British Society for Gene and Cell Therapy Annual Conference University College London Institute of Child Health Friday 15 th April 2016 Conference Abstracts

Session 2. Recent advances in pre‐clinical knowledge, April 15, 2016, 13:15 – 15:15

Mammalian retinas contain two primary types of photoreceptors, cones and rods. The differences between the two cell types are ideal to allow vision under a broad range of light conditions. However, loss of one cell type can severely affect vision. This is particularly true for degeneration of cones, which leaves the retina with slower rod photoreceptors. By using optogenetic tools, we investigated how the two types of photoreceptors interact with each other and whether it is possible to devise optogenetic strategies to improve vision in degenerating retinas.

INV05 Developing an AAV Pipeline and Building a Successful Biotech

Jenkins Annalisa Dr 1

1 Dimension Therapeutics, Cambridge, United States

Session 2. Recent advances in pre‐clinical knowledge, April 15, 2016, 13:15 – 15:15

Biography: Annalisa Jenkins, CEO, Dimension Therapeutics, a gene therapy company developing novel therapies for rare diseases associated with the liver. She has spent 20 years in the biopharmaceutical industry, starting with a career in medicine, a stint in the British Royal Navy, and successful turns in pharma at Bristol Myers Squibb.

Annalisa Jenkins is CEO of Dimension Therapeutics, a gene therapy company developing novel therapies for rare diseases associated with the liver. She has spent approximately 20 years in the biopharmaceutical industry, starting with a career in medicine, a stint in the British Royal Navy, and successful turns in pharma at Bristol Myers Squibb, where she built and led teams across global development and medical affairs for 15 years, and most recently leading global research and development at Merck Serono.

Over the next 10–15 years, the model of translating science to medicines and delivering novel therapeutics to patients globally will continue to evolve in order to deliver meaningful clinical benefit from diverse and innovative pipelines. Using gene therapy as an example, Dr. Jenkins will highlight the fundamental differences between, and the requirement for both, academic research and industry‐sponsored drug development.

Gene therapy recently began its second renaissance, founded on 30+ years of innovation and academic funding. While this first phase of research remains a critical component of successful gene therapy discovery, building medicines requires expertise that often resides outside academia. Dr. Jenkins will share her experiences at Dimension, where she leads efforts to bring breadth and depth to their pipeline by building out the best team of internal capabilities with important partnerships across academia, CROs, and CMOs. From start‐up biotech to large R and D models of pharma, Dr. Jenkins will highlight the importance of building collaborative communities with a shared vision and purpose, keeping patient and science at the center.

INV06 AAV vectors: making something good even better

Kay Mark Professor

Session 2. Recent advances in pre‐clinical knowledge, April 15, 2016, 13:15 – 15:15

Recombinant AAV vectors (rAAV) have provided some successes in early clinical trials. However, when moving from animals to humans a number of unanticipated responses have occurred that resulted in lower than expected efficacy. Based on our published (Lisowski et al., Nature 2014) and more recent unpublished studies, we propose that murine‐human xenotransplant models for muscle and liver represent a robust animal model that may more closely predict clinical trial outcomes. Moreover, such models in combination with multi‐species DNAse‐shuffled AAV capsid libraries have allowed us to select for AAV vectors that show at least 10 times enhanced transduction of human muscle and liver. Current screens for rAAV vectors that show selective transduction of Hepatitis B virus infected cells and increased DNA carrying capacity are ongoing. The episomal nature of the vector genomes in transduced cells restrict classical rAAV‐mediated gene transfer to quiescent tissues. Moreover, even with the low rate of AAV integration, high rates of hepatocellular carcinoma resulting from promoter activation of oncogenic loci in young mice, and loss of episomal AAV genomes (and hence transgene expression) with normal growth and development has raised concerns about treating infants. To circumvent these concerns and provide a viable approach to treating infants, we have developed an AAV promoterless gene targeting approach without the use of nucleases (Barzel et al Nature 2015) and use this tactic to successfully treat mice with hemophilia B. We are now expanding this approach for treating a variety of diseases as well as developing novel methods for increasing gene targeted‐mediated transgene expression. The discovery and characterization of novel AAV vectors as well as their use in both classical genetransfer and genome‐editing approaches broadens their application in both biological discovery and therapeutic applications.

INV07 Liver‐Targeted Gene Therapy using rAAV

Alexander Ian Professor 1

1 Sydney Children's Hospitals Network and Children's Medical Research Institute, Westmead, Australia 2 University of Sydney Medical School, Sydney, Australia 3 University College London, London, Great Britain

Session 3. Clinical experiences and future vision for AAV therapies, April 15, 2016, 15:35 – 17:05

Biography: Professor Alexander is a physician/scientist within the Sydney Children's Hospitals Network and Children's Medical Research Institute, Australia. He is a senior staff specialist in genetic metabolic disease and leads a research group focused on virus‐mediated gene transfer to the liver and haematopoietic compartment and translation through to human clinical trials.

