BSGT 2010 Poster Presentations

1 The Netherlands Cancer Institute, Amsterdam, The Netherlands 2 Erasmus Medical Center, Rotterdam, The Netherlands 3 Max Delbrück Center for Molecular Medicine, Berlin, Germany P 1 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) α- and β-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 has been 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 an essential requirement 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.

1 UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH 2 Unidad de Biología Molecular y Celular y Terapía Génica, CIEMAT 28040 Madrid, Spain 3 BHF Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, G12 8TA P 2 Gene-Corrected Neutrophils from Induced Pluripotent Stem Cells Derived from an X-CGD Disease Model

The near-term potential of induced pluripotent stem (iPS) cell research is in modelling rare human diseases for which there is limited understanding of the basic disease mechanism and/or absence of any suitable disease model. X-linked chronic granulomatous disorder (X-CGD) is a rare inherited immunodeficiency (four affected in a million) arising from a single gene (gp91phox) mutation which results in defective neutrophil function. The outcome of clinical trials employing gene-corrected HSCs have indicated that the future of gene therapy for X-CGD will depend on development of novel therapeutic vectors with improved safety and efficacy profiles. Patient-specific iPS cells could provide a unique experimental platform toward achieving this goal. We have generated iPS cells from adult fibroblasts of an X-CGD mouse model, as well as from CGD patient keratinocytes. The iPS cell lines have been tested for expression of pluripotency markers, and for their ability to form teratoma. Neutrophils generated from the murine iPS cells showed functional defect as expected. We further demonstrated genetic correction in the murine iPS cells by transducing them with self-inactivating lentiviral vectors encoding the codon optimized gp91phox transgene. Neutrophils obtained from these iPS cells stained positively in the NBT test. Similar approaches are being carried out to rectify the genetic defect in the patient-generated iPS cells to obtain functional neutrophils. Our results provide proof of principle that iPS cell technology can be successfully implemented in in vitro modelling of X-CGD and could be potentially exploited for therapeutic vector validation and evaluation of safety profile in gene therapeutic approaches for the treatment of X-CGD.

1 School of Biological Sciences, Royal Holloway – University of London, Egham, Surrey, TW20 0EX P 3 Antisense Oligonucleotide-Induced Multiexon Skipping in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin protein, most commonly as a result of frame-shifting mutations, both deletions and duplications, in the dystrophin gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally-deleted, BMD-like but functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) have been designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript so that gene expression is restored. The rapid steady advances made in this field suggest that it is likely that AO-induced exon skipping will be the first gene therapy for DMD to reach the clinic. However, the different deletions that cause DMD would require skipping of different exons, and personalised molecular medicine may be required. As DMD deletions appear to be concentrated in the region around exons 45 and 55 (65% of all DMD mutations), multiexon skipping has been proposed as a means to treat the maximum number of patients with one formulation of AOs. We describe here studies in cultured human skeletal muscle cells to optimise the skipping of exon 45–55 block, using linked AOs tagged with hnRNP A1 binding sites, and polypyrimidine tract binding protein binding sites. This work will be extended in vitro in cultured DMD patient cells and in the humanised DMD mouse, a transgenic mouse that expresses full-length human dystrophin.

1 Institute of Child Health, Gene Therapy Lab, Molecular Immunology Unit, 30 Guildford Street, London WC1N 1EH 2 Great Ormond Street for Children, Immunology-Camelia Botnar Laboratories, London WC1N 3JH 3 Genethon-Direction Unite' INSERM U951, 1 bis rue de l'Internationale, 91002 Evry, France P 4 Development of a GMP-Grade Transduction Protocol for Treatment of the Wiskott-Aldrich Syndrome by Lentiviral Vector-Mediated Gene Therapy

Wiskott-Aldrich Syndrome (WAS) is a primary immunodeficiency disease, characterised by microthrombocytopenia, recurrent infections and associated with an increased incidence of autoimmunity and lymphoid malignancies. In collaboration with Genethon (France) and TIGET (Milan) we have developed a novel VSVg-pseudotyped w.1.6_hWASP_WPRE(VSVg)lentiviral vector, encoding a WAS transgene regulated by natural regulatory sequences. After rigorous preclinical testing in human and murine cells, a clinical trial has been approved in the UK by both GTAC and MHRA. In preparation for this study we have assessed a GMP-compliant protocol for transduction of human CD34+ haematopoietic stem and progenitor cells. Human CD34+ cells were purified and prestimulated in closed cell culture bags (Miltenyi) in serum free medium (X-VIVO20, Lonza) supplemented with 1% human serum albumin (Baxter), and mitogenic cytokines (SCF, FLT3l 300 ng/ml, IL3 20 ng/ml, TPO 100 ng/ml all GMP-like Peprotech) at a density between 0.5–1 × 10⁶ CD34/ml. After 24 hours, cells were transferred to Retronectin precoated bags (Takara) and containing clinical grade lentiviral vector in a range between 1 × 10⁷ and 3 × 10⁸ i.g./ml. Cells were cultured for a further 16–18 hours under the same conditions. After this period, transduced cells were frozen then thawed and tested for cell viability (trypan blue, 7AAD),transduction efficiency, and reconstitution of WASp function. The transduction efficiency assessed by qPCR was 63.1% (batch H.0108.BPF) and 44% (batch H.0208.BPF). No adverse effects on cell viability and proliferation were observed. Furthermore, cytoskeletal function measured in derived dendritic cells and reconstitution of podosomes were clearly observed in vitro. These results demonstrate effective transduction of human WASp-deficient CD34+ cells using a clinically applicable protocol and establishing a procedure for this new clinical study.

1 Royal Holloway University, School of Biological Sciences, Egham, Surrey, TW20 0EX, UK 2 Department of Cellular and Molecular Neuroscience, Division of Neuroscience and Mental Health, Imperial College London, Hammersmith Hospital Campus London W12 ONN, UK 3 The Dubowitz Neuromuscular Centre, Institute of Child Health, University College London WC1N 1EH, UK P 5 Clinically Applicable Dosing Regimens of Phosphorodiamidate Morpholino Oligomer Profoundly Improve Behaviour, Activity, and Dystrophic phenotype in mdx mice

The administration of antisense oligonucleotides (AOs) to skip one or more mutated exons and correct the reading frame of the transcript, is one of the most promising approaches for the treatment of Duchenne Muscular Dystrophy (DMD). Due to the nature of this approach, chronic administration of AOs for all the life of the patient would be necessary. The choice of the best dosing regimen is a pivotal parameter to minimize the doses and optimize the final outcome. In this study 2 different dosages of phosphorodiamidate morpholino oligomer (PMO), designed to skip the mutated exon 23, distributed in 20 injections in a time of 12 months were administered in mdx dystrophic mice. After 4 months of treatment, muscles showed great histological improvement and increased force strength. At the end of the treatment, mice showed a substantial dose-related amelioration of the pathology. The limb strength and activity were greatly enhanced and normalized to the ones of unaffected mice showing that a partial but widespread dystrophin expression can restore the normal activity in mdx treated mice. Our results show that a chronic long term administration of very low doses of unconjugated PMO, consistently ameliorates the muscle dystrophic phenotype and the activity of affected mice, and support the clinical feasibility of this approach in humans.

1 Molecular Immunology Unit, Institute of Child Health, University College London, London, UK 2 Department of Experimental Hematology, Hannover Medical School, Hannover, Germany P 6 Development of Gene Therapy for HLH Due to Perforin Deficiency

Haemophagocytic lymphohistiocytosis (HLH) is a devastating disorder of early childhood arising from defects of T and NK cell cytotoxicity. Four genetic loci have been identified, the most common form 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 in one vector and in the other vector 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, a cell line lacking perforin expression but able to secrete it through a secretory system analogous to that of cytotoxic lymphocytes. Good transduction efficiency was obtained with both vectors and the cells were used to verify cytotoxicity by chromium release assay. Both vectors were able to induce cytotoxicity in RBL2H3 cells. Next, NK cells derived from perforin deficient mice were transduced with both perforin vectors. Low transduction efficiencies were obtained but nevertheless, when cells were used in a cytotoxicity assay to test perforin functionality, both perforin vectors were able to restore cytotoxicity to NK cells. 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.

1 School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, UK 2 Pierre & Marie Curie University, Myology Institute, Paris, France P 7 Evaluation of the Truncated Products of Multiple Exon Skipping in DMD Therapy

Duchenne's muscular dystrophy (DMD) is a severe muscle wasting disorder affecting 1/3500 male births. DMD is caused by mutations in the DMD gene leading to a lack of dystrophin protein in skeletal muscle resulting in a breakdown of the integrity of the muscle cell membrane. The resultant muscle fibres are highly prone to contraction induced injury. Consequently the progressive rounds of degeneration and regeneration of the muscle lead to the replacement of muscle fibres with non contractile fibrotic tissue and fatty infiltrates. These alterations lead to progressive muscle wasting, weakness and death in late adolescence. A promising therapeutic approach for DMD is antisense-mediated exon skipping using antisense oligonucleotides (AONs) targeting specific exons to restore the DMD reading frame. The products of these therapies are truncated forms of dystrophin, which should restore the integrity of the muscle cell membrane and elevate the degeneration of muscle fibres. An ideal therapy could target multiple exons, thereby treating many more patients whilst still producing a partially functional truncated dystrophin protein product. In order to evaluate the therapeutic value of these therapies, several different forms of truncated human dystrophin were cloned into the pCI plasmid. These truncated forms represent the dystrophins created by skipping different single exons or skipping multiple exons currently being investigated by various labs. The truncated dystrophins were electro-transferred into mdx mice muscle and their expression was assessed.

1 ICH, 30 Guilford St, London 2 Kings College London 3 University of Hamburg P 8 Preparation for a Phase I/II Clinical Trial of T Cell Suicide Gene Therapy Following Haematopoietic Stem Cell Transplantation in Children

Studies in adult patients with haematological malignancies have shown that genetic modification of T cells with retroviral vectors encoding a suicide gene mechanism can be used to reduce the risk of intractable graft versus host disease (GVHD). We have developed a retroviral vector system with a modified Herpes Simplex Thymidine Kinase (HSVTK) gene fused to truncated CD34, a clinically applicable selection marker. The strategy will be tested in a phase 1 study in children undergoing mismatched transplantation. Here we the summarise the process of generating a stable retroviral packaging cells as the prelude to producing a fully characterised master cell bank and manufacturing clinical grade vector supernatant. A retroviral packaging cell line (PG13) was generated by stable transduction with retroviral supernatant transiently produced from a fully characterised 293T cell line. Limiting dilution culture allowed isolation of clonal populations producing GALV pseudotyped vector. The best performing clone was then expanded and a master cell bank (MCB) generated and evaluated in detail to exclude adventitious pathogens or replication competent retrovirus (RCR). Clinical grade vector supernatant was then harvested in X-Vivo 10 from multi-layered cell factories seeded with PG13 cells from the MCB. The vector was then characterised in detail, including screening for RCR and adventitious pathogens. Full scale preclinical testing, including transduction and selection of human lymphocytes under GMP conditions has been completed and the data used to secure regulatory approvals.

