. One of these studies showed that an intact miniature biopsy of human dermis transduced ex vivo by a viral vector encoding a gene for the therapeutic protein could be implanted into SCID mice and produced the protein for several months.

Gene electrotransfer (EGT) is used to internalize DNA plasmids in cells without causing irreversible damages on plasma membranes. A combinations of two types of electric pulses are used: (i) one 100 μs duration, high voltage pulse (HV) which transiently permeabilizes the cell membrane, and (ii) one or several 100 ms duration, low voltage pulse (LV) which exert(s) electrophoretic forces on the DNA and bring(s) this large macromolecule (that does not move by diffusion because of its size) in contact to the electropermeabilized membranes.

In this study, we show that ex vivo skin transduction is possible using a non viral method namely electrotransfer. Whole skin or skin fragments dissociated into dermis and epidermis were electrotransferred with a plasmid containing reporter gene coding for the luciferase. We show that the effective electrical parameters depend on the tissue complexity (whole skin, dermis or epidermis). Interestingly when whole skin is exposed to the electric pulses, both combinations of electric pulses are efficient, however at a lower level than for the isolated dermis or epidermis. Results were confirmed with other reporter genes (β-galactosidase and GFP). A study on the cell types transduced using this non viral method is ongoing.

Viecelli HM Dr 1 Chen ZY Dr 2 Kay MA Dr 2 Harbottle RP Dr 3 Thony B Dr 1

1 Division of Clinical Chemistry and Biochemistry, University of Children's Hospital Zurich, Zurich, Switzerland 2 Pediatrics and Genetics, Stanford University School of Medicine, Stanford, California, United States 3 National Heart and Lung Institute, Imperial College London, London, United Kingdom P 150 Comparison of minicircle production methods for delivering naked DNA and expressing genes in animals

Minicircle-DNA vectors are supercoiled plasmids carrying a minimal expression cassette and are generated via intramolecular recombination to eliminate the undesired plasmid backbone sequences. The advantages of minicircle-DNA vectors include the reduction of CpG motifs and the bacterial backbone which have shown to contribute to silencing of episomal transgene expression and to trigger immunotoxic responses. In this study, we compared two methods for the production of minicircle-DNA with different bacterial recombinase expression systems: A first method uses a bacterial strain (MM294) expressing Cre recombinase under the tight control of the araC regulon mediating the recombination between two partially mutant loxP sites in the parental plasmid (Bigger et al. 2001, J Biol Chem 276: 23018). The residual parental plasmids and excised bacterial vectors are digested by restriction enzyme and the undigested supercoiled minicircle-DNA is purified subsequently by CsCl-gradient centrifugation. A second method uses a bacterial strain (ZYCY10P3S2T) which genome contains ten copies of the phage ØC31 gene encoding the bacteriaphage ØC31 integrase to mediate the recombination event between attB and attP sites. In addition, the genome contains three copies of the I-SceI gene which encodes the restriction enzyme that destroys the plasmid backbone circle. Both genes are co-expressed under the control of the araC/BAD system (Chen and Kay, unpublished). We found that the latter technology is more favourable due to the high yield of pure minicircle-DNA, reduced costs and time. In conclusion, this method is highly suitable for large-scale production, making minicircle-DNA attractive as non-viral vectors for human gene therapy and vaccination.

Koller U Dr 1 Wally V Dr 1 Mitchell LG Dr 2 Klausegger A Mr 1 Murauer EM Dr 1 Mayr E Ms 1 Gruber C Dr 1 Hintner H Dr 1 Bauer JW Dr 1

1 Division of Molecular Dermatology and EB-house Austria, Department of Dermatology, Paracelsus Medical University Salzburg, Salzburg, Austria 2 RetroTherapy, Bethesda, USA P 151 Improvement of RNA trans-splicing based gene correction using a RTM screening system

Background: Since many genes, involved in various diseases such as epidermolysis bullosa, are too large for a therapy with whole gene insertion, the method of “Spliceosome Mediated RNA Trans-splicing” (SMaRT) can be used as an alternative gene repair strategy on pre-mRNA level. The trans-splicing reaction is achieved by “RNA Trans-splicing Molecules” (RTMs) that bind to a target pre-mRNA thereby inducing the gene correction.

Method: By using a fluorescence reporter system we are able to rapidly evaluate the effect of various RTM binding domains on trans-splicing functionality. The screening system is composed of an intron specific target molecule and a RTM library both including split parts of the reporter gene acGFP. Recombination of both pre-mRNAs by trans-splicing lead to the fusion of both acGFP parts manifested in the expression of acGFP in RTM and target molecule transfected HEK293 cells.

Results: Fluorescence microscopy reveals the expression of the reporter molecule acGFP two days post transfection. The most efficient RTMs, identified by “Fluorescence Activated Cell Sorting” (FACS), are able to induce endogenous trans-splicing on pre-mRNA level manifested in the fusion of the RTM with the 3′ exonic sequence of the target gene PLEC.

Conclusion: The screening for most functional RTMs using a fluorescence based screening system should improve endogenous trans-splicing in efficiency and specificity. We anticipate that these experiments should lead us to promising tools for gene therapy in plectin deficient patients.

Kahraman S Ms Dirice E Dr 2 Hapil FZ Miss 3 Ertosun MG Dr 4 Ozturk S Mr 5 Griffith TS Dr 6 Sanlioglu S Professor 7 Sanlioglu AD Dr 3

2 Section of Islet Cell and Regenerative Medicine, Joslin Diabetes Center, Harvard Medical School, Boston MA, 02215, USA. Human Gene and Cell Therapy Center of Akdeniz University,Antalya, Turkiye, 07058 3 Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics, Department of Medical Biology and Genetics, Antalya, Turkiye, 07058 4 Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics, Antalya, Turkiye, 07058 5 Department of Histology and Embryology, Akdeniz University Faculty of Medicine, Antalya, Turkiye, 07070 6 Gene Therapy Center of University of Iowa, Iowa City IA, 52242, USA 7 Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics, Department of Medical Genetics, Antalya, Turkiye, 07058 P 152 In vivo fluorescence imaging of xenogeneic islet graft survival

Background: Pancreatic islet xenotransplantation is an appealing therapy for the treatment for type 1 diabetes because of the potential to provide unlimited donor tissue supply, but it provokes an intense immune response. Also, it is difficult to assess graft mass after transplantation [1]. The aim of the study was to follow up the survival of rat-islet xenografts overtime in diabetic mice using fluorescence imaging system [2].

Method: AdEGFP transduced rat pancreatic islets were transplanted under the renal capsule of streptozotocin induced diabetic mice and non-fasting blood glucose levels were followed. Fluorescence signal emanated from graft was quantified over time using the cooled charge-coupled device. Grafts were examined for morphology and insulin content.

Results: Adenoviral transduction or EGFP expression did not affect the function of islets. Transplantation of AdEGFP transduced islets reversed diabetes in recipients within two days. Fluorescence signals emitted from xenografts were detectable post-transplantation and the signal intensity was gradually decreased as a sing of graft rejection.

Conclusion: It appears that the fluorescence imaging is a useful tool for the quantitative assessment of islet cell viability post-transplantation and it allows earlier detection of graft rejection in rat-to-mouse xenogeneic model.

References

1 Dirice E. et al. Adenovirus-mediated TRAIL gene (Ad5hTRAIL) delivery into pancreatic islets prolongs normoglycemia in streptozotocin-induced diabetic rats. Hum Gene Ther, 2009; 20,10:1177–89. a-1616 2 Kahraman S. et al. In vivo fluorescence imaging is well-suited for the monitoring of adenovirus directed transgene expression in living organisms. Mol Imaging Biol, 2010; 12,3:278–85. a-1617 Pedersen A.G Miss 1 Koefoed M.J Mrs 2 Ulrich-Vinther M. Dr 3 Soeballe K. Professor 3 Dagnaes-Hansen F. Mr 4 Jensen T.G Professor 1 1 Department of Human Genetics, Aarhus University, 8000 Aarhus C, Denmark 2 Department of Human Genetics, Aarhus University, 8000 Aarhus C, Denmark/Department of Orthopedic Surgery, Aarhus University Hospital, 8000 Aarhus C, Denmark 3 Department of Orthopedic Surgery, Aarhus University Hospital, 8000 Aarhus C, Denmark 4 Department of Medical Microbiology and Immunology, Aarhus University, 8000 Aarhus C, Denmark P 153 In vivo gene transfer by non-viral transfection reagents freeze-dried onto mouse femoral allografts

Background: Structural bone allografts lead to limited bone healing and a tendency to fracture. Recombinant adeno-associated virus freeze-dried onto murine femoral bone surfaces can be used to mediate in vivo gene transfer of osteogenic factors, thereby inducing new bone formation. In order to evaluate the efficiency of in vivo gene transfer using non-viral methods, we have freeze-dried complexes of transfection reagents together with plasmids onto the surface of bone allografts.

Methods: DNA complexes were made by combining plasmids with either Lipofectamine 2000 or branched polyethylenimine (PEI)/hyaluronic acid (HA) and were freeze-dried onto the surface of murine femoral allografts. For in vitro measurements, GFP-coated grafts were placed onto a monolayer of 293 cells and gene expression measured after 48 hours using flow cytometry. For in vivo experiments luciferase-coated allografts were inserted intra-muscularly in female Balb/c mice and gene expression was analyzed by a Luciferase Reporter Assay System.

Results: Freeze-dried complexes from the surface of coated allografts lead to significant transfection of 293 cells using both transfection reagents. Within 10 minutes the complexes are released from the grafts. Lipofectamine 2000 was approximately 4 times more efficient than PEI. Also in the tissue samples from mice, Lipofectamine 2000 seems to be more efficient than both the negative controls and the PEI/HA-transfected group.

Conclusion: Freeze-drying DNA-complexes with Lipofectamine 2000 onto bone allografts provides a novel non-viral method for introducing important factors in bone healing (e.g. BMP-2) to the surrounding tissue thereby stimulating the formation of new bone leading to enhanced incorporation of the allograft.

Benabdellah KB Mr 1 Muñoz PM Dr 1 Martín FM Dr 1

1 Andalusian Stem Cell Bank, CSJA-UGR. Granada. Spain P 154 Development of a Tet-On inducible lentiviral system for gene therapy of autoimmune disorder

Lentiviral vectors (LV) are powerful tools for gene therapy and hold great potential as a therapeutic gene therapy strategy for autoimmune disorder. However the controlled delivery of therapeutic genes represents one of the limiting factor of LV. The tetracycline-dependent transcriptional regulatory systems (Tet-systems) are one of the best studied systems with proved efficacy in vitro and in vivo. In the present study, we aimed to develop a highly inducible LV system as a tool for the gene therapy of autoinmune disorder. For this purpose, we used vasoactive intestinal peptide (VIP) [a type-II cytokine with demonstrated therapeutic effect in animal models of several autoimmune diseases], as therapeutic gene model. And experimental autoimmune encephalomyelitis (EAE) as autoimmune disorder model.

We constructed (i) a binary lentiviral-based system (SFFV-TetR, CMV-TetOGFP/VIP) and (ii) several single vectors containing the represor (TetR) and the TetO-regulated transgene (eGFP/VIP) in the same vector. We compared our systems with the previously described inducible single LVs system (pLVCT-tTR-KRAB and pLVPT-tTR-KRAB) in term of leakiness, transgene expression levels and transduction efficiency after Doxycycline (DOX) treatment. The highest transgene expression levels in the presence of DOX and the highest basal activity (leakiness) in the absence of DOX were found in the binary system. However, the PGK-driven (pLVPT-tTR-KRAB) and the CAG-driven (pLVCT-tTR-KRAB) LVs achieved better results in terms of responsiveness to DOX. Both systems show very low leakiness and good inducibility by DOX. We are currently studding potential applicability of this systems on EAE disease and potential therapeutic benefits compared with unregulated system.

Gasparini C Dr 1 Tommasini A Dr 1 Ventura A Professor 1

1 Child Health Institute IRCCS “Burlo Garofolo”, via dell'Istria, 65/1, 34137, Trieste, Italy P 155 Turning expression into advantage: a new tool for regulatory T cell therapy

Background: FOXP3 is a transcription factor with a pivotal role in the function of regulatory Tcells (Tregs), which are responsible for immunetolerance. Although many surface antigens characteristic of Tregs have been described, the selection and expansion of these cells for clinical purposes are still a difficult challenge.

The aim of this research is to give a selective advantage to cells expressing the transcription factor, to allow better selection and expansion.

Methods: We ideated a system in which the promoter sequence of TLR10, which is actively regulated by FOXP3, is cloned into a bidirectional promoter cassette^®. This system will couple the presence of FOXP3 with the expression of a surface protein (NGFR) to allow cell selection, and of a proliferative gene (to be selected) to allow cell expansion. Integration of the vector will be accomplished with the Sleeping Beauty transposon system® (SB). A FOXP3-expressing vector is used to test the system in Jurkat and 293T cells.

Results: We transfected Jurkat cells and 293T cells with the Foxp3-expressing vectors, the bidirectional promoter cassette or the SB. The transfection efficiency was about 5% on Jurkat cells and ≥ 70% in 293T cells, as measured by fluorescence microscopy and flow cytometry. We cloned the TLR10 promoter into the pGL3-basic vector in order to check the activation of the promoter sequence by luciferase assay.

Conclusions: These are very preliminary results. Jurkat cells are a suitable cellular model to test our expression system, since 5% transfection efficiency can allow us to select cells positive for the surface marker NGFR.

Özbaş-Turan S. Dr 1 Akbuğa J. Professor 1 Demir Sezer A. Dr 1 Şalva E. Dr 2

1 1 Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Biotechnology, Istanbul 2 2 Marmara University, Vocational School of Health Related Professions, Pathology Laboratory P 156 Intramuscular Delivery of Antisense Oligonucleotides by Chitosan Nanoparticles

Background: Antisense oligonucleotides (ODN) provide a gene-targeted approach for the prevention and treatment of diseases due to over expression of genes. In the antisense approach, ODNs complementary to specific mRNA sequence can inhibit gene expression. However one of the major drawbacks in antisense therapy is the difficulty in finding an adequate delivery methods for ODNs in relation to their cellular uptake. A number of strategies have been developed to deal with this problem, and use of cationic polymers as carrier for ODNs one of them. Chitosan is potentially safe and useful cationic carriers for gene delivery.

The aim of this study is to investigate the therapeutic potential of ODN-loaded chitosan nanoparticles after intramuscular application.

Method: 15-nucleotide phosphorothioate oligonucleotides (MWG-Biotech) was used as ODN. ODN_loaded chitosan nanoparticles were produced based on the ionic gelation of TPP with chitosan. ODN-chitosan nanoparticles were intramuscularly injected to the Sprague Dawley rats (initially b-gal injected) in different doses 15, 30 and 60μg. After 1, 3, 6, 9 and 12 days of post-transfection, animals were sacrified and samples were taken for b-gal assay. b-gal expression was measured enzymatically.

Results: Highest gene inhibition was measured at 6^th day with nanoparticles. The better b-gal inhibition was obtained using multiple doses nanoparticles than single, and this value was around 89%.

Conclusion: Chitosan nanoparticles are suitable for intramuscular application for antisense oligonucleotides. High gene suppression may be obtained using ODN-loaded chitosan nanoparticles.

Acknowledgement: The authors are grateful for financial support of Marmara University Scientific Research Projects Association (BAPKO).

jakbuga@marmara.edu.tr

Joubert V Dr Schmeer M Dr 2 Schleef M Dr 2 Mir LM Dr

2 Plasmid-Factory, Bielefeld, Germany P 157 Increased efficiency of minicircles versus plasmids under gene electrotransfer suboptimal conditions

Electric pulses are known to permeabilize the cell membrane and they are routinely used to allow drugs or genes entering the cells. In the case of the nucleic acids electrotransfer, combinations of two types of electric pulses can be used: (i) one 100 μs duration, high voltage pulse (HV) which transiently permeabilizes the cell membrane, and (ii) one or several 100 ms duration, low voltage pulse (LV) which exert(s) electrophoretic forces on the DNA and bring(s) this large macromolecule (that does not move by diffusion because of its size) in contact to the electropermeabilized membranes. This electrophoretic effect is very important in vivo, where the extracellular matrix really limits convection movements in the very close proximity of the cell membrane.

We explored whether similar effects can be observed in vitro. We compared the efficacy of plasmids and of the corresponding minicircles, which are circular DNA molecules derived from the plasmids, without the bacterial sequences. These minicircles are thus smaller than the plasmids. We found the minicircles more efficient than the plasmids at identical molar concentration, not only in vitro but also in vivo, as they result in the production of a greater quantity of the protein encoded by electrotransferred reporter genes. We are investigating the possibility that the greater efficiency of the minicircles could be due, at least in part, to an easier passage through the extracellular matrix because of their smaller size.

Dirice E Dr Kahraman S Mrs Elpek O Professor 3 Balci MK Professor Omer A Sanlioglu S Sanlioglu AD

3 Department of Pathology, Akdeniz University Faculty of Medicine, Antalya, Turkiye 07058 P 158 STZ administration leads to a significant rise in TRAIL and DcR1 levels in Non-Obese Diabetic (NOD)

Background: TNF-Related Apoptosis-Inducing Ligand (TRAIL) is an important component of the immune system [1]. It is well known for its preferential toxic effects on tumor cells. Yet its role in T1D development is not clear [2]. Therefore, we analyzed the effects of Streptozotosin (STZ) induction on TRAIL ligand and receptor expression levels in NOD mice.

Materials and Methods: An optimal single dose of 150 mg/kg STZ was administered to a total of 20 NOD mice. Pancreatic tissues collected before diabetes induction and at sequential steps of disease development following induction. Sections were analyzed by immunohistochemistry. Related apoptotic cell counts were detected with TUNEL.

Results: STZ induction provided a significant increase in TRAIL ligand and DcR1 levels at day 14 when 90% of the animals were diabetic. DcR2 and DR5 expressions were not altered significantly. Apoptotic count peaked at day 7, while it decreased in the following days.

Conclusion: Increase in the TRAIL ligand may imply a protective role for TRAIL against the infiltrating lymphocytes in STZ-induced T1D. In addition, increased DcR1 expression may reveal a protective strategy adapted by the pancreatic beta islets against the infiltrating lymphocytes expressing TRAIL.

References

1 Sanlioglu A.D. et al. Molecular mechanisms of death ligand-mediated immune modulation: a gene therapy model to prolong islet survival in type 1 diabetes. J Cell Biochem, 2008; 104,3:710–20. a-1637 2 Dirice E. et al. Adenovirus-mediated TRAIL gene (Ad5hTRAIL) delivery into pancreatic islets prolongs normoglycemia in streptozotocin-induced diabetic rats. Hum Gene Ther, 2009; 20,10:1177–89. a-1638 Kahraman S Mrs Dirice E Dr Elpek O Professor Balci MK Professor Omer A Professor Sanlioglu S Professor Sanlioglu AD P 159 TRAIL expression significantly decreases in CY-accelerated type 1 diabetes in NOD mice

Background: Induction of Type 1 Diabetes (T1D) in NOD mice provides a faster disease development and increased diabetes prevalence. Although various investigators prefer Cyclophosphamide (CY) for T1D induction, there is little information on its effects on the immune system components such as the TNF-Related Apoptosis Inducing Ligand (TRAIL) [1]. TRAIL's role in T1D is not yet clear and further studies are required [2]. Thus, we analyzed the expression profiles of TRAIL ligand and its receptors in CY-accelerated T1D in NOD mice.

Materials and Methods: All mice received a single dose of 200 mg/kg CY. Pancreatic tissues were collected from pre-diabetic and diabetic NOD mice in addition to Non-Obese Diabetes Resistant (NOR) mice prior to immunohistochemical analysis. TUNEL assay was used for the detection of the related apoptotic profiles.

Results: TRAIL expression was significantly decreased in CY-accelerated T1D in NOD mice while no change was observed in the age-matched CY-applied NOR mice. The apoptotic cell count was increased along with diabetes development.

Conclusion: Different effects exerted on the immune system by various diabetes-inducing agents should be taken into consideration in diabetes investigations.

References

1 Sanlioglu A.D. et al. Molecular mechanisms of death ligand-mediated immune modulation: a gene therapy model to prolong islet survival in type 1 diabetes. J Cell Biochem, 2008; 104,3:710–20. a-1639 2 Dirice E. et al. Adenovirus-mediated TRAIL gene (Ad5hTRAIL) delivery into pancreatic islets prolongs normoglycemia in streptozotocin-induced diabetic rats. Hum Gene Ther, 2009; 20,10:1177–89. a-1640 Singh M Dr 1 Ariatti M Professor 1 Sewbalas A Miss 1 1 Discipline of Biochemistry, University of KwaZulu-Natal, Durban P 160 Crown Ethers potentiate Transfection activity of Cationic Lipoplexes in Mammalian Cells

Background: Non-viral gene delivery modalities compatible with application in man are receiving growing attention in light of safety issues surrounding viral vector systems. Amongst the more promising DNA vehicles are liposomes. Those derived from cationic lipids show much potential although transfection levels achieved in vivo are moderate. In this investigation two cholesteryl derived ionophores have been formulated into cationic liposomes and have been examined for their transfection enhancing potential

Methods: Liposome formulations included the cytofectin 3β[N-(N′,N′-dimethylaminopropane) carbamoyl] cholesterol (Chol-T), the co-lipid dioleoylphosphatidylethanolamine and 5% (mole/mole) of the cholesteryl crown ethers RUI-128 (aza-18-crown-6) and RUI-129 (aza-15-crown-5). The remaining preparations were developed for liver targeting by including 5% (mole/mole) cholesteryl-β-D-galactopyranoside/glucopyranoside. Liposome interaction with plasmid DNA was characterized by band shift, ethidium displacement and serum nuclease digestion analyses. Both growth inhibition and transfection activities were determined in the human kidney HEK293 and human hepatoma HepG2 cell lines.

Results: All liposomes showed effective DNA binding and protection against serum nuclease degradation. Liposomes containing the crown ether preparations exhibited slightly higher cytotoxicity but enhanced transfection levels in both cell lines. The preparations containing the cholesteryl-β-D-glycopyranosides however showed poor targeting in the HepG2 cell line.

Conclusion: Liposome preparations showed generally low cytotoxicity levels in both cell lines. Crown ether liposomes may have potentiated transfection through endosome destabilization however the absence of targeting may be ascribed to molecular shielding.

Papanikolaou E Dr 1 Stamateris E Mr 1 Georgomanoli M Miss 1 Wilber A Dr 2 Sankaran VG Dr 3 Hargrove PW Dr 4 Kim YS Dr 4 Orkin SH Professor 3 Nienhuis AW Professor 4 Persons DA Professor 4 Anagnou NP Professor 1

1 Laboratory of Biology, University of Athens, School of Medicine, and Laboratory of Gene Therapy, Biomedical Research Foundation of the Academy of Athens, Athens, Greece 2 Surgery, SIU School of Medicine, Springfield, IL, USA 3 Hematology/Oncology, Harvard Medical School, Boston, MA, USA 4 Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA P 161 Gene therapy for β-thalassemia using lentiviral vectors encoding either γ-globin or BCL11A/shRNA

β-thalassemia results from severely reduced or absent expression of the β-chain of adult hemoglobin (α₂β₂;HbA) causing precipitation of excess α-chains and apoptosis of erythroid precursors. However, increased levels of fetal hemoglobin (α₂γ₂;HbF), such as in hereditary persistence of HbF (HPFH), ameliorate the severity of β-thalassemia. Furthermore, recent data revealed BCL11A as a major repressor of γ-globin expression in adult erythroid cells (Sankaran et al, Science 322:1839, 2008). In the present study, we compared two, self-inactivating, γ-globin lentiviral vectors termed V5m3-400 (Pestina et al, Mol Ther 17:245, 2009) and GGHI (Papanikolaou et al, Mol Ther 16[Suppl.1]:S278, 2008) with a lentiviral vector comprising a U6-regulated shRNA for BCL11A knockdown (BCL11A/shRNA), using steady-state bone marrow CD34⁺ cells from adults with β-thalassemia major. An in vitro model of human erythropoiesis was used to assay for enhanced production of HbF (Wilber et al, Blood 115:3033, 2010). Gene transfer for all three vectors was >80% as determined by PCR analysis of CFUe. The mean levels of HbF produced from each vector, measured by HPLC, were 56.4% (V5m3), 63.4% (GGHI) and 49.4% (BCL11A/shRNA). Cells transduced with V5m3-400 demonstrated an average of 45% reduction in apoptotic cells (Tunnel⁺ assay), while the respective values using Annexin V assay were 20.2% for GGHI and 14.5% for BCL11A/shRNA, suggesting a significant rescue via correction of the globin chain imbalance. These novel data document that both approaches, i.e. gene addition or genetic reactivation, have the potential to provide therapeutic levels of HbF to thalassemic patients with such a degree of β-globin deficiency.

Carmo M Dr 1 Booth C Dr 1 Montiel-Equihua C A Dr 1 Schambach A Dr 2 Baum C Professor 2 Thrasher A J Professor 1 Gaspar H B Professor 1

1 Molecular Immunology Unit, Institute of Child Health, University College London, London, UK 2 Department of Experimental Hematology, Hannover Medical School, Hannover, Germany P 162 Development of gene therapy for haemophagocytic lymphohistiocytosis (HLH) due to perforin deficiency

Haemophagocytic lymphohistiocytosis (HLH) is a devastating disorder of early childhood arising from defects of T and NK cell cytotoxicity. The most common form of this disease is due to mutations in the perforin gene. Current treatment options of HLH are limited, thus the development of gene therapy may have great benefit for such patients.

Two lentiviral vector constructs were designed; with perforin expression being driven by the SFFV promoter or by the PGK promoter. Both vectors induced expression of perforin in the RBL1 mast cell line and confocal microscopy confirmed that perforin expression was confined to the secretory granules.

To test the functionality of the perforin protein, vectors were used to tranduce RBL2H3 cells; both vectors transduced efficiently and were able to induce cytotoxicity in RBL2H3 cells.

Next, NK cells and CD8⁺T cells derived from perforin deficient mice were transduced with both perforin vectors. Low transduction efficiencies were obtained, nevertheless both perforin vectors were able to restore cytotoxicity to NK and CD8⁺T cells.

