British Society for Gene Therapy 2012 March 9,2012 UCL Institute of Child Health,London

P 001

Abstract Withdrawn

Mukherjee Sayandip 1 Pipino Caterina 2 Sung Peggy 1 DeCoppi Paolo 2 Thrasher Adrian 1

1 Centre for Immunodeficiency, Institute of Child Health, UCL, 30 Guilford Street, London WC1N1EH, UK 2 Paediatric Surgery Unit, UCL Institute of Child Health, UCL, 30 Guilford Street, London WC1N1EH, UK P 002 Investigating perturbed haematopoiesis in Down Syndrome patients: an induced pluripotent stem cell approach

Children with Down syndrome exhibit 150-fold excess risk of developing myeloid leukaemia in comparison to normal paediatric population.10% of Down Syndrome (DS) neonates suffer from a self-resolving Transient Myeloid Disorder (TMD) within weeks of their birth resulting in an abnormal proliferation of myeloid blasts in their blood. Between ages 1–4 years, 20–30% of children with TMD develop a severely life-threatening Acute Megakaryocytic Leukaemia (AMKL) involving cytopenia and complex karyotypic abnormalities requiring chemotherapeutic intervention. While mutations affecting the expression of the X-linked GATA1 gene are known to cause AMKL, it is unknown whether GATA1 mutations are solely responsible for triggering TMD and also whether TMD constitutes an initial permissive step for the full-blown acute leukaemia which occurs later in life. A recent study covering a large number of patients suffering from TMD and AMKL showed that although GATA1 mutation is present in almost all cases, there is no significant difference in the mutational spectrum between the two disorders. The type of GATA1 mutation is therefore not a reliable prognostic marker for future susceptibility to AMKL in TMD patients. In the absence of suitable animal models of DS which can recapitulate the pathology of TMD & AMKL, we wish to establish a platform for dissecting the molecular events of this perturbed haematopoiesis by generating induced pluripotent stem cells from amniotic fluid derived cells (cultured from routine amniocentesis) of normal and DS foetuses. These iPS cells would then be subjected to established haematopoietic differentiation protocols and analysed for gene expression signature by various molecular and functional assays at every stage of development.

sayandip.mukherjee@ucl.ac.uk

Rivat Christine 1 Booth Claire 1 Blundell Michael 1 Thrasher Adrian J 1 Gaspar Hubert B 1

1 Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH, UK P 003 Lentiviral vector mediated gene therapy for X-linked lymphoproliferative disease (XLP1) restores humoral and cellular functions

XLP1 arises from mutations in the SH2D1A gene, which codes for SAP, an intracellular adaptor expressed in T and NK cells. The lack of SAP results in decreased T and NK cell cytoxicity, the absence of NKT cell development, defective CD4 T cell help, and consequently abnormal humoral function. Similar defects are seen in SAP deficient mice, which provide a relevant model to test gene therapy strategies. Corrective measures for XLP1 are limited and the only curative option is allogeneic transplantation, which has a high mortality rate in the mismatched donor setting.

We designed a lentiviral vector in which a codon optimised version of the human SAP cDNA is under the transcriptional control of the short form of the EF1- α promoter (EFS). Lin-ve cells from SAP KO mice were transduced with using a cytokine cocktail of SCF, Flt3 ligand and TPO and transplanted into irradiated SAP KO recipients. At 11 weeks after transplant, baseline immunoglobulins levels showed significantly increased IgG, IgM, IgG1 and IgG3 in comparison with SAP KO and control mice receiving GFP transduced cells. Mice were then vaccinated with the T dependent antigen NP-CGG and then sacrificed 10 days later for full analysis. NKT cell development was seen in SAP-GFP transduced mice, and we observed significantly increased NK cell cytotoxicity in these mice in comparison to SAPKO mice or mice receiving GFP transduced cells. T-dependent humoral responses were also restored, with SAP-GFP transduced mice having levels of NP-specific Ig in IgG1 and IgG3 that were significantly higher than control animals.

This study demonstrates reconstitution of both cellular and humoral defects in SAPKO mice through lentiviral vector mediated gene transfer into haematopoietic progenitor cells and provides a proof of concept for developing haematopoietic stem cell gene therapy for XLP1.

c.rivat@ucl.ac.uk

Carr Bob 1 Siupa Agnieszka 1 Hole Patrick 1 Malloy Andrew 1 Hannell Claire 1

1 NanoSight Ltd, Amesbury, Wiltshire, SP4 7RT, UK P 004 Nanoparticle Tracking Analysis for the rapid and direct visualisation, sizing and counting of viruses, phage and VLPs in suspension

Nanoparticle Tracking Analysis is a new optical technique which allows nanoparticles such as viruses and phage in suspension to be directly visualised on an individual basis, their size and number frequency size distribution to be rapidly analysed to high resolution and their number concentration to be determined. Additionally, fluorescently labelled virues in complex backgrounds (e.g. growth media) can be specifically detected, analysed and counted as can their zeta potential on a particle-by-particle basis.

Examples of the use of the recent use of NTA in virus and phage detection and counting will be reviewed.

bob.carr@nanosight.com

Georgiadis C. 1 Semenova K. 1 McGrath J. 2 Di W. 1 Qasim W. 1

1 Institute of Child Health, MIU, IBU, UCL, 30 Guilford Street, London, WC1N 1EH, UK 2 St Johns Institute of Dermatology, Guys Hospital, Great Maze Pond, London, SE1 9RT, UK P 005 Development of lentiviral gene therapy for type VII collagen deficient epidermolysis bullosa

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a severe debilitating genodermatosis characterised by severe blistering of the skin, chronic morbidity and predisposition to malignancy. This autosomal recessive disorder is caused by mutations in the human type VII collagen gene (COL7A1), with loss of expression of collagen protein essential for the anchoring of the epidermis to the dermis. Structurally defective anchoring fibrils compromise the dermal-epidermal junction (DEJ) causing blistering. Experimental cell based therapies have included intradermal allogeneic fibroblast injections and allogeneic bone marrow stem cell therapy. We are investigating ex-vivo correction and administration of autologous cell populations and have designed and constructed a third generation lentiviral platform for the delivery of an optimised COL7A1 transgene. Previous studies have reported difficulties with expression of full length collagen VII from lentiviral configurations, but codon optimisation of the transgene sequence, and removal of cryptic splice sites has addressed these issues.

Collagen VII deficient human keratinocyte (KC) and fibroblast (FB) cells with compound heterozygote mutations were transduced and evaluated for reconstitution of collagen expression. Reconstitution of full length 290kda protein was confirmed by western blot analysis and further characterised by immunocytochemistry techniques. Stability of expression was tracked for one month in culture and in three dimensional organotypic cultures. Furthermore, collagen VII was detectable in supernatant collected from gene corrected cells but not from control transduced collagen deficient lines.

This platform is now being developed for the modification of fibroblasts, keratinocytes and haematopoietic stem cell populations derived from patients with RDEB.

christos.georgiadis.11@ucl.ac.uk

Zhang Fang 1 Knight Sean 2 Collins Mary 2 Takeuchi Yasuhiro 2 Antoniou Michael 3 Thrasher Adrian J 1

1 Molecular Immunology Unit, Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH, UK 2 MRC/UCL Centre for Medical Molecular Virology, Division of Infection & Immunity, UCL Cancer Institute, UCL, Cruciform Building, Gower Street, London, WC1E 6BT, UK 3 Gene Expression and Therapy Group, Department of Medical and Molecular Genetics, Kings College London School of Medicine, Guy's Campus, London Bridge, London, SE1 9RT, UK P 006 Improved safety of Lentiviral vectors containing silencing resistant A2UCOE regulatory elements

Self-inactivating lentiviral vectors (SIN-LV) have been shown to be promising tools for delivering therapeutic gene into cells, including quiescent cells and stem cells. However, loss of gene expression in vivo due to epigenetic mediated gene silencing can compromise efficiency. To tackle this problem, we have developed a SIN-LV incorporating a novel A2UCOE driving transgene expression. We have shown that long term stable transgene expression from A2UCOE is directly due to its resistance to DNA methylation mediated silencing (Zhang et al, 2010). The utility A2UCOE in driving therapeutically relevant gene expression for IL2-RG, Rag1 and Rag2, in comparison with other commonly used promoters, has also been shown from our study and others (Zhang et al. 2007; Pike-Overzet et al. 2011; N. P. van Til et al. unpublished). To improve vector safety, we have recently modified the A2UCOE to abolish existing and cryptic splicing signals as well as stable readthrough transcripts. Using an in vitro assay, in which the mutagenesis occurs due to splicing of vector initiated mRNA transcripts to the growth hormone receptor (GHR) gene (Knight et al. 2010), we have found that these modifications abolish the development of mutant clones in striking contrast to the original wild-type A2UCOE vector. The function of the modified A2UCOEs in driving EGFP expression was also assessed in mouse embryonic carcinoma P19 cells following virus transduction as these are known to rapidly methylate and silence foreign sequences. We found that the levels of EGFP expression from the modified A2UCOE vectors were sustained at a similar level to that for the wild type vector. Furthermore, DNA methylation analysis showed that there was no significant difference in the percentage of methylated CpG sites in the modified A2UCOE elements compared to a low level of methylated CpGs in the wild type A2UCOE. In contrast, a truncated A2UCOE vector was highly susceptible to DNA methylation and to silencing of transgene expression. These modified A2UCOE elements should provide an improved safety profile in clinical gene therapy applications.

sejjfzh@ucl.ac.uk

Boza Maria G 1 Wanisch Klaus 1 Tizzano Eduardo 2 Yáñez-Muñoz Rafael J. 1

1 Genetic Therapy Laboratory. School of Biological Sciences. Royal Holloway University of London, Egham, Surrey, TW20 0EX, UK 2 Department of Genetics and Institute of Biomedical Research, Hospital Sant Pau, Sant Antoni Ma, Claret n° 167, 08025 Barcelona, Spain P 007 Studying spinal muscular atrophy through induced pluripotent stem cell-based models