1 Molecular Immunology Unit, Institute of Child Health, University College London, London, UK 2 Department of Immunology, Division of Infection & Immunity, University College London, Royal Free Hospital, London, UK P 9 Defining an Optimal Promoter for Lentiviral Gene Expression of T Cell Receptor Genes

As an alternative to transplanting T cells between individuals, it is now possible to contemplate the transfer of the genes which encode the pertinent T cell receptors against tumour and virus antigens. Gamma retroviral transfer of T cell receptor genes has already been tested in human studies of melanoma. In order to improve the safety and efficacy of TCR gene therapy, lentiviral-based TCR gene transfer is now being developed. Lentiviral delivery is associated with a lower risk of insertional mutagenesis compared to gamma retroviral vectors and T cells can be transduced with minimal activation. These vectors have a self-inactivating configuration and rely on internal promoters to drive gene expression. Here we have compared three different internal promoters for their ability to express a high affinity T-cell receptor specific for the Wilms' tumour antigen 1 (WT1). Lentiviral vectors derived from HIV-1, and incorporating a central polypurine tract (cPPT) sequence and woodchuck posttranscriptional regulatory element (WPRE) were generated to encode human promoter elements (elongation factor 1-α, EFS, and phosphoglycerate kinase, PGK) and were compared against the highly efficient spleen focus-forming virus (SFFV) promoter. Functional analysis of the transduced T cells demonstrated that they produced IFN³ in a peptide-specific manner and the efficiency of gene transfer and expression was comparable from vectors including SFFV and PGK, but reduced from constructs using EFS. We conclude that the PGK promoter is an efficient and effective alternative to the SFFV for the expression of TCRs in human primary T cells.

1 Department of Immunology, UCL Medical School, Royal Free Hospital, London NW3 2PF P 10 Development of Virus Specific T Cells for the Management of Leukaemia Patients

Bone marrow transplantation is a curative approach for the management of leukaemia patients. However, in order to prevent the rejection of allograft, patient's immune system is often profundly suppressed. Thus, reactivation of viruses, such as EBV or CMV remains a significant cause of morbidity and mortality. EBV reactivation can cause EBV driven post transplant lymphoproliferative disease (PTLD), and CMV reactivation can cause early- and late-onset CMV disease. Adoptive transfer of donor-derived virus-specific CD8+ CTL clones has proven effective in the prevention of reactivation and treatment of viral infection that is unresponsive to antiviral therapy. But this procedure has not been adopted widely because of the significant technical and financial demands for extensive ex vivo T-cell isolation and expansion cultures. In a situation where donor is seronegative for these viruses, it would be impossible to generate virus-specific CTL via above procedures. In the development of new strategies, we have cloned both EBV and CMV-specific T-cell receptor (TCR) genes, and these virus-specific TCR genes can be genetically introduced into patient's T cells thus side-stepping the above mentioned problems. Preliminary experiments have shown that these virus specific TCR genes can be expressed on the surface of human T cells, and these TCR engineered T cells can recoganize and kill CMV or EBV viral antigen expressing target cells. We are now developing an animal model to test the in vivo functions of these TCR gene modified T cells, and get more information about these TCR engineered T cells before using them for clinical trials.

1 Department of Immunology, University College London, Royal Free Hospital, London NW3 2PF 2 Department of Medicine, Centre for Rheumatology, University College London, London W1T 4JF 3 Transplantation Immunology Group, Department of Haematology, University College London, Royal Free Hospital, London NW3 2PF P 11 Adoptive Therapy with Redirected Antigen-Specific Primary Regulatory T Cells Is a Potential Novel Cellular Therapy for Graft-Versus-Host Disease

Alloreactive immune responses directed against malignant cells in recipients of allogeneic haematopoietic stem cell transplants have proven curative potential in treatment of haematological malignancies. However, such immune responses may cause substantial morbidity when directed against healthy recipient tissue, resulting in Graft-versus-Host Disease (GvHD). Regulatory T cells (Tregs) can suppress many different immune cells, making them ideal candidates for the control of GvHD. Whilst adoptively transferred polyclonal Tregs suppress GvHD in several murine models, they may also compromise beneficial immunity against malignancy or infection. The superiority of antigen-specific Tregs has been demonstrated using T-cell receptor (TCR)-transgenic mice. However, clinical translation of targeted antigen-specific therapy with Tregs is hampered by the difficulties of isolating and expanding rare antigen-specific Tregs from the natural polyclonal T-cell repertoire.

We have previously demonstrated two novel strategies to generate antigen-specific Tregs: retroviral transfer of TCR genes into purified CD4+CD25+ T cells, redirecting the specificity of naturally occurring Tregs; and cotransfer of Foxp3 and TCR genes into conventional CD4+ cells, converting them into antigen-specific regulators. We have shown that the cotransfer of Foxp3 alongside TCR genes into CD4+ cells renders them anergic, reducing both proliferation and IL-2 production in response to stimulation by their cognate peptide. We now explore the use of these antigen-specific regulators to suppress an alloreactive response against MHC-mismatched target cells: this opens the possibility of generating large numbers of regulators directed against tissue-specific antigens, allowing targeted suppression of GvHD without systemic suppression of antiviral or antitumour immunity.

1 Department of Genetics, UCL Institute of Ophthalmology, London, UK 2 Developmental Biology Unit, UCL Institute of Child Health, London, UK P 12 Gliosis and Extracellular Matrix Proteins: Their Role in Rod Photoreceptor Transplantation in the Degenerating Retina

Transplantation of rod photoreceptor precursors leads to robust integration in wildtype retinae but significantly fewer cells integrate when transplanted into degenerating retinae. Sizable integration into the degenerating retinae must be achieved for photoreceptor transplantation to be considered as a future therapy. It is important to address factors within the host environment that impede integration and migration of the transplanted cells. Degeneration triggers a Müller cell-mediated trauma response termed gliosis. Gliotic changes in the composition of the extracellular matrix, particularly the chondroitin sulphate proteoglycans, have been shown to inhibit axon and neurite growth in the spinal cord and CNS. Enzymatic digestion using chondroitinase ABC has been shown to improve integration in injury models of the retina. We characterized the progression of gliosis in relation to the expression of chondroitin sulphate proteogylcans and assessed their impact on impeding integration of transplanted rod photoreceptor precursor cells in mouse models of retinal degeneration. Using chondroinase ABC we observed a significant increase in integration in mouse models of degeneration. This suggests that components of the gliotic scar and extracellular matrix present a barrier to the migration and integration of transplanted precursor cells within the degenerating retina.

1 UCL Institute of Ophthalmology, London P 13 Lentiviral Gene Transfer of E2F2 to Induce Corneal Endothelial Cell Replication

The osmotic activity of corneal endothelial cells (CEC) maintains the clarity of the eye's cornea. CECs being nonreplicative in humans, a decrease of CEC density due to aging, injury or inherited disease causes corneal swelling and loss of transparency. E2F2 is a transcription factor regulating progression from G1 to S phase of the cell cycle. We investigate whether gene transfer of E2F2 is able to induce cell proliferation in CEC. We constructed a non-integrating HIV-based lentiviral vector delivering E2F2 under a CMV promoter (LNT-E2F2). A lentiviral vector delivering GFP (LNT-GFP) served as control. In 293T cells, exposure to LNT-E2F2 resulted in a >200-fold increase of E2F2 mRNA compared to uninfected or LNT-GFP infected controls, as detected by quantitative PCR. E2F2 overexpression was detected by Western blot and immunohistochemistry. Samples of human corneal tissue were exposed to either LNT-E2F2 or LNT-GFP or left uninfected. Exogenous E2F2 protein was detectable only in LNT-E2F2 infected samples by Western blot and immunohistochemistry. 5-Bromodeoxyuridine (BrdU) incorporation and Ki-67 immunostaining was used to detect G1 to S phase progression or cells in any active cell cycle phase, respectively. CECs infected with LNT-E2F2 showed both BrdU and Ki67 nuclear staining. Lentivirus mediated E2F2 overexpression could be used to increase CEC density in donor corneas ex vivo before transplantation. Safety measures are now being investigated to enable clinical use.

1 Molecular Immunology Unit, 30 Guilford Street, Institute of Child Health, London WC1N 1EH P 14 Developing an In Vitro Model of Common Gamma Chain Deficiency for Correction by Zinc Finger Nucleases

X-linked severe combined immunodeficiency (SCID-X1) is a disease of the immune system caused by lack or dysfunction of the common cytokine receptor gamma chain (³c). Gene therapy using integrating retroviral vectors has proved to be highly efficient at correcting the cellular defect, although with a risk of insertional mutagenesis manifesting as T-acute lymphoblastic leukaemis (T-ALL) in some patients. An alternative solution is to achieve gene correction by homologous recombination induced by double strand breaks made by targeted zinc finger nucleases (ZFN). In order to study this strategy, we have developed model human T cell lines that do not express ³c. We have packaged the entire IL2RG locus with a common exon 5 SCID-X1 mutation (G691A) into an integrating lentiviral vector and transduced ED7R cells to create a target for homologous recombination. Cell clones have been isolated and characterised for copy number and absence of gene expression. Control cell lines have also been created in which ³c expression has been restored in ED7R cells by transduction with an integrating lentiviral vector encoding the entire wild type locus. These cell clones are functional as demonstrated by the phosphorylation of downstream transcription factor STAT-5 upon stimulation with IL-2. Effectiveness of ZFN-mediated gene disruption will be tested by nucleofection and later by delivery using a non-integrating lentiviral vector platform. Repair of the mutant locus will also be tested by codelivery of homologous sequences. These studies establish a useful model to test effectiveness and toxicity of gene targeting strategies in a human cell background.