The liver is an immensely attractive target for gene therapy as mastery of the biological and technological challenges involved opens the prospect of treating a broad spectrum of organ‐specific and systemic disease phenotypes, both inherited and acquired. Among contemporary vector systems, those based on recombinant AAV offer the most immediate promise for liver targeted therapy, with the added advantage that the system can be exploited for both gene addition and genome editing, alone or in combination. The successful treatment of haemophilia B (FIX deficiency) in adult patients following systemic delivery of AAV2/8 has stimulated immense interest in the AAV system and intensive efforts to further refine the system for other indications. This remains a considerable challenge for a number of reasons. Therapeutic levels of FIX can be achieved with a very low percentage of hepatocytes transduced, provided a sufficiently strong promoter is used to drive FIX expression. By contrast many cell autonomous liver disease phenotypes, such as urea cycle defects, will require that a considerably higher percentage of hepatocytes be transduced, and there is a pressing need to develop and test novel AAV capsids with increased tropism for the human liver. Inter‐species differences in capsid tropism complicate this process and uncertainty remains about the best preclinical models to predict performance in the human liver. Yet another challenge in the context of gene addition approaches is the clearance of episomal AAV genomes in concert with hepatocellular proliferation. This appears unlikely to pose a major challenge in the adult liver provided the initial level of transduction is sufficiently high, but is likely to be a very important consideration in the paediatric liver, particularly in infancy. Conversely, genome editing approaches exploiting AAV‐mediated homologous recombination (HR) may actually be more efficient in the paediatric liver as resolution of Holliday junctions are thought to be favoured by passage through mitosis. Even in the highly proliferative infant liver it is unlikely that the basal rates of HR achievable will be sufficient to treat the majority of theoretically amenable liver disease phenotypes. Here the addition of user‐designed nucleases is likely to be required in combination with AAV‐mediated HR template delivery, and early small animal data using this combinatorial approach is highly encouraging. At present, however, this necessitates the use of two different AAV vector constructs and again emphasises the need to further develop highly liver tropic AAV capsids. In summary, the AAV system is on the cusp of delivering therapeutic benefit in an increasing spectrum of liver disease phenotypes and is uniquely placed to play a central role in ushering in the exciting era of therapeutic genome editing in vivo.

INV08 S100A1 and its therapeutic use for heart failure

Most Patrick Professor 1

1 uniQure GmbH, Heidelberg, Germany

Session 3. Clinical experiences and future vision for AAV therapies, April 15, 2016, 15:35 – 17:05

Biography: Prof. Patrick Most, MD, Managing Director of uniQureGmbH, is Co‐founder of InoCardGmbH and Full Professor for Molecular and Translational Cardiology at Department of Internal Medicine III of University of Heidelberg. He developed uniQure's S100A1 lead program for human heart failure treatment and is a key opinion leader in cardiovascular medicine.

o Professor for Molecular and Translational Cardiology, Im Neuenheimer Feld 410, Heidelberg University Hospital, Heidelberg, Germany

o Managing Director, uniQure GmbH Germany, Im Neuenheimer Feld 410, Heidelberg University Hospital, Heidelberg, Germany

This paper aims to discuss development and translation of molecular therapies against cardiac dysfunction that harness signaling mechanisms of the EF‐hand calcium sensor protein S100A1 in the heart. Originating from its physiological role in cardiac performance regulation and almost exclusive abundance in ventricular cardiomyocytes, the pathophysiological relevance of abnormal S100A1 expression and therapeutic targeted reconstitution for cardiac systolic and diastolic function in cellular and animal models will be highlighted. A focus will be on molecular mechanisms how S100A1 controls a calcium‐driven network in cardiac muscle that regulates contractile performance, muscle growth, and energy homeostasis and rhythm stability.

INV09 Gene Therapy for Heart Failure: The Road Ahead

Hajjar Roger Professor 1

1 Icahn School of Medicine at Mount Sinai, One Gustave Levy Place, United States

Session 3. Clinical experiences and future vision for AAV therapies, April 15, 2016, 15:35 – 17:05

Biography: Dr Hajjar is the Director of the Cardiovascular Research Center, and holds the Arthur & Janet C. Ross Professor of Medicine at Mount Sinai School of Medicine, New York, NY. He received his Bachelor of Science degree in Biomedical Engineering from Johns Hopkins University and his MD from Harvard Medical School and the Harvard‐MIT Division of Health Sciences & Technology. He completed his training in internal medicine, cardiology and research fellowships at Massachusetts General Hospital in Boston. Dr. Hajjar is a scientific leader in the field of cardiac gene therapy.

Congestive heart failure remains a progressive disease with a desperate need for innovative therapies to reverse the course of ventricular dysfunction. A number of genetic mutations have also been associated with cardiomyopathies resulting in heart failure. Recently, Adeno‐Associated Virus (AAV)‐based vectors have emerged as promising gene delivery vehicles for a wide array of diseases, including cardiovascular disorders. Despite early encouraging results, the CUPID (Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease) trial using an AAV serotype 1 vector encoding the sarcoplasmatic calcium ATPase SERCA2a failed to meet both its primary and secondary endpoints. Preliminary results suggest that the disappointing outcome was due to a failure of AAV1.SERCA2a to deliver efficiently the SERCA2a gene. Novel vectors with altered capsids that escape antecedent neutralizing antibodies and de‐target the liver while maintaining cardiac tropism have been developed. The engineered vector AAV2i8 combined with a constitutively active form of protein phosphatase inhibitor 1 will be used in a new clinical trial in patients with advanced heart failure.