Spinal muscular Atrophy (SMA) is an autosomal recessive neuromuscular disease caused by mutations in the Survival of Motor Neuron 1 gene (SMN1), which result in dramatic decreases in the expression of SMN protein. Low levels of SMN lead to the specific degeneration of alpha motor neurons (MNs) in the anterior horn of the spinal cord, with variable degrees of severity associated to the overall amount of the protein present. The pathological mechanism of SMA is highly controversial and until recently it was not possible to obtain human MNs in culture in order to study the disease. The development of induced pluripotent stem cell (iPSC) technology has made it possible to bypass this obstacle and an IPSC-based model of SMA type I has already been validated. Encouraged by this pioneering finding we have produced and characterized iPSCs from several members of a discordant consanguineous family in which four haploidentical siblings share the same homozygous SMN1 mutation, but nonetheless show different phenotypes of the disease. We have been able to differentiate several of these clones into HB9+/ChAT+ MNs and we are currently working on the purification of these cells from the mixed population in which they are produced. Potential disease phenotypes and novel disease modifying factors will be studied in culture through imaging analysis as well as transcriptomic and proteomic approaches. The rescuing ability of therapeutic viral vectors already developed in our laboratory will also be tested once phenotypes are identified.

maria.boza.2009@rhul.ac.uk

Sanchez Noëlia 1 Lao Nga 2 Gallagher Clair 1 Aherne Sinead 1 Doolan Padraig 1 Clarke Colin 1 Clynes Martin 1 Barron Niall 1

1 NICB, DCU, Collins Avenue, Glasnevin, Dublin, Ireland 2 Conway Institute, UCD, Belfield, Dublin, Ireland P 008 MiR-7 as a potential target for control of cell proliferation

MiRNAs are small non-coding RNAs that appear to be key regulators in most cellular pathways. Each miRNA is thought to regulate hundreds of genes in a post-transcriptional manner by translational repression and/or mRNA degradation. MiRNAs act as tumor supressors or as oncogenes in many cancer diseases. Therefore there is a growing interest in miRNAs as potential biological markers for diagnostics or targets for gene therapy in cancer research. MiR-7 is involved in cell proliferation, apoptosis, homeostasis and development. MiR-7 dysregulation promotes tumorigenesis and invasion in many cancer diseases. To better understand the complex role of miR-7 growth regulation, we used transient and stable approaches to regulate miR-7 expression in CHO cells. After transient up-regulation cell growth was significantly reduced. Stable knockdown of miR-7 using a “sponge miR” approach enhanced cellular proliferation. Following miR-7 target gene identification, cell proliferation-, cell cycle- and apoptosis-related genes were found to be the most abundant. These findings suggest that miR-7 plays a crucial role in cell cycle regulation and may be considered as a promising target for cancer gene therapy.

noelia.sanchez2@mail.dcu.ie

Lu-Nguyen Ngoc B 1 Broadstock Martin 1 Yáñez-Muñoz Rafael J 1

1 Royal Holloway University of London, Egham, Surrey, TW20 0EX, UK P 009 Insulin-like growth factor 1-mediated neurotrophic effects for the treatment of Parkinson's disease

Parkinson's disease (PD), 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. Gene therapy approaches delivering neurotrophic factors have offered promising results in both pre-clinical and clinical trials. Although one of the most promising factors, GDNF (glial cell line-derived neurotrophic factor), failed phase II clinical trials, other trophic factors which may retain efficacy in the clinic are being investigated. One such factor, IGF-1 (insulin-like growth factor 1), has offered promising initial results in the treatment of PD. Direct injection of IGF-1 into a rodent model of PD demonstrated impressive protection of dopaminergic neurons against 6-hydroxydopamine toxicity, but clinical protein delivery would require administration by implanted mini-pumps, with associated surgical risks. We have therefore developed lentiviral vectors expressing IGF-1. In a bid to enhance specificity, these vectors were designed to target neurons (synapsin promoter, SYN), glia (glial fibrillary acidic protein promoter, GFAP), or a mixture of both cell types (immediate early cytomegalovirus promoter, CMV). Transduction of lentiviral vectors encoding eGFP driven by these promoters showed cell-type specificity in primary rat ventral mesencephalic cultures, and significant differences in transduction efficiency between integrating and non-integrating vectors. An investigation of neurotrophic effects on primary rat neuronal cultures demonstrated that those neurons transduced by IGF-1 had significantly improved survival upon withdrawal of exogenous trophic factors. We are currently investigating the effects of such vectors in an animal model of PD, the 6-hydroxydopamine-lesioned rat.

Ngoc.LuNguyen.2009@live.rhul.ac.uk

Field Anne-Christine 1 Stauss Hans J 2 Morris Emma C 2 Qasim Waseem 1

1 Molecular Immunology Unit, Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH, UK 2 Department of Immunology, Division of Infection & Immunity, UCL, Rowland Hill Street, Royal Free Hospital, London, NW3 2PF, UK P 010 Comparison on lentiviral and Sleeping Beauty transposon-mediated gene expression of T cell receptor genes

Initial clinical trials using integrating γ-retroviral and lentiviral gene delivery platforms have demonstrated that TCR gene therapy can mediate tumour regression in human studies. Alternative integrating vector systems based on transposon plasmids may allow more rapid screening of candidate TCRs. Here we have compared lentiviral and transposon platforms for delivery of a Wilms' Tumor antigen 1 (WT-1) specific TCR. Sleeping Beauty (SB) transposon systems, in combination with a hyperactive transposase, mediate genomic insertion with unbiased integration profiles compared to viral vectors, and enhanced mutant variants have recently been shown to mediate efficient gene transfer. For lentiviral delivery, alternative internal promoters derived from PGK, EF1α and SFFV were compared in combination with different envelope pseudotypes (VSV-G, RDpro and MV). We found the human PGK an effective alternative to the SFFV promoter for the expression of WT-1 TCR transgenes and VSV-G the most efficient envelope. We showed that SB gene delivery by nucleofection allowed for stable expression of TCR transgenes and confirmed the superior transposition efficiency of the hyperactive SB100 compared to SB11. The viability and efficiency of gene transfer and expression by nucleofection was lower than by transduction in T lymphocytes. However, the efficiency of gene transfer was comparable in CD8+ T cells modified with SB transposons or lentiviral vectors. Their functionality was confirmed by a peptide-specific IFNg production. In conclusion, SB gene therapy offers an alternative to viral vectors for testing tumour-specific TCR genes in human T lymphocytes.

Anne-christine.field@ucl.ac.uk

Munye Mustafa M 1 Howe Steven J 1 Hart Stephen L 1

1 Molecular Immunology Unit, Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH, UK P 011 Towards gene therapy for primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) describes a family of rare genetic disorders affecting ciliary motility. The DNAH5 gene is the most frequently mutated in PCD patients and encodes a large 500kDa protein which forms part of the outer dynein arm complex of the ciliary axoneme and powers ciliary movement through its ATPase activity. Ciliary motility is important in the normal function of different organ systems and ciliary dysfunction leads to a wide variety of symptoms. In patients respiratory defects are of most concern as they can lead to lung failure requiring lung transplantation. Current therapy for PCD therefore focuses on the prevention of progressive lung damage and respiratory gene therapy is an attractive strategy to achieve this. We have optimized a receptor-targeted nanocomplex (RTN) formulation for respiratory cell gene transfer with the aim of rescuing ciliary motility in cells with DNAH5 gene defects. The RTN's are formed using a cationic liposome, plasmid DNA, and a dual-functioning cationic peptide, K16GACSERSMNFCG with DNA condensation (K16) and receptor-targeting sequences (SERSMNF), which has close homology to the ICAM-1-binding capsid protein of human rhinovirus. The optimised RTN vector shows greater luciferase reporter gene activity in both 16HBE bronchial epithelial cell line and primary nasal epithelial cells than the commercial transfection reagent Lipofectamine 2000. Finally, we have cloned the wild-type DNAH5 gene into a mammalian expression vector to allow rescue in mammalian models of PCD. We show both mRNA and protein expression of DNAH5 following transfection of the plasmid into mammalian cells using the optimized RTN formulation.

m.munye@ich.ucl.ac.uk

Montiel-Equihua Claudia A 1 Zhang Lin 1 Knight Sean 2 Saadeh Heba 3 Scholz Simone 4 Carmo Marlene 1 Alonso-Ferrero Maria E 1 Blundell Michael P 1 Monkeviciute Aiste 1 Schulz Reiner 3 Collins Mary 2 Takeuchi Yasuhiro 5 Schmidt Manfred 4 Fairbanks Lynette 6 Antoniou Michael 3 Thrasher Adrian J 1 Gaspar H Bobby 1

1 Centre for Immunodeficiency, Molecular Immunology Unit, Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH, UK 2 MRC/UCL Centre for Medical Virology, Division of Infection and Immunity, UCL Cancer Institute, 72 Huntley Street, London, WC1E 6DD, UK 3 Department of Medical and Molecular Genetics, King's College London, Guy's Hospital Great Maze Pond, London, SE1 9RT, UK 4 German Cancer Research Centre, Im Neuenheimer Feld 280, 69120 Heidelberg Germany 5 Wohl Virion Centre, Division of Infection and Immunity, UCL, Gower Street, London, WC1E 6BT, UK 6 Purine Research Laboratory, St. Thomas Hospital, Westminster Bridge Road, London, SE1 7EH, UK P 012 Development of a gene expression system for enhanced erythroid expression

We have developed a gene expression system in a lentiviral vector whereby use of the β-globin LCR (β -LCR) upstream of the EFS-1α promoter allows for high transgene expression in the erythrocyte lineage, whilst maintaining expression in other hematopoietic lineages. Our vector design could be advantageous in hematopoietic stem cell gene therapy applications that require the systemic delivery of a product to multiple organs, such as enzyme deficiencies.

We demonstrate that our β -LCR vector is able to upregulate transgene expression in erythroid models in vitro and in a murine transplant model, it enabled equivalent levels of expression to a control vector in lymphoid and myeloid lineages but >20× over expression of a reporter (GFP) or therapeutic transgene (ADA) in erythrocytes even up to a year after transplant.