1 Moleclular Immunology Unit, Institute of Child Health and Perinatal Brain Repair Group, Institute of Women's Health, University College London, 30 Guilford Street, London WC1N 1EH 2 Dept of Cell and Develpmental Biology, University College London, Gower Street, London WC1E 6BT 3 Dept of Cell and Develpmental Biology, University College London, Gower Street, London WC1E 6BT 4 Perinatal Brain Repair Group, Institute of Women's Health, University College London, 86-96 Chenies Mews, London WC1E 6HX 5 Moleclular Immunology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH P 15 Non-Integrating Polycistronic Lentiviral Vectors for Use in the Central Nervous System

Polycistronic constructs allow expression of multiple exogenous genes from a single vector. The use of viral internal ribosome entry site (IRES) elements is a common strategy for producing polycistronic expression. However, in our experiments, the signal of EGFP expressed from the EMCV IRES was absent in the neuronal cells both in vivo and in primary cell culture. A variety of mammalian IRES sequences have been described, but they have not been widely employed in vector construction. We tested two mammalian IRES elements derived from the n-myc and x-linked in apoptosis protein (XIAP) genes in neuronal system. Of the two, XIAP IRES showed a superior performance in neurons both in culture and in vivo. Neuronal cells and their processes could be easily visualised using fluorescent microscopy. Strong EGFP signal in axons of the cortical motor neurons of mice was detected as far as the spinal cord. We further characterised XIAP IRES activity in several cell lines. Small size and a high expression level in a variety of cell types make XIAP IRES element an attractive tool for use in lentiviral vectors and an essential tool for use in the CNS. We are currently employing vectors with this element to assess the role of MAP kinase pathway components and inflammatory cytokines on neuronal degeneration and regeneration.

1 Department of Immunology and Molecular Pathology, Division of Infection and Immunity, University College London, Royal Free Campus, Rowland Hill Street, London NW3 2PF 2 UCL/MRC Centre for Medical Molecular Virology, Division of Infection and Immunity, London, UK P 16 Lentiviral Vectors for TCR Gene Transfer: Improved Function of TCR-Transduced T Cells After Non-Activating Cytokine-Mediated Transduction

The ability to manipulate the immune-system to induce protection against tumour, is one of the most fascinating challenges in immunology. In this regards, TCR gene transfer is an attractive and powerful strategy to generate high number of tumour antigen-specific T cells for adoptive transfer treatment. Lentiviral vector construct containing murinised chains of an HLA-A*0201-restricted TCR specific for Wilms' tumour antigen 1, was used for redirecting T-cell specificity. The effects of common gamma chain receptor cytokines IL2, IL7, IL15 and IL21 were investigated on primary T cells for transduction efficiency, proliferative potential, phenotype, and functional activity of the WT1 TCR-transduced T cells. Although all cytokines tested allowed transduction, stimulations with IL15 and IL15 with IL7 or with IL21 promoted a higher efficiency. Expression analysis of CD28 and CD62L showed an important role of IL21 in maintenance of a naïve phenotype. In addition to that, all cytokines promoted maintenance of “quality” of T cells as shown by co-expression of IL2, IFN³ and TNFα after specific stimulation. Therefore, these data demonstrate that gene modification of cytokine exposed T cells by lentiviral vector can improve adoptive transfer therapy for treatment of leukaemia.

1 UCL Institute of Child Health, London, UK 2 Centro de Investigaciones Energeticas Medioambientales y Tecnologicas-CIBERER U714, Madrid, Spain P 17 Lentiviral Mediated LEKTI Expression Reverses the Netherton Syndrome Phenotype

Defective expression of SPINK5, the gene encoding Lympho-epithelial Kazal-type-related inhibitor (LEKTI), gives rise to Netherton syndrome (NS). The disease is characterised by ichthyosiform erythroderma, atopic diathesis and a defective skin barrier and is associated with 10% mortality in the first year of life. Gene-modified autologous skin sheets could provide barrier protection against dehydration in NS and mediate systemic LEKTI protein delivery. Previously we generated self-inactivating HIV-1 based lentiviral vectors encoding a codon optimised SPINK5 transgene. Ahead of possible clinical application, the lentiviral constructs have been revised, with the retroviral SFFV promoter being replaced by human involucrin promoter. Involucrin promoter directs LEKTI expression to the granular layer of the epidermis, where endogenous LEKTI is normally expressed. Primary keratinocytes from NS patient were transduced with the vector and showed LEKTI expression in the differentiated cells within keratinocyte colonies. Similarly, involucrin promoter targeted eGFP and LEKTI to the outer layers of the epidermis in organotypic cultures. As part of the preclinical evaluation of gene therapy for NS, gene modified keratinocytes were tested in a skin-humanized mouse model. Results showed significant correction of NS epidermal phenotype even in grafts where only small numbers of LEKTI expressing keratinocytes could be indentified suggesting a wider bystander benefit occurring around these small populations. We conclude that the involurcin promoter is an effective alternative to SFFV and warrants further development for therapeutic application. Overall, this study confirms the feasibility of an ex vivo gene therapy approach for the correction of inherited skin disorders.

1 Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow G12 8TA 2 Thrombosis Research Institute, Emmanuel Kaye Building, Manresa Road, London SW3 6LR P 18 Modification of the FX Serine Protease Domain Ablates HSGP Engagment by Ad5/FX Complexes

Adenoviruses are commonly used vectors in gene therapy clinical trials, the majority of which are based on serotype 5. Following intravascular administration in vivo Ad5 exhibits high sequestration to the liver and spleen. Recent studies show that hepatocyte transduction by Ad5 is mediated by a high affinity interaction between coagulation factor X (FX) and the adenovirus 5 hexon proteins bridging the adenovirus capsid to the cell hepatocyte surface putatively through interaction with heparan sulfate proteoglycan receptors (HSPGs). Pharmacological blockade of the heparin-binding exosite in the serine protease (SP) domain of FX prevents cell surface binding and gene transfer mediated through FX1. However the exact HSPG-interacting residues in the SP domain of FX require further elucidation. Previous studies have indicated that the basic residues Arg-316, Lys-319, Arg-349, Arg-390, Lys-394, Lys-463, and Arg-467 of FX constitute the exosite in the SP domain2. We therefore sought to generate a FX mutant and assess the importance of this exosite. A human FX plasmid construct with mutations at Arg-316, Lys-319, Arg-349, Arg-390, Lys-394, Lys-463, and Arg-467 was generated and confirmed by sequence analysis. Recombinant FX (mutant and wildtype) was produced by plasmid transfection into 293T cells in the presence of vitamin K with collection of the supernatent. Enzyme-linked immunosorbent assay was used to quantify the rFX. In vitro assays in SKOV3 cells demonstrated that the exosite mutations blocked the ability of FX to enhance transduction of Ad5 thus confirming the importance of this exosite in HSPG engagement.

1 Department of Genetics, UCL Institute of Ophthalmology, London, UK 2 Developmental Biology Unit, UCL Institute of Child Health, London, UK P 19 Immune Responses Affect the Survival of Integrated Photoreceptor Precursors Transplanted to the Adult Murine Retina

Photoreceptor transplantation is a novel strategy to repair the degenerate retina. We previously demonstrated that postmitotic photoreceptor precursors can functionally integrate into the adult wild type and degenerating retina. We sought to characterise the long-term survival of these integrated cells in nondegenerate retinas without the confounding factors of retinal disease. GFP labelled progenitor cells from dissociated murine P3 retinas (H-2s2 × H-2b) were transplanted subretinally into partially matched adult recipients (H-2b) and photoreceptor integration was quantified 1 to 12 months later. We investigated the presence of immune cells at the site of injection and the effect of the immune suppressant cyclosporin A on the number of integrated photoreceptors at 1 and 4 months posttransplantation. In a minority of eyes an acute loss of integrated photoreceptors 1 month post injection correlated with the presence of macrophages at the injection site. Immune suppression did not affect this loss. In the majority of eyes, integrated photoreceptor cells were present in the outer nuclear layer at 2 months posttransplantation; however, the survival of these cells was reduced by 4 months. Macrophages and T cells were present throughout this period indicating a chronic immunological response. Immunosuppressive treatment following transplantation significantly increased the survival of integrated photoreceptors at 4 months. Despite the high level of MHC compatibility between our donor cells and the host retina, long-term loss of integrated photoreceptors after transplantation is associated with a chronic immune response. These results suggest that the development of MHC-matched or iPS-derived photoreceptor precursors may be required for long-term survival.

1 PlasmidFactory GmbH & Co. KG, Bielefeld, Germany P 20 Progress in Minicircle Manufacturing and Performance Testing

Plasmid DNA is commonly used in vaccination, gene- or cell therapy but also as a basic substance in viral vector production. The dissemination of antibiotic resistance genes, as well as the uncontrolled expression of backbone sequences present in such plasmids may have profound detrimental effects. Therefore, an important goal in vector development is to produce supercoiled DNA lacking bacterial backbone sequences: Minicircle DNA. PlasmidFactory's minicircle production technology facilitates the large scale production of highly pure minicircles 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 efficient in vivo recombination; 2) A chromatographic purification technology for the isolation of minicircle DNA. The resulting minicircle DNA only consists of the gene of interest and a tiny residual sequence stretch including one of the recombination sites. The 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., electrogene 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.

1 British Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, Glasgow, UK 2 Division of Clinical Neuroscience, University of Glasgow, Glasgow, UK P 21 Upregulation of Neuroglobin as a Putative Neuroprotectant Following Ischaemia/Reperfusion Injury

Neuroglobin (Ngb) is a recently discovered member of the globin superfamily that is predominantly expressed in neuronal cells. Previous studies determined Ngb to be neuroprotective both in vitro and in vivo. Although its exact mechanism of action is unknown, putative mechanisms include scavenging of reactive oxygen species and nitric oxide synthase detoxification. In the current study we aimed to quantify alterations in endogenous levels of Ngb following hypoxia in neuronal cells in vitro and to construct a lentivirus designed to overexpress Ngb to offer potential therapeutic efficacy assessed using an in vitro hypoxia model and ultimately in vivo following experimental stroke. B50 neuronal cells were exposed to hypoxia (1% oxygen; 3, 6, 9 or 18 h) followed by 24 h re-oxygenation and protein or RNA extracted. Ngb mRNA levels were measured by real-time quantitative PCR (RT-qPCR) and protein levels were assessed by western blotting and immunocytochemistry. In vitro analysis of Ngb mRNA showed a time-dependent increase in expression, significant vs. control at 9 h and 18 h time-points which also correlated to protein expression. Endogenous upregulation following injury was insufficient to protect neurons from hypoxia-induced death. We generated a lentivirus to overexpress Ngb and confirmed functional overexpression of the transgene at mRNA and protein level. Ongoing studies will determine the efficacy of Ngb overexpression using a lentivirus alone and combined with an anti-apoptotic agent in vitro and subsequently in vivo with a view to identifying a novel therapeutic intervention to improve neuronal viability and functional outcome following stroke.