INV10 Clinical gene therapy for choroideremia

Maclaren Robert Professor 1

1 University of Oxford, Oxford, United Kingdom

Session 3. Clinical experiences and future vision for AAV therapies, April 15, 2016, 15:35 – 17:05

Biography: Robert MacLaren is Professor of Ophthalmology at the University of Oxford. He is currently leading clinical trials to treat choroideremia which involve several international sites. He is the founding director of Nightstarx, a gene therapy company based at the Wellcome Trust specialising in developing AAV gene therapy for retinal degenerations.

Recently the long‐term sustainability of AAV retinal gene therapy has been questioned with the results from two trials on Leber congenital amaurosis caused by deficiencies of the RPE65 gene that showed limited effects which were not sustained. We therefore sought to determine the long‐term outcome of AAV gene therapy used to treat choroideremia, which is another retinal degeneration that requires sustained transgene expression in the retinal pigment epithelium.

Six patients were recruited into a phase 1 clinical trial using an AAV2 vector expressing the choroideremia gene sequence driven by the CBA promoter and with a downstream WPRE. Patients (P) 1–5 received 1e10 genome particles (gp) whilst P6 received 6 ×10⁹ gp. The initial results from the study at six months were previously reported (NCT01461213).

At most recent follow‐up 3.5–4 years after the initial gene therapy treatment, the visual acuity improvements seen at 6 months were sustained and degeneration in the untreated fellow eyes during this period led to the treated eyes becoming dominant in all patients receiving the higher dose. P6 who had the lowest dose had a steady decline in visual acuity in both eyes.

The effects of retinal gene therapy for choroideremia appear to be sustained for at least 4 years, assuming the correct dose is applied. Patients with visual acuity close to the physiological maximum at baseline maintained it after gene therapy, despite loss in vision being detected in their unoperated control eyes during this period. Several years may be required to confirm the beneficial effects of AAV gene therapy in chronic retinal degenerations.

Fairbairn Award Nominations

FB01 Dissecting immune responses to HAdV‐5 in wild type and immunocompromised mouse serum reveals a new mechanism of transduction via the coxsackie and adenovirus receptor

Gordo Estrella Lopez Miss 1 Coughlan Lynda Dr 2 Bradshaw Angela C. Dr 1 White Katie M. Dr 1 Denby Laura Dr 3 Nicklin Stuart A. Dr 1 Baker Andrew H. Professor 3

1 Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom 2 Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom 3 Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom

Session 1. Cutting edge vector technology and immunological challenges, April 15, 2016, 10:00 – 11:45

Following intravascular delivery coagulation factor X (FX) interacts with HAdV‐5 hexon mediating liver transduction and protection from immune attack. FX is dispensable for liver transduction in immunocompromised mice.

Here, we investigated the mechanisms for HAdV‐5 immune neutralization in vivo and in vitro, and identified novel pathways mediating HAdV‐5 FX‐independent transduction in vitro.

While intravascular administration of a non‐FX binding HAdV‐5 (AdT*) to C57BL/6 mice produced 6800‐fold (P < 0.05) lower liver transduction than HAdV‐5, it was equivalent in NSG mice, which lack innate and adaptive immunity. Interestingly, AdT* transduction was 23‐fold lower (P < 0.05) in Rag‐2^−/− mice liver, only lacking adaptive immunity. To dissect the underlying mechanisms of AdV neutralization further, in vitro adenoviral transduction was assessed in the presence of C57BL/6 or Rag‐2^−/− serum +/− X‐bp, a FX:HAdV‐5 hexon interaction inhibitor. While C57BL/6 serum inhibited AdT* and HAdV‐5 transduction in the presence of X‐bp, transduction was not inhibited by Rag‐2^−/− serum but, instead, it was increased by 3‐fold (P < 0.05) in high coxsackie and adenovirus receptor (CAR)‐expressing cells (CARhigh). Blockade of CAR with soluble HAdV‐5 fiber knob inhibited mouse serum‐enhanced transduction of CARhigh cells. However, HAdV‐5KO1 (non‐CAR‐binding) transduction with Rag‐2^−/− serum was equivalent to that of HAdV‐5, indicating that HAdV‐5 fiber:CAR interaction is dispensable.

These data suggest that while HAdV‐5 neutralization in vitro requires adaptive immunity, innate immunity is sufficient in vivo. Furthermore, a FX‐independent HAdV‐5 transduction pathway exists mediated via a mouse serum protein that bridges HAdV‐5 to CAR. These findings are important for the development of HAdV‐5 vectors for gene therapy.

FB02 The adenoviral AdΔΔ mutant enhances mitoxantrone‐induced cell death by promoting apoptosis and attenuating autophagy in prostate cancer cells.