A series of genotoxicity assays to test the safety of our new expression platform have been concluded and indicate that, although the β -LCR element is able to activate the expression of about 15% of the genes that surround the insertion site, this does not seem to translate into enhanced mutagenic potential.

h.gaspar@ucl.ac.uk

Delhove Juliette M K M 1 Waddington Simon N 2 Ward Natalie J 2 Jathoul Amit P 3 Pule Martin A 3 McKay Tristan R 1 Buckley Suzanne M K 2

1 William Harvey Research Institute, Queen Mary University of London, London, UK 2 Institute for Women's Health, University College London, London, UK 3 Department of Haematology, UCL Cancer Institute, University College London, London, UK P 013 Development of bicistronic lentiviral vectors for continual in vivo and endpoint analysis of gene and stem cell transfer

Analysis of the biodistribution of gene and stem cell transfer in rodents is performed commonly as endpoint analysis however this limits assessment of expression and distribution dynamics unless multiple groups of animals are treated and sacrificed at different time points. Over the past decade, the adoption of luciferase marker gene, which emits light upon administration of the substrate luciferin, permits quantitation and localisation of expression from gene transfer vectors or transduced cells.

One of the challenges in using luminescence within deep-lying visceral tissues such as the liver using firefly luciferase (emission: 560 nm) is the signal attenuation seen in vivo due to the scatter and tissue absorption of light emitted at this wavelength. In order to maximise photonic output from the liver we generated thermostable firefly luciferase (peak photonic emission at 549 nm) through the introduction of 5 non-synonymous mutations and codon-optimised for mammalian expression. A variant containing a single-base substitution (Ser284Thr) was introduced to allow for a shift in the light emission wavelength further toward the infra-red end of the light spectrum (608 nm), the region at which there is significantly decreased light absorption within haemoglobin-dense tissues.

A second challenge of using firefly luciferase is the relatively poor immunohistochemistry and FACS data obtained using anti-luciferase antibodies versus those against GFP or FLAG. Therefore we developed bicistronic cassettes containing the wild-type or red-shifted firefly luciferase with a 3x FLAG tag for Western blot, and FACS analysis. This cassette was joined to a GFP reporter by means of a 2A linker peptide, which permits the post-translational cleavage of the luminescent and fluorescent reporters. The bicistronic cassettes were introduced into a 2nd generation lentiviral backbone with constitutive gene expression driven by an upstream SFFV promoter. It was possible to quantitate vector titres using FACS analysis of both the FLAG tag and GFP. In vitro comparisons of the wild-type and red-shifted luciferase constructs revealed that wild-type luciferase emits significantly more light than the red-shifted variant. However, in vivo the red-shifted luciferase emitted more photons of light per transduced liver cell than wild-type luciferase. Endpoint analysis by immunohistochemistry was more robust against GFP compared with luciferase. Red-shifted luciferase is therefore proposed to be the optimal light emitting transgene for bioimaging, particularly of haemoglobin-rich tissues such as the liver. In combination with FLAG tags and GFP, effective endpoint analysis is also feasible.

s.waddington@ucl.ac.uk

Hutchinson Lauren 1 Boza-Morán Maria Gabriela 1 Yáñez-Muñoz Rafael J 1

1 School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, TW20 0EX, UK P 014 ^* Generation of a motor neuron-specific lentiviral transfer plasmid carrying SMN1, a therapeutic gene for Spinal muscular atrophy

Lentiviral vectors are capable of transducing non-dividing cells, including motor neurons (MNs), whilst sustaining long-term transgene expression. This is particularly relevant for the development of therapies in MN diseases. Spinal muscular atrophy (SMA) is a neuromuscular disease characterised by the degeneration of lower motor neurons, leading to severe muscle weakness and degeneration. The disease is caused by mutations or deletions in the Survival Motor Neuron 1 gene (SMN1), and the level of SMN protein expression is inversely correlated with the severity of the disease. A published lentiviral reporter construct carries eGFP driven by the MN-specific promoter HB9, which upon transduction can achieve efficient and specific transduction of MNs. Encouraged by this finding, we have replaced the reporter eGFP gene with hSMN1 cDNA in order to create a lentiviral vector which may effectively and specifically express hSMN1 in MNs. For this, the hSMN1 cDNA was amplified from another plasmid via PCR, with the addition of appropriate restriction sites that would allow successful ligation into the lentiviral HB9 transfer plasmid upon removal of eGFP. Restriction analysis and DNA sequencing confirmed that the ligation was successful and the lentiviral transfer plasmid Lenti-HB9::hSMN1 had been generated. This lentiviral transfer plasmid will be used to produce a lentivector for the transduction of MNs derived in vitro from induced pluripotent stem cells from SMA patients, in order to study SMN interaction partners within this disease-relevant cell type. This new approach could help us to better understand the pathological mechanisms of SMA.

Lauren.Hutchinson.2009@live.rhul.ac.uk

Griesenbach Uta 1 McLachlan Gerry 2 Collie David D 2 Innes J Alastair 3 Higgins Tracy E 1 Cheng Seng H 4 Scheule Ronald K 4 Alton Eric W F W 1 Davies Jane C 1 Hyde Steve C 5 Gill Deborah R 5 Porteous David J 3 Boyd A Christopher 3

1 Imperial College London and the UK CF Gene Therapy Consortium, Department of Gene Therapy, Emmanuel Kaye Building, National Heart & Lung Institute, Imperial College, Manresa Rd, London, SW3 6LR 2 The Roslin Institute and Royal (Dick) School of Veterinary Medicine, University of Edinburgh, Easter Bush, Midlothian, EH25 9RG 3 University of Edinburgh, Department of Respiratory & Sleep Medicine, Royal Hospital for Sick Children, Sciennes Road, Edinburgh, EH9 1LF 4 Genzyme Corporation, Framingham, MA, USA 5 Gene Medicine Group, Nuffield Dept. of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU P 015 Toxicology studies in support of the UK CF Gene Therapy Consortium's Multi-dose Clinical Trial

In preparation for a Multi-dose clinical trial to assess whether 12 monthly doses of the CFTR gene can improve CF lung disease we have conducted two toxicology studies (TS). In addition to a GLP-mouse TS study (outsourced to a CRO) we also performed an in-house study in sheep. Mice received 12 doses of pGM169 (a plasmid carrying the CFTR gene) complexed to Genzyme lipid GL67A at fortnightly intervals by inhalation; exposure to the complex was for 0.5 (low=∼5× human dose), 2 (mid=∼ 20× human dose) or 6 hr (high dose=∼60× human dose). Controls were exposed to air for 6 hr. Standard non-invasive assessments were performed at regular intervals and post-mortem toxicology undertaken 2 weeks and 3 months after the last dose. Approximately 600 mice were included in the study. Anesthetised sheep (n=8) received 9 doses (consistent with EMEA guidelines) of aerosolized pGM169/GL67A at monthly intervals. Similar assessments were made as for the mouse TS, in addition to lung function measurements and bronchoalveolar lavage (BAL) at intervals throughout the study. The amount (mg/kg) of pGM169/GL67A delivered to sheep approximately mimics the proposed human dose. Anesthetised controls (n=8) received air only. All animals tolerated the treatment well. The transient and dose-related systemic inflammatory responses and drop in lung function observed in the single-dose phase 1 trial (separate abstract) were not replicated in non-CF sheep and non-CF mice only developed mild systemic inflammation at the highest dose (∼50x human dose) possibly highlighting species-specific differences or an increased response to the lipid/DNA complexes in the inflamed human CF lung. Both TS support progression into a Multi-dose CF gene therapy trial.

u.griesenbach@imperial.ac.uk

Sergijenko Ana 1 Langford-Smith Alexander 1 Langford-Smith Kia J 1 Wynn Robert F 2

1 Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, Manchester, UK 2 Blood and Marrow Transplant Unit, Royal Manchester Children's Hospital, Manchester, UK P 016 Correction of the Mucopolysaccharidosis IIIA mouse via stem cell gene therapy using a CD11b promoter to improve myeloid specific expression in the brain

Mucopolysaccharidosis IIIA (MPSIIIA) is caused by mutations in the sulfamidase (SGSH) gene, leading to cellular accumulation of heparan sulphate and progressive neurodegeneration in patients. Our aim was to compare mammalian promoters in a clinically applicable lentiviral vector using a stem cell gene therapy approach in WT and MPSIIIA mice.

eGFP expressing lentiviral vectors under the ubiquitous PGK or myeloid-specific CD11b and CD18 human promoters were used to transduce lineage depleted haemopoietic cells and transplanted into WT mice. Full chimerism and over 80% transduction were achieved with an average of 5 viral copies/ cell. The CD11b promoter resulted in the highest GFP expression in monocytes and B-cells in blood and spleen, but was weaker than PGK in T-cells. CD18 was more monocyte specific but weak. Significant numbers of GFP positive microglial cells were present in the brain from all groups, with an average of 25% transduced CD11b-positive cells in perfused mice.

We subsequently codon optimised the SGSH gene, significantly improving activity, and transduced lineage depleted WT cells with the CD11b-SGSH-CO vector. These were transplanted into MPSIIIA mice and outcomes measured 6 months later. The treatment resulted in an average of 1 viral copy/cell with complete correction of abnormal behaviour, elevated enzyme activity in brain, liver and spleen to 9%, 70%, and 170% of WT levels respectively. Brain and liver GAG storage was also completely normalised.

Neurological correction of the MPSIIIA mouse is feasible using a clinically relevant vector, but further improvements or higher copies will be required for autologous transplant correction.

Ana.Sergijenko@postgrad.manchester.ac.uk

Jarmin Susan 1 Foster Helen 1 Trollet Capucine 2 Dickson George 1 Foster Keith 1

1 Centre for Biomedical Science, School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, TW20 0EX 2 2UMRS 974, Institut de Myologie, Paris, France P 017 Positive effects on an OPMD mouse model of muscle atrophy by AAV mediated myostatin inhibition

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterised by ptosis, dysphagia and proximal limb weakness. Autosomal dominant OPMD is caused by a short (GCG)8-13 expansions within the first exon of the poly(A) binding protein nuclear 1 (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres.