Perforin vectors were also used to transduce enriched hematopoietic stem cells from perforin deficient mice and cells were differentiated in NK differentiation culture conditions. More than 70% of these cells differentiated into NK cells and from this population 30% of the cells expressed perforin. These cells were also shown to have the cytotoxic activity restored.

The restoration of cytotoxicity of perforin deficient cells allows us to take these vectors to the next step: the correction of a mutant murine model of perforin deficiency.

Cotugno G Miss Annunziata P Miss Tessitore A Miss O'Malley T Mr 2 Capalbo A Miss Bartolomeo R Miss O'Donnell P Miss 2 Wang P Miss 2 Russo F Mr Sleeper M Dr 2 Knox V Dr 2 Fernandez S Mr 2 Levanduski L Mrs 2 Hopwood J Dr 3 De Leonibus E Mrs Haskins M Dr 2 Auricchio A Dr

2 Depts. of Pathobiology and Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA 3 Lysosomal Diseases Research Unit, SA Pathology [at Women's and Children's Hospital], Adelaide, Australia P 163 Long-term amelioration of feline Mucopolysaccaridosis VI after AAV-mediated liver gene transfer

Mucopolysaccaridosis VI (MPS VI) is caused by deficient arylsulfatase B (ARSB) activity resulting in lysosomal storage of glycosaminoglycans (GAGs). MPS VI is characterized by dysostosis multiplex, organomegaly, corneal clouding, and heart valve thickening.

Gene transfer to a factory organ like liver may provide a life-time source of secreted ARSB. To this end, adeno-associated viral (AAV) vectors with serotype 8 capsids (AAV2/8) represent a valuable tool for efficient liver gene transfer. We show that intravascular administration of adeno-associated viral vectors (AAV) 2/8-TBG-felineARSB in MPS VI cats resulted in ARSB expression up to one year, the last time point of the study. In newborn cats, normal circulating ARSB activity was achieved following delivery of high vector doses (6x10e13 gc/kg) while delivery of AAV2/8 doses as low as 2x10e12 gc/kg resulted in higher than normal serum ARSB levels in juvenile MPS VI cats. In MPS VI cats showing high serum ARSB levels, independent of the age of treatment, we observed: i) clearance of GAG storage ii) improvement of long bone length iii) reduction of heart valve thickness, and ìv) improvement in spontaneous mobility. Thus, AAV2/8-mediated liver gene transfer represents a promising therapeutic strategy for MPS VI patients.

To this end, we are evaluating the efficacy of systemic delivery of AAV2/8-TBG-humanARSB in a knock-out mouse made tolerant to human ARSB by transgenic insertion of an inactive human ARSB allele (MPS VI-C91S).

Brunetti-Pierri N Dr Grove N Mr Patel P Dr 3 Palmer D Dr Beaudet A Dr Ng P Dr

3 Department of Pediatric Cardiology, Baylor College of Medicine, Houston, TX, USA P 164 Sustained therapeutic hFIX levels in rhesus macaques following catheter-based delivery of HDAd

Hemophilia B is a good candidate for gene therapy because factor IX (FIX) activity ≥ 1% results in clinically significant improvement of the bleeding diathesis. Helper-dependent adenoviral (HDAd) vectors hold tremendous potential because they mediate long-term transgene expression without chronic toxicity. Administration of first generation adenoviral (FGAd) vectors expressing human FIX (hFIX) in rhesus macaques resulted in short term hFIX expression and induction of high-titer, neutralizing anti-hFIX antibodies. To determine safety and efficacy of HDAd-mediated hemophilia B gene therapy, we administered an HDAd expressing hFIX into rhesus macaques through a balloon catheter strategy we have previously developed and resulting in greater hepatocyte transduction than systemic intravenous administration. In this approach a balloon catheter is percutaneously positioned in the inferior vena cava to occlude hepatic venous outflow and 1×10¹⁰, 3×10¹⁰, 1×10¹¹, or 1×10¹² vp/kg of an HDAd expressing hFIX was injected into the liver via a hepatic artery catheter. Animals injected with 1x10¹¹ and 1x10¹² vp/kg exhibited therapeutic levels of hFIX (>5% of normal FIX activity) for at least 461 days and 793 days, respectively. Sub-therapeutic levels corresponding to ∼0.1-0.5% were achieved in the animal injected with 3x10¹⁰ vp/kg and 1x10¹⁰ vp/kg. Importantly, HDAd administration did not result in the development of neutralizing anti-hFIX antibodies. These results suggest that in contrast to FGAd, HDAd vectors can drive long-term FIX expression, do not result in neutralizing anti-hFIX antibody formation, and 1x10¹¹ vp/kg is the minimal dose to achieve clinically beneficial levels of FIX. Therefore, HDAd are attractive vectors for hemophilia B gene therapy.

Chandler RJ Mr 1 Chandrasekaran S Ms 1 Venditti CP Dr 1

1 National Human Genome Research Institute, National Institutes of Health, Bethesda, MD USA P 165 AAV8 Gene Transfer Rescues a Neonatal Lethal Murine Model of Proprionic Acidemia

Propionic Acidemia (PA) is an autosomal recessive disorder of metabolism caused by a deficiency of propionyl-CoA carboxylase. Despite optimal dietary and cofactor therapy, PA patients still suffer from lethal metabolic instability and experience multisystemic complications. A murine model of PA (Pcca ^−/−) that uniformly perishes within the first 48 hours of life was used to determine the efficacy of adeno-associated viral (AAV) gene transfer as a potential therapy for PA. An AAV serotype 8 vector was engineered to express the human PCCA cDNA and delivered to newborn mice via an intrahepatic injection. Greater than 70% of the Pcca ^−/− mice were rescued after AAV8 mediated gene transfer and survived until day of life 16 or beyond. Western analysis of liver extracts showed that propionyl-CoA carboxylase was completely absent from Pcca ^−/− mice but was restored to greater than wild-type levels after AAV gene therapy. The treated Pcca ^−/− mice also exhibited markedly reduced plasma levels of 2-methylcitrate compared to the untreated Pcca ^−/− mice, which indicates significant propionyl-CoA carboxylase enzymatic activity was provided by gene transfer. At the time of this report, the oldest treated Pcca^−/− mice are over two months of age. In summary, AAV gene delivery of PCCA effectively rescues Pcca ^−/− mice from neonatal lethality and substantially ameliorates metabolic markers of the disease. These experiments demonstrate a gene transfer approach using AAV8 that might be used as a treatment for PA, a devastating and often lethal disorder desperately in need of new therapeutic options.

Follenzi AF Dr 1 Raut SR Dr 2 Merlin SM Dr 1 Sarkar RS Dr 3 Gupta SG Professor 4

1 University of Piemonte Orientale, Medical School, via Solaroli, 17, Novara - Italy 2 3Haemostasis Section, Biotherapeutics Group, National Institute for 3 University of Pennsylvania, Dept of Genetics, Philadelphia, PA, USA 4 Albert Einstein College of Medine, Dept of medicine and Pathology, Bronx, NY, USA P 166 Bone marrow transplantation cures hemophilia A

Identification of cells capable of synthesizing and releasing FVIII is critical for therapeutic development in hemophilia A. Recent studies indicated that endothelial cells, particularly liver sinusoidal endothelial cells, are a major source of FVIII, although the origin of endothelial cells is incompletely defined and FVIII could potentially be expressed in additional cell types. To determine whether endothelial cells, or other cells, derived from bone marrow could produce FVIII, we transplanted healthy mouse bone marrow into hemophilia A mice. We tracked donor cells by genetic markers and analyzed FVIII production, as well as correction of hemophilia by several assays. After bone marrow transplantation, mice survived induction of bleeding and hemophilia A was corrected, although despite hepatic and endothelial injury to recruit transplanted cells, donor bone marrow-derived hepatocytes or endothelial cells were extremely rare, and did not account for these benefits. We discovered that FVIII was produced in mononuclear cells and mesenchymal stromal cells derived from donor bone marrow, which expressed FVIII at mRNA and protein levels. Moreover, injection of healthy mouse Kupffer cells (liver macrophage/mononuclear cells), which predominantly originate from the bone marrow, or of healthy human bone marrow-derived mesenchymal stromal cells resulted in the appearance of FVIII in blood and protected hemophilia A mice from bleeding challenge. Therefore, we concluded that bone marrow transplantation cured hemophilia A through donor mononuclear cells and mesenchymal stromal cells. These insights in cellular origins of FVIII offer new mechanisms for understanding alterations in FVIII synthesis and production and for developing effective therapies in hemophilia A.

Cantore A Mr 1 Ranzani M Mr 1 Damo M Ms 1 Sergi L Sergi Ms Vallanti G Dr 3 Radrizzani M Dr 3 Brown B Dr 4 Montini E Dr Nichols T Dr Naldini L Dr 1

1 San Raffaele Telethon Institute for Gene Therapy and “Vita-Salute San Raffaele” University, Milan, Italy 3 MolMed, Milan, Italy 4 Mount Sinai School of Medicine, New York, USA P 167 Sustained partial correction of hemophilia B in the dog by a microRNA-regulated lentiviral vector

Gene therapy has long held the promise of a cure for hemophilic patients. We previously demonstrated phenotypic correction of hemophilia B after a single administration of a microRNA-regulated lentiviral vector (LV) in a mouse model. We have now tested our gene therapy strategy in hemophilia B dogs. We administered intraportally 1.5x10¹⁰ integration units of highly purified LV to an 8-month old, 20 Kg hemophilia B dog. The infusion was well tolerated and uneventful with only minor self-limiting hepatocellular toxicity. At the current follow up (>1 year post-infusion) the dog is alive and well. Whole blood clotting times have been stably shortened and anti-canine FIX inhibitors tested negative. We have administered a 4 fold higher dose in a second dog. The infusion was aborted due to an acute and severe hypotensive response, likely due to an allergic reaction to an unknown vector component or contaminant, which resolved after arresting the administration. Experiments are now underway in order to elucidate the reason for this. In parallel to those studies, we have validated a number of specific interventions which increased the potency of our strategy, including the use of a codon-optimized transgene, a single administration of a proteasome inhibitor prior to LV infusion or a different LV pseudotype (baculovirus gp64 envelope), which allow the use of lower LV doses. These results indicate that liver-directed gene therapy by LV can provide long-lasting benefit without inducing an immune response to the transgene.

Bortolussi G Dr 1 Zentilin L Dr 1 Giraudi P Dr 2 Dapas M Dr 1 Bellarosa C Dr 2 Giacca M Professor 1 Tiribelli C Professor 2 Muro AF Dr 1

1 ICGEB, Padriciano 99, Trieste, Italy 2 CSF, Basovizza, Trieste, Italy P 168 A new mouse model for Crigler-Najjar syndrome and possible therapeutic approaches

Crigler-Najjar syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphospho-glucuronosyl transferase-1a1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced brain damage unless phototherapy is applied since birth. However, the treatment becomes ineffective during adulthood due to increased thickness of the skin. As a result, liver transplantation is required. We generated a mouse model of the disease by introducing a frame shift mutation in UGT1A Exon 4 creating a stop codon immediately after the deletion. Newborn homozygous mutant mice (UGT1A^−/−) developed severe jaundice 2 days after birth. All mutant animals died within day 7 showing severe neurological damage, mimicking all major features of the human Crigler-Najjar syndrome.

Recombinant AAV9 viral vectors carrying the human UGT1a1 cDNA were IP injected into P2 newborn mutant mice. We were able to rescue 100% of AAV-treated mutant mice, that reached adulthood and were indistinguishable from their control littermates. Bilirubin levels in mutant AAV-treated mice were significantly lower than untreated mutant mice although they did not reach those of heterozygous or wild type littermates. Behavioral and histological analysis confirmed that mutant mice treated with AAV-UGT1a1 had no obvious neurological damage. Real time quantitative PCR showed presence of the AAV-genomic DNA in liver, heart and skeletal muscle after 5 months post injection. However, UGT1a1 protein was detectable only in skeletal muscle by western blot analysis. We are now testing different vectors with liver-specific promoters in order to improve the efficacy of the gene-targeting protocols.

Ferro L Dr 1 Hollyman D Dr Riviere I Dr 2 Spinelli G Dr 3 Maggio A Professor 4 Acuto S Dr 4 Sadelain M Dr 2

1 1275 York Avenue 2 1275 York Ave 3 Viale delle Scienze 4 Via Trabucco P 169 The Sea Urchin Sns5 Insulator Improves Expression of Lentiviral Vector Encoded Human β-Globin Gene

Globin gene therapy for the treatment of b-thalassemia requires sustained and stable expression of the human b-globin gene in the erythroid lineage. Transgene expression following viral-mediated gene transfer may be variable and subject to silencing. Chromatin insulators are genomic elements that may reduce chromosomal position effect and silencing affecting g-retro and lentiviral vectors.

We compared different insulators flanking the TNS9.3 globin lentiviral vector (LV). We constructed LVs flanked with the sns5 sea urchin insulator (in sense and reverse orientation), the 400bp and 1.2kb chicken HS4 insulator or a control sequence of identical size. In panels of single copy murine erythroleukemic (MEL) cell clones, we showed an increase in human b-globin transcript levels when the sns5 insulator flanks the LV in sense orientation. The sns5 insulator also reduced LV siliencing. Unlike the 1.2kb cHS4 insulator, the insertion of sns5 into the LV did not reduce vector titer.

In order to assess the in vivo effect of the sns5 insulator on b-globin expression, lethally irradiated C57BL6Hbb^Th3+/− mice were transplanted with LVs transduced C57BL6Hbb^Th3+/− lin^- bone marrow cells. Human b-globin expression was analyzed in peripheral blood over time in long-term bone marrow chimeras. The sns5 insulator increased transgene expression when compared with the TNS9.3 LV, as well as with LVs flanked with the spacer sequence or the 400bp cHS4 insulator.

Clonal globin expression analysis and microarray studies in CFU-S are currently being perrformed to evaluate the capability of sns5 to reduce endogenous gene trans-activation.

Schuettrumpf J Dr 1 Milanov P Mr 1 Abriss D Ms 1 Lyssy P Ms 1 Tonn T Professor 2 Seifried E Professor 1

1 DRK-Blutspendedienst Baden-Wuerttemberg-Hessen, Frankfurt/Main, Germany 2 DRK-Blutspendedienst Ost, Dresden, Germany P 170 Factor IX gene transfer for the treatment of hemophilia A

Recently, long-term expression following factor IX (FIX) gene transfer has been shown in the first human subject with hemophilia B. Clinical gene therapy for the more common FVIII deficiency (hemophilia A) or for patients with anti-FVIII antibodies, however, is still out of sight. Here, we report a new gene transfer strategy using FIX variants with activity in absence of their co-factor (FVIII). These variants could be easily adapted to any established FIX gene therapy approach. In an initial in vitro screening, we identified a single mutant (K265T) with 6.6% clotting activity (FIX antigen 100%) and a triple variant (V181I/K265T/I383V) with 17% activity in absence of FVIII. Bypassing activity was confirmed in plasma of patients with high titers of inhibitory antibodies. The muteins were introduced into a non-viral vector system and stably expressed in different mouse models. At FIX levels ranging from 7500 to 19000ng/ml partial normalization of the aPTT and of blood loss following tail clip assay (1.5 and 3 mm) were observed in all the variant groups (n = 5-9 mice/group, p < 0.05-0.005), while wild-type FIX expressing mice did not differ from untreated animals. Similar results were obtained in mice with high titers of anti-FVIII antibodies. Further, untreated mice and mice tolerized to wild-type FIX were challenged with wild-type and mutant FIXs. While all untreated mice exhibited a strong immune response against all forms of FIX, no immune response was observed in mice tolerant to the wild type protein. Therefore, the proposed mutants do not break tolerance to the FIX.

Qasim W Dr Semenova E Dr Thrasher A Professor Larcher F Dr 2 Di W Dr

2 Ciemet, Madrid P 171 Preclinical modelling of ex-vivo skin gene therapy for Netherton syndrome

Defective expression of Lympho-epithelial Kazal-type-related inhibitor (LEKTI) gives rise to the inherited skin disorder Netherton syndrome (NS). The disease is characterised by a defective skin barrier with ichthyosiform erythroderma and is associated with severe dehydration and a high risk of mortality in early infancy. LEKTI is a secreted protein and we have shown that HIV-1 based lentiviral vectors encoding the SPINK5 transgene can correct the abnormal architecture of human skin grafted onto immunodeficient mice. Previous results showed notable correction of NS epidermal phenotype even in grafts where only small numbers of LEKTI expressing keratinocytes were detectable, raising the possibility of wider bystander benefit from the secreted protein. We have now revised the vector design to substitute retroviral promoter elements with sequences derived from the human Involucrin promoter . We show in three dimension organotypic cultures that the Involucrin promoter directs gene expression to the granular layer of the epidermis, the same compartment where LEKTI is normally expressed. Grafting of gene modified human NS skin onto nude mice showed revealed robust and durable correction of skin sheets. Interestingly vectors encoding the SPINK5 transgene in combination with the Involurcin promoter were less prone to methylation mediated gene silencing compared to vectors incorporating SFFV. These experiments demonstrate the feasibility of ex-vivo tissue modification and the engineering of grafts to correct inherited skin diseases.

Malgieri A Dr 1 Luchetti A Dr 2 Sanchez M. Dr 3 Bellocchi M. Dr 1 Spitalieri P Dr 1 Murdocca M Dr 1 Novelli G Professor 2 Sangiuolo F Professor 2

1 Department of Biopathology and Diagnostic Imaging, Tor Vergata University, Rome, Italy 2 Department of Biopathology and Diagnostic Imaging, Tor Vergata University, Rome, Italy 3 Istituto Superiore di Sanità, viale Regina Elena, Rome, Italy P 172 Restored SMN1 expression by lentiviral vector in murine Embryonic Stem Cells derived from Spinal Muscular Atrophy (SMAI) mice

Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by the loss of Survival Motor Neuron-1 gene (SMN1). Despite its ubiquitous expression SMN reduction cause the selective death of the alpha motor neurons (MNs). We explored the possibility of transducing by lentiviral vector (LVVs-GFP-hSMN1) two novel cells populations: murine Embryonic Stem Cells derived from SMA I mouse model and Neural Stem Cells (NSC) derived from spinal cord SMAΔ7 model. After 5 days of transduction, mES and NSCs cells were sorted by FACS, based on their GFP expression. Subsequently, molecular and biochemical analyses were performed such as RT-PCR, Western Blot and confocal immunofluorescence microscopy. The increase of SMN1 mRNA in each sample treated was estimated up to 2.5 fold in mES SMA cells and in SMA neurosheres respect to untreated cells. Immunoblotting and “gems” count (63 gems every 100 clones) confirmed these observations. Fluorescence-sorted cells (both mES and NSCs) were further differentiated in vitro into functional MNs, correctly expressing cell-specific markers (Pax6, Olig2, Isl1, Hb9) both in transduced and not transduced cells. Preliminary study in vitro and in vivo are in progress to implant mES and NSCs-derived motor neuron, lentiviral transduced, into SMA mice, in order to evaluate any possible recovery of the pathological phenotype. Teratogenic potential of these committed cells has been preliminary evaluated.

Lentiviral-mediated approach on stem cells represents an attractive source for cells replacement therapy in neurological disorders such as SMA.

Moscatelli I. Dr 1 Thudium C.S 2 Flores C. 1 Henriksen K. Dr 2 Richter J. Professor 1

1 Lund University, Sweden 2 Nordic Bioscience, Denmark P 173 A Human Model of IMO by Lentiviral Mediated shRNA Knock-down of Tcirg1 in CD34+ Cells

The overall aim of this study was to derive functional osteoclasts from CD34+ human cord blood cells and investigate whether lentiviral vectors can be used to overexpress and/or knock down genes in these cells.

First, human cord blood CD34+ cells were cultured with M-CSF, GM-CSF, IL-6, SCF and Flt3L for 2 weeks. They expanded 500 fold, and gradually lost CD34 expression while 50% of cells became CD14 + . Non-adherent cells were then incubated with M-CSF and RANKL on bone for 10 days. Osteoclasts derived from these cells expressed TRAP, released calcium, exhibited an actin-ring and generated resorption pits on bone slices.

Next, CD34+ cells were transduced on day 0 using a SIN lentiviral vector expressing GFP under a SFFV promotor at MOI of 5 resulting in GFP expression in 20-25% of the cells at day 2. Expression was retained throughout differentiation to functional osteoclasts.

Finally, a human model of infantile malignant osteopetrosis (IMO) was generated by transducing cord blood CD34+ cells using lentiviral vectors with the puromycin resistance gene and shRNAs targeting TCIRG1. After transduction and selection with puromycin cells were differentiated to mature osteoclasts. qPCR analysis and western blot revealed decreased Tcirg1 mRNA and protein levels. Osteoclasts had a 40% increase in TRAP expression whereas calcium release, pit formation and CTX-1 levels were all lower, in line with the known phenotype of IMO.

In conclusion the knock-down of TCIRG1 in CD34+ cord blood cells provides a human model of IMO necessary for our on-going gene therapy studies.

Rio P Dr 1 Agirre X Dr 2 Garate L Ms 2 Baños R Ms 1 Alvarez L Ms 1 José-Enériz E San Ms 2 Casado J.A Dr 1 Garin M Dr 1 Prosper F Dr 2 Bueren J Dr 1

1 Hematopoiesis and Gene Therapy Division.CIEMAT/CIBERER.Madrid.Spain 2 CIMA/Departamento de Hematología. Clínica Universitaria de Navarra. Pamplona P 174 Improved growth of Fanconi anemia hematopoietic progenitors by the expression of specific miRNAs

MicroRNAs are important regulators of gene expression. Initial studies from our laboratory in peripheral blood samples from FA-A patients showed that four microRNAs: hsa-miR-133a, hsa-miR-135b, hsa-miR-139, hsa-miR-181c are down-regulated, compared to expression levels determined in cells from healthy donors. Based on in silico studies and previous description of genes up-regulated in FA patients we focused our study in hsa-miR-181c, which has as possible targets TNF-α and IL-1β both of them previously described to be up-regulated in FA cells. Significantly, we observed that the ectopic expression of miRNA181c in FA LCLs decreased the expression of TNF-α in these cells, as determined by qPCR analyses while this was not the case for IL-1β. Similarly, the ectopic expression of this microRNA in BM samples from FA patients induced a modest, though consistent decrease in TNF-α expression, as deduced from flow cytometry studies. Strikingly, a marked increase in the generation of hematopoietic colonies was observed when BM cells from FA patients were transfected with microRNA181c. This effect was even higher to that observed after addition of ethanercept to these samples, suggesting that this microRNA may also interact with other negative regulators of the hematopoiesis in FA patients. Finally, luciferase activity assays showed that hsa-miR-181c can directly recognize the TNF-α 3′UTR, confirming the direct interaction between hsa-miR-181c and the TNF-α mRNA. Based on these observations we propose that the expression of miRNAs specifically down-regulated in cells from FA patients may constitute a new approach to improve the hematopoiesis, and probably other pathological manifestations of FA.

Yannaki E Dr 1 Psatha N Mrs 2 Sgouramalli E Mrs 2 Athanasiou E Dr 3 Bougiouklis D Mr 3 Arsenakis M Professor 4 Fassas A Dr 1 Anagnostopoulos A Dr 1

1 George papanicolaou Hospital, Gene and Cell Therapy Center, Hematology-BMT unit 2 George Papanicolaou hospital, Gene and Cell therapy Center/Aristotle University, Medical School, Dpt of Genetics, Development and Molecular Biology 3 George Papanicolaou Hospital, Gene and Cell Therapy Center 4 Aristotle University, Medical School, Dpt of Genetics, Development and Molecular Biology P 175 Hematopoietic stem cell mobilization with Mozobil (AMD3100) + G-CSF in a thalassemic mouse model

We have previously shown that G-CSF mobilization in the HBB^th-3 thalassemic mouse model is less efficient compared to normal C57Bl6 strain, mainly due to increased trapping of hematopoietic stem (Lin^-sca-1⁺ckit⁺–LSK) and progenitor cells (CFU-GM) in the enlarged thalassemic spleen. We explored here, whether the novel mobilizer Mozobil, either alone or in combination with G-CSF improves the mobilization efficiency of thalassemic mice.

HBB^th-3 mice received G-CSF (250microgr/kgX7days) or AMD (5mg/kgX3days) or their combination (AMD starting at day 5 of G-CSF administration).

Mozobil-alone didn't significantly affect the spleen size and increased the numbers of circulating CFU-GM as compared to G-CSF. The AMD + G-CSF combination resulted in significant improvement of the mobilization efficiency of HBB^th-3 mice over the G-CSF- and AMD-treated group in terms of blood LSK and CFU-GM, completely restoring their mobilizing capacity at levels comparable to G-CSF-treated normal mice. AMD mobilized stem and progenitor cells by “detaching” them from the bone marrow and probably the spleen, in contrast to G-CSF-induced marrow and splenic hyperplasia. Although splenectomy, as expected, restored the impaired G-CSF mobilization of thal mice at levels comparable to normal mice, it didn't further improve the AMD-or AMD + G-CSF mobilization thereby adding support to the concept that AMD may mobilize HSCs from extramedullary sites also.

The combination of AMD3100 + G-CSF restores the inefficient G-CSF mobilization in a thalassemic mouse model without further burden the hyperplastic bone marrow and spleen. This effect may prove beneficial in a gene therapy setting where high numbers of stem cells need to be safely collected for genetic correction.

Aliño SF Professor 1 Herrero MJ Mrs 1 Santamaria P Mr 1 Bodí V Dr 2 Montilla A Mrs 1 Miguel A Mr 1 Noguera I Dr 3 Sabater L Dr 4

1 Dpto Farmacologia, Fac Medicina, Univ Valencia-Spain 2 Hemodinámica, Hospital clínico Universitario, Valencia 3 SCSIE, Univ Valencia-Spain 4 Servicio Cirugía, Hospital Clínico Uniersitario, Valencia P 176 Catheter design and procedures for in vivo hAAT gene transfer to pig liver

Background: Gene transfer by hydrodynamic procedure in mice is now being translated to larger animals, like pigs, by means of a retrovenous injection with interventionist catheterism (hydrocatheterism). Many limitations are being found when transfecting the liver since this is a very big organ with a particular ability to absorb and by-pass great quantities of liquid. This work tries to clarify the best conditions to achieve pig liver transfection with the therapeutic human a1-antitrypsine (hAAT) gene.