Hernandez Carmen Aguirre Ms 1 Halldén Gunnel Dr. 1

1 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, United Kingdom

Session 1. Cutting edge vector technology and immunological challenges, April 15, 2016, 10:00 – 11:45

Prostate cancer (PCa) is the second cancer killer in men globally due to the rapid development of resistance to all available therapies. Late‐stage metastatic disease is incurable. In response to cytotoxic drugs autophagy is frequently induced to rescue cancer cells from the toxic agent and may contribute to the development of drug‐resistance. We previously demonstrated that the oncolytic adenoviral mutant AdΔΔ, in combination with cytotoxic drugs, effectively kills resistant prostate cancer cells but not normal cells. AdΔΔ is deleted in the pRb‐binding E1ACR2‐region and in the anti‐apoptotic E1B19K‐gene for tumour selectivity and apoptosis induction, respectively. AdΔΔ in combination with drugs currently used clinically to treat PCa, mitoxantrone or docetaxel, causes synergistically enhanced cell killing by increasing apoptosis. Here, we investigated whether the increased apoptosis was dependent on attenuation of drug‐induced autophagy.

We found that mitoxantrone and docetaxel potently stimulated the initiation of autophagy by promoting the conversion of LC3I to LC3II in both 22Rv1 and PC3 cells. Completion of autophagy, determined by p62 degradation, was only significant in 22Rv1 cells. Simultaneous infection with AdΔΔ promoted apoptotic cell death while preventing LC3I to LC3II conversion and p62 degradation but did not affect basal autophagy. Addition of the autophagy inhibitor chloroquine further increased the apoptotic cell killing while the autophagy inducer rapamycin decreased cell killing. The AdΔΔ‐mediated attenuation of drug‐induced autophagy was mimicked by overexpression of the small viral E1A12S protein.

In conclusion, our data suggest that inhibition of autophagy in combination with cytotoxic drugs is a promising therapeutic strategy for late‐stage prostate cancer.

FB03 AAV8‐mediated gene therapy rescue of lethal Argininosuccinate lyase‐deficient mice.

Baruteau Julien Dr 1 Hanley Johanna Dr 2 Buckley Suzy M Dr 1 Gissen Paul Prof 2 Howe Steven J Dr 3 Karda Rajvinder Miss 1 Mills Philippa B Dr 4 Ng Joanne Dr 1 Perocheau Dany Mr 1 Suff Natalie Dr 1 Rahim Ahad Dr 5 Prunty Helen Dr 6 Waddington Simon N Dr 1

1 Gene Transfer Technology Group, Institute for Women's Health, University College London, London, United Kingdom 2 MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom 3 Clinical and Molecular Genetics, Institute of Child Health, University College London, London, United Kingdom 4 Wolfson Centre for Gene Therapy, Molecular Immunology Unit, Institute of Child Health, University College London, London, United Kingdom 5 School of Pharmacy, University College London, London, United Kingdom 6 Biochemistry, Great Ormond Street Hospital, London, United Kingdom

Session 2. Recent advances in pre‐clinical knowledge, April 15, 2016, 13:15 – 15:15

Background: The urea cycle is an essential hepatic pathway, which allows detoxification of the highly‐neurotoxic ammonia. Argininosuccinate lyase (ASL) is an enzyme involved in (i) the urea cycle (ii) the synthesis of nitric oxide. ASL‐deficient patients develop argininosuccinic aciduria (ASA), with high mortality and neurological morbidity. Despite best‐accepted treatment, patients are susceptible to acute life‐threatening hyperammonaemic encephalopathy, even as neonates. Therefore, neonatal intervention is essential. Cerebral nitric oxide deficiency could contribute to the poor neurological outcome. An approach for liver and brain delivery of gene therapy was considered.

Methods: Using a hypomorphic mouse model AslNeo/Neo we designed an AAV‐based gene therapy for ASA (ssAAV8‐EFS‐mAsl‐WPRE). Only 20% of affected mice survive beyond 25 days of life. Five AslNeo/Neo mice received a supportive treatment to prevent early death until day 30, after which they received a single intraperitoneal injection of vector. Seven AslNeo/Neo mice received a single neonatal intravenous injection of vector.

Results & discussion: In mice injected at day 30, survival was rescued (p = 0.0027). Growth, fur appearance, several biomarkers assessed at 2 months post‐injection (plasma amino acids, urinary orotate and argininosuccinate) were significantly improved. In 2/5 animals, improvement of the urea cycle function (liver ASL enzymatic assay, liver immunohistochemistry) was persistent one year after injection.

In mice injected neonatally, significant improvement in survival (p = 0.0055) and cerebral ASL activity was significantly increased 9 months after injection.

We were able to demonstrate a sustained improvement of the urea cycle after adult injection and persisting correction of the cerebral ASL activity after neonatal injection.