1 Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, UK OX3 7BN 2 William Dunn School of Pathology, South Parks Road, Oxford, UK OX1 3RE P 22 Development of Artificial Chromosomes in Human Embryonic Stem Cells

We developed a novel system for generating functional artificial chromosomes (HAC) in human embryonic stem cells. This is based on previous work in establishing HAC in human cultured cells using the highly efficient Herpes Virus Simplex-1 amplicon technology for vector delivery (Moralli et al., 2006). Human embryonic stem cells (hESc) were successfully transduced at high efficiency with HSV-1 amplicons containing HAC vector DNA, and novel HAC expressing the gene for green fluorescent protein (GFP) were generated. Compared to introduction of HAC DNA by transfection, HSV-1 delivery was up to 4 × 10² times more efficient at delivering large construct to hES cells. Functional artificial chromosomes which replicated the behaviour of the host chromosomes and bound centromeric protein C (CENP C), were detected in 80% of the hESc HSV-1-amplicon clones, and 9% of the hESc transfected cells. In one clone, HUES 40.2, HAC were stably maintained at a reasonable frequency, and the GFP was highly expressed without selection for four months. Embryoid bodies were successfully generated from HUES 40.2 cells indicating they maintained the hESc phenotype, and exhibited potential to develop into differentiated cell types. Experiments are underway to generate the three embryonic germ layers through teratoma formation in HUES2 40.2 and monitor GFP expression. This is the first report of successful gene expressing de novo HAC which are stably maintained in hES cells and is an exciting step forward in HAC technology.

1 Insitute of Child Health, University College London, Molecular Immunology Unit, London 2 King's College London School of Medicine, Department of Medical and Molecular Genetics, London 3 Insitute of Child Health, University College London, Molecular Immunology Unit, London P 23 A2UCOE Lentiviral Vector Resists DNA Methylation-Mediated Silencing, Conferring Long-Term Expression in HSCs Following Serial Transplantation In Vivo

Loss of gene expression due to epigenetic-mediated silencing following gene transfer has been an obstacle in achieving efficacy in gene therapy. We have previously shown that, the human HNRPA2B1-CBX3 housekeeping gene locus (A2UCOE) can give rise to stable transgene expression in haematopoietic stem cells (HSCs) in vivo. We have recently shown that the stability of A2UCOE is due to its resistance to DNA methylation-mediated silencing. In order to assess the ability of the A2UCOE to confer long-term transgene expression in vivo, lethally irradiated mice received HSCs transduced with a high dose of LV-A2UCOE-eGFP viruses. Our results show that eGFP expression profiles remained consistent between primary (n = 8) and secondary transplant recipients (n = 19) over a 12 month period. Furthermore, DNA methylation analysis has confirmed a lack of CpG methylation of the A2UCOE region from secondary recipients as in primary recipients, illustrating that this element displays a true resistance to epigenetic modification in HSCs. In addition, long-term in vivo studies where the A2UCOE is used to drive expression of a therapeutic gene, common cytokine receptor gamma chain gene (IL2RG), are currently underway. Preliminary results show that the LV-UCOE-IL2RG vector is able to rescue the disease phenotype in a mouse model of SCID-X1 for at least 8 months following engraftment of transduced HSCs. Furthermore, no genotoxicity effects from this LV have been observed to date. These data provide further evidence demonstrating that the A2UCOE provides highly reliable, long-term transcriptional activity in HSCs and reinforces its potential as an excellent regulatory element for gene therapy applications.

1 Molecular Immunology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK 2 Institute for Biomedical Research, Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42, 60596 Frankfurt, Germany 3 Hematopoiesis and Gene Therapy Division, CIEMAT/CIBER-ER and Marcelino Botín Foundation, Madrid, Spain P 24 Targeted Lentiviral Gene Therapy to Rescue the X-CGD Phenotype

X-linked Chronic Granulomatous Disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene encoding the phagocyte NADPH oxidase catalytic subunit gp91phox. A clinical trial for X-CGD using a Spleen Focus-Forming Virus (SFFV)-based retroviral vector has recently shown promising results. However, in three out of four patients the expression of gp91phox therapeutic gene has declined over time, due to methylation of CpG dinucleotides within the viral LTR. This trial suggests that there is need to improve vector design. In this study we aimed at optimising gp91phox expression in neutrophils using myeloid specific promoters in the context of lentiviral vectors. We created a synthetic chimeric promoter that contains binding sites for myeloid transcription factors (e.g., c/EBPs and PU.1) expressed during granulocytic differentiation. Indeed, the chimeric promoter drives higher GFP expression in myeloid than in nonmyeloid cell lines suggesting that is myeloid specific. When compared to the mir 223 promoter, a physiological myeloid specific promoter, the chimeric drives higher expression in granulocytes/monocytes and it is more efficient in rescuing the X-CGD phenotype in transplant experiments. However while the mir 223 promoter drives transgene expression only upon granulocytic differentiation, the chimeric promoter is slightly leaky in hematopoietic progenitors. We are currently investigating the feasibility of adding a microRNA tag to the vector with the chimeric promoter to achieve expression of gp91phox in the granulocytic compartment only.

1 School of Pharmacy, Royal College of Surgeons in Ireland 2 Department of Pharmaceutical & Medicinal Chemistry, School of Pharmacy, Royal College of Surgeons in Ireland 3 Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland P 25 Mannosylated Liposomes for Targeted Delivery of siRNA to Alveolar Macrophages

Targeted local delivery of siRNA to the lungs via inhalation offers a unique opportunity to treat a range of previously untreatable respiratory conditions. However, progress has been greatly hindered by safety and delivery issues. Disadvantages associated with naked siRNA, viral vectors, and highly cationic liposomes have lead to interest in the development of targeted neutral liposomes. Alveolar macrophages are the first line of defence against inhaled pathogens and are essential for the initiation of the inflammatory response. Targeting these cells with siRNA provides a means of manipulating the pulmonary immune response. Therefore we have set out to investigate liposome formulations, for the transfection of alveolar macrophages with siRNA after local delivery via inhalation. Specific targeting of alveolar macrophages will be achieved via the mannose receptor by the incorporation of cholesten-5-yloxy-N-(4-((1-imino-2-a-thioglycosylethyl)amino)butyl)formamide (Mann-C4-Chol) and two analogous mannosylated cholesterol derivatives Mann-C2-Chol and Mann-C6-Chol into the liposome formulations. Liposomes were prepared consisting of DOPC, DOTAP, cholesterol and mannosylated cholesterol (60:10:22.5:7.5 and 70:0:22.5:7.5 molar ratios) by dehydration-rehydration. Average diameters of 174 nm ± 19 were determined by dynamic light scattering. The encapsulation efficiency of siRNA was optimised to 36% by the inclusion of a small percentage of a DOTAP, sufficient for neutralisation only. In vitro uptake of rhodamine-labelled liposomes by differentiated THP-1 (alveolar macrophage-like) cells was significantly increased by inclusion of mannosylated cholesterol, with Mann-C4-Chol facilitating the most significant uptake ( p < 0.001). Uptake of siRNA into THP-1 cells was significantly increased when encapsulated in mannosylated liposomes compared to siRNA alone as determined by spectrofluorimetry ( p < 0.05) and fluorescent microscopy.

1 Laboratory for Molecular Virology and Gene Therapy, Catholic University of Leuven, Belgium 2 Biomedical NMR Unit/ MoSAIC, Catholic University of Leuven, Belgium 3 Division of Nuclear Medicine, Catholic University of Leuven, Belgium 4 University Hospital Gasthuisberg, Department of Gynaecology and Obstetrics, Leuven, Belgium P 26 Evaluation of Different rAAV Serotypes and Their Tropism for the Lung Using Non-Invasive Imaging in a Fetal Mouse Model

Background: The fetal mouse lung is typically targeted by intra-amniotic (IA) injection. We evaluated the potential of intratracheal (IT) delivery by comparing IT with IA injection using fluorescent molecules. Next, we injected different rAAV serotypes (rAAV6.2, rAAV5 and rAAV9) encoding FLUC and β-gal to assess their efficacy targeting the fetal lung. BLI was combined with MRI to obtain a more accurate localization of gene expression in vivo.

Methods: Fetuses of pregnant NMRI were injected IT or IA at E18 with 30 ¼L fluospheres (n = 5) or rAAV (n = 3 per serotype). Combined BLI-MRI of injected pups was performed at 1 week. For co-registration, BLI images were overlaid with whole-body MRI using newly developed software. Mice were sacrificed at 1 and 4 weeks. X-gal staining was performed on frozen sections to localize gene expression.

Results: IT injection was more efficient in delivering fluospheres to the fetal lung compared to IA. BLI-MRI co-registration showed FLUC expression in the lung region after IT delivery of rAAV6.2, in the lung region and nose after rAAV5 and predominantly in the abdomen after rAAV9. Confirmation of transduction was obtained after X-gal staining on tissue sections. rAAV6.2 mainly targeted the lung, rAAV5 showed β-gal positive cells in lung and nose and rAAV9 targeted the lung and liver.

Conclusions: We developed a novel fetal mouse model for lung transduction. Combining BLI with MRI allowed for in vivo evaluation of different rAAV serotypes and their tropism for the lung. In the future, this fetal mouse model can be used to investigate gene therapy for genetic disorders.

1 Oxford BioMedica (UK) Ltd, Medawar Centre, Oxford Science Park, Oxford, OX4 4GA 2 Oregon Health & Science University, Mailcode 111, Baird Hall 1027, 3181 SW Sam Jackson Park Rd, Portland, OR 97239-0398 P 27 Development of EncorStat^®: Corneal Tissue Genetically Engineered to Resist Graft Rejection

The cornea is the most successfully transplanted tissue with approximately 60,000 grafts in the USA and EU every year, with the most common indications for this type of transplant (penetrating keratoplasty) being keratoconus and bulbous keratopathy. Nevertheless, corneal graft failure occurs in about 10% of these indications. Neovascularisation following corneal transplantation is a major risk factor for graft failure. The cornea is a transparent tissue which transmits light to the retina and the presence of even the smallest blood vessels impairs this process. EncorStat is a cornea transduced with an Equine Infectious Anaemia Virus (EIAV)-based lentiviral vector delivering two genes, endostatin and angiostatin. These proteins have been shown to be potently angiostatic, targeting different stages of the vascularisation process. This therapy aims to prevent neovascularisation in transplanted corneal grafts by transducing corneas ex vivo; these engineered corneas subsequently express these two angiostatic proteins which prevent neovascularisation and subsequent graft rejection. To assess gene transfer, we transduced fresh non-human primate (NHP) corneas with an EIAV-CMV-eGFP vector in Optisol (the current storage medium used in American eye banks), EB MEM 2% FBS medium (organ culture medium used in many European eye banks), and MegaCell DMETM medium, a serum free medium also shown to be suitable for corneal storage. We showed significant and persistent expression of GFP following the transduction of whole NHP corneas in the endothelial cell layer in these three media. This result paves the way for efficacy studies in a rabbit model of corneal graft rejection.