In the transgenic mouse model of OPMD (A17.1) a severe and progressive muscular atrophy is associated with a reduction in muscle strength from the expression of the mutated PABPN1 protein and its nuclear inclusions.

Myostatin is a member of the transforming growth factor-β (TGF-β) superfamily and is produced almost exclusively in skeletal muscle tissue where it is secreted and acts as a negative regulator of muscle mass. It is naturally inhibited by its own propeptide and naturally Myostatin mutants exhibit a ‘double muscling’ phenotype.

Adeno-associated virus-8 (AAV8) myostatin propeptide vector (AAv8-ProMyoFc) was systemically introduced in 8 week old and 6 month old A17.1 and FvB (wt) mice. After 10 weeks, ProMyo expression was stable and a significant increase in muscle mass was seen as well as a restoration in physical activity. The use of Myostatin inhibition in gene therapeutic strategies for age-related or disease-related muscle loss is being evaluated through this model.

keith.foster@rhul.ac.uk

Vink Conrad A 1 Howe Steven J 1

1 Molecular Immunology Unit, Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH, UK P 018 Epigenetics of integration-deficient lentiviral vectors

The epigenetic status of vector DNA influences the level of transgene expression. A number of authors have reported lower transgene expression from integration-deficient lentiviral vectors (IDLVs) relative to integration-proficient lentiviral vectors (IPLVs). As these vectors should be identical in primary DNA sequence, differences in transgene expression are presumably due to epigenetic factors. At present we are characterising DNA methylation of IPLVs and IDLVs at the HIV-1 packaging signal and the internal promoter and plan to study other epigenetic marks including the acquisition of nucleosomes and histone modifications.

c.vink@ucl.ac.uk

Steichen Clara 1 Corbineau Sébastien 1 Pucéat Michel 2 Weber Anne 1 Dubart-Kupperschmitt Anne 1

1 Inserm U972, Hôpital Kremlin-Bicêtre, 94276, France 2 UMR-S 633 Inserm, University Paris Descartes, Campus Génopole 1, Evry, 91030, France P 019 Hepatic differentiation of simian pluripotent stem cells: a tool for cell therapy applications

Pluripotent stem cell based therapy carries great potential in the treatment of liver disease since stem cell-derived hepatocyte transplantation represents an alternative to orthotopic liver transplantation, which is limited by the lack of donor and the difficulty to expand hepatocytes in vitro. However, before clinical application is realized, the safety, efficacy and feasibility of this therapeutic approach must be established especially in large animal models, including non-human primate models. Among primates, the macaque is physiologically and phylogenetically very close to human. Despite the fact that many protocols already exists describing the differentiation of human embryonic stem (ES) cells into hepatocyte-like cells, they cannot be directly transferred to monkey ES cells and little is known about the hepatocyte differentiation potential of these cells. By using rhesus monkey ES cell lines derived from in-vitro produced blastocysts (Ormes cells), the aim of this work is to generate hepatic progenitor cells and hepatocytes and to characterize them in vitro for their phenotype markers and hepatic functions. We have set up chemically defined conditions devoid of serum, to differentiate rhesus monkey ES cells into endodermal cells expressing specific markers such as SOX17, GATA4, CXCR4. We are currently defining conditions to further differentiate these endodermal cells into hepatic progenitors and more mature hepatocyte-like cells. In parallel, we are reprogramming simian fibroblasts into iPS cells. The final goal of these experiments would be the use of simian pluripotent stem cell-derived hepatocytes for transplantation.

clara.steichen@inserm.fr

Alonso-Ferrero Maria E 1 Bartolovic Kerol 1 Kinnon Christine 1 Mata Marcia 1 Til Niek P Van 2 Howe Steven J 1

1 Molecular Immunology Unit, Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH, UK 2 Hematology, Erasmus University Medical Center, Rotterdam, 3015 GE, The Netherlands P 020 Transient manipulation of haematopoietic stem cells with integrative deficient lentiviral vectors for improved cell expansion, survival and engraftment

Haematopoietic Stem Cells (HSCs) are successfully used for the treatment of many blood disorders. However the limited number of cells capable of reconstitution that are available from sources such as umbilical cord blood is restrictive.

HSCs and progenitor populations can be expanded in vitro with cytokine cocktails but this leads to partial differentiation into more mature cells, reducing their potential to home and engraft into the bone marrow space and self-renew. To improve the survival and expansion of progenitors in vitro and hence increase the percentage of chimaerism in vivo after injection, we designed integration-deficient lentiviral vectors (IDLV) to express genes involved in HSC self-renewal, proliferation and maintenance of multipotency. As IDLV-delivered genes dilute out of dividing populations, expression is transient and could promote short-term changes in cells whilst avoiding insertional mutagenesis and undesirable constitutive over-expression of the transgene.

The homeobox family member HOXB4 is a transcription factor involved in HSC homeostasis. ANGPTL3 is a glycoprotein structurally similar to the angiopoietins recently described to stimulate expansion of long-term repopulating HSCs. To determine whether transient expression of HOXB4 or Angptl3 benefits self-renewal, proliferation and survival of progenitors, these genes were delivered to Lin-, Sca1+, c-Kit+ bone marrow cells with IDLV and tested by in vitro clonogenic assays. We have also performed in vivo competitive repopulation experiments in mice to determine the engraftment capacity of the transiently-transduced progenitors. Preliminary results suggest that expression levels of HOXB4 from IDLV is lower than from integrating vectors and that optimisation is required to improve the dose-dependent effect of this gene.

m.alonso-ferrero@ucl.ac.uk

Alonso-Ferrero Maria E 1 Langford-Smith Alexander 2 Burke Derek 3 Heales Simon 3 Bigger Brian W 2 Thrasher Adrian 1 Gaspar Bobby 1

1 Molecular Immunology Unit, Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH, UK 2 Stem Cell and Neurotherapies, School of Biomedicine, Faculty of Medical and Human Sciences, University of Manchester, Manchester, M13 9PT, UK 3 Clinical & Molecular Genetics, Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH, UK P 021 Gene Therapy for Lysosomal Storage Diseases using an enhanced lentiviral vector delivery system

LSDs arise from specific enzyme defects which lead to the accumulation of toxic storage compounds that affect normal cellular function in different organ systems both peripherally and in the brain. The key to effective disease correction is dependent on successful delivery of enzyme to both the CNS and peripheral organs but current therapeutic strategies have a limited effect on CNS (ERT) or don't provide sufficiently high enough enzyme levels for disease correction (allogeneic HSCT).

Gene therapy of autologous HSCs has been shown in recent pre-clinical studies to provide supra-physiological levels of enzyme and CNS correction through gene modified myeloid cell microglial differentiation, but current vector designs are limited by their ability to correct both CNS and peripheral abnormalities.

We have developed lentiviral vectors expressing transgenes under the transcriptional control of the EFS promoter, which drives expression in all hematopoietic lineages, and which also encodes the essential elements of the locus control region of the β-globin gene thereby upregulating transgene expression specifically in erythroid cells.

With the aim of demonstrating the effectiveness of this vectors in LSDs such as mucopolysaccharoidosis type I, we carried out in vitro assays using these design expressing α-L-iduronidase. Our experiments have shown that these vectors are able to restore the enzymatic activity in patient cell lines and also provide enhanced levels of expression in erythroid lineage. To prove the effectiveness of these engineered erythrocytes as systemically enzyme releasers and evaluate the therapeutic effect of the corrected HSCs in CNS, we are carrying out in vivo studies in α-L-iduronidase deficient mice.

m.alonso-ferrero@ucl.ac.uk

Vandermeulen Gaëlle 1 Uyttenhove Catherine 2 de Plaen Etienne 2 van den Eynde Benoît 2 Preat Véronique 1

1 University of Louvain, Louvain Drug Research Institute, Pharmaceutics and Drug Delivery, 1200 Brussels, Belgium 2 Ludwig Institute for Cancer Research, Brussels Branch, 1200 Brussels, Belgium P 022 Generation of protective immune response against the P815 tumor by DNA electrotransfer

The mastocytoma P815 expresses several tumor antigens and is a relevant model to test different vaccination modalities. This study aimed to construct DNA vaccines expressing the mouse P1A tumor antigen and to generate a protective immune response against the P815 tumor by DNA electrotransfer. Several DNA vaccines expressing either the P1A tumor antigen or P1A specific epitopes were constructed and DBA/2 mice were immunized by injection of these plasmids into their tibial cranial muscles. Then, 8 square-wave electric pulses (200 V/cm 20 ms 2 Hz) were delivered through plate electrodes. Two boosts were similarly applied two and four weeks after the priming. The lymphocytes, isolated two weeks after the last boost, were cultured and lytic activity was measured in a chromium release assay. Three weeks later, mice were challenged with P1A expressing P815 cells and mice survival was daily evaluated. Highest CTL activity was observed when pVAX2-P1A plasmid was used for DNA electrotransfer and a moderate CTL response with plasmid encoding the LPYLGWLVF antigenic peptide (pVAX2-miniP1A). Surprisingly, co-delivery of IL-12 or delivery of plasmids encoding both antigen and immunostimulant sequences failed to improve the immune response. A 13 days tumor growth delay was obtained after challenge when mice were immunized with pVAX2-P1A and electrotransfer appears therefore as a promising delivery method for such vaccine.

gaelle.vandermeulen@uclouvain.be

Grimshaw Benjamin D 1 Pule Martin A 1 Linch David C 1

1 Pule Group, Research Department of Haematology, Cancer Institute, University College London, London, WC1E 6BT, UK P 023 Creating a 'null' T cell for use in adoptive immunotherapy