FB04 Novel therapeutic approaches for childhood parkinsonism

Ng Joanne Dr 1 Zhen Juan Dr 2 Karda Raj Ms 1 Barral Serena Dr 3 Meyer E Dr 3 Suff Natalie Dr 1 Massaro Giulia Ms 4 Hughes Michael Mr 4 Cuka S Ms 4 Baruteau Julien Dr 1 Perocheau Dany Mr 1 Buckley Suzanne Dr 1 Pope S Dr 5 Heales Simon Professor 5 Reith Martin Professor 2 Rahim Ahad Dr 4 Kurian Manju Dr 3

1 Gene Transfer Technology Group, UCL‐Institute for Women's Health, United Kingdom 2 Psychiatry, New York Medical School, USA 3 Molecular Neurosciences, UCL‐Institute Child Health, UK 4 Pharmacology, UCL‐School of Pharmacy, UK 5 Neurometabolic Unit, National Hospital Neurology and Neurosurgery, UK

Session 2. Recent advances in pre‐clinical knowledge, April 15, 2016, 13:15 – 15:15

Background: Dopamine transporter (DAT) deficiency syndrome is a neurotransmitter disorder caused by loss‐of‐function mutations in SLC6A3 encoding the Dopamine transporter. It is characterised by progressive infantile onset dystonia‐parkinsonism with raised cerebrospinal fluid dopamine metabolite, homovanillic acid. There are no disease modifying therapies for this life‐limiting disorder. This study aims to evaluate the clinical disease spectrum and develop a gene therapy approach using the DAT Knockout^(−/−) mouse model.1

Methods: Patients with childhood onset dystonia‐parkinsonism had SLC6A3 gene sequenced. In vitro functional studies were undertaken for identified missense mutations. We phenotyped DAT^−/− motor behaviour and evaluated effects of hDAT gene therapy delivered by intracerebroventricular injection to neonatal DAT^−/− mice as a proof of concept study.

Results: We identified 10 patients with novel recessive SLC6A3 mutations. CSF immunoblotting studies showed reduced levels of dopaminergic synaptic proteins. In vitro functional characterisation revealed loss of mutant DAT function, with abnormal DAT trafficking, glycosylation, substrate recognition and uptake function.

The DAT^−/− mouse clearly recapitulates many features of human disease, including reduced survival, early hyperkinesia with later parkinsonism, raised homovanillic acid and neurodegeneration. Gene therapy resulted in improved growth, survival rate and amelioration of motor phenotype.

Conclusion: We report an expanding disease spectrum with clinical presentation mimicking both cerebral palsy and juvenile parkinsonism. Genotype‐phenotype correlation is evident, with in vitro functional studies demonstrating greater residual DAT function in later‐onset milder forms of disease. Viral hDAT gene therapy can rescue the DAT^−/− phenotype.

1. Giros B. et al. Nature 1996;379:606–612.

Keynote Speaker

KN01 Directed Evolution of New Viruses for Therapeutic Gene Delivery

Schaffer David Professor 1

1 University of California, Berkeley, Berkeley, United States 2 4D Molecular Therapeutics, Emeryville, United States

Keynote, Kennedy Lecture Theatre, April 15, 2016, 17:15 – 18:00

Biography: David Schaffer is a Professor of Chemical and Biomolecular Engineering, Bioengineering, Molecular and Cell Biology, and Neuroscience at University of California, Berkeley, where he also serves as the Director of the Berkeley Stem Cell Center. He also co‐founded 4D Molecular Therapeutics for the clinical development of optimized AAV vectors.

Strong basic and translational efforts in the gene therapy field have culminated in successes in an increasing number of human trials involving viral vectors, particularly ones based on adeno‐associated virus (AAV). These include trials for hemophilia B, Leber's congenital amaurosis, Sanfilippo B, and lipoprotein lipase deficiency (LPLD), and a gene therapy for LPLD became the first to receive regulatory approval in 2012 (in the European Union). AAV is thus capable of safe and therapeutic gene delivery to some targets; however, vectors face a number of challenges that limit their efficacy. These include anti‐vector neutralizing antibodies, low transduction of some therapeutically relevant cells in vitro or in vivo, difficulty in overcoming cellular and physical barriers within complex tissue structures, and an inability for targeted delivery to specific cells. These challenges are not surprising, as nature did not evolve viruses for our convenience to use as human therapeutics, and thus “off the shelf” natural viruses do not meet a range of clinical needs. In most situations there is insufficient mechanistic knowledge of underlying virus structure‐function relationships to empower rational design to improve such vectors; however, directed evolution has been emerging as a strategy to engineer novel viral variants that meet specific biomedical needs. We were the first to develop and have since been implementing directed vector evolution – the iterative genetic diversification of a viral genome and functional selection for desired properties – to address a number of problems with AAV. Genetic diversification has included the random diversification of peptide sequences at defined locations in the capsid, random point mutagenesis of the cap gene, and recombination of cap genes from a number of parental serotypes to create random chimeras. Using a range of in vitro and in vivo selection strategies, we have evolved AAV for evasion of neutralizing antibodies, enhanced biodistribution and spread within a target tissue, greatly improved delivery efficiency, and targeted delivery in vitro and in vivo, thereby improving the vectors' capacity to meet human therapeutic needs.