1 Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London NW1 0TU 2 Imperial College Cerebrovascular Research Unit, Charings Cross Hospital, Fulham Palace Rd, London W6 8RF P 28 mdx Mice Show Changes in Muscle Fiber Profile Number with Age

The mdx mouse is used extensively to investigate the pathogenesis of and potential treatments for Duchenne muscular dystrophy (DMD). However their phenotype is milder than that of DMD boys and it is thought that mdx mice do not exhibit significant muscle fiber loss. Many studies investigating treatments to restore the absent dystrophin protein use estimates of muscle fiber number to calculate the proportion of dystrophin-positive fibers. This study investigated whether fiber profile number stays constant throughout the lifetime of the mdx mouse. TA, EDL and soleus muscles were collected from mdx mice of a range of ages. Sections were immunostained with an anti-perlecan antibody as staining for an ECM protein facilitated the counting process. All 3 muscles exhibited significant changes in fiber profile number but in different patterns. Fiber profile number in the EDL dropped progressively from 3 weeks to 24 months, and was significantly lower at 12 and 24 months than at 3 weeks. Fiber profile number in the soleus was significantly higher at 6 months than at 3 weeks, 12 months or 24 months. At 24 months the number was significantly lower than at 3 weeks. Fiber profile number in the TA increased slightly between 3 and 10 weeks then dramatically between 10 weeks and 10 months. The fiber profile number at 10 months was significantly higher than at 10 weeks and 17 months. These changes in fiber profile number should be taken into account when assessing the effects of treatments to avoid under- or over-estimating their significance.

1 Department of Immunology, University College London, Royal Free Hospital, London NW3 2PF P 29 Enhancing the Efficacy of TCR Gene Therapy by Co-Transfer of CD3 Molecules

TCR gene transfer is an efficient strategy to redirect the specificity of T lymphocytes. Efficient cell surface TCR expression requires the formation of a stable TCR-CD3 complex. A limited number of CD3 molecules, may result in reduced cell surface expression of the introduced TCR. Although current TCR gene transfer protocols have successfully manipulated CD8+ T cells, less data is available on CD4+ T cells; CD4+ T cells may be essential in providing help for anti-tumor effects of CD8+ T cells. In TCR deficient 58-/- cells, co-transfer enhanced expression of a Flu-TCR (NP-specific); and bound NP tetramers more efficiently than cells transduced with TCR. Our data demonstrate that transduction of both CD8+ and CD4+ T cells with MHC-class I restricted Flu-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 functional avidity of these cells to their specific peptide. Our preliminary data suggest that TCR-CD3 co-transduced T cells may eradicate tumor faster than TCR-only transduced cells when transduced CD8+ T cells are adoptively transferred into NP-expressing tumor bearing mice. Using in vivo imaging, it was demonstrated that increased efficiency in tumor eradication may be linked to faster tumor infiltration by TCR-CD3 co-transduced T cells compared to TCR-only transduced T cells. Data suggest that co-transduction of CD8+ and CD4+ T cells with TCR and CD3 enhances functional avidity of cells without resulting in toxicity in vivo.

1 BHF Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow, G12 8TA P 30 Development of Cardiac Hypertrophy Regulated Expression Cassettes Using miRNA Targeting Sequences

The heart is a target for gene therapy in disorders including heart failure and myocardial infarction (MI). Regulated gene delivery, activated in response to pathophysiological stimuli would be desirable to improve efficacy and safety. MicroRNAs mediate post-transcriptional control of gene expression in a spatial, temporal and disease-regulated manner. Cardiac remodelling and hypertrophy, which occur in heart failure and post-MI, are associated with a signature of up and down-regulated miRNAs. We generated expression cassettes under the control of a disease-regulated miRNA to test the concept that this would provide regulated transgene expression in cardiomyocytes undergoing hypertrophy. Exposure of H9c2 cardiomyocytes to angiotensin II (AngII), resulted in a significant increase in cell size ( p = 3.8 × 10⁻⁶). Expression of a panel of candidate miRNAs in H9c2 cardiomyocytes in the presence or absence of AngII was quantified. Only miR-133 was significantly down-regulated following AngII treatment ( p = 0.001). Expression cassettes were generated in which destabilized luciferase was transcribed from the chicken beta-actin cytomegalovirus (CAG-CMV) promoter, +/− miR-133 targeting sequences inserted into the 3′ untranslated region. Following transfection into H9c2 cardiomyocytes, CAG-CMV induced luciferase expression in the presence or absence of AngII. In the absence of AngII, CAG-CMV/miR133 had reduced luciferase expression, however addition of AngII increased its expression by 50%. In summary, AngII treated H9c2 cardiomyocytes undergo hypertrophy with concomitant decreased miR-133 expression. Inserting miR-133 targeting sequences into an expression cassette provided regulated luciferase expression in cells undergoing hypertrophy. Incorporating miRNA target sequences into expression cassettes may enable the development of disease-regulated gene therapy in cardiovascular disease.

1 King's College London School of Medicine 2 University of Witten Herkecke, Germany P 31 Evaluation of Several DNA Expression Constructs for the Level and Longevity of Expression of the Pancreatic Transcription Factor pdx1 in Rat Liver

In vivo transdifferentiation of hepatocytes and pancreatic exocrine cells to pancreatic β cells has been achieved in mice by delivery of multiple pancreatic transcription factors to the liver and pancreas, using adenovirus vectors. The use of nonviral approaches requires expression of the correct combination of transcription factors at appropriate levels for a sufficient time. We have evaluated several DNA constructs, using rat pdx1 as the expressed gene. These were delivered to rat liver hydrodynamically, using the IVC segment approach, and pdx1 expression was assayed quantitatively using Taqman PCR at days 1, 3, 14 and 28. The pEPI plasmid, which has a CMV promoter and the ∼2 kb Scaffold/Matrix Attachment Region (S/MAR) of β interferon, gave very transient pdx1 expression, levels at day 3 being ∼150-fold lower than day 1. Substitution of the CMV promoter in pEPI with a short alpha1-antitrypsin promoter/hepatic locus control region, or with ∼10 kb of the genomic alpha1-antitrypsin promoter, greatly reduced the rapid suppression of pdx1 expression. Addition of a ∼4 kb Ubiquitous Chromatin Opening Element (UCOE) was not of further benefit. A CpG depleted construct (Invivogen Inc) also delayed suppression of pdx1 expression. Levels of pdx1 expression at day 28 with these latter constructs were ∼2% to 12% of those at day 1, in comparison with 0.03% with unmodified pEPI. The CpG depleted construct, but none of the pEPI constructs, gave expression of insulin-2 (at day 14). Differences between the CpG depleted and pEPI constructs, and the degree of pancreas differentiation, are currently being evaluated.

1 Molecular Immunology Unit, Insitute of Child Health, 30 Guilford St, London, WC1N 1EH P 32 Development of Gene Therapy for X-Linked Lymphoproliferative Disease

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterised by severe immune dysregulation, typically following infection with Ebstein Barr Virus (EBV), lymphoproliferative disease and autoimmune phenomena. Treatment options are limited and currently haematopoietic stem cell transplant is the only curative option. XLP is caused by mutations in the SH2D1A gene, encoding SAP (SLAM associated protein), a small intracellular adaptor expressed in NK, T and NKT cells and transduces signals from the SLAM family receptors. SAP deficiency results in disrupted lymphocytic signalling pathways and reduced NK and T cell cytotoxicity, especially towards EBV-infected B cells. SAP deficient mice have been shown to have a similar immunophenotype to XLP patients.

Our aim is to generate a SIN lentiviral vector for gene therapy as an alternative therapeutic strategy for XLP. We transduced Lineage negative murine bone marrow cells with SIN lentiviral vectors encoding cDNA for human SAP with expression driven by the endogenous human elongation factor 1α (EFS) promoter. 24 hours after transduction cells were injected into lethally irradiated SAP−/− mice. After 12 weeks, animal tissue was analysed to determine immune reconstitution and expression of SAP. Despite highly efficient transduction of SAP−/− Lin negative cells, copy number data supportive of integration of the vector and reporter gene expression in multiple immune cell lineages, SAP expression and correction of XLP phenotype could not be demonstrated in animals 12 weeks after reconstitution. As SAP mRNA was detected in splenic NK cells this could suggest a defect in post-translational modification resulting in the lack of correction.