Adoptive immunotherapy with engineered T-cells is showing promise. However, this approach is limited by the requirement to generate autologous T-cells. A methodology which would allow the generation of universal T-cells would be highly desirable: We are developing an expression cassette which will (1) introduce a CAR, (2) a sort-suicide gene, (3) knock-down HLA, (4) knock-down or block TCR function and (5) inhibit recipient NK activity. First, we have compared a variety of methods to knockdown HLA-expression. We compared seven siRNAs designed against beta-2-microglobulin, Golgi retention sequences attached to scFvs targeted to beta-2-microglobulin: one scFv extracted from the BBM.1 hybridoma and two scFvs extracted from the BB7.7 hybridoma. Finally, we tested two viral proteins, US11 and ICP47. We found that ICP47 and US11 are the most effective and give knock-down of 88% and 91% of HLA expression respectively when tested in donor PBMCs. We have attempted similar strategies to block TCR expression. SEKDEL retention sequences attached to OKT3 and BMA031 only gave modest knockdown when tested in donor PBMCs. Introduction of CD3 components attached to the retention signals KKAA or the E19 adenovirus protein resulted in a slight knockdown by CD3epsilon. To address rejection of HLA negative cells by NK cells, HLA-G has been cloned and expressed in cell lines and donor PBMCs. Testing HLA knockdown in MLRs and testing genome-editing TALENs for TCR knockdown are the next steps to be approached. These are the first steps to create an “off-the-shelf” cellular therapeutic.

benjamin.grimshaw.10@cancer.ucl.ac.uk

Ingrao Dina D I 1 2 3 Galy Anne A G 1 2 3 Fenard David D F 1 2 3

1 Généthon, Evry, F-91002, France 2 INSERM U951, Généthon, Evry F-91002 France 3 University of Evry Val d'Essonne, Evry, France P 024 Adaptation of a FRET-based fusion assay to concomitantly study lentiviral vectors binding, fusion and transduction in hematopoietic progenitor CD34+ cells

Lentiviral vectors (LVs) are used for various gene transfer application, notably for hematopoietic gene therapy. Various envelope pseudotypes can be used although it is not entirely understood how cellular entry mechanisms contribute to cellular transduction. To study the fusion step of non replicative LVs with human hematopoietic target cells, we adapted a fluorescence resonance energy transfer (FRET)-based fusion assay, initially designed to study replicative HIV-1 virions (Cavrois et al., Nat.Biotech., 2002). The assay utilizes recombinant LVs containing beta-lactamase (BLAM)-Vpr chimeric proteins (BLAM-LVs) to measure fusion with target cells via delivery of BLAM-Vpr into the cytoplasm, which is detected by flow cytometry using the CCF2-AM substrate. We successfully generated high-titer, infectious recombinant BLAM-LVs pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) and encoding the surface marker deltaLNGFR (truncated form of low affinity nerve growth factor receptor) using 6 plasmids co-transfection. The adapted FRET fusion assay using this VSV-G BLAM-LV is specific, sensitive (detection of fusion events as low as 1%), vector dose-dependent, and shows that transduction correlates with fusion in HCT116 cells. This fusion assay was used to characterize the effect of new transduction enhancers of hCD34+ cells called Vectofusins. Furthermore, The LV binding step was studied by incubating hCD34+ cells at 4°C with LVs in presence of Vectofusins before the fusion engagement. Data have shown that Vectofusins enhance VSV-G binding and fusion with target cell membranes. In conclusion, this LV-based fusion assay is a powerful and versatile tool to concomitantly study LVs adhesion, fusion and transduction in primary cells.

dingrao@genethon.fr

Aitchison Katherine L 1 Rahim Ahad A 2 Hughes Derralynn 3 Kinnon Christine 1 Waddington Simon N 2 Howe Steven J 1

1 Molecular Immunology Unit, Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH, UK 2 Institute for Women's Health, UCL, 86–96 Chenies Mews, London, WC1E 6HX, UK 3 Lysosomal Storage Disorders Unit, Royal Free Hampstead, Pond Street, London, NW3 2QG, UK P 025 Developing lentiviral vectors for gene therapy of Type I Gaucher Disease

Type I Gaucher Disease (GD) is a lysosomal storage disorder, caused by a mutation in the glucocerebrosidase (GBA) gene, which is currently treated with fortnightly infusions of GBA enzyme to provide replacement therapy. The goal of this work is to develop a gene therapy for GD using lentiviral vectors carrying a functional copy of the GBA gene. We have created a variety of lentiviral vectors carrying the GBA gene including constructs which carry a version of GBA fused to the protein transduction domain of HIV-1 TAT. Here we present results showing that the constructs express functional copies of the GBA gene product and that fusion to the TAT domain has no effect on enzyme activity or expression levels. We show that the functional protein is expressed from both the wild type and fusion constructs. And that it is not only present within the transduced cell but is also secreted into the extracellular environment which provides evidence that these vectors may be suitable for cross-correction of neighbouring, untransduced cells. We also demonstrate preliminary work with bone marrow from GBA-deficient mice which demonstrates that the vectors are capable of correcting the enzyme deficiency present in this model. Together this work provides the first steps in developing a vector for gene therapy of Type I GD.

katherine.aitchison.10@ucl.ac.uk

Locanto Elisabetta 1 Hitchman Richard B 1 King Linda A 1

1 Oxford Brookes University, Gipsy Lane, Oxford, UK P 026 BacMam vector-mediated gene delivery for ischaemia reperfusion-injury

Virus vectors offer a unique way to deliver protective genes to organs to ameliorate disease conditions. The baculoviridae family comprise a unique group of insect-specific DNA viruses. Over a decade ago, baculoviruses were modified to contain mammalian cell-active promoters upstream of the target gene, now referred to as BacMam vectors. Their non-replicative nature and high safety profile in mammalian cells, low cytotoxicity and ability to accommodate multiple genes or large DNA inserts make them an attractive alternative to mammalian virus vectors such as Adenovirus. This BacMam system has evolved rapidly in recent years and is currently also being used for recombinant expression of therapeutic proteins in vitro. Organ transplantation is a field that is becoming increasingly relevant for gene therapy applications. Ischaemia reperfusion injury (IRI) occurs during organ transplantation and is associated with hypoxia, free radical formation and organ failure. Previous studies by other workers suggested the possibility to ameliorate the effects of IRI by introducing anit-oxdidant, anti-apoptotic and cytoprotective genes using virus vectors. In this study BacMam was tested as a novel vector for delivery of mitochondrial manganese superoxide dismutase (Mnsod), bcl-2 and heme-oxigenase 1 (ho-1) to ameliorate IRI in human kidney cells. An in vitro ischaemia model was developed and optimized in human kidney cells using Antimycin A, mitochondrial respiration inhibitor, in combination with a non-metabolizable 2-deoxyglucose and calcium ionophore. BacMam viruses containing a cytomegalovirus (CMV) immediate-early promoter upstream of targets gene were constructed and the protective effect of the target gene on IRI was investigated in vitro.

elisabetta.locanto-2010@brookes.ac.uk

Chan Lucas 1 Ma Yu-Qian 1 Krishnamurthy Pramila 1 Darling David 1 Giunti Giulia 1 Audit Muriel 1 Bhalla Joti 1 Ingram Wendy 1 Kordasti Shahram 1 Kizilors Aytug 1 Barber Linda 1 Mufti Ghulam J 1 Farzaneh Farzin 1

1 Rayne Cell Therapy Suite (MA/MS IMP), Department of Haematological Medicine, Guy's, King's and St. Thomas' School of Medicine, The Rayne Institute, 123 Coldharbour Lane, London, SE5 9NU, UK P 027 A lentiviral vector manufactured under GMP for a Phase I study in Acute Myeloid Leukaemia – manufacture summary, safety testing, application and post-administration safety-monitoring

RFUSIN2 is a third generation self-inactivating lentiviral vector expressing the immune modulators (CD80/IL-2); it is cleaved endogenously by the Golgi endonuclease – furin. RFUSIN2 was manufactured under GMP in a Grade A isolator environment by transient transfection of 293T cells using multi-layered cell factories and a total of 40 litres was produced. Concentration by centrifugation resulted in a total batch size of 1.5×1011 infectious units with an overall yield of 115%. Extensive safety characterization demonstrated the absence of RCL, partial recombinants, mobilisable vectors or evidence of secondary transfer.

RFUSIN2 transduced leukaemia blasts have been generated as a Whole Cell Vaccine for Acute Myeloid Leukaemia. To date, two patients have undergone vaccination with up to 10⁷ modified cells. We have not found evidence of any adverse events including immunological toxicity manifested as GvHD, over-expression of IL-2 and evidence of vector dissemination in patients' serum, blood or bone marrow samples.

lucas.chan@kcl.ac.uk

Zech Mathias 1 Velica Pedro 1 Holler Angelika 1 Chakraverty Ronjon 1 Stauss Hans 1

1 Department of Immunology, Rowland Hill Street, Royal Free Hospital, NW3 2PF, London P 028 Manipulating mTOR to enhance T cell mediated tumour immunity in vivo

T cell based strategies to treat cancer, especially redirecting specificity through TCR gene transfer, represent an effective therapeutic option. However, tumour associated immune suppression poses a limitation to this approach. One mechanism of inhibiting T cell responses in a tumour context is by depleting amino acids, especially Arginine or Tryptophan, through the action of Arginase I and/or IDO. This results in an inhibition of mTOR activation which is crucial for the integration of T cell activation signals. The aim of this work is to design a strategy to enhance mTOR signalling in T cells in an amino acid low environment. BW cells transduced with a key regulator of the mTORC1 pathway were able to activate the mTORC1 pathway under Arginine low conditions. The same strategy was applied to T cells to test their function and phenotype under Arginine low conditions. Additionally, the ability of these cells to engraft in vivo was tested and their CD62L/CD44 profile was evaluated. Results so far show that mTORC1 signalling can be increased in T cells. Activation of mTOR seems to support T cell engraftment and result in a CD62L-low/CD44-high T cell phenotype upon adoptive transfer. Future work will dissect in detail the phenotype and functional profile of adoptively transferred T cells in murine tumour models.

m.zech@ucl.ac.uk

Ward Natalie 1 Buckley Suzanne 1 Rahim Ahad 1 McVey John 2 Thrasher Adrian 3 Waddington Simon 1

1 Institute for Womens Health, UCL, 86–96 Chenies Mews, London, WC1E 6HX 2 Thrombosis Research Institute, Manresa Road, London, SW3 6LR 3 Institute of Child Health, 30 Guilford Street, UCL, London, WC1N 1EH P 029 Codon optimised FVIII constructs for treatment of haemophilia A

Haemophilia A is a compelling candidate for treatment with gene therapy as therapeutic benefit only requires a modest increase in the endogenous coagulation factor level, response to treatment can be easily monitored, and FVIII expression can be mediated by many cell types in vivo. B-domain deleted (BDD) FVIII protein retains full procoagulant function and is expressed at higher levels than wild type FVIII. However a partial deletion of the B-domain leaving an N-terminal 226 amino acid stretch (N6) has been previously reported to increase in vitro secretion of FVIII tenfold compared to BDD-FVIII.