Methods: Pigs were anaesthetized and catheterized through the jugular vein testing two conditions: a) 11F fitted catheter with 125ml injections to different neighbour small areas, b) 9-11F balloon catheter with 1-3 injections of 200ml to larger areas. Plasmid containing hAAT gene in saline solution (20μg/ml) was injected at 20ml/s. Plasma samples were taken for ELISA to determine hAAT protein.

Results: In catheter fitted model, the best results were obtained with a plasmid total concentration of 15-20mg/ml, regarding the whole volume injected, achieving up to 1mg/ml of hAAT protein in pig plasma. With this total concentration, in the balloon catheter experiments we obtained (11F catheter, 2 injections with 8 total mg of plasmid), 1.6mg/ml hAAT protein, maintaining this level at least 15 days post-injection.

Conclusion: We settled the best conditions (catheter type, perfusion flow, size of the territories involved and total plasmid concentration) amongst our different experiments. Despite this, 500 times higher protein production is needed to achieve therapeutic levels, so more studies are required to identify the barriers for a better transfection. Supported by SAF-2007-64492.

Garcia M Dr 1 Gostynski A 2 Llames S 3 Escamez MJ 1 Martinez-Santamaria L 1 Pasmooij M 2 Azon A 4 Mir S 5 Meana A 3 Larcher F 1 Jonkman M 2 Rio M Del 1

1 Regenerative Medicine Unit, CIEMAT and Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER U714) Madrid, Spain 2 Department of Dermatology, University Medical Center Groningen,University of Groningen, Groningen, The Netherlands 3 Tissue Engineering Laboratory, CCST-PA and Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER U714) Oviedo, Spain 4 Department of Dermatology, University Hospital Sant Joan de Reus, Reus, Spain 5 Plastic Surgery Department, Hospital Plato, Barcelona, Spain P 177 A pre-clinical cell therapy approach for RDEB with COL7A1 revertant keratinocytes

Revertant mosaicism, also referred to as “natural gene therapy”, has been described for a number of genetic diseases of haematopoiesis, muscle, liver and skin. Until recently revertant mosaicism had not been described for COL7A1. A Recessive Dystrophic Epidermolysis Bullosa (RDEB) patient homozygous for the frame shift mutation COL7A1: c.6527insC, a prevalent mutation with founder effect in Spanish RDEB families, exhibited presented with a patch of clinically healthy revertant skin. In revertant keratinocytes a second site mutation (c.6528delT) was present, resulting in correction of the reading frame and expression of wild-type type VII collagen polypeptide (Pasmooij et al, JID online 24 June 2010). Such keratinocytes can be used for autologous transplantation of “naturally corrected” cells to treat affected skin.

In this study we assessed the in vivocapacity of COL7A1 revertant keratinocytes to attain long-term skin regeneration in a pre-clinical setting. Transplantation of revertant keratinocytes as part of bioengineered skin equivalents onto immunodeficient mice resulted in the engraftment and sustained regeneration of disease-free human skin. Persistence of collagen VII protein at the dermo-epidermal junction 10 weeks post-grafting suggests that the therapeutic effect was achieved due to engraftment of long-lived revertant epidermal stem cells.

Our preliminary data represents a proof of principle of revertant cell therapy (natural gene therapy) for RDEB.

Herrero MJ Mrs 1 Mayol A Mrs 2 Sabater L Dr 2 Miguel A Mr 1 Montilla A Mrs 1 Sánchez M Mrs 1 Abargues R Dr 3 Aliño SF Professor 1

1 Dto Farmacologia, Fac Medicina, Univ Valencia-Spain 2 Servicio Cirugía, Hospital Clínico Universitario, Valencia-Spain 3 Instituo Ciencia Materiales, Univ Valencia-Spain P 178 Gene delivery and gold nanoparticle distribution in human ex vivo liver segments after catheterism

Background: In the translation of hydrodynamic gene transfer from mice to human application, different problems must be adressed, including molecular characterization of gene transfer and histological differences between species, that represent a barrier for the plasmid entry into cells.

Methods: Human hepatic segments from chirurgically fresh resected livers were obtained and weighed. A catheter was introduced through the suprahepatic vein, reaching an area as large as possible and inflating the catheter balloon to avoid the flow back. A volume of aprox.1/5 or 1/10 of the segment weight, of 20μg/ml plasmid solution containing the green fluorescent protein gene (EGFP) was injected at 10-20ml/s flow rate.The same procedure was performed, but injecting a solution of 15-20nm gold nanoparticles (1.8x10¹² Au particles/ml) for electron microscopy observations.

Results: EGFP gene expression was confirmed by fluorescent microscopy. RT-PCRq shows a better (more than one magnitude order) delivery index (exogenousDNA copies/100pg total DNA) at 20 ml/s flow rate but equivalent results of transcription index (exogenousRNA copy/100ng total RNA) employing either 20 or 10 ml/s, reaching a better intrinsec efficacy (exogenousRNA/exogenous DNA copies) with 10ml/s. Electron microscopy shows that after injection and fixation, the gold nanoparticles remain under or inside the endothelia but never reach the hepatocytes.

Conclusion: Milder conditions in flow rate during injection achieve better transfection results than stronger ones. However, the electron microscopy reveals that human endothelia does not behave like mouse and gold particles remain trapped in the vessels, not reaching the hepatocytes, due to its hermetic characteristics. Supported by SAF 2007-64492.

Daugaard L Ms 1 Luo Y Mr 1 Christensen R Ms 2 Jensen TG Professor 1

1 Department of Human Genetics, Aarhus University, DK-8000 Aarhus C, Denmark 2 Department of Clinical Genetics, Aarhus University Hospital, Aarhus Sygehus, DK-8000 Aarhus C, Denmark P 179 Microarray-based analysis of phenylalanine metabolism in retrovirus-transduced human keratinocytes

The aim of this study is to characterize the overall gene expression of cells with high levels of phenylalanine metabolism. Phenylalanine can be converted to tyrosine by phenylalanine hydroxylase (PAH). PAH requires a cofactor, tetrahydrobiopterin (BH₄). The first step in the BH₄ synthesis is catalyzed by GTP cyclohydrolase 1 (GCH1). Primary human keratinocytes co-transduced with PAH and GCH1 are capable of phenylalanine hydroxylation, as shown in previous studies. Co-transduction with GHC1 was required because overexpression of PAH alone did not lead to significant phenylalanine clearance.

In the present study, expression profiles of keratinocytes with increased phenylalanine metabolism were obtained using Affymetrix GeneChip HG-U133 Arrays. The data were normalized and filtered using Bioconductor packages. Compared to controls we identified 102 differentially expressed genes. GCH1 is known to take part in other processes as well as the phenylalanine catabolism. Therefore, by excluding genes differentially expressed by GCH1 alone and PAH alone we obtained a group of genes differentially expressed as a result of metabolically active PAH. This resulted in 42 remaining genes. Gene Ontology analysis revealed immunological response genes and carbohydrate binding genes to be upregulated as a result of transduction with PAH and GCH1 compared to the neomycin-resistance (neo) expressing controls. This result will be verified by qPCR on selected genes and we will investigate the effect on the phenylalanine metabolism of knocking down expression of selected genes.

Our results are important for future attempts to improve gene therapy strategies of phenylketonuria (PKU) where phenylalanine metabolism is impaired.

Bisgin A Dr Terzioglu E Professor Erbasan F Dr Aydin C Mrs Sanlioglu S Professor

P 180 The importance of sTRAIL in patients with rheumatoid arthritis

Background: TRAIL is an apoptosis-inducing agent in transformed cells but not in normal tissues [1]. It has been reported that TRAIL and TRAIL receptors might be involved in the pathophysiology of autoimmune diseases [2, 3]. The secreted form of TRAIL namely soluble TRAIL (sTRAIL) has recently been linked to the severity of SLE [4]. The goal of this study was to investigate whether or not a similar connection could be established in RA patients as well.

Methods: Serum sTRAIL levels of 40 RA patients and 20 healthy controls were measured by ELISA.

Results: Only those DMARD treated RA Patients (n = 20) had elevated levels of sTRAIL compared to those newly diagnosed RA patients who did not receive DMARDs and control group.

Conclusion: Serum sTRAIL levels were useful in distinguishing newly diagnosed RA patients and healthy control individuals from those who received DMARD treatment.

References

1 Sanlioglu A.D. et al. High levels of endogenous tumor necrosis factor-related apoptosis-inducing ligand expression correlate with increased cell death in human pancreas. Pancreas, 2008; 36,4:385–93. a-1771 2 Terzioglu E. et al. Concurrent gene therapy strategies effectively destroy synoviocytes of patients with rheumatoid arthritis. Rheumatology (Oxford), 2007; 46,5:783–9. a-1772 3 Sanlioglu A.D. et al. Molecular mechanisms of death ligand-mediated immune modulation: a gene therapy model to prolong islet survival in type 1 diabetes. J Cell Biochem, 2008; 104,3:710–20. a-1773 4 Lub-de Hooge M.N. et al. Soluble TRAIL concentrations are raised in patients with systemic lupus erythematosus. Ann Rheum Dis, 2005; 64,6:854–8. a-1774 Suchkova M.V Miss 1 Misyurin A.V Dr 1 Soldatova I.N Mrs 1 Stakhina O.V Mrs 1 Chelysheva EYU Dr 1 Turkina A.G Professor 1 Khoroshko N.D Professor 1 1 National Research Center for Hematology, Moscow, Russian Federation P 181 There are different subclones with differing mutations in patients with CML

Background: The bcr-abl cancer gene produces a protein that leads to chronic myeloid leukemia (CML). Point mutations within the ABL kinase domain of the BCR-ABL fusion protein are a major underlying cause of resistance. The aim is to show that there are different subclones, that, apparently, keep different mutations.

Method: We analysed 2 patients at the moment of detection simultaneously two mutations E255V, T315I. We are cloning product RT-PCR in pGEMTEasy plasmid vector and transformed in strain Escherichia coli DH5 alpha. The received clones analysed with PCR. PCR fragments were purified with 6% PAAG and then directly sequenced using the same forward and reverse primers.

At first One of the patients has only one mutation E255V on treatment imatinib. After translated him on second-line treatment TKIs, bosutinib, we detected T315I along with E255V. The patient has been translated on the combined therapy hydrea and interferon alpha. Mutation E255V comes to light only.

Results: The analysis of the received clones has shown presence only one of two mutations – E255V in either case.

Conclusion: We confirmed presence of the different subclones bearing different mutations. Different subclones are selectively destroyed in the course of therapy.

Hapil FZ Ms Bisgin A Dr Aydin C Mrs Undar L Professor 3 Sanlioglu AD Dr Sanlioglu S Professor

3 Department of Hematology of Akdeniz University Faculty of Medicine, Antalya, Turkiye 07058 P 182 The outcome of trail presentation on T cells in patients with multiple myeloma

Background: Multiple Myeloma (MM) develops due to the accumulation of malignant plasma cells in the bone marrow. Since TRAIL and TRAIL receptors are involved in cell death/survival pathways leading to carcinogenesis, TRAIL and TRAIL receptor profiles on tumor cells might serve as a prognostic marker for many cancer types [1, 2]. Cytotoxic T lymphocytes (CTLs) exhibit anti-tumor activity and are one of the major sources of TRAIL in blood. Thus, we hypothesized that an alteration or a defect in TRAIL expression might influence MM progression.

Methods: TRAIL expression profiles of T lymphocytes were investigated via EpicsAltra Beckman-Coulter flow cytometer, and statistical analysis was performed using SPSS.

Results: We first analyzed surface TRAIL expression on T cells of 16 sex and age matched control individuals. TRAIL expression was detected on both primary CD4 + helper T and CD8 + cytotoxic T cells where TRAIL expression on CTLs was higher than that of helper T cells.

Conclusion: Our next step is to recruit MM patients to analyze surface TRAIL expression on CTLs to check if the profile is connected to MM prognosis.

References

1 Sanlioglu A.D. et al. TRAIL death receptor-4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma. Int J Radiat Oncol Biol Phys, 2007; 69,3:716–23. a-1783 2 Koksal I.T. et al. Tumor necrosis factor-related apoptosis inducing ligand-R4 decoy receptor expression is correlated with high Gleason scores, prostate-specific antigen recurrence, and decreased survival in patients with prostate carcinoma. Urol Oncol, 2008; 26,2:158–65. a-1784 Aydin C Mrs Bisgin A Dr Sanlioglu AD Dr Pestereli E Professor Erdogan G Dr Ozbudak IH Dr Simsek T Professor 4 Sanlioglu S Professor 4 Department of Gynecology & Obstetrics, Akdeniz University Faculty of Medicine, Antalya, Turkiye 07058 P 183 Distinctive expression profiles of trail and trail receptors in patients with endometrial carcinoma

Background: Endometrial cancer is the most common gynecological malignancy and the fourth most common cancer type in women. TRAIL and TRAIL receptor profile has recently been tested for its potential to be used as a prognostic marker in cancer [1, 2]. However, how TRAIL and TRAIL receptors contribute to endometrial carcinogenesis is not known. Thus, we investigated TRAIL and TRAIL receptor expression profile during endometrial carcinogenesis to evaluate its potential as a prognostic marker and to predict the feasibility of TRAIL-mediated gene therapy approach.

Method: We analyzed TRAIL and all four TRAIL receptors in 100 patients with endometrial carcinoma including 18 normal endometrial tissue using immunohistochemistry. TUNEL assays were deployed to determine the cell death.

Results: Patients with endometrial carcinoma displayed a distinctive expression profile of TRAIL and TRAIL receptors compared to healthy controls. A decrease in TRAIL and DR4 death receptor expression but an increase in both of the decoy receptor expressions was detected.

Conclusion: Distinctive expression profile of TRAIL and its receptor expression in patients with endometrial carcinoma suggests that TRAIL signaling might be important during the endometrial carcinogenesis process.

References

1 Sanlioglu A.D. et al. TRAIL death receptor-4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma. Int J Radiat Oncol Biol Phys, 2007; 69,3:716–23. a-1786 2 Sanlioglu A.D. et al. High TRAIL death receptor 4 and decoy receptor 2 expression correlates with significant cell death in pancreatic ductal adenocarcinoma patients. Pancreas, 2009; 38,2:154–60. a-1787 Aydin C Mrs Bisgin A Dr Sanlioglu AD Dr Pestereli E Professor Erdogan G Dr Ozbudak IH Dr Simsek T Professor Sanlioglu S Professor P 184 Endometrial hyperplasia features distinctive trail and trail receptor expression profiles

Background: Endometrial cancer (EC) is known to be a common worldwide malignancy. It is often preceded by endometrial hyperplasia, whose management and risk of neoplastic progression vary. Currently, there is no reliable method to determine which hyperplasia will progress to cancer, due in part to the lack of reliable indicators of hyperplastic subvariants. Here we profiled TRAIL and its receptor expression to subcategorize endometrial hyperplasia as it is done with other types of hyperplasia [1] and/or cancer [2, 3].

Method: The expression pattern of TRAIL and its receptors in endometrial hyperplasia was revealed using immunohistochemistry. Apoptotic cell death was assessed using TUNEL assay.

Results: Hyperplastic lesions displayed higher levels of DR5, DcR1 and DcR2 expression but lower levels of DR4 compared to that of normal endometrium. Intriguingly, complex atypical hyperplasia samples exhibited higher levels of DR4 and DcR2 in comparison to complex hyperplasia.

Conclusion: TRAIL and TRAIL receptor expression exhibited distinctive expression pattern in normal versus hyperplastic lesions.

References

1 Sanlioglu A.D. et al. Differential expression of TRAIL and its receptors in benign and malignant prostate tissues. J Urol, 2007; 177,1:359–64. a-1788 2 Sanlioglu A.D. et al. TRAIL death receptor-4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma. Int J Radiat Oncol Biol Phys, 2007; 69,3:716–23. a-1789 3 Sanlioglu A.D. et al. High TRAIL death receptor 4 and decoy receptor 2 expression correlates with significant cell death in pancreatic ductal adenocarcinoma patients. Pancreas, 2009; 38,2:154–60. a-1790 Wang J Dr 1 Friedman G Mr 1 Doyon Y Dr 1 Wang N Mr 1 Li J Ms 1 Miller J Dr 1 Hua K Mr 1 Holmes M Dr 1 Gregory P Dr 1 1 Sangamo BioSciences, 501 Canal Blvd., Suite A100, Richmond, CA 94804 P 185 Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme

Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene addition, respectively. Here we report that ZFNs can be engineered to induce a site-specific DNA single-strand break (SSB) or nick. This nicked target site stimulates gene addition using a homologous donor template, but fails to induce the small insertions and deletions characteristic of repair via NHEJ. Gene addition by such zinc finger nickases (ZFNickases) occurs in both transformed and primary human cells at efficiencies of ∼1-20%. Introduction of a site-specific nick at an endogenous locus provides an important tool for the study of DNA repair. Moreover, the ability of a SSB to direct repair pathway choice (i.e. HDR but not NHEJ) may prove advantageous for certain therapeutic applications such as the targeted correction of human disease-causing mutations.

Appelt JU 1 Giordano FA 1 Jeltsch KS 2 Rausch T 3 Blake J 3 Koegler G 4 Maier P 5 von Kalle C 1 Bennes V 3 Laufs S 1

1 Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany 2 Clinical Cooperation Unit Molecular Oncology of Solid Tumors, DKFZ (German Cancer Research Center), Heidelberg, Germany 3 European Molecular Biology Laboratory, Heidelberg, Germany. 4 Institute for Transplantation Diagnostics and Cell Therapeutics, University Medical Center Duesseldorf, Germany 5 Department of Radiation Oncology, Mannheim Medical Center, University of Heidelberg, Mannheim, Germany P 186 Transcriptome Sequencing of Lentivirally Transduced Unrestricted Somatic Stem Cells

Gene therapy using integration-proficient retroviral vectors was found associated with flanking gene de-regulation. Expression analysis of genes located in genomic “windows” around insertion sites or transcriptional profiling using microarrays only focus on known or array-spotted genes, respectively.

To provide deeper insights into vector-related alteration of the transcription profile, we used mRNA of lentivirally transduced human unrestricted somatic stem cells (USSC) for ultra-deep shotgun sequencing (Genome Analyzer II; Solexa/Illumina).

Transcriptome sequencing yielded 2x10⁶ of mRNA sequence reads per sample (untransduced and transduced USSC). Reads were unambiguously aligned to the human genome (GRCh37 draft) using the TopHat software at recovery rates of nearly 90 %. Using the Cufflinks pipeline we detected a total of 42 345 individually transcripts in untransduced USSC and 42 555 in the transduced sample.

In parallel by linear amplification-mediated PCR (LAM-PCR) and 454 pyrosequencing we obtained a total of 34 924 insertion site sequences of the lentiviral vector in the genomic DNA of USSC. Insertion site analysis yielded a total of 5 528 non-redundant and 4 413 (78 %) intrageneic insertion sites. Thus, we were able to establish a detailed integration profile for lentiviral vectors in human mesenchymal stem cells.

One goal of further analysis is the assessment of the capability of lentiviral vectors activating adjacent genes by identifying vector-genome read-throughs. Thus, using the combination of transcriptome sequencing and high throughput insertion site analysis a detection of a vector-dependent alteration of the transcription profile lentivirally transduced versus non-transduced cells should be provided.

Ramirez JC Dr 1 Torres R Mr 1 Garcia A Ms 1 Garate Z Ms 1 Vijayaragavan K Dr 2

1 National Center for Cardiovascular Research (CNIC) Viral Vector facility 2 Cardiovascular Research (CNIC) Regenerative Cardiology Department P 187 Non-Integrative Lentivirus Drives High-Frequency cre-Mediated Cassette Exchange in Human Cells

Recombinant mediated cassette exchange (RMCE) is a second-order (two-step) process leading to genetic modification in a specific genomic target sequence. It is necessary for future development of regenerative and genetic therapies and for human developmental studies. The process involves insertion of a docking genetic cassette in the genome followed by DNA transfer of a second cassette. Although the minimal requirements are the presence of compatible recombination signals and expression of recombinase, efficiency relies at least on cell viability upon transfection, expression of the enzyme, and the ability to target cell types of different origins. To overcome such drawbacks, we developed an RMCE assay that uses an integrase-deficient lentivirus (IDLV) in the second step, thus avoiding undesired random integration of the vector sequences. We show that combining the lentiviral vector with promoterless trapping of double selectable markers during RMCE makes for an efficient, safe, and simple strategy with expected wide applicability. Additionally, recombinase that is encoded in the exchangeable cassette is self-limiting as a result of its own recombination, thus avoiding toxicity. Our approach is based on IDLV vectors and provides proof-of-principle of a novel strategy modeled on a human cell line with randomly integrated copies of a genetic landing pad. This strategy does not present foreseeable limitations for application to other cell systems modified by homologous recombination. Safety, efficiency, and simplicity are the major advantages of our system, which can be applied in low-to-medium throughput strategies for screening of cDNAs, non-coding RNAs during functional genomic studies, and drug screening.

Noske NN Mrs 1 Harms NH Mrs 1 Ehrhardt AE Mrs 1

1 Max von Pettenkofer-Institute, Pettenkofer Str.9a, 80336, Munich, Germany P 188 Design of novel PhiC31 Integrase fusion proteins for improving efficacy and safety of transgene insertion

Based on site-directed recombination the bacteriophage derived PhiC31 integrase system provides outstanding opportunities for transgene integration into the host genome. Due to unpredicted insertion events mediated by PhiC31, the optimization of activity and directing integration into specific target sites of the genome are important goals for the usage in therapeutic applications.

We constructed several fusion proteins of the integrase and different DNA binding domains (DBD) like the synthetic polydactyl zinc finger E2C and the AAV/Rep protein. The motifs were either fused N- or C-terminal to the integrase separated by a short TS- or a long GGS₅-linker. Immunoblot and flow cytometry analyses revealed that the fusion proteins were expressed in comparable amounts after transfection into 293-cells. Catalytic activity retained up to 80% of the wildtype integrase (WT) when binding motifs were attached to the C-terminus connected by the long interdomain. We also generated fusion mutants lacking the native DBD of the integrase (ΔDBD), which were inactive when measuring recombination efficiencies between WT PhiC31 integrase recognition sites. After performing initial colony forming assays to detect integration mediated by the ΔDBD fusion constructs, we observed 30% activity of the WT.

In our previous study we could identify critical amino acids of the integrase involved in the insertion process and also hyperactive variants showing 5.5-fold increased integration efficiency. Mutants lacking their native DNA binding properties may be restored by fusing to known DBDs. In combination with hyperactive PhiC31 variants this approach could lead to a more specific and efficient PhiC31 integrase system.

Schenkwein D. Ms 1 Turkki V. Mr 1 Parviainen S. Ms 2 Niskanen E. Dr 3 Lesch H. Dr 4 Määttä A-M Dr 4 Ahlroth M. Dr 5 Airenne K. Professor 6 Ylä-Herttuala S. Professor 7

1 Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, 70211 Kuopio, Finland, Ark Therapeutics Ltd, FIN-70210 2 Cancer Gene Therapy Group, Biomedicum, University of Helsinki, FIN-00290 Helsinki,Finland 3 Department of Biological and Environmental Science University of Jyväskylä Ambiotica FIN-40014 Jyväskylän yliopisto, Finland 4 Ark Therapeutics Ltd, FIN-70210 Kuopio, Finland 5 Suomen Reumaliitto Ry, FIN- 00120 Helsinki, Finland 6 University of Eastern Finland, PO Box 1627, 70211 Kuopio 7 Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, PO Box 1627, 70211 Kuopio, Finland P 189 Site-specific genome cleavage by a vector-incorporated HIV-1 integrase fusion protein

To meet the well established need for a gene transfer vector bearing a safe integration profile, several approaches are being studied, including the development of zinc finger nucleases and harnessing the mechanisms of DNA-modifying elements. Despite the good progress already achieved, a set of difficulties related to the safe yet efficient delivery of the DNA-modifying proteins and elements still remains. We have previously shown that lentivirus vectors (LVVs) can be modified to contain both cytotoxic and inert integrase (IN) fusion proteins. To test the feasibility of this cis- packaging system to promote cellular delivery of a site-specific meganuclease, we fused I-PpoI into the C-terminus of IN. The DNA cleaving ability of the IN-I-PpoI fusion protein was first studied in vitro, after which LVVs carrying the fusion protein were produced and tested in cultured cells. Vectors containing IN-I-PpoI were able to promote specific cleavage of I-PpoI's target site found in the nucleolar rRNA genes in two studied human cell lines. The vector-contained IN molecule profile was next optimized with regard to fusion protein derived cytotoxicity and integration efficiency to allow integration site selection (ISS) studies. Our results show that by altering the IN-composition of LVVs it is possible to 1) deliver a functional dimerization-requiring endonuclease into transduced cells nuclei 2) retain integration proficiency of the modified LVVs 3) affect the ISS of LVVs. These findings may prove useful in the quest for safe site-specific gene addition and genome modification, and show the potential of LVVs as diverse genome modifying tools.

Jensen NM Miss 1 Dalsgaard T Ms 1 Jakobsen M Ms 1 Jensen TG Professor 1

1 Department of Human Genetics, Aarhus University P 190 A novel assay for measuring gene repair efficiency in the mouse PAH gene

Targeted gene repair of single-base mutations can be used as an alternative to cDNA-based gene transfer. Promising techniques for this type of gene therapy include the use of Zinc Finger Nucleases (ZFNs) and Adeno-associated Viral Vectors (AAVs). PCR-based assays are currently used to screen the efficiency of gene repair - however the reliability of such assays can often be questioned due to possible PCR artifacts. Therefore, a highly sensitive non-PCR based assay which can be employed in a variety of cells is important for reliable establishment of the repair efficiencies. We are working on ZFNs and AAV specifically targeting the missense mutation in exon 7 of the active site in the phenylalanine hydroxylase (PAH) gene of phenylketonuria (PKU) mice (pah(enu2)). We have inserted 4 bp in exon 7 resulting in translational stop and disturbance of the reading frame. A fusion protein is produced where a GFP gene is fused to exon 1-6, the mutated exon 7 and a truncated exon 8 resulting in an assay where GFP is only expressed upon repair of the stop mutation by homologous recombination mediated by ZFNs or AAV. The construct is subsequently cloned into a Flp-vector which is applicable in a variety of cell types containing a Flp-site (Flp-cells). As only a single copy of the gene construct encoding the fusion protein is site-specifically inserted into these cells, it enables a simple way to compare various ZFN-pairs as well as AAV serotypes.