Poster Presentations

PO01 Intravenous AAV8‐Estrogen related receptor gamma gene transfer improves pathology in mdx mice

AL‐Siyabi Muzna Miss 1 Foster Helen Dr 1 Eilers Wouter Dr 1 Matsakas Antonios Dr 2 Patel Ketan Professor 1 Foster Keith Dr 1

1 University of Reading, Reading, United Kingdom 2 University of Hull, Hull, United Kingdom

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Duchenne muscular dystrophy (DMD) is the most severe form of muscular dystrophy. Loss of dystrophin causes progressive loss of muscle fibre which leads to cardiac and respiratory failure and ultimately death within the third decade.

Estrogen related receptor gamma (ERRγ) is an orphan nuclear receptor. It is considered to be the master switch of oxidative and angiogenic factors. ERRγ is down‐regulated in mdx along with its target metabolic and angiogenic genes. Transgenic overexpressing ERRγ in mdx mice, improve sarcolemmal integrity and muscle perfusion with restoration of metabolic and angiogenic genes.

Previously, we showed intramuscular administration of (5×1010 vg) into Tibialis anterior (TA) of mdx mice result in increasing the expression of ERRγ and VEGF‐165 significantly. Pathological analysis of Oxidative capacity, centrally nucleated and regenerated fibres were improved significantly. We have further evaluated ERRγ overexpression following intravenous injection of (1×10¹² vg) into 6 week old mdx mice (n = 7). A selection of muscles were recovered 4 weeks post administration. Creatine kinase assay did not showed any significant difference between the treated mice and the control. Quantitative analysis of ERRγ showed significant increase of 3, 5 and 10 folds in extensor digitorum longus (EDL), diaphragm and gastrocnemius, respectively. However, only gastrocnemius muscle showed a significant increase in the transcript level of VEGFβ by 1.4 fold. In gastrocnemius, succinate dehydrogenase analysis demonstrated a significant increase in oxidative muscle fibres (24–37%, p = 0.001). H&E analysis showed a significant reduction in the centrally nucleated fibres (7% reduction, p = 0.007) with no significant difference in regeneration or myosin heavy chain fibre typing.

PO02 Comparison of CAG and synapsin promoter for viral transduction in the central nervous system

Nieuwenhuis Bart Mr 1 Haenzi Barbara Ms 1 Verhaagen Joost Mr 2 Fawcett James Mr 1

1 University of Cambridge, Cambridge, United Kingdom 2 Netherlands Institute for Neuroscience, Amsterdam, The Netherlands

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

The central nervous system (CNS) is known to be challenging for gene manipulation. Therefore, it is essential to determine the best promoter and serotype for adeno‐associated viruses (AAVs) mediated delivery of genes into the cell type of interest. The choice of promoter is particularly important if the inserted gene is large since the packaging size of AAV is limited. Here, we performed a study with the small promoters CMV early enhancer/chicken β actin (CAG) and synapsin (SYN). We aim to target the neurons of origin of the corticospinal tract in V of the cortex. We compared 1) AAV1‐SYN‐GFP and AAV5‐SYN‐GFP to highlights the best serotype to transduce layer V cortical neurons (transduction efficiency); (2) the effect of promoters by the expression level of GFP using AAV5‐CAG‐GFP and AAV5‐SYN‐GFP; (3) the double infection efficiency of co‐injection of AAV5‐CAG‐GFP and AAV5‐CAG‐mCherry in these neurons. Our data provides insight into the best promoter and serotype for selective layer V neuron transduction in the cortex. In the future, we will expand our study by comparing additional promoters.

PO03 Development of Salmonella typhimurium VNP20009 as a Delivery Vector of the Novel Neisserial Anti‐tumor Protein Laz

Mansour Manar Dr 1 Ismail Shehab Associate professor 2 Abou‐Aisha Khaled Associate professor 1

1 German University in Cairo, New Cairo, Cairo, Egypt, Egypt 2 The Cancer Research Institute CRUK Beatson Institute, Glasgow, UK

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Salmonella typhimurium VNP‐20009 (VNP) is a non‐pathogenic attenuated strain, which, as a facultative anaerobe, preferentially accumulates in hypoxic regions of solid tumors. Here, VNP was exploited as a bacterial delivery vehicle of the anti‐tumor protein Laz (Lipidated azurin), Laz, is a surface‐exposed protein and possesses in its N‐terminus a 39 aa, H.8 epitope, which has been suggested to play a role in disrupting an entry barrier in brain tumor cells. Laz has been demonstrated to induce apoptosis through an interaction with the tumor suppressor protein p53.

In this study, the entire ORF of the laz gene, including ∼17‐bp signal sequence, was cloned downstream of a hypoxia inducible promoter (HIP‐1), before being electroporated into VNP. Successful expression and export of the Laz protein by VNP was confirmed by Western Blot of the cell‐free culture medium. Effective expression of Laz by VNP was investigated in glioblastoma cell lines: LN‐229 and U‐373, with the latter cell line carrying a mutated version of p53; and in the breast cancer cell line MCF‐7. Cytotoxicity of the VNP‐Laz combination was assessed by determining the levels of the apoptotic markers caspases 3/7 using fluorometry. Compared to the purified Laz, the VNP‐expressing Laz, significantly induced apoptosis in LN‐229 and, to a lower extent in U‐373. Fluorescence levels determined for the VNP‐Laz delivery system were higher than those attained using either agent, VNP or the purified Laz protein, alone.