Previously, our group has shown that expression of FVIII protein from a codon optimised cDNA sequence increases expression in comparison to the wild type sequence over 30-fold in vivo yielding over 200% normal human FVIII levels in neonatal mice after injection of a SIN lentiviral vector. A longstanding goal for treatment of haemophilia A using gene therapy is to maintain sustained production of FVIII in the absence of an immune response in adult mice. To achieve this we have produced an optimised vector system including our codon-optimised FVIII N6 cDNA sequence under control of the liver specific promoter LP1 to prevent off target expression and maximise expression in hepatocytes. Further to this, we have also included target sequences for the hematopoietic specific microRNA, miR-142-3p, to eliminate off target expression in antigen presenting cells. With this approach we hope to achieve stable long term expression of FVIII in adult FVIII knock-out mice.

natalie.ward@ucl.ac.uk

Rahim Ahad A 1 Wong Andrew M 2 Hofer Klemens 2 Buckley Suzanne M 1 Mukherji Srishti 1 Cooper Jonathan D 2 Waddington Simon N 1

1 Institute for Womens Health, UCL, 86–96 Chenies Mews, London, UK 2 Kings College London, Coldharbour Lane, London, UK P 030 Intravenous administration of AAV2/9 to fetal and neonatal mice leads to extensive transduction of visceral organs and intestinal epithelia

A number of systemic diseases affect a wide range of visceral organs. A gene therapy approach to treating such diseases would require a vector that efficiently transduces a wide range of organs.

We intravenously administered fetal (E15) and neonatal (P1) mice with AAV2/9 carrying the GFP reporter gene. 1 month after injection, the mice were examined for visceral organ transduction. Stereoscopic fluorescence microscopy revealed extensive transduction of a range of organs and tissue including the liver, spleen, kidney, heart, lungs, muscle, intestines and also evidence of bone. Immunohistology confirmed transduction and immunofluorescence and confocal microscopy identified specific cell types expressing GFP. Interestingly, intestinal epithelia were efficiently transduced and clearly expressed GFP. The relative concentration of GFP in the various organs was measured by ELISA and qPCR was conducted to quantify genome numbers.

AAV2/9 has already been shown to efficiently transduce the CNS following intravenous injection. This, in combination with our findings here, indicates that AAV2/9 may be a suitable vector for treating systemic diseases that affect both the CNS and the viscera.

a.rahim@ucl.ac.uk

Biferi M G 1 Roda M 1 Barkats M 1

1 Equipe Biothérapie des Maladies Neuromusculaires (Groupe « SNC/Motoneurone »); UPMC-CNRS-AIM-INSERM UMRS 974, Institut de Myologie, 105, Bd de l'Hôpital - 75013 Paris P 031 Amyotrophic Lateral Sclerosis gene therapy using recombinant AAV9 vectors to silence the mutant SOD1 gene

Amyotrophic Lateral Sclerosis (ALS) is an incurable neuromuscular disease characterized by progressive degeneration of MNs leading to muscle atrophy, paralysis and premature death. 20% of the familial ALS (fALS) forms are consequence of mutations in the Superoxide Dismutase1 (SOD1) gene. SOD1 mutations cause a toxic gain of function of the enzyme and mice overexpressing human mutated form of SOD1 resemble the human pathology (SOD1G93Amice).

RNA interference (RNAi) mediated silencing of SOD1 has arisen as a promising therapy for SOD1-linked fALS. The immediate challenge now facing this therapeutic strategy is the widespread delivery of the silencing instructions to the most pertinent target cells. Recently we have identified self-complementary (sc)-AAV9 as a vector of choice for systemic gene transfer to central nervous system cells, including MNs (Barkats. Patent PCT/EP2008/063297, 05-10-2007). This vector had a remarkable capacity to transduce both neurons and astrocytes in neonate and adult mice following intravenous delivery, suggesting efficient crossing of the mature BBB (Duque et al., 2009). Since scAAV9 transduces principal histotypes involved in ALS (MN, glia, muscle cells), we propose to combine the use of RNAi with the scAAV9 vector to treat SOD1-fALS mice. Specifically, we produced recombinant scAAV9 carrying the SOD1-shRNA or one CTL-shRNA. Each vector allowed co-cistronically expression of the shRNA and EGFP, regulated by CMV or PGK promoters.

In the next future, neonatal (PND1) (presymptomatic) and 90 day-old (onset of symptoms) SODG93A or control mice will be intravenously injected with these vectors to assess the effects of the treatment on disease onset and progression.

mg.biferi@institut-myologie.org

Acosta-Saltos Carolina 1 Gonitel Roman 2 Acosta-Saltos Alejandro 1 Chavda Seetal 3 Thrasher Adrian 2 Anderson Patrick 4 Raivich Gennadij 1

1 Institute for Women's Health, UCL, 86–96 Chenies Mews, London, WC1E 6HX 2 Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH 3 Institute of Ophthalmology, UCL, 11–43 Bath Street, London, EC1V 9EL 4 Cell and Developmental Biology, UCL, Anatomy Building, Gower Street, London, WC1 6BT P 032 Lentiviral GMCSF is neuroinflammatory and proregenerative

Disparity in regeneration after axonal injury between peripheral and central nervous system has been associated with differences in the mounted inflammatory response. Regeneration competent peripheral neurons avidly recruit microglia, central neurons do not. We have created non-integrating lentivirus (NILV) containing GMCSF and eGFP (GMCSF/eGFP/NILV), to stimulate microglia around the injured neurons of rat and mouse CNS, so as to mimic peripheral injury. Most of the transfected cells were neurons and astroglia, the microglia stayed eGFP-negative.

Titration studies in SpragueDawley rats revealed dose dependent microglial activation with titers of 10⁴/ml and 10⁵/ml producing moderate, and 10⁷/ml maximum effects, in terms of activation and appearance of brain resident phagocytes. In rats that experienced spinal cord injury and were transfected 10⁴/ml, 10^5.5/ml and 10⁷/ml GMCSF/eGFP/NILV or with the 10⁷/ml eGFP only/NILV (control), those treated with 10^5.5/ml revealed a particularly high number of axons close to the site of injury, as well as high density of neuronal end bulbs, compared with other titers or with the control virus.

To explore strain and gene involvement in the neuroinflammatory and regenerative response, outbred and inbred mice were injected with GMCSF/eGFP NILV (10⁷ TU/mL) at the striatum. The outbred CD1 mice showed a variable neuroinflammatory response. Amongst the inbred strains, 129/SVJ exhibited weak microglial activation, Balb/C moderate microglia activation that was widespread throughout the cortical hemisphere, and FVB the appearance of numerous local brain phagocytes and tissue damage. The results suggest that GMCSF overexpression may be double-edged sword promoting regeneration at moderate, but increasing damage at high dosage.

francia.acosta-saltos.09@ucl.ac.uk

Pantoglou Julia 1 Abdul-Razak Hayder H 1 Rocca Céline 1 Howe Stephen J 2 Gregory Philip D 3 Holmes Michael C 3 Kinnon Christine 2 Thrasher Adrian J 2 Yáñez-Muñoz Rafael J 1

1 School of Biological Sciences, Royal Holloway University of London, Egham, Surrey 2 Institute of Child Health, UCL, 30 Guilford Street, London, WC1N 1EH 3 Sangamo BioSciences Inc., Richmond, California, USA P 033 ^* Specificity of a zinc-finger nuclease targeting mouse Prkdc: preparation of samples for analysis of off-target cutting

Zinc-finger nucleases (ZFNs) are artificially engineered enzymes able to introduce a double strand break at a specific site in genomic DNA. Such ZFNs have many potential uses, including gene editing in gene therapy strategies. A major concern for clinical use of these enzymes is off-target cutting, which can mediate geno- and cytotoxicity. We have developed a model to study gene repair in the Prkdc scid mouse, affected by DNA-dependent protein kinase catalytic subunit (DNA-PKcs) deficiency. We produced a ZFN targeting Prkdc and demonstrated target cutting and gene correction. In the current study we have started to address potential off-target cutting by the Prkdc ZFN. The study is based on the introduction of INDELs (insertion/deletions) by the cellular DNA repair machinery when ZFN-induced double strand breaks are repaired by nonhomologous recombination (end-joining). The consensus target site for the Prkdc ZFN was determined by SELEX analysis. Subsequent bio-informatic analysis allowed the identification of the 20 most likely off-target sites for the ZFN in the mouse genome. We have PCR-amplified the ten most likely off-target sites from four different mouse scid samples: mock-treated fibroblasts and haematopoietic progenitors, and ZFN-treated fibroblasts and haematopoietic progenitors. The PCR products have been verified by standard DNA sequencing. We are now ready to study the specificity of the Prkdc ZFN by Survey or assay and deep sequencing of the off-target PCR products.

julia.pantoglou.2008@live.rhul.ac.uk

Djelti Fathia 1 Hudry Eloïse 1 Varin Jennifer 1 Bieche Ivan 1 Vidaud Michel 1 Aubourg Patrick 1 Cartier Nathalie 1

1 UMR745, Faculté des sciences Pharmaceutiques, Univerity Paris-Descartes, Paris P 034 In vivo inhibition of cholesterol-24-hydroxylase induces a sequential and progressive neuronal loss in hippocampus of C57BL/6 mice

The abnormalities of cholesterol metabolism are closely related to neurodegenerative disorders. The neurons cannot degrade cholesterol by themselves and blood-brain barrier restricts its export out of the brain. The major mechanism of cerebral cholesterol clearance involves the conversion of cholesterol into 24S-hydroxycholesterol by neuronal cholesterol 24-hydroxylase, an enzyme encoded by the CYP46A1 gene. 24S-hydroxycholesterol can freely cross the blood-brain barrier and is degraded in the liver. Intracerebral injection of AAV5 vector encoding CYP46A1 in a mouse model of Alzheimer disease (APP23) decrease amyloid pathology and improve cognitive function.