Dalsgaard T Mrs 1 Jensen N M Miss 1 Jensen T G Mr 1

1 Department of Human Genetics, University of Aarhus, Denmark P 191 Development of gene therapy of PKU

Phenylketonuria (PKU) is an inherited disease resulting in elevated levels of phenylalanine. In most patients this is due to defects in the enzyme phenylalanine hydroxylase (PAH). PKU positive children are treated with a lifelong diet with low concentrations of phenylalanine preventing the development of neurological and psychological changes especially intellectual impairment. The main goal of the project is to improve the current techniques in gene therapy and to target the PKU-disease.

We have constructed lentiviral vectors containing the functional PAH gene and a secreted reporter gene. These are injected in PKU mice to develop in vivo selection of PAH expressing cells.

We are testing site-specific zinc finger nucleases (ZFN) targeted against the PAH gene (adjacent to the disease causing mutation found in more than 50% of the Danish PKU patients). The ZFN leads to specific double stranded breaks stimulating homologous recombination. The ZFN´s are cotransfected with a donor plasmid containing the functional PAH gene. So far we have used the ZFNs to insert a selection marker gene in the PAH gene by non-homology end joining in the doublestranded break in tissue cultured mouse cells. We are currently using H2AX antibodies to measure the amount of double stranded breaks in ZFN transfected cells.

Bourdel A Miss 1 Jollet A Mr 1 Gouble A Dr 2 Pâques F Dr 2 Danos O Dr 1

1 INSERM U845, Universite Paris Descartes Hôpital Necker Enfants Malades, 156 rue de Vaugirard 75015 Paris 2 Cellectis SA, 102 route de Noisy, 93340 Romainville P 192 A quantitative luciferase assay for DNA double strand break-induced homologous recombination

Meganucleases (MN) are site specific endonucleases, with 12 to 45 bp DNA recognition sites. They generate double-strand breaks that can be repaired by homologous recombination if a DNA template is present. MN may be engineered for custom recognition of any genetic locus and used for gene targeting. Our interest is to develop efficient means of targeting transgenes at specific loci for the purpose of gene therapy.

Our working model is a single chain MN derived from I-CreI that recognizes a sequence within the human Rag-1 gene. Available systems for the detection of MN activity in mammalian cells are based on reporter genes (lac Z, GFP) inactived by a short duplication. The MN target sequence is placed in the middle of the duplication and upon double strand break, the reporter is repaired by recombination. We have constructed such a recombination detection system using luciferase as a reporter, in order to obtain a more quantitative assay that could also be used for detecting the MN activity after delivery into live animals. A duplication-inactivated thermostable Firefly luciferase gene was constructed and used in different assay format in human 293 cells. This luciferase-based system is fast, sensitive and quantitative, and will be useful for the detection of MN activity into experimental animals.

Turan S. Mr 1 Heinz N. Mr 1 Schambach A. Dr 1 Bode J. Professor 1

1 Hannover Medical School (MHH), OE6960-Experimental Hematology P 193 Chromosome-based vectors: Novel routes to predictable gene expression

The mammalian genome is organized into independently-regulated chromatin domains. Each domain is largely shielded from its surroundings and prevented from heterochromatization by bordering elements, most notably S/MARs. During recent years we could refine our gene transfer procedures to enable • Re-targeting of endogenous stable, highly expressed chromosomal loci after initial characterization (RMCE approach); increase efficiencies by the inclusion and strategic localization of S/MAR elements. • Design and verify novel sets of heterospecific 48 bp FRT sites to enable enhanced RMCE approaches like multiplexing (simultaneous targeting of two or more specified genomic loci) or the simultaneous deletion of several FRT-flanked (“flirted”) genomic inserts in the absence of unwanted genomic rearrangements. • Create replicating nonviral episomal units (minicircles, MCs) based solely on a transcribed gene and a single S/MAR. • Combine both procedures: create MC derivatives after their establishment in the nuclear architecture All our approaches obey the pFAR (free of antibiotic resistance) principle in a straightforward manner. We will discuss routes leading to the predictable establishment and differentiation of ES- and iPS cells.

References

Broll S , Oumard A , Hahn K , Schambach A , Bode J. 2010. Minicircle Performance Depending on S/MAR-Nuclear Matrix Interactions. J. Mol. Biol., 395,5:950–965. https://dx-doi-org.web.bisu.edu.cn/10.1016/j.jmb.2009.11.066 Turan S , Kuehle J , Schambach A , Baum C , Bode J . 2010. RMCE-multiplexing: versatile extensions of the Flp-Recombinase-Mediated Cassette-Exchange technology. J. Mol. Biol.In press. Shiau AL Professor 1 Chen SY Dr 2 Wang CR Dr 3 Wu CL Professor 2

1 Department of Microbiology and Immunology, National Cheng Kung University Medical College 2 Department of Biochemistry and Molecular Biology, National Cheng Kung University Medical College 3 Department of Internal Medicine, National Cheng Kung University Medical College P 194 Suppression of Collagen-Induced Arthritis by Intraarticular Lentiviral Delivery of TLR7 shRNA

Background: Knockdown or inhibition of the Toll-like receptor (TLR) pathway from the surface or intracellular receptors to downstream signaling molecules is a novel therapeutic strategy for the treatment of rheumatoid arthritis (RA). TRL7, the receptor of single strand RNA, is highly expressed in the RA joints. Here we examined the effects of lentiviral vectors expressing small hairpin RNA (shRNA) targeting TLR7 on amelioration of collagen-induced arthritis (CIA).

Methods: Lentiviral vectors expressing TLR7 shRNA (Lt.shTLR7) or scrambled TLR7 shRNA (Lt.scramble) were injected intraarticularly to rats with CIA and their treatment responses were monitored by measures of clinical, radiologic, and histologic changes. Microvessel density as well as levels of VEGF, pro-inflammatory cytokines, matrix metalloproteinases (MMPs), and T cell infiltrates in the synovial tissues of ankle joints were determined.

Results: Expression of TLR7 was upregulated in the ankle joint during the disease progression of CIA in rats. Treatment of Lt.shTLR7 significantly reduced ankle circumference, articular index scores, radiographic scores, and histologic scores, as compared with Lt.scramble treatment. Moreover, microvessel density, VEGF, IL-1β, and IL-6 contents, as well as T-cell infiltrates within the synovial tissue were significantly lower in the Lt.shTLR7-treated rats. Whereas the level of MMP-2 was unaffected by the Lt.shTLR7 treatment, the expression of MMP-9 was decreased in the Lt.shTLR7-injected ankle joints.

Conclusion: Our results demonstrate for the first time that disruption of intraarticular TLR7 signaling can suppress arthritis in rats with CIA. Our findings implicate manipulation of TLR7 molecule as a potential target in clinical therapy of RA patients.

Vogel MA Mr 1 Frank D Dr 2 Müller OJ Dr 1 Chorianopoulos E Dr 1 Katus HA Professor 1 Hecker M Professor 3 Frey N Professor 2

1 University Hospital Heidelberg, Internal Medicine III, Heidelberg, Germany 2 University Hospital Schleswig-Holstein, Department of Cardiology, Kiel, Germany 3 University of Heidelberg, Institut of Physiology and Pathophysiology, Heidelberg, Germany P 195 Gene therapy with AAV9-Calsarcin-1 avoids Angiotensin-II mediated contractile dysfunction

Cardiac remodelling caused by sustained pressure and/or volume overload leads to the development of cardiac hypertrophy. One molecular pathway that is involved in hypertrophic growth is mediated by the calcium- and calmodulin-dependent serine/threonine phosphatase calcineurin, which plays a key role in transducing calcium-dependent signals from the cytosol to the nucleus.

In previous studies we have shown that mice lacking Calsarcin-1 (CS1), a calcineurin-interacting protein, are sensitized to calcineurin signaling. Conversely mice overexpressing CS1 blunt hypertrophic growth when exposed to chronic angiotensin-II (Ang-II) infusion.

To use the protective effect of CS1 we first generate an adeno-associated virus serotype 9 (AAV9) that encompasses the CS1 full length coding sequence under the control of the 0.26kb cardiac myosin light chain promoter fused to the cytomegalovirus immediate-early enhancer (CMV(enh)/MLC0.26). AAV9-CS1 or a Renilla-luciferase control-AAV9 (AAV9-Ren) where systemically injected via the tail vain (dose 2x10¹¹ Vg) in 8-weeks-old male C57Bl/6 wild type (WT) mice. One week after intravenously AAV9 injection mice were subjected to long-term stimulation with Ang-II (1000ng/kg per min) or 0.9% NaCl respectively using subcutaneous minipumps for two weeks.

Ang-II stimulated and AAV9-Ren treated WT mice showed a contractile dysfunction with a significant reduction of the fractional shortening (FS) assessed by echocardiography and a strong trend to a dilation of the left ventricle. AAV9 mediated overexpression of CS1 leads in contrast to a complete removal of LV-dysfunction.

Taken together these results give a further hint that CS1 influence the cardiac remodelling after hypertrophic stimulation in a positive way.

Kang JK Miss 1 Malerba A Dr 1 Popplewell LJ Dr 1 Foster K Dr 1 Dickson GJ Professor 1

1 Royal Holloway, University of London, Egham, Surrey, TW20 0EX P 196 Antisensehyphen;induced myostatin exon skipping leads to muscle hypertrophy in mice following Octa-guanidine oligomer treatment

Myostatin is a negative regulator of muscle mass, and several strategies are being developed to knock down its expression to improve muscle wasting conditions. Use of anti-myostatin antibodies and natural binding partners, have resulted in increase in muscle mass in test animals but they have low sustainability. The strategy using short hairpin RNAs offers long term expression but is accompanied by viral vectors-associated risks of uncontrolled insertion into the human genome. In this study we report for the first time the use of antisense oligonucleotides (AOs) to manipulate myostatin pre-mRNA splicing by exon skipping. AOs of both 2′O-methyl RNA (with a phosphorothioate backbone) and Phosphorodiamidate morpholino (PMO) chemistries lead to the disruption of the reading frame of myostatin transcript in vitro through exon skipping as shown by RT PCR analysis of RNA from transfected C2C12 cells. This was exemplified by increased cell proliferation in treated cultures. Preliminary intramuscular studies in vivo showed that 2′O-methyl RNA and PMO AOs successfully induced exon skipping that was observed until after 4 weeks of treatment. Systemic in vivo studies using vivo-PMO (octa-guanidine PMO) showed myostatin exon skipping led to a significant increase in the mass and cross sectional area of soleus muscle of treated mice. AO-induced myostatin exon 2 skipping provides the potential for a controlled and safe therapy for the increase in muscle mass in muscle wasting conditions including diseases such as Duchenne Muscular Dystrophy as the dosage can be controlled, monitored and re-administered safely as the need arises.

Robert M-A Mr 1 Bendjelloul M Mr 1 Zeng Y Dr 1 Guérin C Mr 2 Larochelle N Dr 3 Massie B Dr 1 Nalbantoglu N Dr 3 Gilbert R Dr 1

1 Vecteurs de Génomique et de Thérapie Génique, Institut de Recherche en Biotechnologie du Conseil National de Recherche du Canada, Montréal, QC, Canada 2 Neuromuscular Research Group, Montreal Neurological Institute (McGill University) 3 Neuromuscular Research Group, Montreal Neurological Institute (McGill University), Montréal, QC, Canada P 197 Efficient muscle-specific expression after gene transfer with promoters derived from troponin I gene

Gene replacement therapy for muscle diseases using viral vectors requires strong and muscle specific expression of a therapeutic gene product. To achieve this goal, we generated regulatory cassettes by linking three (ΔUSEx3) or four (ΔUSEx4) copies of the truncated upstream enhancer (ΔUSE) of the slow troponin I gene. To evaluate the activity of ΔUSEx3 and ΔUSEx4 in the context of viral vectors, we constructed helper-dependent adenoviruses (HD) expressing β-galactosidase (β-gal) regulated by ΔUSEx3, ΔUSEx4 or the very potent hybrid CMV enhancer/β-actin (CAG) promoter. We also constructed AAV and lentiviral vectors (LV) expressing the green fluorescent protein (GFP) regulated by ΔUSEx3, ΔUSEx4, or CMV. The β-gal activity of HD-ΔUSEx3(β) and HD-ΔUSEx4(β) was 100 fold lower than HD-CAG(β) in non-muscle cell lines, but only 5 to 10 fold lower in differentiated myotubes. After intravenous delivery in mice, HD-ΔUSEx3(β) and HD-ΔUSEx4(β) were 100 fold weaker than HD-CAG(β) in the liver, lung and spleen. In contrast, after intramuscular injection in dystrophic (mdx) mice, the strength of our best muscle-specific HD (HD-ΔUSEx3(β)) reached 20-40% the strength of HD-CAG(β). Notably, under comparable experimental conditions, the transduction efficacy of AAV-ΔUSEx4(GFP) in mdx skeletal muscle was similar to AAV-CMV(GFP). In culture of non-differentiated myoblasts, GFP expression was 5 to 20 fold lower with LV-ΔUSEx3(GFP) compared to LV-CMV(GFP). However, after differentiation into myotubes, GFP expression became higher with LV-ΔUSEx3(GFP). In summary, the strength, muscle specificity and relative small size (<600-bp) of ΔUSEx3 and ΔUSEx4 render them very attractive for gene transfer application in skeletal muscle.

Supported by CIHR.

Kotimaa A Mr 1 Kärkkäinen A-M Ms 1 Huusko JM Mrs 1 Hämäläinen E Ms 1 Heinonen SE Mrs 1 Kholova I Dr 1 Dijkstra MH Ms 1 Stedt H Ms 1 Mäkinen PI Mr 1 Turunen M Dr 1 Ylä-Herttuala S Professor 1

1 Neulaniementie 2 P 198 VEGF-D transgenic mice created by lentiviral transgenesis method

Vascular endothelial growth factor D (VEGF-D) contributes to the growth and formation of blood- and lymphatic vessels, and has shown promising results in gene therapy in ischemic conditions. However, the normal physiology of VEGF-D is still somewhat unclear, since the knockout mice showed no clear phenotype.

Transgenic mice expressing the mature form of human VEGF-D (hVEGF-D^ΔNΔC) under the constitutive human phosphoglyserate kinase (hPGK) promoter were produced. In order to study the targeted expression of VEGF-D in epithelium only, also transgenic animals expressing hVEGF-D^ΔNΔC under the mouse endothelial promoter Tie1 were created. The transgenic mice were generated by lentiviral perivitelline injection technique where the zona pellucida covering the oocytes is left in place. This results to a significantly higher number of implanted embryos and transgenic founders compared to traditional plasmid microinjection.

Transgenic mice were followed up to F3-F5. The VEGF-D transgenic mice showed enhanced blood capillary density and improved post ischemic muscle regeneration. No major changes in lymphatic capillary density were found, which suggests that hVEGF-D^ΔNΔC has no lymphangiogenic effect. The transgenic mice showed an increased susceptibility to tumor formation and a reduced life span. The expression of transgene was observed in several tissues; however the transgene expression decreased in advanced generations. When the expression was targeted to the epithelium only, no lymphangiogenic effects were detected either but more different tumor types were observed. Lentivirus perivitelline injection has proven to be a very efficient transgenesis method. VEGF-D could be a potent factor in gene therapy for treating ischemic diseases.

Yilmazer AY Ms 1 Al-Jamal WAJ Dr 1 Van den Bossche JVB Dr 1 Kostarelos KK Professor 1

1 Nanomedicine Laboratory, Centre for Drug Delivery Research,The School of Pharmacy, University of London, London WC1N 1AX, United Kingdom P 199 The Interaction between Artificially Enveloped Adenovirus and Blood Components

Gene therapy with human adenovirus type 5 (Ad5) has been extensively explored for the treatment of diseases resistant to traditional therapies. However, following systemic administration of Ad, the resulting innate and adaptive immune responses dramatically affect the kinetics and toxicity profile of the vector. Building on our previously reported technology of artificial envelopment of Ad, we designed a variety of artificial lipid bilayer envelopes around the viral capsid in order to develop safe and efficient Ad vectors for gene therapy. Considering the critical role of host blood cells and plasma proteins in systemic administration of Ad, we studied the interaction of enveloped Ad in cationic non-pH-sensitive (DOTAP:Chol) or anionic pH-sensitive (DOPE:CHEMS) lipid bilayers with several different blood components. In vitro experiments showed that uptake of DOTAP:Chol-enveloped Ad in HepG2 cells was not affected by the presence of any blood factors, whereas treatment with blood factor X, but not IX or XI, increased cellular uptake and gene expression levels of DOPE:CHEMS enveloped Ad. Different blood cells were isolated from human or Balb/c mouse blood and their interactions with artificially enveloped Ad were investigated ex vivo. Artificial envelopment of Ad with DOTAP:Chol bilayers resulted in significantly higher binding to human and murine red blood cells (RBC) compared to DOPE:CHEMS envelopes, which highlighted the importance of the characteristics of the lipid envelopes. These results suggest that artificial envelopment of Ad dramatically alters the affinity towards blood components that will affect the virus tissue-distribution following systemic administration.

P 200 Evaluation of AAV6-microutrophin expression in dystrophic mouse models and the non-human primate

Abstract Withdrawn

Varadi K. Mr 1 Michelfelder S. Dr 2 Trepel M. Dr 2 Katus H. Professor 3 Kleinschmidt J. Dr 1 Müller O. Dr 3

1 Department of Tumor Virology, German Cancer Research Center, Heidelberg, Germany 2 Department of Molecular Oncology, University of Hamburg-Eppendorf, Hamburg, Germany 3 Internal Medicine III, University Hospital Heidelberg, Heidelberg, Germany P 201 Efficient transduction of human umbilical veins ex vivo using vectors selected from an AAV9 library

In our previous studies we have shown the potential of an AAV2 random peptide display library to select for AAV2 vectors with improved efficiency for endothelial gene transfer. A lower prevalence of neutralizing antibodies in humans and a more efficient gene transfer in several animal models, however, might render AAV9 more suitable for this purpose than AAV2.

Hence, our goal was the generation of an AAV9-based randomized heptapeptide library to select for efficient and specific gene transfer vectors on human endothelium.

Using a highly diverse plasmid library (1.25E + 9 motives) harboring randomized oligonucleotides in the AAV9 cap gene, we generated an AAV9 peptide display library, which ensures that the displayed peptides correspond to the packaged genomes. The library was biopanned for 4 rounds on human coronary artery endothelial cells (HCAEC) and transduction efficiencies of AAV9 vectors displaying the enriched peptide motives were compared to wtAAV9. While wtAAV9 resulted in a transduction efficiency of 2.3 ± 1.6%, motives selected from the AAV9 library enabled efficiencies up to 92.8 ± 3.1% (p < 0.001). In order to analyze the potential of endothelial-targeted vectors to transduce endothelial cells in the vascular context, we incubated human umbilical veins in situ with wt or mutant AAV9 vectors. FACS analysis of endothelial cells isolated from these umbilical veins (HUVEC) revealed an improvement of transduction efficiency from 0.4 ± 0.7% to 80.3 ± 11.9%, respectively (p < 0.001).

In summary, selection of our AAV9 library has yielded motives with strong transduction of HCAEC in vitro and HUVEC ex vivo, rendering these vectors candidates for future clinical approaches.

Wu CL Professor Chen SY Dr Shiau AL Professor Wang CR Dr

P 202 Amelioration of Collagen-Induced Arthritis through Apoptosis of CD4+T Cells and Reduction of Synovial IL-17 Production

Background: Indoleamine 2,3-dioxygenase (IDO), one of the initial and rate-limiting enzymes involved in the catabolism of tryptophan, has immunomodulatory activities and is an emerging therapeutic target in autoimmunity-related arthritis. Here we investigated the therapeutic effect IDO gene delivery on collagen-induced arthritis (CIA) in rats.

Methods: The reatment responses of adenoviral vectors encoding IDO (AdIDO) in the arthritic rats were examined. The therapeutic effects of AdIDO on ankle circumferences, articular index, and radiographic and histological scores were evaluated in the treated rats. We further investigated the underlying mechanism of action.

Results: Reduction of ankle circumferences, articular index, and radiographic and histological scores were observed in AdIDO-treated ankles, as compared with those injected with control vectors. Furthermore, IDO gene transfer led to decreased infiltrating CD4⁺ T cells with enhanced apoptosis, reduced CD68⁺ macrophage numbers, increased kynurenine (a downstream tryptophan metabolite) levels, lower IL-17 concentrations, and decreased expression of RORγt, a transcription factor important for the development of Th17 cells, within the ankle joints. In addition, IDO gene delivery diminished type II collagen-specific IL-17 production and RORγt expression in CD4⁺ T cells from draining lymph nodes of CIA rats.

Conclusion: Our results demonstrate for the first time that intraarticular delivery of IDO gene ameliorates arthritis symptoms in rats with CIA by induction of CD4⁺ T cell apoptosis and reduction of synovial IL-17 production through the supplement of kynurenine. Taken together, these findings implicate IDO gene delivery as a therapeutic strategy for the treatment of patients with rheumatoid arthritis.

Suga T Mr 1 Kimura E Mr 1 Morioka Y Ms 2 Ikawa M Mr 2 Uchino K Mr 1 Koide T Mr 1 Uchida Y Mr 3 Yamashita S Mr 1 Maeda Y Mr 1 Li S Mr 4 Chamberlain JS Mr 4 Uchino M Mr 1

1 Department of Neurology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan 2 Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 3 Laboratory of Pharmacology, Division of Life Science, Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan 4 Department of Neurology, University of Washington School of Medicine, Seattle, WA, USA P 203 Muscle fiber type-predominant promoter activity in lentiviral-mediated transgenic mouse

Variations in gene promoter/enhancer activity in different muscle fiber types after gene transduction was noticed previously, but poorly analyzed. The murine stem cell virus (MSCV) promoter drives strong, stable gene expression in hematopoietic stem cells and several other cells, including cerebellar Purkinje cells, but it has not been studied in muscle. We injected a lentiviral vector carrying an MSCV-EGFP cassette (LvMSCV-EGFP) into tibialis anterior muscles and observed strong EGFP expression in muscle fibers. We also generated lentiviral-mediated transgenic mice carrying the MSCV-EGFP cassette and detected transgene expression in striated muscles. MSCV promoter activity in skeletal muscle is fiber-type-dependent when delivered directly by lentiviral infection as well as in transgenic mice generated by lentiviral infection. Further analysis of this promoter may have the potential to achieve certain gene expression in severely affected muscles of mdx mice. The Lv-mediated transgenic mouse may prove a useful tool for assessing the enhancer/promoter activities of a variety of different regulatory cassettes.

Ksienzyk J Mr 1 Raake P Dr 1 Schlegel P Mr 1 Shan C Dr 1 Weber C Mr 1 Kleinschmidt JA Dr 2 Katus HA Professor 1 Müller OJ Dr 1

1 University Hospital Heidelberg, Internal Medicine III 2 German Cancer Research Center, Applied Tumor Virology P 204 In vitro aided identification of AAV6 as the best vector for gene transfer into porcine myocardium

Background: Adeno-associated virus (AAV) mediated gene transfer into diseased myocardium holds high promises. While efficient transfer is achieved in rodents, gene delivery to the hearts of larger animals appears to be limited. Our aim was the development of in vitro models to facilitate vector development for cardiac gene transfer in large animals and for future clinical trials.

Methods: To identify suitable models, luciferase reporter constructs driven by the CMV-promoter have been crosspackaged into the capsids of AAV serotypes 1-6, 8 and 9. These constructs have been tested in cardiomyocytes and/or organotypic myocardial slices of mice, rats and pigs. We then analyzed porcine myocardial gene transfer with the two most promising vectors and AAV9 after application via retroinfusion into the coronary veins.

Results: Cardiomyocytes and organotypic slices gave very similar results showing strong luciferase activity and pronounced variation between the serotypes. Comparison with published in vivo data revealed a good correlation for AAV1 to 6 but not for 8 and 9 which partially failed in vitro. Comparison of reporter activities in the pig in vivo revealed that AAV6 enables the most efficient cardiac gene transfer followed by AAV5, while AAV9 was inefficient, confirming the predictions of our model.

Conclusion: We have developed predictive models for the efficiency of cardiac gene delivery, enabling strongly improved transgene delivery to the porcine heart. The correlation with in vivo data underlines the feasibility of both models for vector assessment. The predictions unveiled two AAV serotypes more suited for porcine myocardial gene transfer than AAV9.

Koo TY Dr 1 Shin JH Dr 2 Athanasopoulos T Dr 1 Takashi O Dr 2 Foster H Dr 1 Shin'ichi T Professor 2 Dickson G Professor 1

1 The Biological Sciences, Royal Holloway University of London, Egham, Surrey, UK 2 National Centre of Neurology and Psychiatry, Tokyo, Japan 2 National Centre of Neurology and Psychiatry, Tokyo, Japan P 205 Canine microdystrophin results in remarkable expression and improvement in muscle pathology in a canine model of Duchenne Muscular Dystrophy following AAV gene transfer

Duchenne muscular dystrophy (DMD) is a severe inherited muscle-wasting disorder caused by mutations in the dystrophin gene. Recombinant adeno-associated virus (rAAV)-mediated gene therapy to transfer dystrophin genes into muscle has been attempted in mouse and dog models of DMD. However, low and transient expression of dystrophin in dog models suggests that improved functional microdystrophin configurations are essential and need to be tested in large animal models prior to clinical trials for DMD patients. In this study, we generated recombinant adeno-associated viral pseudotyped 2/8 (rAAV2/8) vectors expressing a canine-specific and codon-optimized microdystrophin driven by a muscle-synthetic promoter (Spc5-12). The microdystrophin configurations were subjected to comparative evaluation by intramuscular injection of 9 weeks old Canine X-linked Muscular Dystrophy (CXMD) dogs. Dystrophin expression was exhibited in approximately 50% to 80% of all injected muscles and displayed functional association with the dystrophin associated Protein (DAP) Complex. Moreover, codon-optimized canine-specific microdystrophin improved muscle function and protected the muscle from damage. Our results suggest that administration of canine-specific codon optimized microdystrophin can increase microdystrophin expression in a large animal model of DMD, requiring reduced titers of gene transfer vectors enhancing the objective of developing safe and effective muscle gene therapy protocols appropriate to move forward towards clinical trials.