1 BHF Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow, G12 8TA 2 Harvard Medical School, Beth Israel Deaconess Medical Center, E / CLS - 1047, 330 Brookline Avenue, Boston, MA 02215 P 33 In Vitro Evaluation of Hexon Modifications on the Capacity of Ad5 to Evade Neutralising Antibodies

Despite their exceptional promise for clinical gene therapy applications, vectors based on Ad5 show limited efficacy due to high levels of pre-existing anti-vector immunity.1 Pseudotyping Ad5 with fibers from rare serotypes represents a promising means to evade pre-existing Ad5 immunity,2 however, it is widely regarded that the hexon protein represents the major site of antibody mediated neutralisation. Recent evidence also documents an important role of hexon in mediating hepatic transduction.3 We sought to evaluate whether mutations introduced into Ad5 hexon protein to abrogate FX interactions also facilitate evasion of Ad5 antibodies in vitro. Using sera from a Scottish cohort, we evaluated the effects of hexon modification on the ability of Ad5 to evade Ad5 antibodies in vitro, using transgene expression to gauge neutralisation. We observed lower levels of neutralisation against Ad5 than previously documented, with 21/63 (33.3%) sera efficiently neutralising Ad5 expression by >90%. Interestingly a significant proportion of sera (26/63, 41.3%) augmented Ad5 mediated transduction by >50%. No sera neutralised Ad48, confirming the low seroprevalence rates for this serotype. Hexon modified, FX-binding ablated vectors Ad5HVR48, Ad5HVR26(5 + 7), and Ad5T* demonstrated patterns of neutralisation similar to Ad5, with neutralisation observed in 25/63, 33/63, and 25/63 sera respectively. Together these data indicate that a proportion of Ad5 antibodies are directed against non-hexon capsid proteins, possibly reflecting overexpression and secretion of fiber protein in airway epithelial cells that facilitates viral escape following natural infection.4

1 MPS Stem Cell Research Group, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK 2 Blood and Marrow Transplant Unit, Royal Manchester Children's Hospital, Manchester, UK 3 Genetic Medicine, St. Mary's Hospital, Manchester, UK P 34 Optimisation of Lentiviral Transduction Conditions of Haematopoietic Stem Cells for Treatment of Mucopolysaccharidosis Type IIIA (MPSIIIA)

MPSIIIA is caused by a dysfunctional substrate degradation pathway in lysosomes due to the lack of sulfamidase enzyme. We are developing a lentiviral gene therapy to overdrive sulfamidase expression in donor haematopoietic stem cells (HSCs) prior to transplantation. We have demonstrated preclinical efficacy of this approach in a mouse model of MPSIIIA. A significant increase in enzyme levels and reduction in substrate storage in all tissues, including the brain, were observed. The objectives of the project are to generate a clinically applicable vector, develop an efficient transduction protocol for HSCs, and assess vector safety. We have generated high titers (2 × 10⁸ IU/ml) of a new lentiviral vector with a clinically approved backbone incorporating a human promoter. Transduction conditions have been optimised using cytokines, BSA, and chemicals, such as MG132 and valproic acid to improve infectivity and stemness of mouse HSCs. The addition of MG132 with cytokines and BSA improved transduction by 10%, while maintaining cell viability. Valproic acid showed no toxicity and increase in transduction in a short term study. It is now being tested as an SCF substitute in a long-term CFC assay. This will be translated to human CD34+ cord blood-derived HSCs. The risk of vector genotoxicity was assessed using an in vitro immortalisation assay. After 4 weeks in culture, no clones were detected when using a low dose of the lentiviral vector. This is a promising therapy for which we have already shown preclinical efficacy in a mouse model of MPSIIIA disease. Future experiments are aimed at translating this mouse study into a clinical trial for human stem cell gene therapy.

1 School of Biological Sciences, Royal Holloway-University of London, Egham 2 Institute of Human Genetics and NESCI, Newcastle University 3 Hospital de la Santa Creu y Sant Pau, Barcelona, Spain P 35 Human-Induced Pluripotent Stem Cell-Derived Models for the Study of Spinal Muscular Atrophy

Spinal muscular Atrophy (SMA) is an autosomal recessive neurodegenerative disease caused by mutations in the Survival of Motor Neuron 1 gene (SMN1), which result in dramatic decreases in the levels of SMN protein. Low levels of SMN lead to degeneration of alpha motor neurons (MNs) in the anterior horn of the spinal cord. Since human MNs cannot be obtained from living individuals, most human research has been performed with surrogate cells of limited relevance to SMA. However, the recent development of iPS (induced pluripotent stem) cell technology, has made it possible to derive ES (embryonic stem)-like cells from somatic cells. For this a combination of transcription factor-encoding transgenes are introduced into cells. Subsequently, iPS cells can be induced to differentiation into many cell types, including MNs. We are keen to generate iPS cell models from a cohort of Spanish SMA patients, with three major aims: (i) to improve our understanding of SMA; (ii) to test potentially therapeutic vectors; and (iii) to produce clinically applicable patient-specific iPS cells. For the latter case, it would be crucial to avoid stable integration of the iPS-inducing transgenes, which we hope to achieve with the use of integration-deficient lentiviral vectors (IDLVs). We have demonstrated efficient transduction of human fibroblasts with IDLVs expressing eGFP. We have then compared expression levels of iPS cell-inducing transgenes from IDLVs or standard integrating lentivectors driven by the EF1alpha, CMV or CAG promoters. Optimum combinations of vectors have thus been defined and used in preliminary attempts of iPS cell generation.

1 School of Biological Sciences, Royal Holloway-University of London, Egham P 36 Transcriptional Targeting of Integration-Proficient and Integration-Deficient Lentiviral Vectors for the Treatment of Parkinson's Disease

Parkinson's disease results from the gradual loss of neurons within the dopaminergic substantia nigra pars compacta, and its projections to the striatum. Current pharmacotherapies aim to restore lost striatal dopamine; however, such therapies do little to halt the underlying neurodegeneration; and are plagued with side-effects and suffer a loss of efficacy over time. Whilst genetic therapies offer exciting therapeutic strategies for the treatment of PD, there remain limitations regarding safety and efficacy as such therapies currently do not allow specific targeting of neurones or glia. Optimised targeting to disease-relevant subpopulations of neurones would enhance specificity and may reduce potential side-effects of CNS gene therapy, whilst transduction of astrocytes may be more efficient for supplying secreted neurotrophic factors. The aim of this study is to develop targeted lentivectors for the efficient transduction of either neurones or astrocytes. To achieve this aim we will determine whether the well-characterised neuronal (synapsin-1) and glial-specific (GFAP) promoters are efficiently expressed from both integrating and non-integrating vectors, by the use of eGFP as a marker gene. These vectors will additionally be pseudotyped with either: VSV-G (control); Ross River (glial) or Rabies (neuronal) envelope proteins, in a bid to further enhance cell-specificity. The vectors will then be stereotaxically injected into either the substantia nigra or the striatum. The expression of eGFP will be monitored at different time points post-vector injection to determine cell-type specificity and vector spread.

1 School of Biological Sciences, Royal Holloway-University of London, Egham P 37 Efficient Transduction of the Central Nervous System with Integration-Deficient Lentiviral Vectors

Parkinson's disease, the second most common neurodegenerative disease, results from the loss of dopaminergic neurons in the substantia nigra pars compacta and subsequent depletion of striatal dopamine. Current symptomatic treatments (e.g., medication, surgery, transplantation of foetal stem cells, etc.) do not halt Parkinson's disease progression. Gene therapy approaches delivering neurotrophic factors have offered promising results in neuroprotection and neurorestoration both in vitro and in vivo in animal models, but GDNF (glial cell line-derived neurotrophic factor) delivery has failed in phase II clinical trials. One novel factor that may overcome this failure is IGF-1 (insulin-like growth factor 1). IGF-1 leads to neuroprotection in various degenerative disease models. Additionally, IGF-1 receptors are found in the substantia nigra and decline in serum levels of IGF-1 have been recorded in elderly patients with cognitive impairment. We are developing integrating and integration-deficient lentiviral vectors (IDLVs) encoding IGF-1 for therapeutic application in Parkinson disease models. We have initially cloned eGFP under the control of the GFAP promoter to test efficiency of astrocytic-specific gene expression with integrating lentivectors and IDLVs. We are currently developing IGF-1-expressing lentivectors under the control of the GFAP promoter to similarly test expression efficiency. A variety of lentiviral pseudotypes will be used to improve astrocyte-specific gene expression. Optimal vectors will be tested in vivo in rat models of Parkinson's disease.

1 BHF Glasgow Cardiovascular Research Centre, Division of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, G12 8TA 2 Department of Pharmacology and Program in Vascular Signaling and Therapeutics, Boyer Centre for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut, USA 3 Molecular Immunology Unit, UCL, Institute of Child Health, London, UK P 38 Analysing the Effect of Nogo-B Overexpression in Vascular Smooth Muscle Cell Proliferation and Migration Induced by Non-Integrating or Integrating Lentiviral Vectors

Proliferation and migration of vascular smooth muscle cells (VSMCs) is a hallmark of neointima formation associated with acute vascular injury. Previous studies have demonstrated that adenoviral-induced overexpression of Nogo-B reduced neoinitma formation after acute vascular injury in animal models. This effect was mediated through inhibition of VSMCs proliferation and migration. Lentiviral vectors offer additional potential for vascular gene delivery because they are efficient at targeting VSMCs and can mediate long-term transgene expression. Here, we have assessed non-integrating and integrating lentiviral-induced overexpression of Nogo-B in vitro, as a potential therapeutic strategy for the prevention of neointimal formation in vivo. Immunofluorescence and Western Blot analysis indicated that non-integrating and integrating lentiviral gene transfer of Nogo-B efficiently increased Nogo-B expression in VSMCs, compared to lentivirus expressing GFP controls. Assessment of proliferation demonstrated that non-integrating and integrating lentivirus expressing Nogo-B, led to a significant decrease in VSMC proliferation compared to lentivirus expressing GFP controls (at a MOI of 25: 0.14 ± 0.0084 absorbance (abs) at 570 nm vs. 0.28 ± 0.01 abs, p < 0.05 and 0.24 ± 0.003 abs vs. 0.31 ± 0.006 abs, p < 0.05, respectively). Results from a wounding assay demonstrated that lentivirus delivery of Nogo-B significantly reduced the migration of VSMCs, compared to lentivirus expressing GFP control (at a MOI of 50: 31 ± 4.5 ¼m vs. 107.2 ± 5.5 ¼m, p < 0.05). Taken together, our study demonstrates that lentiviral vectors are efficient in vascular cell gene transfer and that overexpressing Nogo-B in VSMCs leads to phenotypic effects on migration and proliferation using either non-integrating or integrating lentiviral vectors.

1 School of Biological Sciences, Royal Holloway University of London, UK 2 Immunology Group, Imperial College London, UK 3 Immunobiology, Kings College London School of Medicine, UK 4 Hybrid Therapeutics Ltd, UK 5 Development Unit, TheraJect, Inc, USA 6 Vaccine Immunology Statistical Center-VISC, Fred Hutchinson Cancer Research Center, USA 7 VLP Development, London, The National Institute for Medical Research -NIMR, UK 8 Division of Retrovirology, National Institute for Biological Standards and Control, UK P 39 Development and Assessment of Thermo-Stable Adenovirus (Ad)-Based Vaccines Expressing Ubiquitinated and Genetically Fragmented HIV/SIV Genetic Components

Development, evaluation and optimisation of HIV-1/SIV genetic vaccine components towards broadening the immune response constitute a cohort of strategies in HIV-1/SIV vaccination studies. We have previously identified sugar mixtures that give optimal preservation of adenoviral-based vaccines infectivity whilst maintaining the strength and rigidity in a micro-needle array compartment. Vectors retained substantial infectivity (i.e., >55% eGFP+ cells) for up to 3 months trough a temperature range tested (−200°C to +400°C). In vivo stability/infectivity studies have been successfully conducted via transcutaneous injections of dessicated Ad-OVA-GFP vectors (4 × 10⁹ vg) in C57BL/6 mice and footpad-mediated migration of Ad5-CMV-eGFP to lymph nodes. We originally aimed to induce immunity to HIV-1 infection by developing a cohort of vectors in which HIV-1 genetic components have been engineered to stimulate broader CTL responses. Full-size gag genes including an across clade conserved epitope were engineered, fused to mono- or tetra-ubiquitin (Ub) sequences and further tested on DC and non-DC cell lines by using plasmid, in vitro synthesised mRNA, or adenoviral vectors. We subsequently constructed rAd vectors carrying genetically fragmented ubiquitinated fusions for either HIV or SIV gag genes in an attempt to reduce antigenic competition and alter epitopic dominance. We are currently extending our studies using an in vitro CTL epitope-mapping system employing human monocyte-derived DCs from healthy and HIV-1+ individuals alongside mouse. In vivo plasmid prime and Ad boost studies impacting the antigen ubiquitination and genetic fragmentation on the quality of the generated immune responses. SIV constructs will be forward tested in non-human primate models (cynomolgus macaques).