These results might represent a new approach of targeted gene delivery and suggest a potential application in brain tumor therapy.

PO04 Generation of light‐producing somatic‐transgenic mice using adeno‐associated virus vectors

Karda Rajvinder Miss 1 Perocheau Dany P Mr 1 Buckley Suzanne MK Dr 1 Delhove Juliette MKM Ms 2 Hughes Michael Mr 3 Rahim Ahad Dr 3 Johnson Mark R Professor 4 Ng Joanne Dr 1 Suff Natalie Dr 1 Martin Nuria Palomar Ms 5 Henckaerts Els Dr 5 McKay Tristan R Dr 6 Waddington Simon N Dr 1

1 Institute for Women's Health, University College London, London, United Kingdom 2 Institute for Child Health, University College London, London, United Kingdom 3 Department of Pharmacology, University College London, London, United Kingdom 4 Institute of Reproductive and Developmental Biology, Imperial College London, London, United Kingdom 5 Kings College London, London, United Kingdom 6 Manchester Metropolitan University, Manchester, United Kingdom

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Germ line light producing transgenic mice, where luciferase expression is controlled by a surrogate promoter downstream of tandem, synthetic, transcription factor binding elements, are used to provide an in vivo readout of disease processes. However, germ line transgenics are expensive to maintain.

We have developed a novel technology for the generation of light emitting somatic transgenic animals using lentiviral vectors. This allows signalling pathways in diseased organs to be monitored in a non‐invasive manner.

In this study, we aimed to deliver NFκB driving a luciferase reporter construct to the nervous system of neonatal mice to generate somatic‐transgenic mice using AAV vectors.

AAV8 serotyped vector was injected intracranially or intravenously to outbred CD1 neonatal (P1) mice and luciferase expression was monitored continually by whole body bioluminescence imaging of conscious mice.

Intracranial injection of AAV8 NFκB biosensor showed a widespread luciferase expression. We also administered AAV8 NFκB biosensor intravenously at P1 and observed a widespread transduction.

Previous studies show AAV8 serotype transduces many systemic tissues through intravenous administration; we observed luciferase expression predominantly in the brain and spine.

Using a standard Gateway^® cloning system we have established a library of more than 25 lentivirus biosensors where some have been tested in vitro and in vivo. We plan on incorporating these response elements into the AAV backbone described above. Therefore, enabling the generation of somatic‐transgenic mice. This will complement existing germ line transgenic, light producing technology by maximising the use, and reducing the numbers, of animals used in biomedical research.

PO05 Gene therapy treatment abrogates intranuclear inclusions and normalizes muscle strength in a mouse model of oculopharyngeal muscular dystrophy

Malerba Alberto PhD 1 Klein Pierre Mr 2 Bachtarzi Houria PhD 1 Jarmin Susan PhD 1 Ferry Arnaud Prof 2 Graham Michael PhD 3 Suhy David PhD 3 Dickson George Prof 1 Trollet Capucine PhD 2

1 School of Biological Sciences, Royal Holloway, University of London, UK, Egham, United Kingdom 2 Sorbonne Universitós, UPMC Univ Paris 06, UM76, INSERM U974, Institut de Myologie, CNRS FRE3617, 47 bd de l'Hôpital, 75013, Paris, France, Paris, France 3 Benitec Biopharma, Sydney, Australia, Sydney, Australia

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Oculopharyngeal muscular dystrophy (OPMD) is a muscle specific, autosomal dominant, late‐onset degenerative disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. OPMD is caused by a mutation in the Poly‐adenylate Binding Protein Nuclear 1 (PABPN1) gene that results in an N‐terminal expanded polyalanine tract. PABPN1 controls several biological processes including the length of mRNA poly(A) tails, mRNA export from the nucleus and the alternative poly(A) site usage. OPMD is characterized by filamentous intranuclear inclusions (INIs) of aggregated expanded PABPN1 (expPABPN1), fibrosis and muscle atrophy. Here we demonstrate that treating mice affected by OPMD over 4 months with a single‐dose gene therapy strategy based on silencing the endogenous expPABPN1, combined with the expression of a healthy sequence‐optimized human PABPN1, significantly reduced the amount of INIs in affected muscles, decreased the intramuscular fibrosis and reverted the muscle strength to the level of healthy wild‐type muscles. These results are obtained in a relevant mammalian animal model of OPMD and pave the way for the clinical development of a gene therapy approach to treat OPMD in human.