Trani M. Miss 2 Mujagic E. Dr 1 Gianni-Barrera R. Dr 2 Gürke L. Professor 1 Heberer M. Professor 2 Wolff T. Dr 1 Banfi A. Dr 2

1 Vascular Surgery, Department of Surgery, Base University Hospital 2 Cell and Gene Therapy, ICFS, Department of Biomedicine, Basel University Hospital P 206 Dose-dependent angiogenesis by VEGF is species-specific

Background: Therapeutic angiogenesis by vascular endothelial growth factor (VEGF) gene delivery is a promising approach for the treatment of ischemic diseases. However, over-expression of murine VEGF₁₆₄ can induce either normal or aberrant angiogenesis depending strictly on its dose in the microenvironment around each producing cell in vivo. On the other hand, the dose-dependent effects of human VEGF₁₆₅, which is the real target of therapeutic angiogenesis approaches, are currently unknown. Therefore, here we took advantage of a highly controlled cell-based gene delivery platform to rigorously determine the in vivo dose-dependent effects of hVEGF₁₆₅.

Methods: Primary mouse myoblasts were retrovirally transduced to express either mVEGF₁₆₄ (mV) or hVEGF₁₆₅ (hV). Clonal populations were then isolated that homogeneously produced specific levels of each VEGF and implanted into skeletal muscles of SCID mice.

Results: Four weeks after implantation, low levels (35-40 ng/10⁶ cells/day) of either factor induced only normal angiogenesis. However, while high levels (130 ng/10⁶ cells/day) of mVEGF₁₆₄ caused widespread aberrant structures, as expected, similar or even higher levels (130 and 250 ng/10⁶ cells/day) of hVEGF₁₆₅ induced only normal capillaries and angiomas were never observed. All expression levels were confirmed by ELISA and qRT-PCR, both in vitro and in vivo over 2 weeks.

Conclusions: Further studies are ongoing to determine the underlying mechanism, e.g. differential potency in activating VEGFR-2. Nevertheless, these results suggest that murine pre-clinical models cannot accurately predict the therapeutic window of hVEGF₁₆₅ in patients and highlight the need to test strategies for microenvironmental dose control in rigorous stage I clinical trials.

Makarevich PI Dr 1 Tsokolaeva ZI Dr 2 Shevchenko EK Mr 3 Beloglazova IB Dr Shevelev AYA Dr Vlasik TN Dr Parfyonova YEV Professor 4 Tkachuk VA Professor 5

1 pavel.makarevich@gmail.com 2 tsokolaevazoya@mail.ru 3 mazhorrr@gmail.com 4 yeparfyon@mail.ru 5 tkachuk@fbm.msu.ru P 207 Peripheral artery disease; combined approach in therapeutic angiogenesis

Peripheral artery disease (PAD) is one of leading causes of disability in Western societies. Gene and cell therapy can be used to induce angiogenesis in ischemic skeletal muscle to prevent major necrosis and amputations in affected patients. Benefit from gene transfer and cell transplantation was shown in many experimental and clinical studies but development of a novel combined approach in this field is still an important point in medical science.

Our group focused on animal testing of several plasmid constructs with human VEGF165, HGF, angiopoietin-1 and mouse uPA to find combinations which could amplify angiogenic efficacy of gene transfer. We found that co-transfer of 2 growth factors (VEGF + HGF, VEGF + Ang-1 or HGF + Ang-1) renders a higher angiogenic effect than sole plasmids, which was shown by laser Doppler imaging and histologic methods.

Another important point assessed in our studies was use of AAV-transduced adipose derived stromal cells (ADSC) for induction of angiogenesis in mouse ischemic limb and Matrigel plug model. Recombinant AAV was used to induce overexpression of VEGF165 in human ADSC and modified cells showed higher angiogenic efficacy than GFP-transduced or unmanipulated ones. Cell transplantation gave a higher and more stable increase in limb perfusion than plasmid gene transfer. Our findings indicate that gene and cell therapy efficacy in PAD might be increased using combined methods: 1) gene transfer of several growth factors, 2) modified cells transplantation or, possibly, using both which is still to be tested in appropriate animal models.

Akizawa Y Dr 1 Kanno H Dr 2 Kawamichi Y Dr 1 Hamada T Miss 3 Matsuda Y Professor 4 Ohta H Professor 5 Matsui H Professor 4 Fujii H Professor 6 Saito K Professor 3

1 Institute of Medical Genetics and Department of Gynecology and Obstetrics, Tokyo Women's Medical University, Tokyo, Japan 2 Institute of Medical Genetics and Department of Transfusion Medicine and Cell Processing, Tokyo Women's Medical University, Tokyo, Japan 3 Institute of Medical Genetics, Tokyo Women's Medical University, Tokyo, Japan 4 Department of Gynecology and Obstetrics, Tokyo Women's Medical University, Tokyo, Japan 5 Sanno Medical Center, International University of Health and Welfare, Tokyo, Japan 6 Department of Transfusion Medicine and Cell Processing, Tokyo Women's Medical University, Tokyo, Japan P 208 MYOD1-transduced placental MSCs: The feasible cell therapy of neuromuscular disorders

Background: Mesenchymal stem cells are one of the somatic stem cells, which could be induced into several tissues derived from mesoderm. We have shown that human dystrophin was able to be expressed in the muscle of mdx-scid mice after local administration of human placenta derived mesenchymal stem cells (hpMSCs) (Kawamichi et al. 2010). In order to enhance the myogenic properties of hpMSCs, we introduced human MYOD1 cDNA into hpMSCs using the lentiviral vector, and examined how the muscle-specific gene expression was enhanced.

Methods: The MYOD1 cDNA and pLenti6/UbC/V5-DEST were obtained from the RIKEN and Invitrogen, respectively. Amniotic mesoderm derived cells (AMCs) were isolated from human preterm placentas. AMCs were transduced with the MYOD1 vector, and total RNA was extracted after 7days culture. The mRNA levels for MYOD1, MYF5, MYOG and DMD were determined by qPCR using the Universal Probe Library system (Roche). Relative gene expression levels were calculated using the normal human skeletal muscle myoblast cells (Lonza) as a control.

Results: The mRNA levels of untreated and MYOD1-transduced hpMSCs as follows: MYOD1, 0.101 ± 0.053% and 4.77 ± 2.02%; MYF5, 0.013 ± 0.001% and 5.61 ± 3.82%; MYOG, &!lE; 0.000% and 0.125 ± 0.006%; DMD, 2.76 ± 1.01% and 19.9 ± 5.9%.

Conclusion: Myogenic factor 5, myogenic differentiation 1 and myogenin are the essential transcription factors during the skeletal muscle development, and designated as the myogenic regulatory factors (MRFs). In this study, we showed the simultaneous upregulation of the MRF genes as well as DMD in the MYOD1-transduced hpMSCs, suggesting that the MYOD1-hpMSCs seem to be a promising tool for cellular therapy for neuromuscular disorders.

Pettersson OJ Mr 1 Larsen J Mr 1 Thomsen R Mr 1 Damgaard CK Dr 2 Corydon TJ Dr 1 Jakobsen MV Ms 1 Jensen TG Professor 1

1 Aarhus University, Department of Human Genetics, Wilhelm Meyers Allé 4, DK-8000 Aarhus C 2 Aarhus University, Department of Molecular Biology, C. F. Møllers Allé 3, DK-8000 Aarhus C P 209 A new model for DM1 based on the genetic reprogramming of primary fibroblasts to myocytes

Background: Type 1 myotonic dystrophy (DM1) is the most prevalent form of muscular dystrophy among adults. The disease is characterized by expanded CTG-repeats in the myotonic dystrophy protein kinase (DMPK) gene leading to accumulations (foci) of mutant mRNA in the nucleus. These accumulations of expanded repeats of the mutant mRNA lead to altered activity of splicing factors, resulting in several events of miss-splicing ^{1 –3} . In order to investigate novel treatment modalities (oligonucleotides ^4–5 and siRNA ⁶ ) we have used lentiviral delivery of the gene myoD ⁷ to convert human primary dermal DM1-fibroblasts to myocytes.

Methods: Cells were analyzed by immunostaining for myoD and myogenin and in situ hybridization FISH with a Cy3-labeled (CAG)10-repeat probe targeting the expanded CUG-repeat.

Results: RNA FISH revealed 2.4 ± 1.4 foci and 3.2 ± 1.6 distinct foci per cell, respectively, in 2 different DM1 cell lines whereas no foci were present in normal cells. MyoD-transduced cells were myoD- and myogenin positive. Interestingly, RNA foci could be found both in the nucleus and cytoplasm. Currently, we are investigating the possible co-localization of RNA foci with enzymes involved in RNA degradation.

Conclusion: MyoD-induced myogenesis can be used to study the pathogenesis of DM1.

References

1 Kaliman P et al. 2008. Cell Signal, 20:1935–41. a-1943 2 Taneja KL et al. 1995. J Cell Biol, 1995; 128; 6:995–1002. a-1944 3 Davis BM et al. 1997. Proc Natl Acad Sci U S A, 94,14:7388–7393. a-1945 4 Wheeler TM et al. 2009. Science, 325:336–339. a-1946 5 Mulders SA et al. 2009. Proc Natl Acad Sci USA, 106:13915–13920. a-1947 6 Langlois MA et al. 2005. J Biol Chem, 280:16949–16954. a-1948 7 Davis RL et al. 1987. Cell, 51:987–1000. a-1949 Freudenthaler H Dr 1 Küpcü S Dr 2 Gornikiewicz A Dr 1 Zommer A Mrs 1 Sára M Dr 2 Sleytr U Professor 2 Brostjan C Dr 1 1 Medical University of Vienna, Department of Surgery, General Hospital of Vienna, Austria 2 University of Natural Resources and Life Sciences, Department of NanoBiotechnology, Vienna, Austria P 210 Targeted gene transfer to proliferating endothelium: viral versus non-viral approaches

Specific transfer of “therapeutic genes” into proliferating endothelial cells would constitute a valuable tool for the treatment of disorders involving the sprouting of new blood vessels such as tumor angiogenesis. Thus, specific endothelial surface molecules induced during the process may serve to target gene transfer vectors to the proliferating endothelium. We have focused our approach on E-selectin (CD62E), an activation marker largely restricted to endothelial cells in angiogenic and inflammatory settings, and we have compared viral versus non-viral vector systems for targeted gene transfer. The viral approach involves a chimeric retrovirus, incorporating a single chain antibody fragment directed against CD26E in the retroviral envelope protein to mediate host cell specificity and entry. Envelope proteins were derived from Mo-MLV, SNV and 4070A retrovirus. The non-viral gene transfer variant was based on particles of cationic liposomes and plasmid DNA covered by a chimeric S-layer-streptavidin fusion protein engineered to bind the E-selectin antibody via a biotin-streptavidin bridge. Both systems were tested in vitro with proliferating human microvascular endothelial cells (stimulated to express E-selectin) for selective transfer of a reporter gene. In either case, target specificity was attained but resulted in a considerable reduction of gene transfer efficiency as compared to unmodified viral or liposomal particles. Both systems are currently further tested for stable versus transient trans-gene expression and for modifications to increase transfer efficiency.

Jungmann AJ Dr 1 Welzel MW Mrs 1 Ogris MO Dr 2 Wagner EW Professor 2 Katus HAK Professor 1 Hecker MH Professor 3 Müller OJM Dr 1

1 University of Heidelberg, Cardiology, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany 2 Pharmaceutical Biotechnology, Center for System-based Drug Research, Department of Pharmacy, Butenandtstraße 5-13, Building D, 81377 München, Germany 3 University of Heidelberg, Institut of Physiology and Pathophsiology, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany P 211 Biotinylated AAV as tool for attaching nucleic acids on the capsid surface

Background: Adeno-associated viruses (AAV) enable an efficient transduction of myocardium. Aim of our study was to develop an approach for cardiac delivery of nucleic acids attached to the AAV surface combining viral and non-viral gene transfer techniques.

Methods: We loaded plasmid DNA on polylysin coupled to streptavidin. These DNA/polylysin complexes were attached to AAV2 and AAV9 vectors genetically biotinylated using a biotin acceptor protein (BAP) motif within the cap-sequence of AAV and cotransfection of biotin ligase within the vector production.

Results: Complete biotinylation decreased vector tropism and efficacy. Thus, we determined the amount of biotinylation of capsids that enables the best compromise between preservation of AAV tropism and transfer efficiency of plasmid-DNA by comparing different ratios of wildtype (wt) and BAP-modified capsids for vector production. Plasmid DNA encoding a red fluorescent reporter protein was linked via streptavidin to the vectors harboring an EGFP reporter gene within their genome. A ratio of one biotinylated capisd per three wt capsids showed the highest transduction efficacy and plasmid transfer in HEK293 cells up to 10% transfer.

For in vivo delivery, biotinylated AAV9 vectors were used for packaging a luciferase reporter. 4 weeks after injection in adult mice, luciferase expression in representative organs was determined. Although the in vivo cardiac tropism of AAV9 wt decreased after biotinylation by about 50%, the heart was still the main target.

Conclusion: Conditions were established for transfer of plasmid-DNA via biotinylated AAV2 in HEK293 cells. Genetically biotinylated AAV9 enabled a preserved cardiac transduction pattern of myocardium in vivo.

Ruotsalainen J Mr 1 Martikainen M Mr 1 Vuorinen K Dr 1 Vähä-Koskela M Dr 2 Jääskeläinen J Professor 3 Bell J Professor 2 Laakkonen P Professor 1 Hinkkanen A Professor 1

1 A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland 2 Ottawa Health Research Institute, Ottawa, Canada 3 Neurocenter, Kuopio University Hospital, Kuopio, Finland P 212 Semliki Forest virus VA7 infects human glioma derived slice cultures and cell explants whereas having only limited replication in healthy macaque brain tissue

Background: Semliki Forest virus (SFV) is a non-pathogenic alphavirus, which replicates efficiently in a number of different cancer cell lines. In our recent study intravenously administered SFV VA7 completely eliminated subcutaneous and intracranial U87 human glioma xenografts in nude mice. Here we demonstrate the susceptibility of a human malignant glioma biopsy derived tissue slice culture and a cancer stem cell explant culture to VA7 mediated infection and oncolysis, and limited replication in healthy Macaque brain tissue culture.

Method: Live tissue slice cultures were established from a human malignant glioma biopsy and a healthy macaque brain respectively, and they were infected with oncolytic SFV VA7 alphavirus vector. VSV Δ51-EGFP and wtVSV-EGFP viruses were used as controls in these infections. The CD133⁺ cancer stem cells were derived from another human malignant glioma biopsy using commercial isolation column (Miltenyi Biotec). The cells and spontaneously forming tumour spheres were infected with VA7 virus with or without human IFNβ pretreatment.

Results: VA7 infected human malignant glioma tissue slice cultures and eradicated biopsy derived malignant glioma stem cell explants. The replication was either restricted or delayed in the cancer stem cell explant cultures after human IFNβ pretreatment. VA7 had only limited and focal replication profile in healthy macaque brain tissue slice culture.

Conclusion: Here we demonstrate that SFV VA7 is capable of infecting live human malignant glioma tissue cultures and destroying human malignant glioma derived cancer stem cells. The limited replication profile in the healthy macaque brain supports the evidence of SFV safety for primates.

Zhang Y Miss 1 Fielding A Dr 1

1 University College London, Royal Free Campus, Rowland Hill Street, London NW3 2PF P 213 Mechanisms of oncolytic measles virus-induced neutrophil-mediated cytotoxicity against tumour

Numerous studies implicate neutrophils in eliminating tumour cells following cancer treatments, most prominently after antibody therapies. We previously showed that neutrophils play a role in regression of human B-cell tumour xenografts in immunodeficient mice following oncolytic measles virus (MV) treatment. Here we sought, using normal human donor neutrophils, to identify potential mechanisms for oncolytic MV-induced neutrophil cytotoxicity against leukaemia targets. We showed that neutrophils were efficiently infected by MV in-vitro; they became activated and lived longer after infection. TRAIL (TNF-related apoptosis inducing ligand) was secreted from these infected neutrophils without concomitant increase in intracellular TRAIL or up-regulation of TRAIL mRNA. Inhibiting protein synthesis with cycloheximide did not reduce MV induced TRAIL secretion. However, treatment with monensin (a azurophil granule release stimulator) increased TRAIL secretion, suggesting that the TRAIL secreted did not come from de novo synthesis, but may be released from pre-formed granules within neutrophils. An up-regulation of CD63 (an azurophil granule degranulation marker) confirmed MV induced neutrophil degranulation. Cytotoxicity assays showed that MV infected neutrophils could specifically lyse both uninfected and MV-infected Jurkat target cells, while neutrophils alone can only kill MV-infected target cells. Despite the MV mediated effect on the TRAIL system, antibody-blocking experiments suggested that the in-vitro cytotoxicity was not TRAIL mediated. Transwell experiments showed that direct cell-cell contact was required for MV-induced neutrophil cytotoxicity and the addition of anti-MV anti-serum significantly enhanced the cytotoxicity, suggesting that neutrophil-mediated antibody dependent cellular cytotoxicity (ADCC) plays a role in oncolytic MV-induced neutrophil cytotoxicity.

Ekblad M Dr 1 Oberg D Dr 1 Lemoine N Professor 1 Hallden G Dr 1

1 Centre for Molecular Oncology and Imaging, Institute of Cancer, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, UK P 214 Development of an oncolytic adenovirus expressing the prodrug-converting enzyme CD/UPRT (AdΔΔCU)

Replication selective oncolytic adenoviruses (Ad) are genetically engineered viruses that target cancer cells. We generated AdΔΔ (Ad5 with E1A∆CR2 and E1B∆19K) that exhibits cytoxic potency in prostate cancer cells and significantly reduced replication in normal cells. Ad∆∆ synergistically enhanced tumour cell killing in combination with chemotherapeutics (i.e. mitoxantrone and docetaxel). To further improve on efficacy, the prodrug-converting enzyme CD/UPRT was inserted into the E3B region generating AdΔΔCU. CD/UPRT converts the prodrug 5-fluorocytosine (5-FC) to the cytotoxic 5-fluorouracil (5-FU) in infected cells, resulting in high local concentrations while systemic toxicity is avoided. The aim of this study was to identify cellular mechanisms involved in the enhanced cancer cell killing of AdΔΔCU and 5-FC. The Ad∆∆CU virus was characterised for cell killing (MTS), replication (qPCR), infectivity (FACS) and in vivo efficacy. Combination treatments of AdΔΔCU and 5-FC greatly increased cell killing efficiency in prostate cancer cells compared with virus alone (set to 0%) (PC3: 84.5 + /-9.0%, 22Rv: 91.7 + /-0.6%). The time to tumour progression (1.44cm²) was significantly prolonged in a PC3 xenograft model in athymic mice from 27 + /-9 days (PBS control) to 41 + /-22 days (AdΔΔCU alone) and ≥ 97 days (AdΔΔCU + 5-FC). A potential mechanism for the improved anti-tumour efficacy is the increase in viral uptake induced by 5-FU followed by higher early viral protein expression (E1A). However, viral replication was attenuated in the presence of drug indicating a complex interaction between the virus and drug. In conclusion, AdΔΔCU is a promising mutant as a viral gene therapy treatment for prostate cancer.

Mazzacurati L Dr 1 Marzulli M Dr 1 Reinhart B Dr 1 Hong C Dr 1 Uchida H Dr 1 Cohen J Dr 1 Glorioso J Dr 1 Grandi P Professor 1

1 Department of Microbiology and Molecular Genetics, University of Pittsburgh, PA USA P 215 Design and application of a new HSV vector for treatment of Glioblastoma

Oncolytic vectors (OV) are attenuated lytic viruses that depend on characteristic defects in the anti-viral response of cancer cells to preferentially replicate and spread in tumors. OV derived from herpes simplex virus type 1 (oHSV) have shown promise in the clinic, but their efficacy is limited due to poor replication in tumor cells. We sought to develop a new class of oHSV vectors which can replicate essentially as unattenuated virus in glioma cells, but are blocked for replication in normal neurons. To Achieve this goal, (i) we developed suitable methods for targeting infection of specific cell types by directing vector attachment to unique cell surface receptors; (ii) we engineered the essential viral proteins ICP4 and ICP27 which will be express only in the absence of specific miRNAs that are down-regulated in gliomas. expression is strictly controlled by naturally occurring micro-RNAs (miR) that are differentially expressed in normal brain neurons, neural precursor cells (NPCs) and tumor cells. Unlike current oncolytic viruses, our vectors doesn't have any defective genes and our preliminary data showed a dramatically improved virus replication in tumor cells without raising toxicity for normal tissue.

Cherubini G Dr 1 Kalllin C Miss 1 Mozetic A Miss 1 Hammaren-Busch K Miss 1 Muller H Miss 1 Lemoine N Professor 1 Halldén G Dr 1

1 Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Institute of Cancer and CR-UK Clinical Centre, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ P 216 Investigating a new potent and selective oncolytic adenovirus (AdΔΔ) in combination with chemotherapy

Patients suffering from pancreatic cancer face a poor prognosis. The only treatment option currently available is Gemcitabine, but tumour resistence rapidly develops. Replication-selective oncolytic adenoviruses (Ad) represent a novel anti-cancer approach with the additional advantage of improved anti-tumour efficacy when combined with chemotherapy. We previously demonstrated that the deletion in the adenovirus anti-apoptotic gene E1B19K greatly enhances Gemcitabine-induced apoptosis in pancreatic cancer cell lines and in tumour xenografts compared to the corresponding wild-type virus (Leitner et al., 2010). The E1B19K deletion was therefore included in the replication selective E1AΔCR2 mutant to generate AdΔΔ, a new oncolytic Ad. We demonstrate that AdΔΔ replicates as efficiently as wild-type virus on a panel of pancreatic cancer cells, whilst being severely compromised on normal primary cells (NHBE). BrdU incorporation confirmed that AdΔΔ has impaired S-phase induction in normal cells, explaining tumour selectivity. Importantly, AdΔΔ retains the capacity to enhance tumour cell-killing in combination with cytotoxic drugs (e.g. Gemcitabine). Specifically, we observed 2-4 fold reductions in EC50 values for AdΔΔ when combined with Gemcitabine, Irinotecan and Cisplatin, while no sensitisation was observed in NHBE cells. Ongoing studies are investigating the mechanism of action for the enhanced cell death; preliminary findings indicate that induction of aberrant mitosis is important to lead to the observed sensitisation. We suggest that the AdΔΔ virus is a promising new agent for the treatment of pancreatic cancer and potentially other forms of cancer, based on its specific replication and induction of cell death in cancer cells in combination with chemotherapy.

Brueggemann S Miss 1 Franco-Guo R Dr 2 Sandaradura de Silva U Mrs 1 Akguel B Dr 1 Pfister H Professor 1 Stoff-Khalili MA Dr 1 Mathis JM Professor 2

1 University of Cologne, Institute of Virology, Fuerst-Pueckler-Str. 56, 50935 Cologne, Germany 2 LSU Health Sciences Center, Dept. of Cellular Biology and Anatomy,1501 Kings Highway, Shreveport, LA 71130, USA P 217 Generation of a 5/3 adenovirus incorporating transcriptional and translational control elements

Virotherapy employing conditionally replicating adenoviruses (CRAds) represents a promising tool for a wide array of neoplastic diseases. Critical for a therapeutic index is selective killing of tumor cells while avoiding killing normal cells. Herein, we introduce a novel CRAd targeting strategy for cancer, incorporating three control elements: 1) transductional, 2) transcriptional and 3) mRNA translational targeting. A modified CRAd (Ad5/3-CXCR4-UTR) incorporating a serotype 5/3 chimeric fiber, the CXCR4 promoter and the 5′-untranslated region (UTR) from the Fibroblast Growth Factor-2 mRNA to regulate E1A transcription and translation, was generated. We hypothesized that this novel vector would improve the therapeutic index by selective targeting of cancer cells.

Methods: Cytotoxicity of the constructed CRAd, Ad5/3-CXCR4-UTR, was tested in several breast cancer cell lines and primary cells by MTT-assay and crystal violet analysis. E1A transcription and viral replication were assessed in cells by measuring E1A mRNA levels and E4 copy number. Expression of E1A protein was examined by immunoblot analysis.

Results: The Ad5/3-CXCR4-UTR virus demonstrated a gain of specificity from normal cell killing compared to wild-type and dual-level CRAds (two control elements). However, in breast cancer cells although the triple level virus showed similar oncolytic activity compared to wild-type, it was less oncolytic than the dual-level CRAds.

Conclusion: Our conceptually novel approach did not lead to the expected gain of oncolytic activity in breast cancer cells, suggesting that Ad5/3 cell binding may induce a change of the intracellular signal cascade influencing the translation activity of the 5′-UTR sequence. Further investigations will address this proposition.

Allaume X. Mr 1 El-Andaloussi N. Dr 1 Leuchs B. Miss 1 Rommelaere J. Professor 1 Marchini A. Dr 1

1 Infection and Cancer Program, German Cancer Research Center (DKFZ) and Inserm U701, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany P 218 Genetic Retargeting of Oncolytic H-1 Parvovirus

H-1 rat parvovirus (H-1PV) attracts high attention as an anticancer agent, because of its oncosuppressive properties while not showing obvious side-effects in humans. The viral oncotropism relies on a more efficient H-1PV replication in cancer cells but the virus can enter both normal and cancer cells. For viral oncotherapy this constitutes a problem as uptake by normal cells sequesters a significant portion of the viral dose administered away from the tumor target.