1 School of Biological Sciences, Royal Holloway-University of London, Egham 2 Wolfson Centre for Age-Related Disease, King's College London   3 Centre for Clinical Neuroscience, St George's University of London P 40 Importance of Neurogenesis in a Rodent Model of Stroke

Stroke is commonly caused by occlusion of cerebral blood vessels resulting in widespread cell death. It is a common cause of death and results in debilitating disabilities in survivors. Therapeutic options are limited and developments leading to new treatments would be of significant importance. Animal models have suggested a possible implication of neurogenesis in stroke recovery, but endogenous adult neurogenesis is very limited in humans. The goal of this project is to further study the importance of neurogenesis for stroke recovery in an animal model. We are using pharmacological treatment (CB2 cannabinoid agonist) to enhance neurogenesis and a genetic system (cell cycle inhibition through a dominant-negative cyclin D1) to inhibit the process. We have adopted the MCAo (middle cerebral artery occlusion) model of stroke and are validating the surgical technique, histochemical detection and a panel of appropriate behavioural tests. Our preliminary results with the MCAo model will be presented. We have also produced integration-deficient lentivectors (IDLVs) expressing eGFP to label and track neurogenesis in the rodent brain. Furthermore, we have developed lentiviral vectors encoding dominant-negative cyclin D1 to block dividing cells in G1 and thus block neurogenesis in vitro and in vivo. We are currently testing for cell cycle arrest after transduction of neuroblastoma cells with dominant-negative cyclin D1-encoding integrating lentivectors and IDLVs.

1 Department of Clinical Pharmacology, Roosevelt Drive, University of Oxford, OX3 7DQ P 41 MicroRNA Regulation of Adenovirus E1A Viral Protein

Replication-competent viruses used for selective lysis of cancer cells can be attenuated on a tissue-specific basis inserting microRNA binding sites into viral genomes, allowing regulation of viral gene expression by endogenous microRNAs. A selectively-replicating adenovirus was previously engineered with binding sites for the liver-specific microRNA (miR-122) in the 3′UTR of the E1A gene. A mir122-expressing hepatocellular carcinoma cell line Huh7 was infected with wild-type adenovirus (Ad5WT) and microRNA-regulated adenovirus (Ad5mir122); an MTS assay revealed that more Huh7 cells were killed when infected with Ad5WT than with Ad5mir122 (40% survival compared with 111% survival). Adenoviruses were constructed in which E1A was fused to luciferase with and without mir122 binding sites (Ad5Lucmir122 and Ad5Luc). Ad5Lucmir122 showed less luminescence than Ad5Luc in miR122-positive cells; a 28-fold decrease in primary human hepatocytes from 2 × 10⁴ RLU to 9.45 × 10² RLU, and in Huh7 cells a 14-fold decrease from 3 × 10⁶ RLU to 2 × 10⁵ RLU, but similar levels of luminescence in miR122-negative HepG2 cells. Real-time RT-PCR analysis of RNA extracted from murine livers infected with Ad5WT and Ad5mir122 revealed 6.87 × 10⁵ copies of viral E1A RNA (per ng of total RNA) in Ad5WT infected mice whereas Ad5mir122 showed a 7-fold decrease to 1.01 × 10⁵ copies of E1A RNA. This explains previous observations that there was less hepatotoxicity in mice administered miR-122 adenovirus than in those infected with wild-type virus.

1 School of Biological Sciences, Royal Holloway-University of London, Egham 2 Wolfson Centre for Age-Related Disease, King's College London P 42 Efficient Transduction of the Spinal Cord with Integration-Deficient Lentiviral Vectors

Spinal Muscular Atrophy (SMA) is a neuromuscular disorder characterised by degeneration of motor neurons from the ventral horn of the spinal cord, caused by loss or mutation of the survival of motor neurons gene SMN1. We are interested in investigating new gene therapy techniques for treatment of SMA and other neurodegenerative diseases. We have previously demonstrated that integration-deficient lentiviral vectors (IDLVs) based on HIV-1 are as efficient as standard integrating lentivectors for gene expression in quiescent tissues in vivo, including eye, brain, and muscle. We have now extended these observations to spinal cord tissues. Intraspinal injection of VSV-G-pseudotyped IDLVs or integrating lentivectors expressing eGFP from the CMV promoter showed similar transduction levels of cervical and lumbar motor neurons. Much more restricted eGFP expression was noticed in astrocytes. Transduction of purified motor and dorsal root ganglia (DRG) neurons reached similarly high levels regardless of the vector integration proficiency. Astrocytes were very susceptible to transduction with both IDLVs and integrating vectors, while microglia were more efficiently transduced by the latter. Similar observations were made with Rabies pseudotyped lentivectors. In embryonic motor neuron cultures we have also demonstrated that IDLVs and integrating vectors expressing glial cell line-derived neurotrophic factor (GDNF) are equally efficient at rescuing cells deprived from exogenous neurotrophic factors. We have now developed IDLVs for delivery of potentially therapeutic transgenes to animal models of SMA. Integrating lentivectors and IDLVs encoding mouse SMN led to similar levels of expression after transduction of cultured DRG neurons. In vivo experiments in SMA mice are on-going.

1 School of Biological Sciences, Royal Holloway-University of London, Egham 2 Institute of Child Health, University College London 3 Department of Medical and Molecular Genetics, King's College London 4 Sangamo BioSciences, Inc. , Richmond, California, USA P 43 Towards Correction of the scid Mouse by Gene Targeting

Gene repair at endogenous loci is the ideal goal for gene therapy of inherited diseases. Albeit traditionally considered a low-efficiency process, homologous recombination-mediated gene targeting frequencies have recently been drastically improved through two technical developments: (i) efficient DNA delivery platforms, including adeno-associated virus (AAV) vectors and integration-deficient lentiviral vectors (IDLVs); and (ii) designer nucleases, able to induce double-strand breaks at the target locus. However, no animal model of inherited disease has yet been corrected by gene repair. We are developing an ex vivo system to correct the classical scid mouse, a model of human DNA-dependent protein kinase catalytic subunit (PRKDC) deficiency. Scid mice have a point mutation in Prkdc. We have produced targeting constructs to correct the scid Prkdc mutation and incorporated them into plasmids and IDLVs. We have also produced standard and optimised, obligatory heterodimer zinc-finger nucleases (ZFNs) directed against Prkdc and included the ZFN genes into plasmid and lentiviral vectors. Obligatory heterodimer ZFNs delivered with plasmid and lentiviral vectors have highest activity; they increased about 100-fold the frequency of G418-resistant colonies obtained from Tert-immortalised scid fibroblasts using a Prkdc plasmid targeting construct including a neo gene. We have optimised transduction of mouse haematopoietic (HSC) progenitors with eGFP-expressing integrating lentivectors and IDLVs. eGFP MOIs of 25 and 50 are optimal, respectively, for transduction of HSCs with integrating lentivectors and IDLVs. We are currently performing ZFN cutting assays in HSCs, prior to gene targeting experiments combined with transplantation to attempt correction of the scid phenotype in vivo.

1 Royal Free & University College Medical School, London 2 Immunology, Royal, Free & University College Medical School, London 3 School of Biological Medicine, University of Edinburgh 4 Immunology, Royal Free & University College Medical School, London 5 Immunology, Royal Free & University College Medical School, London P 44 Improving T Cell Functional Avidity Through CD8 Co-Receptor Modification: A Novel Mechanism to Enhance TCR Gene Therapy

TCR gene transfer can be used to generate tumour antigen-specific T cells for adoptive immunotherapy. Following TCR gene transfer, transduced T cells usually display the same functional avidity as the parental clone from which the TCR was isolated. However, tumour-antigen specific T cells typically recognise overexpressed self antigen and are often of low/moderate avidity. It is known that optimal recognition of target cells by CTL requires binding of the cognate peptide MHC class I complex by both TCR and the CD8 co-receptor. Some CD8b chain mutations have been shown to increase CD8 binding affinity with pMHC and enhance T cell effector function. We have generated murine CD8b chain mutants, affecting both MHC binding sites (L58R, S53L, S54V, and L58R/I25A) and glycosylation sites (T120A, T121A T124A, T121A/T124A). The mutated CD8b molecules have been introduced into murine splenocytes using retroviral vectors together with the two MDM TCR that recognise pMDM100, derived from the tumour antigen MDM2 presented by H2b molecules. Using CD8αα T cells obtained from CD8β knockout mice, we have introduced CD8β mutants or control CD8β wild type (WT) chains. All T cells were co-transduced to express the MDM TCR. T cells expressing CD8β glycosylation mutants were as efficient as CD8β WT transduced cells with respect to peptide specific IFN-³/IL-2 production, proliferation and killing. One MHC binding mutant (L58R/I25A) demonstrated better IFN-³ production in response to relevant peptide. We are in the process of showing that this mutant may be associated with improved killing characteristics affording better tumour protection.