To further characterize in vivo the role of CYP, we studied the effect of cholesterol 24-hydroxylase gene down-regulation through RNA interference strategy.

The injection of AAV5-shCYP in the hippocampus of three months old C57BL/6 mice induces to a major inhibition of CYP46A1 expression and leads to a progressive neuronal loss which was associated with electrophysiological and lipid profiles abnormalities. The emergence of hippocampal lesions was accelerated in APP23 mice and was associated with 3-fold increase of Aß40/42 peptides.

These results confirm the crucial role and therapeutic possibilities of CYP46A1 in the hippocampus.

fathia.djelti@inserm.fr

Baoutina Anna 1 Coldham Thosapon 1 Fuller Brodie 1 Emslie Kerry R 1 Zheng Maolin 2 Alexander Ian E 2

1 National Measurement Institute, Bradfield Rd, West Lindfield, NSW, Australia, 2070 2 The Children's Hospital Westmead and Children's Medical research Institute, Locked Bag 4001, Westmead, NSW, Australia, 2145 P 035 Optimisation of protocols for vector detection in biodistribution and persistence studies in gene therapy and gene doping testing

Vector biodistribution and persistence studies are essential in the development of gene transfer medicine. To ensure protocols for vector detection that are used in these studies provide reliable and accurate data, they must be optimised and validated. We addressed several parameters that affect detection of gene therapy vectors and optimised protocols for their analysis in blood.

Using a model in vitro system based on the human erythropoietin gene, we developed several real-time PCR assays to target the complementary DNA for the gene. Each assay was optimised, validated and ranked according to sensitivity and specificity in detecting the transgene in a background of human genomic DNA. The ability of the assays to detect the transgene in nonviral (naked plasmid) or viral (recombinant adeno-associated virus) vectors was assessed. Additional steps in DNA sample preparation prior to PCR such as plasmid linearisation or viral vector lysis were tested. A large number of DNA extraction protocols were evaluated on small volumes of blood and a method selected which provided high, reproducible and preferential recovery of PCR-quality transgenic DNA from nonviral or viral vectors.

The adoption of the described optimal and validated protocols may facilitate generation of more informative and reliable results from vector biodistribution and clearance studies in gene therapy and more meaningful comparison of results from different laboratories. The developed methodology for improved vector detection is discussed with regard to its application in detecting gene transfer in gene doping in sport, a new form of misuse of gene transfer technology.

anna.baoutina@measurement.gov.au

Tanguy Yannick Y T 1 Marais Thibault T M 1 Roda Marianne M R 1 Astord Stéphanie S A 1 Besse Aurore A B 1 Barkats Martine M B 1

1 Biotherapies of Neuromuscular Disease Team (Group « CNS/Motoneuron »), UPMC-CNRS-AIM-INSERM UMRS 974, Institut de Myologie, 105, Bd de l'Hôpital - 75013 Paris P 036 Post-symptomatic scAAV9-mediated gene therapy in a murine model of severe SMA

Spinal muscular atrophy (SMA) is a neurodegenerative disorder characterised by the selective loss of α motor neurons of the spinal cord, leading to a progressive muscle weakness, which progresses to paralysis and ultimately death in the most severe cases. In 93% of SMA cases, the disease is caused by an homozygous deletion of the “Survival Motor Neuron” gene (Smn1). We and others recently demonstrated that intravenous (IV) injection of a self-complementary adeno-associated virus vector of serotype 9 (scAAV9), carrying an optimized human Smn1 expression cassette (SMNopti), could dramatically rescue SMNdelta7 mice, a model of SMA. These animals were injected just after birth, before the appearance of the clinical signs. We recently proposed to investigate the potential benefits of a post-symptomatic restoration of SMN expression in another mouse model of SMA (Smn-/- ; hSmn2+/-) which manifests the disease during embryonic development. We already demonstrated that postnatal IV and intracerebroventricular (ICV) injections of scAAV9-SMNopti significantly prolong lifespan of the SMA mice and improve the body weight loss phenotype. The analysis of post-symptomatic Smn1 gene therapy effect on neuromuscular junction size and functionality is in progress. As SMN is mainly involved in the biogenesis of snRNPs, we will also evaluate the recovery of the spinal motor neuron transcriptome using an approach of laser microdissection coupled with microarray analysis in scAAV9-SMNopti treated SMA mice. This experiment would allow to evidence mysregulation of crucial genes involved in SMA pathogenesis, and to possibly identify new therapeutic targets.

y.tanguy@institut-myologie.org

Touati Walid 1 Seguin Johanne 2 Gallian Claire 1 Baillou Claude 3 Lemoine François 3 Beaune Philippe 1 de Waziers Isabelle 1

1 INSERM, Unité Mixte de Recherche 775, Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, 45 rue des Saints-Pères, 75006 Paris, France 2 Unité de Pharmacologie chimique, génétique et Imagerie, INSERM U1022, CNRS UMR 8151, Faculté de Pharmacie, 4 avenue de l'observatoire, 75006 Paris, France 3 Unité Mixte de Recherche Centre National de la Recherche Scientifique 7211/INSERM U959, Groupe Hospitalier Pitié-Salpêtrière, 83 boulevard de l'Hôpital, 75013 Paris, France P 037 Sensitization of lung tumors cells to cyclophosphamide by gene therapy: from in vitro to in vivo

Gene-directed enzyme prodrug therapy (GDEPT) is an emerging strategy against cancer consisting in selective delivery to the tumor of a gene able to metabolize a prodrug into cytotoxic metabolites. Our approach consisted of using cyclophosphamide (CPA) as a prodrug and a modified cytochrome P450 2B6 (CYP2B6TM) / NADPH cytochrome P450 reductase (RED) fusion gene as produg-activating gene, vectorized in VSV-G pseudotyped lentivirus. Given the relatively low affinity of CYP2B6 for CPA (Km=4 to 5 mM), we have modified CYP2B6 by site directed mutagenesis, to increasing by 10 fold its catalytic efficiency to metabolize CPA into cytotoxic metabolite (CYP2B6TM). Due to heterogenous expression of RED in tumor cells, a fusion gene (CYP2B6TM-RED) was constructed allowing the expression of both proteins at high levels in tumor cells. Human pulmonary cancer cell line (A549) and mouse pulmonary cell line (TC1), both resistant to CPA, were infected by a lentivirus expressing CYP2B6TM-RED. Expression of the fusion gene CYP2B6TM-RED allowed a sensitization of both cell lines to low doses of CPA (IC50 about 0,5 mM).

First results in C57 Bl/6 mice sub-cutaneously injected with TC1 cells expressing CYP2B6TM-RED showed a rapid regression of the tumor, 48 hours after CPA injection (140 mg/Kg, ip). In control mice injected with TC1 cells without fusion gene expression, CPA was without any effect.

walid.touati@parisdescartes.fr

Abdul-Razak Hayder H 1 Rocca Céline 1 Howe Steve J 2 Estévez Javier F Molina 1 Gan Chih Hao Victor 1 Roberts Alison 3 Gregory Philip D 4 Holmes Michael C 4 Kinnon Christine 2 Thrasher Adrian J 2 Yáñez-Muñoz Rafael J 1

1 School of Biological Sciences, Royal Holloway University of London, Egham, TW20 0EX, UK 2 Institute of Child Health, UCL, 30 Guildford St, London, WC1N 1EH, UK 3 Department of Medical and Molecular Genetics, King's College London, UK 4 Sangamo BioSciences Inc., Richmond, California, 94804, USA P 038 Correction of mouse Prkdc scid deficiency by ZFN-driven gene targeting

An improved approach to treat both dominant and recessive genetic disorders would be to repair the mutant gene by homologous recombination-mediated gene targeting. Recently, gene targeting frequencies have dramatically increased through the development of (i) efficient DNA delivery tools, including integration-deficient lentiviral vectors (IDLVs); and (ii) designer nucleases, able to induce specific double-strand breaks at their target locus. To demonstrate therapeutic gene repair in a pre-clinical model of immunodeficiency we have developed an ex vivo system to correct the classical scid mouse, a model of human DNA-dependent protein kinase catalytic subunit (DNA-PKcs, PRKDC) deficiency. We have produced donor templates to correct the mutation, obtained and optimised a zinc finger nuclease (ZFN) that targets mouse Prkdc close to the scid mutation, and incorporated ZFN genes and donor templates into IDLVs. We have demonstrated specific ZFN activity in scid fibroblasts and haematopoietic progenitors using a Cel-I assay, which detects modifications introduced at the target site upon repair by nonhomologous end-joining. We have observed ZFN-mediated gene targeting of the scid mutation via the incorporation of a diagnostic restriction site from the donor template into the targeted locus. In fibroblasts, we have shown rescue of DNA-PKcs activity and increased resistance to DNA damage upon gene correction. In haematopoietic progenitors we have detected gene correction when the ZFN genes are delivered by integrating lentivectors, and we are currently optimising IDLV-mediated gene targeting.

hayder.abdul-razak.2008@live.rhul.ac.uk

Tordo Julie 1 Eckenfelder Agathe 2 Goyenvalle Aurélie 3 Danos Olivier 1

1 UCL Cancer Institute, UCL, London, WC1E 6BT, UK 2 Inserm U845, Hôpital Necker-Enfants Malades, Faculté de Médecine, Paris 75015, France 3 MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3QX, UK P 039 Optimization of snRNA cassettes for the expression of therapeutic antisense sequences

Synthetic antisense oligoribonucleotides (AONs) can be used to modulate gene splicing by masking motifs on the pre-mRNA required for spliceosome assembly. Transfected AONs display a transient activity whereas stable expression is obtained using chimeric small RNAs containing the antisense sequences, which are delivered by gene transfer vectors. Here we try to maximize the amount of active antisense sequences delivered by creating new and tissue-specific expression cassettes for these small RNA shuttles.