To increase the efficacy of parvovirus-based treatments and to provide additional safety against eventual side-effects on normal cells, we aim to genetically reprogram H-1PV to selectively target human tumor cells. By analogy with the closely related Minute Virus of Mice (MVM), we developed an in silico 3D model of the H-1 wt capsid. Based on this model, we identified the aminoacids in H-1PV involved in cell membrane recognition and entry. In situ mutagenesis of these residues significantly reduces the binding and entry of H1 into its originally permissive cells. We further engineered the entry-deficient virus by inserting into its capsid a double cyclic RGD peptide that targets α_vβ₃ and α_vβ₅ integrins over-expressed in cancer cells and angiogenic blood vessels. While viral capsid and DNA packaging are not affected by these modifications, insertion of the RGD peptide rescues viral infectivity towards cells over-expressing α_vβ₅ integrins and infection with the reengineered virus efficiently kills these cells. This work demonstrates that H-1PV can be genetically retargeted through modification of its capsid and holds strong promise for a more efficient use of this virus in cancer therapy.

Whilding L Miss 1 McNeish I Professor 1 Öberg D Dr 1

1 Centre for Molecular Oncology and Imaging, Institute of Cancer, Barts and the London School of Medicine and Dentistry, Queen Mary University of London P 219 Oncolytic vaccinia virus expressing decorin as a novel anti-cancer agent

We aim to enhance the anti-tumour efficacy of oncolytic vaccinia virus by expressing murine decorin from a selectively replicating Lister strain (Lister-mDCN). Decorin is a small leucine-rich proteoglycan whose protein core interacts with numerous extracellular matrix proteins, growth factors and their receptors. It is downregulated in many tumour cells, including breast, ovarian and pancreatic cancers, and has both autocrine and paracrine actions on cells to downregulate the EGFR, HER2 (ErbB2) and c-met receptor. Additionally, the interaction of decorin with collagen fibrils within the tumour microenvironment is proposed to enhance virus spread within the tumour.

Lister-mDCN and a control virus were constructed and characterised in prostate, pancreatic and ovarian cancer cell lines. Expression of decorin protein from the virus was confirmed by western blotting and ELISA. The effect of decorin on vaccinia virus replication, cell proliferation and virus cytotoxicity was subsequently examined by TCID50 and MTS assay. Lister-mDCN was shown to replicate and cause cytotoxicity to a similar or greater degree than both the wild-type and control viruses. Lister-mDCN was also able to transiently reduce tumour burden by >70%, and significantly reduce the rate of tumour growth thereafter, in an in vivo model of ovarian cancer. A variety of in vitro experiments suggest that the mode of cell death by Lister-mDCN is unaltered compared to infection with the control virus. Replication and cytotoxicity abilities are also comparable. Current work therefore aims to elucidate the mechanisms behind the efficacy of Lister-mDCN in vivo, and its effects on the tumour microenvironment.

Martikainen M Mr Ruotsalainen J Mr Vähä-Koskela M Dr Hinkkanen A Professor

P 220 Low interferon-β sensitivity of tumour cells correlates to good response to systemically administered oncolytic VA7 virotherapy in mouse glioma model

Background: In our previous studies, intravenously administered attenuated Semliki Forest virus VA7 effectively infects and eradicates orthotopic U87 gliomas in nude mice. However, in treatment of syngeneic GL261 gliomas in immunocompetent mouse model, VA7 failed to infect the tumour cells and had no positive effect on survival. Combination therapies with immunosuppressive cyclophosphamide and rapamycin were tested. As virally induced cytokines are powerful inducers of antiviral response, possibly explaining the poor infectivity in vivo, we studied our cell lines for their interferon-β (IFN-β) sensitivity.

Method: GL261 tumour-bearing B57BL/6 mice were subjected to combination therapies with VA7 and cyclophosphamide or rapamycin. Infectivity and IFN-β sensitivity of cultured U87 and GL261 glioma cells was studied in vitro.

Results: Cyclophosphamide enhances virus replication drastically in healthy mouse brain tissue but not in tumour in vivo. While rapamycin did not have beneficial effect on VA7 therapy in vivo, it enhances virus replication in vitro. GL261 cell line is sensitive to IFN-β, and pre-treatment with IFN-β effectively hampers VA7 infectivity in GL261 cells in vitro. As compared to GL261 cell line, U87 cell line shows no sensitivity to mouse IFN-β and only low sensitivity to human IFN-β.

Conclusion: Our findings suggest that tumour cell infectivity in vivo and good response to systemically administered oncolytic VA7-treatment correlates to low sensitivity to vector-induced interferons in the targeted cells. Different outcome in our previous animal studies can be explained by difference in IFN-β sensitivity in the used glioma cells. Strategies to safely overcome vector immunity are needed.

Boccalatte F Dr 1 Gentner B Dr 1 Restuccia U Dr 2 Saini M Mr 1 Mavilio F Professor 3 Bachi A Professor 2 Naldini L Professor 1

1 San Raffaele Telethon Institute for Gene Therapy, Via Olgettina 58, 20132 Milan 2 San Raffaele Scientific Institute, Biomolecular Mass Spectrometry Unit, Via Olgettina 58, 20132 Milan 3 San Raffaele Scientific Institute, Gene Expression Unit, Via Olgettina 58, 20132 Milan P 221 Unveiling biological readouts of miRNA-regulated gene therapy vectors

MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that regulate gene expression by translational inhibition or degradation of target messenger RNA sequences complementary to their seed region. Since some miRNAs are expressed in a tissue-specific manner, recent gene therapy approaches have employed miRNA target (miRT) sequences to de-target transgene expression from unwanted tissues. However, there is concern that expressing excess miRT may saturate miRNA activity and offset the regulation of its natural targets. Because miRNA natural targets are mostly unknown and the regulation exerted on them is often subtle, these concerns are difficult to address without characterizing the changes induced by individual miRNAs on the cell transcriptome and proteome. To this aim, we titered up copies of lentiviral vectors overexpressing miRT sequences into cells expressing the cognate miRNA until saturation of miRNA activity and then analyzed the effects on previously validated targets and at the transcriptomic and proteomic level. To test the system we stably knocked-down miR-223 activity in U937 cell line and detected significant (p < 0.05) changes in the expression of 120 transcripts and 190 proteins. Functional annotation and network analysis revealed a consistent regulation of phagocytic activity, confirming a role for miR-223 in inflammatory response regulation. Another current study is characterizing the natural targets of miR-126, a miRNA with a promising role in stem cell gene therapy because of its selective expression in hematopoietic stem and progenitor cells. Our findings will provide relevant biological readouts of miR-126 activity to be used for validating the safety of miR-126 regulated vectors.

Dannemann ND Miss 1 Bednarski CB Miss 1 Dreyer AKD Miss 1 Cathomen TC Professor 1

1 Carl-Neuberg-Str. 1, 30625 Hannover; Germany P 222 Transient cold shock enhances zinc-finger nuclease-mediated gene disruption - Rule or exception?

Designer zinc-finger nucleases (ZFNs) are a promising tool for precise genome editing. They consist of the catalytic domain of the FokI endonuclease and an engineered DNA binding domain. Recently, Doyon et al. (Nat Methods 7, 459-60; 2010) reported that transient hypothermia robustly increases ZFN-induced gene disruption. Here, we aimed at working out the mechanism behind enhanced ZFN activity at 30°C and to elucidate whether hypothermia is a generally applicable strategy to increase ZFN-based genome editing. We employed six different ZFN pairs – Sangamo and OPEN-based ZFNs directed against the human AAVS1, CCR5, IL2RG, HOXB13 and CFTR loci as well as the EGFP gene – and compared their activities at 30°C and 37°C in vitro and in human cell lines. In vitro cleavage assays revealed no difference in ZFN activity when comparing ZFNs of both platforms at 30°C vs. 37°C, implying that neither DNA binding nor the catalytic activity is affected by the temperature shift. Incubation of human cells at 30°C induced an M phase arrest and increased ZFN protein levels in transfected cells. Although we observed increased gene disruption at the AAVS1 locus at 30°C, the higher enzyme levels did not correlate with increased ZFN activity at the other analyzed genomic loci in three human cell lines. In summary, our data suggest that although mild hypothermic conditions let to a robust increase in ZFN protein levels, enhanced gene disruption activity of ZFNs at 30°C may not be a general phenomenon but rather dependent on the cell type and the genomic target locus.

Haase R Dr 1 Magnusson T Mrs 1 Wagner E Professor 1 Ogris M Dr 1

1 Department of Pharmacy, Center for System based Drug Research, Pharmaceutical Biotechnology, Ludwig-Maximilians-University, Butenandstr. 5-13, D-81377 Munich, Germany P 223 Generation of the novel, synthetic hybrid SCE-promoter for gene-therapeutical applications

Background: A major factor for successful gene therapy is strong and enduring expression of the transgene. Commonly used promoters for expression of transgenes are the CMV-immediate early (CMV-IE), chicken β-actin, ubiquitin C or the EF1α promoter, each with their own advantages and disadvantages.

Method: Here we present the generation of novel synthetic hybrid promoter, based on the CMV-IE and the EF1α promoter. It was obtained by generating shuffled consensus sequences of both promoters, which were analyzed by promoter prediction software. One sequence, the shuffle CMV/EF1α promoter (SCE), was synthesized and tested in vitro and in vivo. The SCEP sequence features an identity of 83% with the CMV and 85% with the EF1α promoter. The sequence was CpG-free designed to circumvent inflammatory reactions towards CpG isles and promoter methylation leading to silencing.

Result: In several cell lines a 2-7 fold stronger expression of the SCE promoter compared to the EF1α was observed; when compared to CMV-IE, SCE promoter activity was almost similar. All promoters were within a CpG-free plasmid featuring a human CMV-IE enhancer and luciferase as transgene. In immunocompetent Balb/c mice we observed a ∼3 fold stronger transgene expression of SCEP compared to the EF1α promoter after hydrodynamic gene delivery to the liver for a time period of 30 days.

Discussion: Hence the SCE promoter is qualified for further applications in biotechnology or gene therapy, when strong and enduring transgene expression and evasion of promoter silencing is needed.

Abdullah S Dr 1 Siew Ching N Miss 1 Rosli R Dr 1 Ramasamy R Dr 2

1 Molecular Genetics Laboratory, Faculty of Medicine and Health Sciences, 43400 UPM Serdang, Selangor. 2 Immunology Laboratory, Department of Pathology, Faculty of Medicine and Health Sciences, 43400 UPM Serdang, Selangor P 224 Prevention and Reversion of Transgene Silencing in HSCs Transduced with Lentiviral Vector

Lentivirus (LV) is an attractive tool for gene therapy because it can transduce hematopoietic stem cells (HSCs) efficiently. Unfortunately, few studies have shown that the duration of transgene expression by LV is transient. To substantiate this, we transduced HSCs with LV carrying GFP and the levels of expression were measured at different time points. As expected, GFP expression declined by day 2 post-transduction. This may due to the de novo methylation of the transgene and chromatin modifications at the transgene-integrated region. We hypothesized that 5-azacytidine (5-azaC), a DNA demethylating agent, and Trichostatin A (TSA), an inhibitor of histone deacetylase, would be able to improve the level of transgene expression. First, either 5-azaC or TSA was added to HSCs a day after transduction or on the day following GFP silencing. 5-azaC was able to prevent and reverse the silencing effect. However, TSA was unable to reverse the GFP silencing, but it prevented the GFP silencing at high concentration. Next, we investigated the effects of 5-azaC and TSA combinations on GFP expression. Both drugs were added a day after transduction or on the day following GFP silencing. Combination of both drugs prevented GFP silencing when high concentration of TSA was used but there was no effect in reversing the silenced GFP. Here we suggest that transgene silencing can be prevented and reversed by 5-aza-C, demethylation is a prerequisite for the reversion of silencing and the combination of both drugs did not have a positive synergistic effect on the reversion of silencing.

Salmons B Mr 1 Dangerfield J Mr 2 Brandtner L 2 Toa P 2 Jin Tan W 2 Gunzburg W 2

1 Austrianova Singapore Pte Ltd, Centros, Biopolis, Singapore, 138668 2 Austrianova Singapore Pte Ltd, Centros, Biopolis, Singapore, P 225 A method for protecting therapeutic cells and microenvironment containment in patients for gene and cell therapies: a clinically proven enabling cell encapsulation technology

We have developed a cell encapsulation technology as an enabling, platform technology for the treatment of a wide variety of diseases. Cells producing therapeutic products such as enzymes, growth factors, antibodies etc are encapsulated in polymers of cellulose sulphate and implanted in the body thereby achieving long term release of the therapeutic. The cellulose sulphate is inert and biocompatible and the capsules are robust and stable. The capsule protects the cells from the immune system (even if they are from a different species) and localises them by physically confining them but is porous so that therapeutic products can be released. The capsule also protects the body from release of cells (such as stem cells) that may cause pathology if they become lodged at non target sites. Safety and efficacy has been demonstrated in clinical trials to treat pancreatic cancer, using a cell based suicide gene/prodrug strategy (Lohr et al., Lancet 357, 1591-2). Large scale GMP production of an encapsulated cell product has been achieved and approved by the German authorities (BioProcessing Journal 4, 36-43). Around 20 different cell types have been successfully encapsulated, including stem cells, hybriomas, fibroblasts, lymphoid and epithelial cells. Encapsulated cells have been successfully implanted at various sites in the body e.g. subcutaneous, intraperitoneal, intramuscular, into blood vessels, peri-tumoral etc. The technology can be used to treat diseases as diverse as cancer, cardiovascular and neurodegenerative diseases, enzyme deficiencies, diabetes and other metabolic diseases and data supporting a number of different uses of the technology will be presented.

Muñoz P. Dr 1 Real P.J Dr 1 Benabdellah K. Dr 1 Menéndez P. Dr 1 Martín F. Dr

1 Andalusian Stem Cell Bank, Center for Biomedical Research, University of Granada, Spain P 226 WASp-promoter driven LVs specific expression during hematopoietic differentiation of human embryonic stem cells (hESCs)

Human embryonic stem cells (hESCs) are useful tools for studying early human embryonic development. Using optimized conditions it is possible to promote hematopoietic differentiation that can be followed by the expression of different hematopoietic markers. The aim of the present work was to study the behaviour of WASp-promoter-driven LVs (WE and AWE) during hematopoietic differentiation of hESCs. We transduced AND-1 (hESCs line established at the Andalusian Stem Cell Bank) with WE and AWE LVs, expanded and induced to hematopoiesis differentiation by embryoid bodies (EBs) formation and with the addition of Flt-3L, SCF, IL-3, IL-6, G-CSF and BMP4 for 22 days. eGFP transgene expression and endogenous wasp mRNA levels were analyzed at different time points. eGFP mRNA was only detected upon hematopoietic induction (day 3). This expression increased over time reaching plateau at days 15-22. Interestingly this expression pattern follows very closely the expression profiles of the endogenous was gene. FACS analysis demonstrated that all eGFP + cells were also positive for CD45, CD33 and CD31, typical hematopoietic markers. Interestingly the all CD45 negative population didn't express eGFP, indicating that both vectors were restricted to the hematopoietic cell population. This study contributes to the better knowledge of wasp expression during human hematopoietic differentiation and demonstrates the value of WE and AWE vectors backbone for hematopoiesis basic research. In addition, the demonstration that the WASp-promoter driven LVs follow endogenous WASP expression pattern in this relevant cellular model, point out their potential use as physiologically-regulated vectors for gene therapy of Wiskott-Aldrich Syndrome.

Yang GH Dr 1 Touboul T Dr 1 Corbineau S Dr 1 Martinet C Miss 1 Goulinet-Mainot S Mrs 1 Groyer MT Mrs 1 Bouillé P Dr 2 Vallier L Professor 3 Dubart-Kupperschmitt A Professor 1 Weber A Professor 1

1 U972, Inserm, Le Kremlin-Bicêtre, France 2 Vectalys, Toulouse, France 3 Surgery department, University of Cambridge, Cambridge, United Kingdom P 227 Lentiviral vector mediated purification of hepatic progenitors differentiated from human embryonic stem cells

Cell therapy is now an alternative to orthotopic liver transplantation for the treatment of life-threatening liver metabolic diseases. However this approach is restricted by the lack of donors and limited number of cells since hepatocytes cannot be expanded in vitro. Therefore new sources of stem cells need to be defined and human embryonic stem cells (hESCs) still appear as the gold standard to generate differentiated cells. We previously developed chemically defined culture conditions to differentiate hESCs into hepatic progenitors that could be amplified 6-9 times. We now define an approach to purify hepatic progenitors, using lentiviral vectors in which GFP reporter gene expression is driven by the human apolipoprotein A-II (APOA-II) promoter. Conditions for lentivirus transduction were set up using a EF1a-GFP vector on undifferentiated and differentiated cells yielding 60% transduced cells at MOI 10 and 70% at MOI 30. Cells transduced with APOA-II-GFP vector were differentiated for 16 days, and GFP⁺ cells, representing 30% of the population, were purified by Flow cytometry, yielding 99% GFP⁺ cells. CK19 and PCNA expression showed that sorted cells were proliferating progenitors. Amplification and differentiation conditions were tested after passaging the cells on different matrixes. After 23 days of differentiation, we found that most of the cells expressed albumin. The cells also produced high level of urea and were able to uptake and excrete indocyanin green.

Our data indicate that hepatic progenitors can be purified using hepatic specific promoters in lentiviral constructs. These cells also can be amplified and differentiated further into more mature hepatocytes.

Spitalieri P Dr 1 Quitadamo M.C Dr 1 Orlandi A Dr 1 Guerra L Dr 2 Giardina E Dr 1 Casavola V Professor 2 Novelli G Professor 1 Sangiuolo F Professor 1

1 Via Montpellier,1 Roma 2 Via Amendola, 165 Bari P 228 Engraftment capacity of human embryonic stem cells into bleomycin and silica lung-damaged mice

Respiratory diseases are the leading causes of morbidity and mortality worldwide. Until now no treatments resulted to be resolutive to cure or reverse the disease, consequently, there is a pressing need for the development of novel therapies that facilitate the regeneration of alveolar epithelium pneumocytes (ATIICs) destroyed by acute and chronic lung diseases.

The objective of this study was to assess: (i) the capacity of human embryonic stem cells (HUES-3) to differentiate in vitro into ATIICs; (ii) the ability of committed HUES-3 cells (named HUES-3-ATIICs) to counteract in vivo lung damage in two pulmonary fibrosis disease models, obtained by Bleomycin or Silica inhalation in mice.

HUES-3-ATIIC cells developed in vitro an alveolar phenotype, as evidenced by the presence of multilamellar body formation and by the expression of SP-B, SP-C, and CFTR markers.

Transepithelial conductance of CFTR channel was also measured, demonstrating the formation of a multilayered epithelial structure in which CFTR is functionally expressed.

After injection of HUES-3-ATIICs into both Silica and Bleomycin-damaged mice, microscopic and biomolecular analyses revealed a significant recovery of inflammation process and fibrotic damage. Moreover, a normal blood arterial oxygen saturation and weight recovery confirmed these data. Finally injected cells remained engrafted over four weeks post transplantation, as evidenced by the presence of human SP-C and human nuclear antigen (HuNu).

Molecular analysis further confirmed the presence of human DNA within murine lung.

In conclusion HUES-3 cells have to be considered a promising way to recover lung functionality by reducing inflammatory and fibrotic changes, through a cell therapy approach.

Falahati R. Dr 1 Zhang J. Dr 1 Flebbe-Rehwaldt L. Dr 1 Gerson S.L Dr 2 Gaensler K.L Dr 1

1 Department of Medicine, University of California, San Francisco, 505 Parnassus Ave., Box 1270, San Francisco, CA 94143 2 2Department of Hematology and Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44122, USA P 229 Non-Ablative Allogeneic Neonatal Transplantation and Correction of β-Thalassemia following In Vivo Positive/Negative Selection

Sibling or matched, unrelated allogeneic hematopoietic stem cell transplants remain the only curative therapy for many hereditary disorders. However, the toxicity of preparative regimens, risks of graft vs. host disease (GVHD), infectious complications of immunosuppression, and limited donor availability, restrict application of this approach. The feasibility of allogeneic transplantation, without myeloablation or post-transplant immunosuppression, was tested using in vivo chemoselection of allogeneic hematopoietic stem cells (HSC) after transduction with a novel tricistronic lentiviral vector (MAGIT). This vector contains P140K-O⁶-methylguanine-methyltransferase (MGMT^P140K), HSV-thymidine kinase (TK^HSV), and eGFP enabling a) in vivo chemoselection of HSC by conferring resistance to benzylguanine (BG), an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU and, b) depletion of proliferating cells such as malignant clones or donor T cells mediating GVHD, by expression of the suicide gene TK^HSV and Ganciclovir administration.

Non-myeloablative transplantation of transduced, syngeneic, lineage depleted (Lin^-) BM in neonates resulted in 0.7% GFP⁺ mononuclear cells in peripheral blood. BG/BCNU, administered 4 and 8 weeks post-transplant, produced 50-fold donor cell enrichment. Transplantation and chemoselection of MHC-mismatched MAGIT-transduced Lin^- BM produced similar expansion for >40 weeks. The efficacy of this transplantation approach was then successfully demonstrated in Hbb^th3 heterozygous mice by correction of beta-thalassemia intermedia, without toxicity or GVHD. Negative selection, by administration of Ganciclovir resulted in donor cell depletion. Re-expansion of donor cells could be achieved with additional BG/BCNU treatment. These studies show promise for developing non-ablative transplant approaches using in vivo positive/negative selection.

Anton M Dr 1 Wübbenhorst D Mrs 1 Dumler K Mrs 1 Wexel G Mrs 1 Imhoff A Professor 2 Gansbacher B Professor 1 Schütze N Professor 3 Vogt S Dr 2

1 Inst. f. Exp. Onkologie u. Therapieforschung, Klinikum rechts der Isar der TU München, Germany 2 Abteilung für Sportorthopädie, Klinikum rechts der Isar der TU München, Germany 3 Orthopedic Department, Orthopedic Center for Musculoskeletal Research, University of Würzburg, Würzburg, Germany P 230 Tet-regulated, lentivirally mediated BMP-2 expression in mesenchymal stem cells

Mesenchymal stem cells (MSC) have gained considerable attention in view of their potential use in regenerative medicine and tissue engineering. The combination with gene therapy allows to provide MSC with new traits for enhanced/improved tissue repair. Bone marrow derived MSCs are able to differentiate into different cell types among them osteocytes and chondrocytes and are therefore considered for the treatment of osteochondral defects. Regulated gene expression of growth factor bone morphogenetic protein 2 (BMP-2) or WNT1-inducible-signaling pathway protein 3 (WISP3), a CCN-protein known to be involved in development, homeostasis and repair of mesenchymal tissues was achieved using the Tet-on system, delivered by VSV-G pseudotyped lentiviral SIN vectors (LV). Cells were infected with Tet-on constructs containing the BMP-2 or WISP-3 cDNA. The expression was induced by doxycycline (dox).

Transduction efficiency of rabbit MSCs by eGFP-expressing LV was 65%. Transduction of MSC with the BMP-2 Tet-on vector resulted in inducible BMP-2 expression with a 36-fold increase of BMP-2 expression on RNA level as well as a 113-fold increase on protein level compared to uninduced cells. BMP-2 induction level was 2-fold higher than in primary rabbit chondrocytes. After lentiviral infection cells retained differentiation potential with enhanced chondrogenesis after induction of BMP-2 expression. WISP-3 regulation was demonstrated by RT-PCR.

The lentivirally delivered Tet-on system allows for regulated expression of BMP-2 and WISP3 after induction with doxycyline in mesenchymal stem cells. Induction and switching-off of gene expression on demand will constitute new and exciting therapeutic option for cartilage defects.

Helmrich U Ms Melly L Dr 2 Christ L Ms Scherberich A Dr 3 Heberer M Professor 2 Martin I Professor 3 Banfi A Dr

2 Cell and Gene Therapy, ICFS, Department of Biomedicine, Basel University Hospital/Cardiac Surgery, Department of Surgery, Basel University Hospital 3 Tissue Engineering, ICFS, Department of Biomedicine, Basel University Hospital P 231 VEGF-expressing MSC for rapid vascularization of tissue-engineered bone grafts

Rapid vascularisation of tissue-engineered grafts is a major obstacle in the development of regenerative medicine approaches. Vascular Endothelial Growth Factor (VEGF) is a powerful angiogenic factor. However its dose must be controlled in the microenvironment around each producing cell to avoid toxic effects. To achieve controlled expression in vivo, we developed a high-throughput method to rapidly purify genetically-engineered progenitors secreting a desired level, by linking VEGF expression to that of a FACS-quantifiable cell-surface marker (truncated CD8a). Mesenchymal stromal/stem cells (MSC) are rich in osteoprogenitors, but their differentiation is gradually lost during expansion. Therefore, we sought to genetically modify MSC with minimal expansion to generate bone grafts with intrinsic vascularisation potential.

Primary human MSC were retrovirally transduced to express VEGF-CD8 or just CD8. Transgene expression was verified by ELISA and by FACS. Proliferation and multilineage differentiation potential were assessed in vitro, while bone formation and vascularisation were determined histologically 8 weeks after in vivo implantation.

An optimized protocol allowed MSC to be transduced with high efficiency (80-95%) during the first plating, so that no extra expansion was necessary. Neither CD8 nor VEGF expression impaired MSC proliferation and differentiation potential in vitro. In 2 donors VEGF actually appeared to improve osteogenic differentiation. In vivo vascularization potential was significantly increased in VEGF-expressing MSC compared with control cells. Bone formation by the different populations is currently being determined.

Optimized transduction allowed the genetic modification of primary MSC with minimal manipulation and no loss of biological potential, leading to improved in vivo vascularisation.

Rozemuller H Dr 1 van Til N Dr 2 Moerer P Mrs 3 Martens AC Dr 3 Wagemaker G Professor 2 Wulffraat N Dr 1

1 University Medical Center Utrecht, Department of Pediatrics, Utrecht, The Netherlands 2 Erasmus University Medical Center Rotterdam, Department of Hematology, Rotterdam, The Netherlands 3 University Medical Center Utrecht, Department of Immunology, Utrecht, The Netherlands P 232 Lentiviral transduction of MPS1 bone marrow cells results in high production of alpha-L-Iduronidase

Mucopolysaccharidosis type I (MPS1, Hurler syndrome) is caused by a deficiency of the enzyme alpha-L-iduronidase (IDUA) and is characterized by glycosaminoglycan (GAG) accumulation causing multi-organ failure. Aim of this study is to obtain complete phenotype correction of disease in MPS1 mice, including central nervous system and skeletal defects, by overexpression of IDUA in hematopoietic cells.