1 Prenatal Cell and Gene Therapy Group, Institute for Women's Health, University College London, London, WC1E 6HX, UK 2 Surgery Unit, Institute of Child Health, University College London, London, WC1N 1EH, UK 3 King's College London School of Medicine, Nuclear Biology Group, Department of Medical and Molecular Genetics, London, SE1 9RT, UK 4 Department of Paediatric Immunology, Institute of Child Health, University College London, London, WC1N 1EH, UK P 45 Congenic Fetal Delivered Lentivirus UCOE-Luciferase Transduced Amniotic Fluid Stem Cells Preferentially Engraft in the Liver

Prenatal stem cell-based gene therapy has recently been shown as a possible strategy to treat congenital diseases which cause fetal and neonatal morbidity and mortality. However, allogeneic cells can be rejected in non-immunocompromised animals. Autologous cell/gene therapy offers an alternative potential treatment option. Mouse amniotic fluid (AF) contains around 1% c-Kit(+) amniotic fluid stem (AFS) cells, which have the ability to differentiate into the three germ layers. We present here our first attempts to use lentiviral vector (LV) transduced AFS cells as an in utero congenic transplantation model in mice. Amniotic fluid was collected from 12 pups in one wild type dam (MF1) at E13.5. c-Kit(+) cells were transduced with an LV containing a UCOE promoter driving luciferase transgene expression and, after 24 hours, 50,000 of these cells were injected via the vitelline vessel to each of 8 fetuses from two MF1 dams at E14.5. Five out of 8 (62.5%) injected pups survived to term and demonstrated strong and persistent luciferase activity when analysed by the Caliper Life Sciences in vivo imaging system until at least 12 weeks of age. At 16 weeks, two out of 5 (40%) mice showed clear evidence of UCOE-luciferase positive cell engraftment in peripheral blood as analysed by ex vivo luminometry. These initial studies show a feasible way forward for prenatal cell and gene therapy using AFS cells for regenerative medicine applications.

1 Department of Haematological Medicine, King's College London, London SE5 9NU, UK 2 Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3 RA, UK P 48 Metabolic Biotinylation of Lentiviral Vectors

The aim of the study described here was to develop a suitable strategy for affinity tagging of lentiviral vectors for subsequent avidin mediated purification or targeting applications. We have previously described a new 293T cell based cell line (BL15), that metabolically produces spontaneously biotinylated lentivirus vectors allowing the affinity mediated capture of virus. In BL15 cells a membrane anchored Low Affinity Nerve Growth Factor Receptor (LNGFR) which is fused to a biotin acceptor peptide (BAP) is expressed on the cell surface. The additional expression of bacterial birA (biotin ligase) in these cells enables the covalent metabolic biotinylation of a specific lysine residue in BAP. The biotinylated protein is transported to the cell surface as a membrane anchored “affinity-tag” and incorporated onto the membrane of the lentiviral particles produced by BL15 cells. These cells produce surface biotinylated lentiviral vectors in the usual manner, requiring only the presence of biotin in the culture medium to allow the concentration of the vector by several orders of magnitude using streptavidin conjugated paramagnetic particles. Capture of the biotinylated retroviral vectors by this strategy is highly efficient, but does require the removal of excess biotin containing medium prior to virus harvest. In addition, the high affinity of biotin for avidin/streptavidin makes it unsuitable for processes requiring the subsequent elution of virus from the streptavidin beads. In order to address these issues we have cloned and expressed a bacterial desthiobiotin synthase in the BL15 cells. The new 293T based cells, convert the non-avidin binding precursor 7,8-Diaminopelargonic acid (7-DAPA) to desthiobiotin, which is then coupled to BAP by biotin ligase, thus generating desthiobiotin tagged lentiviral vectors in medium containing no free biotin/desthiobiotin. Here we demonstrate monomeric avidin based column purification of such desthiobiotin tagged lentivirus preps in a process that allows high throughput and high titer purification of lentivirus vectors.

1 King's College London School of Medicine, Nuclear Biology Group, Department of Medical and Molecular Genetics, Guy's Hospital, London, UK 2 Institute for Women's Health, University College London, London, UK 3 Molecular Immunology Unit, Institute of Child Health, University College London, London, UK P 49 Development of In Utero Lentiviral Vector Gene Therapy Approaches for Inherited Diseases

An in utero gene therapy approach for the treatment of inherited diseases such as haematological disorders (thalassaemia, haemophilia, SCID) and body-wide muscular dystrophies (e.g., DMD) in principle offers distinct advantages over postnatal methods employing either an in vivo or ex vivo strategy. This includes treatment delivered in time to prevent irreversible end-organ damage, avoidance of treatment-associated morbidity of postnatal bone marrow transplantation, the presence of highly proliferative stem and progenitor cell compartments; fetal cells are readily transduced and have a competitive engraftment advantage; the human fetus is only 30 to 60 grams between 12 to 14 weeks, allowing delivery to proportionally more cells with lower vector doses than that required postnatally; and the immunological naivete of the early gestation fetus produces tolerance to foreign antigens. We have been assessing the feasibility of an in utero approach for haematological disorders in mice as a model system using lentiviral vectors (LVs). LVs containing transgenes under control of the HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE) were administered to 14–16 day fetuses via the vitelline vessel and vector presence and expression monitored in tissues postnatally out to 6 months of age. Our data show very good marking and stable expression from the adult liver. We also readily detected vector presence and expression in peripheral blood lineages suggesting that haematopoietic stem cells in the fetal liver were also transduced. Our data suggest that LVs are a promising system to deliver effective in utero gene therapy once issues of safety are also addressed.

1 Inserm U781, Hôpital Necker-Enfants Malades, Bât M. Lamy, 149, rue de Sèvres, 75015 Paris 2 INSERM UMRS974, Institut de Myologie, bd de l'Hôpital, 75013 Paris 3 Cancer Institute, University College London, London P 50 Optimization of U7snRNA Cassettes for Expression of Therapeutic Antisense Sequences

Pre-mRNA maturation can be modulated using antisense oligonucleotides. These approaches have been recently used in clinical trials where exon-skipping is induced on a mutated DMD pre-mRNA in order to reestablish the translational reading frame. Stable delivery of antisense sequences can be obtained with modified snRNAs such as U1 or U7, expressed from viral vectors. The main limitation remains the amount of snRNA expressing vector which can be produced and administered to patients with Duchenne Muscular Dystrophy. One way to increase the therapeutic index of these vectors is to optimize the expression level of the snRNA shuttle. In this aim, we have replaced the RNA polymerase (RNAP) II promoter of U7 with RNAPIII promoters such as those used for the transcription of U6 and 7SK genes which are considered the most active for small RNA expression. A U7 shuttle containing antisense sequences known to efficiently mediate skipping of exon 51 on the human DMD pre-mRNA was placed under the control of these promoters and the new cassettes were introduced into lentiviral vectors. No snRNA expression (nor exon 51 skipping) could be detected with these new cassettes. Further modifications have therefore been introduced in the U7 sequence, primarily in order to avoid premature termination of RNAPIII caused by runs of Ts in the sequence. In a second approach, we have introduced a strong muscle specific enhancer (MHCK7, a synthetic fusion between the MCK α-MHC enhancers) upstream of U7 shuttles driven by their cognate RNAPII promoter. These new designs have been applied to U7 carrying antisenses sequences for the skipping of human exon 51 or murine exon 23, and both lentiviral or AAV2/1 vectors are currently tested in human myoblasts or following direct injection into the muscle of mdx mice.

1 Biocentre, Pro-Cure Therapeutics Ltd, United Kingdom 2 Dept of Clin Pharm, OCRB, University of Oxford, United Kingdom 3 YCR Cancer Research Unit, Dept of Biology, University of York, United Kingdom 4 Cherwell Innovation Centre, Hybrid Systems Ltd, United Kingdom P 52 Human Prostate Stem Cells Support Replication of a Prostate-Targeted, Conditionally Replicating Adenovirus

The cancer stem cell hypothesis states that tumour growth is sustained by a population of invasive self-renewing stem cells (SC). In the prostate, SC can be isolated using CD133 and α2β1 integrin expression. These SC constitute <0.1% of the tumour population and are particularly resistant to chemo- and radiotherapy, implying they drive tumour recurrence. If oncolytic adenoviruses (Ad) are to become viable cancer therapeutics, it is essential that they can infect and replicate in SC. We have tested the ability of wild-type Ad (Adwt) and a prostate-restricted oncolytic Ad, Ad-I/PPT-E1a, to infect and replicate in patient-derived prostate SC. Prostate SC were cultured from 3 patients who had undergone a transurethral prostate resection. Immunofluorescence and microarray studies showed that SC expressed the coxsackie-adenovirus receptor. To test their ability to support Ad replication, SC or a positive control prostate cancer cell line (LNCaP) were incubated with Ad-I/PPT-E1a or Adwt. Quantitative-PCR was used to calculate the Ad genomes present directly post infection (PI) and at 65 hr. Equivalent levels (5–10 copies/cell) of all Ads were associated with SC directly PI; this was comparable to levels measured in LNCaPs. By 65 hr, both Adwt (up to 1042 copies/cell) and Ad-I/PPT-E1a (up to 158 copies/cell) achieved replication in the SC, although yields were higher in the LNCaP cell line. Initial data shows that similar replication is not supported by peripheral blood CD34+ or CD133+ cells. Elimination of cancer recurrence requires the destruction of cancer SCs. Oncolytic adenoviruses, such as Ad-I/PPT-E1a, could be a successful and novel treatment for prostate cancer, as they are capable of replication in prostate stem cells. This work was funded by the FP6 GIANT programme.

1 Department of Medical Oncology, Paterson Institute for Cancer Research, University of Manchester, UK 2 Institute for Women's Health, University College London, UK 3 Department of Gene Therapy & Immunology, Institute of Child Health, London, UK 4 Institute of Cancer, Barts and the London School of Medicine and Dentistry, Charterhouse Square, London 5 North West Embryonic Stem Cell Centre, Core Technology Facility, 46 Grafton Street, University of Manchester, Manchester, UK P 53 Gene Delivery of a Mutant TGFβ3 Reduces Markers of Scar Tissue Formation After Cutaneous Wounding

The transforming growth factor-β (TGFβ) family plays a critical regulatory role in repair and co-ordination of remodeling after cutaneous wounding. TGFβ1 mediated chemotaxis promotes the recruitment of fibroblasts to the wound site and their resultant myofibroblastic transdifferentiation which is responsible for elastic fiber deposition and wound closure. TGFβ3 has been implicated in an antagonistic role regulating overt wound closure and promoting regulated wound closure. We generated a mutant form of TGFβ3 (mutTGFβ3) by ablating its binding site for the Latency Associated TGFβ Binding Protein (LTBP-1). Thus the cytokine is not sequestered in an inactive form at the Extra Cellular Matrix (ECM) and is activated by normal intracellular and extracellular mechanisms which include integrin-mediated activation. We show that localized intradermal transduction using a lentiviral vector expressing the mutTGFβ3 in a mouse wounding model reduced re-epithelialization density and fibroblast/myofibroblast transdifferentiation within the wound area, both indicative of reduced scar tissue formation.