Stable delivery of antisense sequences can be obtained with modified snRNAs such as U7, expressed from viral vectors. In the murine model of Duchenne Muscular Dystrophy a chimeric U7 snRNA (U7Dtex23) induces skipping of the mutated exon 23 and restores the Dystrophin mRNA reading frame. 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 study we used the muscle-specific enhancer MHCK7, a synthetic fusion between the muscle creatin kinase (MCK) and the α-myosin heavy chain (α-MHC) enhancers, to drive the expression of the U7Dtex23 cassette. AAV vectors were tested in myoblast cultures and in vivo on mdx mice and our results show that the enhancer improves chimeric U7snRNA expression and subsequently increases dystrophin exon 23 skipping both in vitro and in vivo.

j.tordo@ucl.ac.uk

BSGT 2012 Abstract Author Index

Abdul-Razak, HH, P 033, P 038

Acosta-Saltos, A, P 032

Acosta-Saltos, C, P 032

Aherne, S, P 008

Ahouansou, O, Or 08

Aitchison, KL, P 025

Alexander, IE, P 035

Alonso-Ferrero, ME, Or 02, P 012, P 020, P 021

Alton, EWFW, Or 05, P 015

Anderson, P, P 032

Anjos-Afonso, F, Or 01

Antoniou, M, P 006, P 012

Arumugam, P, Or 02

Astord, SSA, P 036

Athanasopoulos, T, Or 13

Aubourg, P, Or 08, P 034

Audit, M, P 027

Baillou, C, P 037

Baoutina, A, P 035

Barber, L, P 027

Barkats, M, P 031

Barkats, MMB, P 036

Barron, N, P 008

Bartolovic, K, P 020

Baum, C, Or 02

Beaune, P, P 037

Beltrami, AP, Or 10

Bendle, GM, Or 04

Bennett, C, Or 09

Besse, AAB, P 036

Bhalla, J, P 027

Biagi, E, Or 01

Bièche, I, Or 08

Bieche, I, P 034

Bies, L, Or 04

Biferi, MG, P 031

Bigger, BW, P 021

Biondi, A, Or 01

Blundell, M, P 003

Blundell, MP, P 012

Bonnet, D, Or 01

Booth, C, P 003

Boyd, AC, P 015

Boza, MG, P 007

Boza-Morán, MG, P 014

Broadstock, M, P 009

Buckley, S, P 029

Buckley, SM, P 030

Buckley, SMK, P 013

Burke, D, P 021

Carmo, M, Or 02, P 012

Carr, B, P 004

Cartier, N, Inv 03, Or 08, P 034

Carzaniga, R, Or 05

Cathomen, T, Or 12

Cedrone, F, Or 11

Chakraverty, R, Or 09, P 028

Chan, L, P 027

Charton, K, Or 03

Chavda, S, P 032

Cheng, SH, Inv 01, Or 05, P 015

Clarke, C, P 008

Clynes, M, P 008

Coldham, T, P 035

Collie, DD, P 015

Collins, M, P 006, P 012

Cooper, JD, P 030

Corbineau, S, P 019

Daniele, N, Or 03

Dannemann, N, Or 12

Danos, O, P 039

Darling, D, P 027

Davies, JC, P 015

de Plaen, E, P 022

de Waziers, I, P 037

DeCoppi, P, P 002

Delhove, JMKM, P 013

Dewar, A, Or 05

Di, W, P 005

Dickson, G, Or 11, Or 13, P 017

Djelti, F, P 034

Doolan, P, P 008

Dubart-Kupperschmitt, A, P 019

Duclert, A, Or 11

Eckenfelder, A, P 039

Emslie, KR, P 035

Estévez, JFM, P 038

Fairbanks, L, P 012

Fallah-Arani, F, Or 09

Farzaneh, F, P 027

Fenard, DDF, P 024

Ferrer, I, Or 08

Field, A-C, P 010

Finney, H, Or 01

Flutter, B, Or 09

Foster, H, P 017

Foster, K, Or 13, P 017

Fouquet, F, Or 08

Fourcade, S, Or 08

Fuller, B, P 035

Gallagher, C, P 008

Gallian, C, P 037

Galy, AAG, P 024

Gan, CHV, P 038

Gaspar, B, Or 02, P 021

Gaspar, HB, P 003, P 012

Georgiadis, C, P 005

Ghorashian, S, Or 09

Gicquel, E, Or 03

Gill, DR, P 015

Giunti, G, P 027

Gonitel, R, P 032

Goyenvalle, A, P 039

Gregory, PD, P 033, P 038

Griesenbach, U, Or 05, P 015

Grimshaw, BD, P 023

Gubernator, M, Or 07, Or 10

Guidoux-Boralévi, S, Or 08

Hannell, C, P 004

Hart, SL, P 011

Heales, S, P 021

Higgins, TE, P 015

Hitchman, RB, P 026

Hofer, K, P 030

Hole, P, P 004

Holler, A, Or 09, P 028

Holmes, MC, P 033, P 038

Howe, S, Or 12

Howe, SJ, P 011, P 018, P 020, P 025, P 033, P 038

Hudry, E, P 034

Hughes, D, P 025

Hutchinson, L, P 014

Hyde, SC, P 015

Ingram, W, P 027

Ingrao, DDI, P 024

Innes, JA, P 015

Jarmin, S, P 017

Jathoul, AP, P 013

Jordan, MB, Or 02

Katare, R, Or 07, Or 10

King, LA, P 026

Kinnon, C, Or 12, P 020, P 025, P 033, P 038

Kizilors, A, P 027

Knight, S, P 006, P 012

Koo, T, Or 11

Kordasti, S, P 027

Krishnamurthy, P, P 027

Kymäläinen, H, Or 11

L'Homme, B, Or 08

Langford-Smith, A, P 016, P 021

Langford-Smith, KJ, P 016

Lao, N, P 008

Lassailly, F, Or 01

Laurendeau, I, Or 08

Leclerc, X, Or 11

Lemoine, F, P 037

Linch, DC, P 023

Linnemann, C, Or 04

Locanto, E, P 026

Lu-Nguyen, NB, P 009

Ma, Y-Q, P 027

Madeddu, P, Or 07, Or 10

Malik, P, Or 02

Malloy, A, P 004

Marais, TTM, P 036

Marin, V, Or 01

Mata, M, P 020

McGrath, J, P 005

McKay, TR, P 013

McLachlan, G, P 015

McVey, J, P 029

Mitchell, K, Or 07

Mitchell, KJ, Or 10

Monkeviciute, A, P 012

Montiel-Equihua, CA, Or 02, P 012

Morris, E, Or 09

Morris, EC, P 010

Mouly, V, Or 11

Mufti, GJ, P 027

Mukherjee, S, Or 06, P 002

Mukherji, S, P 030

Munkonge, FM, Or 05

Munye, MM, P 011

Naldini, L, Or 12

Pallant, C, Or 12

Pantoglou, J, P 033

Pape, T, Or 05

Paques, F, Or 11

Pesce, M, Or 07

Pipino, C, P 002

Pizzitola, I, Or 01

Popplewell, L, Or 11

Porteous, DJ, P 015

Portero-Otin, M, Or 08

Prandi, F, Or 07

Preat, V, Or 13, P 022

Pucéat, M, P 019

Pujol, A, Or 08

Pule, M, Or 01

Pule, MA, P 013, P 023

Qasim, W, Or 12, P 005, P 010

Rahim, A, P 029

Rahim, AA, P 025, P 030

Raivich, G, P 032

Richard, I, Or 03

Risma, K, Or 02

Riu, F, Or 07, Or 10

Rivat, C, P 003

Roberts, A, P 038

Rocca, C, P 033, P 038

Roda, M, P 031

Roda, MMR, P 036

Rogers, A, Or 05

Ruffert, K, Or 08

Saadeh, H, P 012

Sanchez, N, P 008

Schambach, A, Or 02

Scheule, RK, P 015

Schmidt, M, P 012

Scholz, S, P 012

Schulz, R, P 012

Schumacher, TN, Or 04

Seguin, J, P 037

Semenova, K, P 005

Sergijenko, A, P 016

Singh, C, Or 05

Siupa, A, P 004

Sivakumaran, J, Or 09

Smart, N, Inv 02

Smith, SN, Or 05

Song, J-Y, Or 04

Spinetti, G, Or 07

Stauss, H, P 028

Stauss, HJ, Or 09, P 010

Steichen, C, P 019

Sung, P, Or 06, P 002

Takeuchi, Y, P 006, P 012

Tanguy, YYT, P 036

Thrasher, A, Or 12, P 002, P 021, P 029, P 032

Thrasher, AJ, Or 02, Or 06, P 003, P 006, P 012, P 033, P 038

Til, NP Van, P 020

Tizzano, E, P 007

Tordo, J, P 039

Touati, W, P 037

Trollet, C, P 017

Trundley, A, Or 13

Uyttenhove, C, P 022

van den Eynde, B, P 022

Vandermeulen, G, Or 13, P 022

Varin, J, P 034

Velica, P, P 028

Vidaud, M, P 034

Vink, CA, P 018

Voit, T, Or 11

Waddington, S, P 029

Waddington, SN, P 013, P 025, P 030

Wanisch, K, P 007

Ward, N, P 029

Ward, NJ, P 013

Weber, A, P 019

Wong, AM, P 030

Wynn, RF, P 016

Xue, S-A, Or 09

Yáñez-Muñoz, RJ, Or 11, Or 13, P 007, P 009, P 014, P 033, P 038

Zech, M, P 028

Zhang, F, P 006

Zhang, L, P 012

Zheng, M, P 035