Lentiviral vectors with the spleen focus forming virus- (SF) and phosphoglycerate kinase- (PGK) promoters driving codon optimized human IDUA cDNA (IDUAco) expression were constructed. These vectors were used to transduce male lineage-negative MPS1 bone marrow cells, which were subsequently transplanted in 3-week-old 6 Gy irradiated MPS1 female mice. Blood and urine samples were collected monthly. Long-term stable, on average 80% male cell chimerism was observed in peripheral blood containing 30% IDUA positive cells up to 7 months after transplantation. IDUA levels in blood were 500- and 80-fold that of normal wild type levels for the SF-IDUAco and PGK-IDUAco, respectively. Analysis of GAG secretion in urine revealed normalization to wild type levels for all transplantation groups. At month 7, the mice were sacrificed and assessment of GAG levels in spleen, liver, lung, heart, and kidney demonstrated significant reduction of GAGs to normal levels. Most importantly, GAG accumulation was significantly decreased in brain as well. Skeletal characteristics are currently studied by CT analysis.

We conclude that the IDUAco vectors can provide long-term high expression and concomitant reduction in GAG secretion in urine, and other organs, including brain. This warrants further development towards clinical application.

Duffy G Dr 1 Kavanagh C Dr 2 Coleman C Dr 2 Murphy M Dr 2 O'Brien T Dr 2 Barrett HH Professor 3 Barry F Professor 2 Liu Z Dr 3

1 Regenerative Medicine Institute (REMEDI), National University of Ireland Galway, Ireland, Department of Anatomy, Royal Collage of Surgeons in Ireland, Dublin, Ireland 2 Regenerative Medicine Institute (REMEDI), National University of Ireland Galway, Ireland 3 Department of Radiology, University of Arizona, Tucson, Arizona P 233 Tracking Mesenchymal Stem Cell Homing to the Infarcted Heart after Intracoronary, Intravenous and Intramyocadial Delivery

Acute myocardial infarction with subsequent ventricular remodeling is the leading cause of congestive heart failure and death in developed countries. Mesenchymal stem cell therapy is being investigated to promote regeneration of infarcted myocardium. Many aspects of the delivery protocol will critically influence the outcome, including transplantation route, dose and time of delivery post-infarct. The optimal method of stem cell delivery is currently unknown. The objective of this study was to radiolabel MSCs with ¹¹¹In-oxine and assess myocardial homing of ¹¹¹In-MSCs administered via three delivery routes in rats: intravenous (IV), intramyocardially (IM), and intracoronary (IC).

Myocardial infarction was induced in Fischer 344 rats by ligating the LAD artery for 45 minutes. ¹¹¹In-MSCs were administered via IV, IC, and IM. Using FastSPECT II, scintigraphic images were acquired from 1 hour up to 7 days after ¹¹¹In-MSC injection. Dual-isotope imaging of ¹¹¹In-MSC/99mTc-sestamibi was performed 24-48 hours after MSC transplantation to assess location of transplanted cells within the myocardium.

IV delivery resulted in high lung uptake initially and at 24 hours the lung activity had shifted toward other organs. ¹¹¹In radioactivity remained locally detectable in infarcted hearts up to 6-7 days after IV, IMC and IC delivery. The radioactivity detected in the hearts that received ¹¹¹In-MSCs via IM and IC delivery was significantly higher than that after IV delivery. The activity in the hearts after IC injection was more widely distributed in the ischemic areas. In conclusion, IC delivery of MSCs may offer advantages over IM or IV in terms of cell-homing and engraftment.

engraftment.

Gmyzina A. Mrs 1 Sysoeva V. Dr 2 Parfyonova YE Professor 3 K. K. Dergilev Mr 4 Rubina K. Dr 5 Akchurin R. Professor 6 Tkachuk V. Professor

1 anna_gmz@mail.ru 2 veroniks@mail.ru 3 yeparfyon@cardio.ru 4 dk@cardio.ru 5 rkseniya@mail.ru 6 ackchurin@cardio.ru P 234 The population of C-kit positive cells in the heart contains mast cells and cardiac progenitors

Stem cell therapy is a new promising tool to regenerate damaged myocardium. Human heart contains stem cells showing cardiac regeneration potential in vivo. The aim of the present study is to characterize cardiac stem cells in the human heart tissue.

Methods: Tissue samples of appendix of the right atrium harvested during coronary artery bypass grafting and autopsy samples of left ventricular tissue were analyzed using fluorescence-activated cell sorting and immunohistochemical staining for the presence of C-kit-positive cells, hematopoietic cells (CD34, CD45), blood lineage (Lin) and tryptase. C-kit cells were harvested using explant culture and anti-c-kit antibody. Co-culture of isolated c-kit cells with neonatal rat cardiomyocytes was used to prove their cardiomyogenic potential.

Results: C-kit + cells represented 0,79 + 0,32% of the total cell population of appendix and were CD34- and Lin-. Two populations of C-kit cells were identified: 60% of C-kit cells were CD45 + , assuming that these cells were of bone marrow origin and about 40% of C-kit cells were CD45-. Immunohistochemical staining of autopsy samples of left ventricular tissue showed that majority of C-kit cells are positive for CD45 and tryptase, suggested that they are mast cells. Only a small population of C-kit (+) CD45(-) triptase(-) cells represented human cardiac stem cells.

Using magnetic cell sorting c-kit positive cells could be successfully isolated from human heart tissue and expanded in vitro. C-kit cells undergo cardiomyogenic differentiation when co-cultured with neonatal rat cardiomyocytes.

Conclusions: Appendix of the right atrium could be an alternative source of autologous cardiac stem cells.

Thieme S. Dr 1 Stopp S. Mrs 2 Ugarte F. Dr 1 Wobus M. Dr 2 Bornhäuser M. Professor 2 Brenner S. Dr 1

1 Department of Pediatrics, University Clinic Dresden 2 Medical Clinic and Policlinic I, University Clinic Dresden P 235 Downregulation of MCAM/CD146 in human mesenchymal stromal cells reduces hematopoietic progenitor cell potential

The surface molecule MCAM/CD146 is a novel marker for mesenchymal stromal cells (MSC), which defines osteoprogenitor cells with enhanced self-renewal capacity and supportive effects towards hematopoietic stem and progenitor cells (HPC; Sacchetti et al., 2007). Similarly, Notch signalling has been implicated in the formation of the hematopoietic microenvironment by regulating differentiation of MSC.

We have found that activation of Notch signalling by lentiviral overexpression of Notch Intracellular Domain (NICD) enhances the surface expression of MCAM in human MSC. This effect was partially blocked by co-expression of a dominant negative form of Mastermind1 (dnMAML1). These observations indicate Notch signalling as a novel regulator of MCAM expression in human MSC.

Next, we aimed to modulate MCAM expression in MSC to analyze its impact on primary HPC in vitro. Preliminary results suggest that knock down of MCAM in MSC enhances maturation and proliferation of HPC in co-culture experiments. In addition, MCAM knock down in MSC has a negative effect on the formation of cobblestone area forming cells.

In summary, we demonstrate that Notch signalling in MSC regulates MCAM expression, and MCAM expression itself affects HPC potential. MCAM appears to be a valuable factor in the support of hematopoietic stemness.

Kalinina N Dr 1 Lopatina T Dr 2 Efimenko A Ms 3 Rubina K Dr Parfyonova YE Professor Tkachuk V Professor

1 n_i_kalinina@mail.ru 2 t.lopatina@gmail.com 3 efimenkoan@gmail.com P 236 Adipose-derived Mesenchymal Stem Cells Enhance Tissue Regeneration by Inducing Growth of Blood Vessels

Transplantation of adipose-derived stem cells (ASCs) induces tissue regeneration by accelerating the growth of blood vessels and nerve sprouts. Previously we demonstrated that ASCs stimulate blood vessel growth by producing angiogenic growth factors as well as enhancing vessel maturation. However mechanisms of their action on the growth of nerve fibers are only partially understood. Here we show that transplantation of ASCs stimulates a repair of motor and sensory nerves as well as induces nerve sprouts growth in matrigel implants. Transcriptome analysis has revealed that ASCs express mRNAs for growth factors and extracellular matrix proteins specific for the outgrowth of nerve sprouts and myelination. Exposure of ASCs to a combination of retinoic acid with 5-azacytidine up-regulates BDNF production in these cells as well as their ability to induce nerve fiber growth in matrigel implants. BDNF neutralizing antibodies have abrogated stimulatory effect of ASCs on the growth of nerve sprouts. These data suggest that ASCs induce nerve repair and growth via BDNF production and this stimulatory effect can be further enhanced by cell exposure to neural differentiation medium prior to transplantation.

Cosnuau L Mrs 1 Mbiguino LC Mrs 1 Vincent JP Dr 2 Dubrana F Professor 2 Gunepin FX Dr 3 Ferec C Professor 4 Delepine P Dr 5

1 INSERM U613 2 CHU BREST 3 HIA BREST 4 EFS Bretagne- Brest / CHU BREST / INSERM U 613 5 EFS Bretagne - Site de BREST P 237 Development of a meniscal substitute by combining an inert scaffold and mesenchymal stromal cells

The objective of this Program is to develop a meniscal substitute by combining an inert scaffold and mesenchymal stromal cells (MSCs).

Methodology: The colonisation process of a inert scaffold is currently evaluated. To achieve this goal, various seeding options were tested to colonise the scaffolds with MSCs. The phenotype status of the cells was also considered to define if a differentiation of the MSCs occurs depending on the culture conditions. Immunohistochemistry and flow cytometry were used to characterise the cells. A scanning electron microscopy study was performed to observe cellular distribution inside the scaffold. In parallel, a transfection protocol of the MSCs was initited to define the impact of the transgene on MSCs differentiation.

Results: Cell proliferation inside and on the surface of the matrix appears to be related to the culture media. Platelet lysate allows a better proliferation than fetal bovine serum can do. Some expansion factors and some molecules involved in differenciation process (TGF B3), also have a positive effect on the scaffold's colonisation. Depending on culture conditions, an initiation of differenciation was observed, in particular with a collagen II expression.

Conclusion: These promising results are the first steps of the development of a cellularised meniscal substitute. The long term behaviour of the MSCs in the scaffold remains to be studied. Mechanical and physical resistances of the cellularised scaffold have also to be studied. This would allows to conclude on the improvment of the performance of the scaffold currently commercialised and implanted without cells.

Anghel A Professor Tamas L Dr Samoila C Dr Seclaman E Dr Paunescu V Professor 2 Lighezan D Dr 3 Tatu C Dr 4 Bujor C Dr Draghici D Dr 3

2 Physiology Department, University of Medicine and Pharmacy Victor Babes, Romania 3 Cardiology Department, University of Medicine and Pharmacy Victor Babes, Romania 4 Biology Department, University of Medicine and Pharmacy Victor Babes, Romania P 238 Circulating endothelial progenitor cells (EPCs) in metabolic syndrome therapy

Background: Circulating endothelial progenitor cells (EPC) are derived from more immature, undifferentiated circulating stem cells, being involved in repairing of the damaged endothelium. The aim of this study was to quantify the circulating stem cells (CD34⁺) and EPC (CD34⁺/VEGFR2⁺), in two groups of metabolic syndrome patients (MetSyn), with and without specific therapy (statins), in order to evaluate the therapy efficiency.

Methods: The CD34⁺ cells and CD34⁺/VEGFR2⁺ were quantified in peripheral blood samples from 20 patients with MetSyn, divided in two groups: patients newly diagnosed with no therapy ( n = 10, age 42 ± 10 ) and patients with at least 3 month of statins therapy (n = 10, age 47 ± 6). 10 mL venous blood sample was used for the isolation of mononuclear cells in Ficoll density gradient. CD34⁺ enriched populations were obtained from peripheral blood mononuclear cells by immunomagnetic techniques (Miltenyi Biotec GMBH). The isolated subpopulations of cells were labeled with antibodies: anti CD34-FITC human and anti VEGFR2/KDR-PE human according with the manufacturer's recommendations and analyzed by flow cytometry.

Results: The study has shown that the number of both CD34⁺ and CD34⁺/VEGFR2⁺ cells in the group of metabolic syndrome patients following statins therapy was higher comparative with untreated MetSyn patients: 38.73% increase for CD34⁺ cells (1283 ± 1232 vs. 786 ± 570) and 54.05% increase for CD34⁺/VEGFR2⁺ cells (407 ± 357 vs. 187 ± 159)

Conclusion: These preliminary results have shown that the statin therapy administration have improved endothelial repair capacity by increasing the number of circulating stem cells CD34⁺, the increase being more accentuated for EPC CD34⁺/VEGFR2⁺ cells.

Sevim H Miss 1 Akbay E Miss 1 Gurpinar Ö.A Dr 1 Gunaydin S Professor 2 Onur M.A Professor 1

1 Hacettepe University, Faculty of Science, Department of Biology, Beytepe, 06800, Ankara, Turkey 2 Kırıkkale University, Faculty of Medicine, Department of Thoracic and Cardiovascular Surgery, Kırıkkale, Turkey P 239 Differentiation and Characterization of Cardiomyocytes from Rat BM-MSCs For Myocardial Regeneration

Background: Cell transplantation which aims to introduce healthy myogenic cells into the myocardium of the diseased heart, is showing a bright future in myocardial regeneration therapy. The differentiation potential of bone marrow mesenchymal stem cells (BM-MSCs) can be key role for myocardial regeneration. (1). 5-Azacytidine (5-Aza) is a cytosine analogue and demethylating agent. Recent studies showed that 5-Azacytidine can be used as a differantiation factor for cardiomyocyte differentiation. The present study investigated the replication lifespan and chemical-induced cardiomyogenic differentiation of rat MSCs in vitro.

Method: BM-MSCs isolated from rats and characterized with immunofluorescence staining method for specific cell surface proteins CD13 and CD29. BM-MSCs at P2 and P3 were sellected for cardiomyocyte differentiation. BM-MSCs were exposed to different concentrations of 5-Aza for 24 hours, untreated cells used as the control group. Differentiated cells were incubated for 4 weeks and stained with cardiomyocyte specific markers desmin and cardiac troponin-T by using TR and FITC fluorochroms and cardiac troponin-I levels was measured with ELISA method for the cardiomyocyte characterization.

Results: BM-MSCs at different pasages (P2 and P3) can differentiate into cardiomyocytes with 5-Aza treatment. Differentiated cells started to form cell colonies and expecially these colonies stained with desmin and cardiac troponin-T. In respect of ELISA method results 3 μM and 5 μM 5-Aza dilution displayed the highest amount of cardiac troponin-I.

Conclusion Differentiated mesencyhmal stem cells are considered to have significant applications in cellular approaches for regeneration of heart muscle losses.

Reference

1 Wakitani S. Muscle Nerve, 1995; 18:1417–1426. a-2117 de Elvira MC Ruiz Dr 1 1 12th Floor, Guy's Tower, GSTT, London SE1 9RT, UK P 240 The EBMT Registry: A possible model for Cellular Gene Therapy

The European Group for Blood and Marrow Transplantation (EBMT) is a non-profit organisation established in 1974 in order to allow scientists and physicians involved in clinical bone marrow transplantation to share their experience and develop co-operative studies. From the 70's onwards, clinical data started being collected into what is now known as the EBMT Registry.

When the EBMT started collecting data, transplantation was not a main stream procedure. Today it is for some indications and we believe that the continuous collection of Registry data has widened the experience and improved the outcome of transplanted patients. The Registry currently contains clinical data on more than 315,000 patients, submitted from more than 50 countries.

There is a growing desire on the part of donor registries, health departments, cord blood banks, study groups and other outcome registries to be able to access the content of the EBMT Registry and we developeding closer collaborations.

The EBMT overlaps with the community involved in cellular therapies. A Cellular Therapy Med-A (“minimum essential data”) was designed by the Cellular Therapy committee and has been in use for the last 2 years.

We believe that the EBMT could offer its expertise on data collection and part of the Registry infrastructure to kick start a similar data collection exercise in the Cellular Gene Therapy community. We are looking to further enhance our data collection system, to better serve our centres, improve collaboration with existing partners and create new partnerships. An invitation to do just that is offered to the Cellular Gene Therapy community.

Arens A. Miss 1 Gustafson D. Mr 1 Bartholomae C. Miss 1 Gabriel R. Mr 1 Appelt U. Mr 1 Giordano F. Dr 1 Deichmann A. Dr 1 Glimm H. Dr 1 Laufs S. Dr 1 von Kalle C. Professor 1 Schmidt M. Mr 1

1 National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany P 241 Genome Wide Bioinformatical Analysis of Vector Integration Sites

To address the biosafety and to uncover vector induced side effects of gene transfer approaches, genome wide integration site profiles of the different vector types used in preclinical and clinical studies need to be determined. Combining standard or non-restrictive (nr) linear amplification-mediated (LAM) PCR and next generation sequencing (454/Roche) provides a time- and cost-efficiency tool for integration site retrieval. Our analysis tools, Quickmap (www.gtsg.org) and HISAP (Paruzynski A et al. Nature Protocols 2010), use the sequencing data output to identify relevant integration sites characterized by the chromosomal locus and further genomic information such as distance and orientation to the next genes or retrieval frequency of individual integration sites.

Besides providing an automated, standardized and easy to handle output for non-bioinformaticans, integration site analysis software needs to scope with a vast amount of different vector types (e.g. high frequent integrating oncoretro- and lentiviral vectors, low-frequent integrating integrase deficient lentiviral and AAV vectors as well as transposons such as sleeping beauties) used in various areas of gene transfer approaches. This requires a hitherto unknown flexibility of integration site analysis programs and relevant downstream programs to answer the specific scientific questions beyond. Here, we will present some aspects of our ‘downstream′ bioinformatical data analysis system that concentrate on these requirements, including (i) precision and mechanisms of vector integration, (ii) sequence homology comparison e.g. for targeted integration approaches, (iii) clonal constitution and dynamics of gene-modified cell populations in preclinical and clinical gene transfer settings and and others.

Thomaschewski M Mr 1 Warlich M Mr 2 Weber K Dr 1 Volz T Mr 2 Wege H Dr 2 Dandri M Dr 2 Benten D Dr 2 Fehse B Professor 1

1 Research Dept. Cell and Gene Therapy, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany 2 Internal Medicine I, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany P 242 Highly efficient lentiviral ex vivo gene transfer into primary liver cells

Hepatocellular carcinoma (HCC) is the fifth common cancer and characterised by a very poor prognosis. Additionally for many metabolic liver diseases there is no curative therapy option other than liver transplantation. Efficient ex-vivo gene transfer into isolated hepatocytes facilitates both the investigation of HCC mechanisms as well as the development of potential gene therapy approaches for metabolic diseases. We used the novel LeGO vector platform developed in our lab to establish highly efficient short-term protocols for lentiviral transduction of cultivated and primary hepatocytes. For transplantation experiments, we made use of the uPA-SCID mouse model. We first transduced different liver cell lines, in particular human telomerase-immortalised foetal liver cells (FH-hTERT). We found high transduction rates and stable transgene expression in all cells tested indicating strong activity of the used SFFV promoter in hepatocytes. After transplantation of transduced FH-hTERT cells in uPA-SCID mice we observed stable transgene expression for at least six months in vivo. We went on with the transduction of primary hepatocytes. In order to preserve vitality and prevent dedifferentiation a 1-hour ex-vivo transduction protocol was established. Using this protocol we obtained transduction rates of >60% for primary murine hepatocytes. Transduced murine hepatocytes engrafted at remarkable rates after intrasplenic transplantation in hemizygote uPA-SCID mouse. The new protocol allows high-efficiency transduction while preserving the phenotype and engraftment potential of primary hepatocytes. It thus will be a very useful tool for investigating the roles of various genes in HCC genesis and the development of ex-vivo gene therapy approaches.

Sgualdino J Dr Merella S Dr Naldini L Professor 2 Montini E Dr

2 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy P 243 Characterization of mRNA splicing perturbation in genes targeted by lentiviral vector integration

Oncogenesis induced by insertional mutagenesis with gene therapy vectors occurs mainly by deregulation of proto-oncogenes found at or nearby the insertion site. Proto-oncogene activation occurs by an enhancer-mediated mechanism or by a process of splicing capture which generates chimeric transcripts comprising portions of vector and cellular mRNAs. Although the activation of oncogenes may be reduced by the use of self-inactivating design and moderate cellular promoters, how to reduce genotoxic splicing capture events and aberrant transcript formation triggered by vector integration is still unclear. In this perspective, we developed a new PCR technique named Linear Amplification-Mediated PCR on cDNA (cLAM-PCR) which allows retrieving aberrantly spliced mRNAs that contain LV sequences fused with cellular transcripts.

We applied cLAM-PCR on lentiviral vector (LV)-transduced cell lines and primary human HSCs and identified several established and previously unknown splice sites within the LV backbone that participate in the aberrant splicing process with variable efficiency. Preliminary results with different LV designs show that the integrated LV can perturb the processing of cellular transcripts by interacting with the cellular splicing machinery and fusing with its own splice sites to cellular splice sites both upstream and downstream the integration site. Moreover, qPCR on different LV portions allowed us identifying different splice sites as major o minor contributors to the aberrant splicing process. This strategy will allow characterizing the mechanism and genetic features that modulate vector-induced aberrant splicing. In a biosafety perspective, elimination of splice sites within the lentiviral backbone will be instrumental in enhancing the safety of LVs.

Moiani A Dr 1 Sartori D Dr 1 Miccio A Dr 2 Cocchiarella F Dr 2 Cattoglio C Dr 1 Mavilio F Professor 1

1 Laboratory of Gene Expression, Istituto Scientifico H. San Raffaele, Milan 2 Center for Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy P 244 LV integration in human genes generates abnormal transcripts through the usage of HIV splice sites

HIV-derived, self-inactivating (SIN) lentiviral vectors (LVs), depleted of LTR enhancer and promoter regions, provide an efficient and versatile gene delivery system that overcomes most of the risks of insertional gene activation observed with wild-type LTR, MLV-derived retroviral vectors. Nevertheless, since LVs integrate preferentially into active genes, they have the potential to de-regulate gene expression at the post-transcriptional level, by interfering with the normal splicing and polyadenylation of primary transcripts. To test this hypothesis, human T cells were transduced with a specifically designed, “splice trap” LV, and cloned for the expression of a promoter-less GFP gene placed downstream of the constitutive HIV GAG splice acceptor site. In parallel, T-cells and keratinocytes were transduced with a conventional SIN-LV and randomly cloned. Integration sites were mapped by LM-PCR in 70 T-cell and 31 keratinocyte individual clones, and chimeric transcripts identified by RACE PCR and exon-specific RT-PCR. Abnormal, chimeric transcripts were identified in more than 50% of the LV target genes in both cell types. Semi-quantitative RT-PCR revealed that fusion transcripts were represented at low to high levels compared to constitutively spliced, wild-type transcripts. Fusion transcripts were generated through aberrant splicing caused by the usage of both constitutive and cryptic splice sites located in the viral intron and the U5 portion of the 5′ LTR. These data point out a potential risk of post-transcriptional genotoxicity of LVs in human cells.

Ranzani M Dr 1 Schmidt M Dr 2 Sanvito F Dr 3 Benedicenti F Dr Bartholomä C Dr 2 Cantore A Dr 1 Sergi L Sergi Dr Cesana D Dr 1 Doglioni C Professor 3 VonKalle C Professor 2 Naldini L Professor 1 Montini E Dr

1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; San Raffaele University, Milan, Italy 2 National Center for Tumor Diseases, Heidelberg, Germany 3 Pathology, San Raffaele Scientific Institute, Milan, Italy P 245 Self inactivating lentiviral vectors show undetectable genotoxicity in sensitive mouse models of hepatocarcinogenesis

We setup sensitive assays for liver genotoxicity by systemic injection of the genotoxic lentiviral vector (LV) LV.ET.LTR in Cdkn2a^−/− tumor prone mice or models of chronic liver injury. LV.ET.LTR injection triggered hepatocellular carcinomas (HCC) in 30% of Cdkn2a^−/− mice (p < 0.01). Remarkably, in chronic liver injury models, such as the liver-specific Pten deficient mice or wild type (WT) mice treated with CCl₄, LV.ET.LTR induced HCCs in 25% and 75% of mice respectively (p < 0.05). Integration retrieval in tumors led to the identification of genes repeatedly targeted in independent tumor. These genes are oncogenes because they are able to induce HCC upon liver gene transfer and have a role in human pathology as they were found to be overexpressed and/or map in genomic regions that are amplified in human HCCs.

We exploited these models of hepatocarcinogenesis to test the safety of SINLVs developed for the therapy of hemophilia B. A SINLV that express factor IX transcript under the control of hepatospecific enhancer with target sequences for microRNA 142 (SINLV.ET.FIX.142T) was injected into newborn Cdkn2a^−/− (N = 39) or WT mice (N = 24). The survival and HCC incidence was monitored and compared to control mice (Cdkn2a^−/− N = 19, WT N = 23) injected with the genotoxic LV.ET.LTR. SINLV.ET.FIX.142T did not induce HCCs as assessed by autoptical analysis neither in tumor prone nor WT mice. On the contrary, the mice injected with matched doses of the genotoxic LV developed HCCs at frequency similar to previous experiments. These data provide encouraging results on the biosafety profile for SINLVs-based liver gene therapy.

Cesana D. Dr 1 Ranzani M. Dr 1 Bartholomä C. Dr 2 Volpin M. Dr 1 Benedicenti F. Dr 1 VonKalle C. Professor 2 Schmidt M. Dr 2 Naldini L. Professor 1 Montini E. Dr 1

1 Regenerative Medicine Stem Cells and Gene Therapy, San Raffaele Telethon Institute for Gene Therapy, Italy 2 NCT-Heidelberg, National Center for Tumor Diseases, Germany P 246 Systemic injection in a tumor prone mouse unravels the residual genotoxicity of self-inactivating lentiviral vectors

Our previously validated Cdkn2a^−/− lineage- transduction/transplantation mouse model revealed that a lentiviral vector (LV) containing the spleen focus-forming virus (SF) enhancer/promoter sequences within the LTRs (LV.SF.LTR) was genotoxic with respect to mock-controls. Intriguingly, a self-inactivating (SIN)LTRs LV containing the SF sequences in internal position (SIN.LV.SF) did not cause any tumor acceleration. The lower genotoxicity of this design coupled to the background oncogenesis of this mouse model could hamper the detection of residual vector genotoxicity. Thus, alternative in vivo assays to stringently test the safety of SINLVs are still an unmet need. Here, we report that systemic injection in newborn Cdkn2a^−/− mice of LV.SF.LTR induces an earlier hematopoietic tumor onset with respect to mock-controls or mice transplanted with lineage- Cdkn2a^−/− cells transduced with LV.SF.LTR. From these tumors we identified 18 Common Insertion Site (CIS), among which Braf (68 integrations in a genomic region of 4 kb), Sfi1 and Mef2c were already identified as CIS by retroviruses or transposon integrations studies. These data indicate that insertional mutagenesis is the mechanism responsible for the accelerated tumor onset. Remarkably, when we used this new system to reassess the putative genotoxicity of SIN.LV.SF, newborn Cdkn2a^−/− mice systemically injected with this vector developed tumors at a significantly earlier onset with respect to untreated mice. Our data show for the first time that by this approach it is possible to detect the residual genotoxicity of a SINLV containing an enhancer in internal position and establish a new in vivo genotoxicity assay to test improved versions of SINLVs.

Artus A Dr 1 Duros C Dr 1 Botbol Y Dr 2 Schultz S Dr 3 Schmidt M Dr 3 von Kalle C Dr 3 Lavigne M Dr 2 Cohen-Haguenauer O Y Dr 4

1 LBPA/ CLINIGENE, Ecole Normale Superieure (ENSC), 94235 Cachan Cedex, France 2 Laboratory of Structural Virology, Pasteur Institute, 75015 Paris, France 3 Laboratory of Translational Oncology, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany, 69120 4 LBPA/ CLINIGENE, Ecole Normale Superieure (ENSC), 94235 Cachan Cedex, France, 4Hôpital Saint-Louis, 75475 Paris cedex 10, France P 247 Directed integration of insulated lentiviral vectors to the heterochromatin towards safer gene transfer to stem cells

We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing integration to heterochromatin. We have previously identify a specific GIE combination in collaboration with Nic Mermod, which translates into high titers and boundary effect in both gammaretro and lentivectors (DCaro4). In target cells, the expression profile becomes homogenous; its level is strictly conditioned by the promoter. These data remain stable in both HeLa cells and cord blood HSCs for over three months, irrespective of the multiplicity of infection (MOI). Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, DCaro4 has been further tested and compared to its non-insulated counterpart, with chimeric HIV-1 derived integrases targeting heterochromatin through either histone H3 or methylated CpG islands (ML6 & ML10 chimeras, respectively). With DCaro4 and ML6 chimeras, a homogeneous expression is sustained over time. With the control, GFP expression is just over background double-mutant in catalytic and ledgf binding-sites while expression can be induced with HDAC. In CD34+ cells from cord-blood, these data are recapitulated with the ML6 chimera. High throughput integration sites analysis reveals a distinct profile with DCaro4-ML6. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent genotoxicity.

This work has been performed with the support of the EC-DG research within the FP6-Network of Excellence, CLINIGENE: LSHB-CT-2006-018933.

Gijsbers R Dr 1 Ronen K Dr 2 Vets S Ms 1 Malani N Mr 2 De Rijck J Dr 1 McNeely M Ms 1 Bushman F Professor 2 Debyser Z Professor 1

1 Division of Molecular Medicine, Katholieke Universiteit Leuven, Kapucijnenvoer 33, VCTB + 5, Leuven, B-3000, Belgium 2 Department of Microbiology, University of Pennsylvania School of Medicine, 402C Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104, Pennsylvania, USA P 248 LEDGF/p75 fusion proteins to retarget lentiviral integration away from genes

Correction of genetic diseases requires integration of the therapeutic gene copy into the genome of patient cells. Retroviruses are commonly used as delivery vehicles because of their precise integration mechanism, but their use has led to adverse events in which vector integration activated proto-oncogenes and contributed to leukemogenesis. Here, we show that integration by lentiviral vectors can be targeted away from genes using an artificial tethering factor. During normal lentivirus infection, the host cell–encoded transcriptional coactivator lens epithelium-derived growth factor/p75 (LEDGF/p75) binds lentiviral integrase, thereby targeting integration to active transcription units and increasing the efficiency of infection. We replaced the LEDGF/p75 chromatin interaction-binding domain with CBX1. CBX1 binds histone H3 di- or trimethylated on K9, which is associated with pericentric heterochromatin and intergenic regions. The chimeric protein supported efficient transduction of lentiviral vectors and directed the integration of lentiviral vectors outside of genes, near CBX1 binding sites, regions generally disfavored for integration in wild-type cells. Despite integration in regions rich in epigenetic marks associated with gene silencing, lentiviral vector expression remained efficient. Thus, these results establish LEGDF/75 as the dominant targeting factor for lentiviral integration and demonstrate that engineered LEDGF/p75 fusion proteins provide technology for controlling integration site selection.

Heckl D Dr 1 Meyer J Dr 1 Knoss S Dr 1 Krause J Dr 1 Rudolph C Dr 2 Schlegelberger B Dr 2 Baum C Professor 1 Modlich U Professor 1

1 Department of Experimental Hematology, Hannover Medical School, Hannover, Germany. 2 Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany Heckl Dirk 1 Meyer Johann 1 Knöss Sabine 1 Krause Johanna 1 Rudolph Cornelia 2 Schlegelberger Brigitte 2 Baum Christopher 1 Modlich Ute 1 1 Department of Experimental Hematology, Hannover Medical School, Hannover, Germany 2 Institute of Cell and Molecular Pathology, Hannover Medical School, Hannover, Germany P 249 Lentiviral vector induced leukemia in a murine bone marrow transplantation (BMT) model

The induction of leukemias by insertional mutagenesis has been observed in mouse models and clinical trials when using gammaretroviral vectors with strong viral enhancer/promoters. Lentiviral insertional leukemias have only been described in tumor-prone mouse models. With the aim of evaluating lineage-specific expression, we transplanted C57Bl/6 mice with BM cells transduced with lentiviral vectors expressing EGFP from the human GPIba promoter. Seven months after BMT one mouse succumbed to leukemia with leukocytosis and hepatosplenomegaly. Leukemic cells were donor derived, EGFP + with a surface marker phenotype of early B cell progenitors (B220 + CD43 + IgM−). Southern blot analysis confirmed a clonal leukemia with two vector insertions one of which was in the 8th intron of the Early B cell factor 1 (Ebf1) in forward orientation. Splice sites in the GPIba promoter were involved in splice products from the Ebf exon 8 leading to an early transcriptional termination (5-8% of the allele's RNA). Furthermore RNAs were detected that spliced from the vector into exon 9 and 10. B-cell differentiation was enhanced by overexpressed full length Ebf1 and unaltered by the truncated form. No chromosomal aberrations were detected by SKY. A further mouse developed granulocytic proliferation 5 weeks after BMT with cells transduced with a lentiviral SIN vector driving Ebf1 from the SFFV promoter. A single intronic insertion in the Pdgf C gene upregulated expression ∼100×. Our observations represent rare events detected in >200 transplanted mice but demonstrate the potential of lentiviral vectors to insertionally deregulate gene expression with significant functional consequences.

Cassano M Dr 1 Quattrocelli M Dr 1 Dellavalle A Dr 2 Salvade A Dr 3 Ronzoni F Dr 4 Cossu G Dr 5 Sampaolesi M Dr 6

1 Laboratory of Translational Cardiomyology, Stem Cell Interdepartmental Institute, KU Leuven, Herestraat 49 O&N1 bus 814, 3000 Leuven, Belgium 2 Division of Regenerative Medicine, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy 3 Laboratory of Cell Therapy “Stefano Verri,” Pediatric Department, “University of Milano-Bicocca, Monza, Italy 4 Human Anatomy, University of Pavia, Via Forlanini 8, 27100 Pavia, Italy. 5 Division of Regenerative Medicine, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; Department of Biology, University of Milan, Via Celoria 28. 20129 Milan 6 Laboratory of Translational Cardiomyology, Stem Cell Interdepartmental Institute, KU Leuven, Herestraat 49 O&N1 bus 814, 3000; Human Anatomy, University of Pavia, Via Forlanini 8, 27100 Pavia, Italy P 250 Alpha sarcoglycan is required for FGF dependent myogenic progenitor cell proliferation in vitro and in vivo

Background: Mice null for a-sarcoglycan (Sgca-null) develop a progressive myopathy and serve as a model for human limb girdle muscular dystrophy 2D. In mice the myopathy is very severe; a-sarcoglycan expression can be restored by adenovirus-mediated gene delivery and the dystrophic phenotype has been rescued by cell therapy as shown by previous studies.

Methods: In an attempt to characterize the cellular basis of the disease, we cloned myogenic progenitor cells (mpc), the resident post-natal muscle progenitors from dystrophic and wt mice analyzing their proliferative potency and differentiation ability. We further examined the ability of donor mpcs to proliferate and properly colonize muscle in vivo.

Results: Mpc cells from Sgca-null mice gave rise to smaller clones than wt or mdx dystrophic mice. The proliferation impairment of Sgca-null myogenic precursors was confirmed by single fiber analysis. This difference correlates with the expression of Sgca during the proliferation of mpc: in the absence of dystrophin and associated proteins, which are only expressed after differentiation, a-SG forms a complex with and stabilizes the FGF receptor. Ablation of the gene leads to FGF receptor loss from the membrane and impaired proliferation of mpc. The low proliferation rate of Sgca-null mpc was not affected by basic FGF administration but rescued by transduction with Sgca-expressing lentiviral vectors. When transplanted in dystrophic muscle, Sgca-null mpcs exhibited reduced homing and proliferation.

Conclusion: The reduced proliferation ability of mpcs explains the severity of this muscular dystrophy and why donor progenitor cells engrafted efficiently and consequently ameliorated the disease.

Uchida H Dr 1 Chan J Dr 1 Nakano K Dr 2 Kwon H Dr 3 Goins WF Dr 1 Kumagai I Dr 4 Kuroki M Dr 5 Grandi P Dr 1 Cohen JB Dr 1 Glorioso JC Dr 1

1 University of Pittsburgh, PA 2 Kyushu University, Japan 3 Korea Institute of Radiological and Medical Sciences, South Korea 4 Tohoku University, Japan 5 Fukuoka University, Japan, P 251 Hyperactive glycoprotein B mutations augment fully retargeted HSV infection

Entry of HSV-1 is initiated by the binding of glycoprotein D (gD) to one of its receptors, herpesvirus entry mediator or nectin-1. This interaction causes a conformational change in gD that induces activation of the fusion effectors gB and/or gH required for virus penetration. To retarget virus entry exclusively to epidermal growth factor receptor (EGFR)-bearing cells, we mutated or deleted gD residues essential for binding to the natural receptors and inserted EGF or an EGFR-specific single-chain antibody (scFv) near the amino terminus. Although ablation of the natural tropism was achieved, entry through EGFR was inefficient. In an attempt to isolate gain-of-function mutations that could generally stimulate entry, we passaged a nectin-1-ablated gD-mutant virus repeatedly through cells harboring a gD-binding-impaired nectin-1 until plaques appeared. Surprisingly, each of the purified isolates contained the same double mutation in gB, D285N/A549T. The gB mutations in a wild-type virus background enabled entry into cells that are normally resistant to HSV infection due to the absence of authentic gD receptors. The gB-mutant virus was also capable of entering cells through nectins other than nectin-1, and entry into nectin-1-bearing cells was markedly accelerated compared to wild-type virus. We combined the gB mutations with the EGFR-retargeted gD alleles and found that these viruses entered EGFR-transduced cells 100-fold more efficiently than the wild-type-gB versions, without detectably raising off-target entry. These viruses entered various tumor lines expressing EGFR with similar efficiencies as wild-type virus utilizing the natural receptors, and the retargeted infection was completely blocked by an anti-EGFR antibody. We also inserted a carcinoembryonic antigen (CEA)-specific scFv into detargeted gD and observed efficient CEA-dependent entry, supporting the broader applicability of our retargeting system. Our observations demonstrate that hyperactivity of a downstream component of the entry cascade can greatly enhance the infectivity of retargeted HSV vectors.

Moreno P Dr 1 Blomberg KE Dr 1 Berglöf A Dr 1 Lundin KE Dr 1 Asplund L Dr 1 Bestas B Dr 1 El-Andalussi S Dr 1 Wengel J Dr 2 Smith E Professor 1

1 Department of Laboratory Medicine, Clinical Research Center, Karolinska Institute, 141 86 Huddinge, Stockholm, Sweden. 2 Nucleic Acid Centre, Department of Physics and Chemistry, University of Southern Denmark, Denmark P 252 Splice switching in hematopoietic cells — a novel strategy to correct BTK deficiency derived from aberrant splicing

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disease, where the gene, Bruton's tyrosine kinase (BTK), is mutated. XLA-patients suffer from recurrent infections due to a blockage of B-cell development resulting in almost absent antibody production. Current treatment options are consisting of life-long periodic administrations of immunoglobulines intravenously and antibiotics. This option is only partially effective, expensive and can lead to long-term complications. We have previously found a new mutation in intron 4 of the BTK gene responsible for an aberrant splicing pattern in a Swedish family with XLA subjects. The mutation, which causes a GAàGT change, forms a novel 5′ cryptic splice site resulting in the creation of a pseudo-exon of 118 bp.

Splice switching by the use of antisense oligonucleotides (ONs) is a novel therapeutic method with great potential for several types of genetic disorders. Clinical trials are currently being conducted in Duchenne's muscular dystrophy (DMD) with the aim of convering DMD to the milder Becker dystrophy. For XLA-patients, who incorporate BTK pseudo-exons, it is in theory possible to generate normal transcripts by treatment with antisense ONs blocking the aberrant cryptic splice site. We initially scanned a set of 2′-O-methyl-based splice switching ONs (SSONs) for their capacity to redirect the cryptic splice in intron 4 of BTK using different reporter constructs. A set of highly active SSONs was identified. These SSONs have been further optimized, in both length and composition, using substitution with Locked nucleic acid (LNA) bases. In order to develop a relevant in vivo-model for experimental treatment, we have generated bacterial artificial chromosome- (BAC) transgenic mice. These animals carry either the normal, or the intron 4-mutated, entire human genomic BTK locus and their B-lymphocytes therefore utilize the endogenous human BTK promoter and make the corresponding pre-mRNA transcript. We observed that BAC-transgenic mice faithfully express the same splice-defective transcript as seen in XLA-patients with this mutation. Importantly, we found that ex-vivo treatment with SSONs converted the full-length, defective human BTK transcript in B-lymphocytes from these mice to the normal transcript. Thus, the BAC-transgenic mice, bred on a mouse-BTK-deficient background, will serve as an adequate preclinical in vivo-model for the development of a new therapy for XLA and will give important insights into ON targeting for hematopoietic cells. Also very important we have been successful in correcting the mutated BTK transcript in the XLA patient monocytes (obtained by apheresis and purified by elutriation) and BTK protein production was restored. Thus, we show for the first time that B lymphocytes are amenable for splice correction therapy and that we can revert the human mutated BTK transcript to its correct form thereby restoring B cell function. This work provides the basis for a new therapy for selected genetic mutations, potentially leading to a new treatment option for the patients (in a personalized medicine scenario) The BAC-transgenic mice, bred on a mouse-BTK-deficient background, will serve as an adequate preclinical in vivo model not only for the development of a new therapy for XLA, but will also serve as a versatile model for studying splice modulation and ON targeting in vivo in the hematopoietic cell lineage.

Adachi N Yokohama City University

P 253 Towards 100% gene-targeting efficiency in human cells

An ideal approach to gene therapy would be to repair the defective gene by means of gene targeting via homologous recombination. Although the frequency of gene targeting (or gene correction) is extremely low in human cells, recent studies clearly support the effectiveness of using nucleases or viral vectors as tools to increase absolute gene-targeting frequency. Yet, as for the latter, unfamiliarity with viral manipulation techniques might be a barrier at least for some researchers. Targeting efficiency can actually be obtained by inactivating certain DNA repair proteins; however, perturbing cellular DNA repair machinery may be capable of causing unexpected genomic instability involving off-target effects, which perhaps might also be the case for nuclease-boosted gene targeting.

We have recently shown that a human precursor B cell line, Nalm-6, allows for high-efficiency gene targeting (typically ∼1–5% efficiency), even when conventional (non-viral) replacement-type vectors are used, and the efficiency can be increased 2–3-fold simply by maximizing the electroporation condition for gene transfer. More importantly, with the use of exon-trapping-type vectors, the targeting efficiency has jumped to more than 50%. We are currently trying to extend this technology to other human cell lines. We have also developed a simple and rapid system to construct any exon-trapping-type vectors within one week, without the need for ligation reactions or restriction mapping. Although such vectors may only be applicable to human genes with relatively high levels of expression in the cell, we anticipate that our technology will eventually make it possible to disrupt (or correct) a gene of interest with nearly 100% efficiency.

Colin M 1 Lecolle K 1 Caillierez R 1 Begard S 1 Zommer N 1 Augert F 2 Blum D 1 Hantraye P 3 Deglon N 3 Buee L 1

1 Inserm, U837, Place de Verdun, 59045 Lille, France. 2 Université Lille 2, Faculté de Médecine, IMPRT, JPARC, Place de Verdun, 59045 Lille, France. 3 CEA, Institute of Biomedical Imaging (I2BM), Molecular Imaging Research Center (MIRCen), F-92265 Fontenay-aux-Roses, France P 254 Overexpression of a wild-type human Tau leads to neurofibrillary degeneration in a new non-transgenic rat model

Background: In Alzheimer's disease (AD), neurofibrillary degeneration (NFD) is a brain lesion characterized by intraneuronal aggregation of Tau. It is also encountered in a number of neurodegenerative disorders referred to as Tauopathies. The available transgenic mouse models take advantage of mutations on tau gene found in rare forms of fronto-temporal dementia. However, in AD, no Tau mutation is found and NFD develops through specific cortico-cortical pathways, starting in the hippocampal formation, which is not reproduced in transgenic animal models. Tau spreading process remains unknown probably because the mechanism of Tau pathology differs between AD and Tau mutation cases.

Methods: Targeting brain regions usually affected by NFD in AD requires the induction of localized expression of Tau. Lentiviral vectors (LV) are suitable tools to deliver gene in a specific region as they can be easily injected into the brain to mediate expression in neurons. In the present work, we developed a new non-transgenic rat model after intracranial injections of LV encoding a wild-type (WT) or a mutated form of Tau into the hippocampal formation.

Results: The two constructs mediated an age-dependent NFD which is more pronounced with the mutated form and lead to altered neurochemical profile and subsequent cognitive impairments as soon as 8 months LV post-injection. Moreover, for both constructs, cortico-cortical Tau projections were observed but with different pathways suggesting that the spreading process are different. These results emphasized that development of models based on WT form of Tau is now crucial to understand its ‘prion-like′ propagation to the brain.

Namgung R 1 Singha K 1 Jon S 2 Park IK 3 Kim WJ 1

1 Department of Chemistry, BK21 program, Polymer Research Institute, Pohang University of Science and Technology, San 31, Hyoja-dong, Pohang 790-784 (Korea) 2 Department of Life Science, GIST, 1 Oryong-dong, Buk-gu, Gwangju 500-712 Korea 3 Department of Biomedical Sciences, Chonnam National Univ. Medical School, Gwangju 501-746 Korea P 255 Hybrid superparamagnetic iron oxide-branched polyethylenimine magnetoplexes for gene delivery

The work demonstrated the development of thermally cross-linked superparamagnetic nanomaterial which possessed polyethylene glycol moiety and covalently linked branched polyethylenimine (BPEI), and exhibited highly efficient magnetofection even under serum conditioned media. The study showed its high anti-biofouling, cell viability and serum stability and thus revealed a potential magnetic nanoparticle-mediated targeted gene delivery system. This superparamagnetic particle mediated rapid and efficient transfection in primary vascular endothelial cells (HUVEC) successfully inhibits expression of PAI-1 which is responsible for various vascular dysfunctions such as vascular inflammation and atherosclerosis and thereby provides a potential strategy to transfect highly sensitive HUVEC. The sequential steps for the enhanced magnetofection had been studied by monitoring cellular uptake with the aid of confocal microscopy.

Nakao A 1 Yamamura K 1 Takeda S 1 Nomoto S 1 Sahin T 1 Kanzaki A 1 Sugimoto H 1 Shirota T 1 Kodera Y 1 Kasuya H 1

1 Nagoya University Graduate School of Medicine P 256 Herpes Oncolytic Virus Elicits Greater Anti-Tumor Immunity

Oncolytic virus therapy is becoming a promising anti-cancer therapeutic, and oncolytic viruses have been shown to elicit anti-cancer immunity. We evaluated the effects of the herpes oncolytic virus R3616 on the host immune response compared to a representative chemotherapy drug, 5-FU. R3616 or 5-FU was directly injected into the subcutaneous tumors of non-immunized mice. R3616 induced a greater number of tumor-infiltrating T cells, macrophages, and dendritic cells than 5-FU. MC26 cells plus R3616 or 5-FU as an adjuvant were frozen, thawed, and then used to immunize mice. After immunization, the adaptive immune response suppressed subcutaneous tumor growth and prolonged the survival rate of re-challenged mice. The group immunized with MC26 and R3616 had the greatest tumor suppression and the longest survival rate. We implanted 5 mm cubic MC26 tumors subcutaneously into each group of immunized mice, and then monitored the differences in the number of infiltrating CD8- and CD4-positive lymphocytes by immunofluorescence. The mice immunized with MC26 cells and R3616 showed the greatest number of infiltrating T cells in the implanted tumor among the immunized groups. These results indicate that the oncolytic herpes virus R3616 has the potential to elicit host anti-tumor immunity. we will describe together with our clinical data of HF10 herpes oncolytic virus therapy human trial as a back ground date of anti cancer immunity that was elicited by oncolytic virus.

ESGCT 2010 Abstract Author Index

Aartsma‐Rus, A, Or 3

Abad, X, P 132

Abargues, R, P 178

Abboud, Miguel, Or 16

Abdullah, S, P 91, P 224

Abel, T, Or 88, Or 106

Abken, H, P 71

Abriss, D, P 170

Acosta‐Sanchez, A, Or 36, Or 72

Acuto, S, P 169

Adachi, N, P 253

Addamo, RR, P 102

Adigüzel, D, P 86

Agirre, X, Or 23, P 174

Agostoni, V, P 59, P 65, P 67

Ahlroth, M, P 189

Ahmadi, M, P 49, P 74

Ahmed, SO, P 89

Airenne, K, P 189

Aires da Silva, F, P 28

Aiuti, A, Or 17, Or 70, Or 75, Or 86, P 57, P 115, P 120, P 134

Akbay, E, P 239

Akbuğa, J, P 42, P 45, P 156

Akchurin, R, P 234

Akdis, CA, P 145

Akguel, B, P 217

Akizawa, Y, P 208

Al‐Herz, Waleed, Or 16

Al‐Jamal, WAJ, Or 28, P 147, P 199

Alazard‐Dany, N, P 2

Alba, R, P 3, P 19

Albella, B, Or 23

Aldea, M, P 146

Ali, RR, Or 109

Ali, Robin, Or 10

Alici, E, P 56

Aliño, SF, P 176, P 178

Allaume, X, P 218

Allay, J, Or 15

Almåsbak, H, P 64

Alonso‐Ferrero, ME, Or 23, P 144

Alvarez, L, P 174

Alves, PM, P 92, P 94

Ambrosi, A, Or 86

Amendola, M, P 121

Anagnostopoulos, A, P 175

Anagnou, NP, P 161

Andolfi, G, P 142

Andreu, N, P 22

Angell‐Manning, D, P 76

Anghel, A, P 117, P 238

Anghel, M, P 117

Anguille, S, Or 100

Anliker, B, P 81

Annoni, A, Or 69, Or 72, Or 73

Annunziata, P, P 163

Anton, M, P 86, P 230

Antonelli, A, Or 32

Appelt, JU, P 186

Appelt, U, Or 27, P 241

Arata, L, P 2

Arce, F, P 137

Arens, A, Or 27, Or 43, Or 72, P 108, P 110, P 241

Ariatti, M, P 160

Ariga, T, P 113

Ariza, L, P 6, P 85

Arnould, S, Or 52

Arsenakis, M, P 175

Artus, A, P 247

Arzberger, S, P 135

Asin Pardo, L, P 86

Asplund, L, P 252

Astone, D, P 9

Astrologo, L, P 83

Athanasiou, E, P 175

Athanasopoulos, T, P 205

Aubourg, P, Or 30, P 108, P 148

Audit, M, P 108

Augert, F, P 254

Aumann, J, P 32

Auregan, G, P 78

Auricchio, A, Or 108, Or 111, P 163

Aydin, C, P 145, P 180, P 182, P 183, P 184

Ayuk, F, P 109

Ayuso, E, Or 96, P 22

Azon, A, P 177

Azzouz, M, Or 94, P 80

Bacchetta, R, Or 69, P 121

Bachi, A, P 221

Bagnis, C, P 95

Bagnoli, M, Or 21

Baier, R, P 97, P 106

Baiker, A, P 11

Bailly, P, P 95

Bainbridge, JW, Or 109

Baker, AH, P 3

Balci, MK, P 143, P 158, P 159

Baldassarre, G, Or 21

Bali, P, P 113

Ball, C, P 110

Ball, Claudia R, Or 16

Belmonte JCI, Or 13

Baños, R, P 174

Banerjee, Pinaki P, Or 16

Banfi, A, Or 104, P 206, P 231

Bankiewicz, Krystof S, Or 93

Bannister, R, P 100

Baranov, V, P 35

Baranovicova, E, P 46

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