PO06 Novel Therapeutic Strategies in NBIA: A Gene Therapy Approach for PLA2G6‐associated Neurodegeneration (PLAN)

Cuka Sam Miss 1 Ng Joanne Dr 2 Seino Ken Dr 3 Waddington Simon Dr 2 Kurian Manju Dr 4 Rahim Ahad Dr 1

1 Department of Pharmacology, School of Pharmacy, UCL, London, United Kingdom 2 Gene Transfer Technology Group, UCL Institute of Women's Health, UCL, London, United Kingdom 3 Division of Bioregulation Research, Institute of Medical Science, St Marianna University School of Medicine, Kanagawa 4 UCL Institute of Child Health, London, United Kingdom

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Infantile neuroaxonal dystrophy (INAD) is a debilitating, intractable and ultimately lethal neurodegenerative disorder. It is caused by mutations in the PLA2G6 gene that encodes for phospholipase A. Patients present neurodegeneration‐associated symptoms between six months and three years of age. Severe spasticity, progressive cognitive decline, and visual impairment typically result in death during the first decade. There is no disease‐modifying treatment available and palliative care focuses on quality of life. There is an overwhelming need to develop novel therapies to treat INAD patients.

We aim to conduct a preclinical AAV‐mediated gene therapy study to prevent neurodegeneration and rescue a mouse model of INAD from premature death. We will use recombinant adeno‐associated virus serotype 9 vector (AAV9) to deliver therapeutic human PLA2G6 gene to the neonatal INAD mouse CNS via either intracranial or intravenous administration. The human PLA2G6 and control GFP gene were cloned into an AAV9 backbone plasmid, driven by the synapsin‐I promoter and used to produce high titre viral preparations. Furthermore, the INAD mouse model recapitulates many features of the human phenotype. Investigating indices of neuropathology will not only provide us with readouts against which to gauge therapeutic efficacy but also increase our understanding of the underlying disease mechanisms.

PO07 CRISPR‐based genome editing to correct models of rare pulmonary disease

Foster Robert Mr 1 2 Mali Girish R. Dr. 1 Keighren Margaret Mrs. 1 Boyd A. Christopher Dr. 2 Mill Pleasantine Dr. 1

1 MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom 2 Center for Genomics and Experimental Medicine, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Rare diseases affect up to 7% of the UK population and are a significant burden on society, healthcare institutions and the individuals involved. Whilst NextGen sequencing technology has accelerated candidate gene identification for many rare diseases, development of effective therapies and treatments for these conditions still lags behind, due to the small number of patients in relation to the exorbitant costs involved. In contrast, the CRISPR‐Cas9 system for genome editing could prove to be a potent therapeutic toolkit which can be adapted to correct the mutations behind many rare genetic disorders in‐situ. It is a highly modular system centred on the Cas9 endonuclease, which is easily re‐purposed for different mutations by simply interchanging a short guide sequence, which directs targeting, and the repair template, to correct the mutation. This renders the system simple and affordable to adapt for the “personalised” correction of numerous genetic diseases.

Major bottlenecks in the field of therapeutic gene editing include the targeting of specific tissues and the biasing of gene editing events towards repair. We have developed a fluorescent reporter system in mouse which will allow us to optimise the in‐vivo delivery of Cas9 and repair templates by tracking gene editing events, discriminating between non‐homologous end joining and the desired homology‐directed repair. Using the advances gained from our reporter system to drive repair in the postnatal airways, we will attempt to correct a mouse model of Primary Ciliary Dyskinesia (PCD), a rare genetically heterogeneous disorder which disrupts the correct development of motile cilia.

PO08 Intravenously administered gene therapy for the treatment of neuronopathic Gaucher Disease

Massaro Giulia Ms 1 Perocheau Dany Mr 2 Baker Rob Dr 3 Karlsson Stefen Professor 4 Cheng Seng Dr 5 Waddington Simon Dr 2 Rahim Ahad Dr 1

1 UCL School of Pharmacy, 29–39 Brunswick Square, London WC1N 1AX, United Kingdom 2 Gene Transfer Technology Group, UCL Institute for Women's Health, United Kingdom 3 Lysosomal Storage Disorder / Molecular Lab, Haematology Department, Royal Free Hospital, United Kingdom 4 Division of Molecular Medicine and Gene Therapy, Lund University, Lund, Sweden 5 Genzyme, A Sanofi Company, USA

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Gaucher Disease (GD) is a lysosomal storage disorder caused by mutations in the GBA gene encoding the enzyme glucocerebrosidase (GCase). Deficiency of GCase causes the accumulation of glucosylceramide in macrophages within visceral organs and brain. Neuronopathic forms of GD (type II and III) are characterised by neuronal loss, astrocytosis and microglial proliferation. Enzyme replacement therapy is successfully used to ameliorate the visceral pathology, however there is no treatment available for the lethal neurodegeneration. Our research focuses on GD type II, the most acute neuronopathic form, in which neuropathology results in death in early infancy. The aim of this project is to intravenously administer an adeno‐associated viral vector serotype 9 (AAV9) carrying the functional GBA gene to a GCase‐deficient mouse model of GD type II and assess improvement in lifespan, behaviour, brain and visceral pathology.

The untreated knock‐out mice die 12–14 days after birth. AAV9 carrying the functional human GBA gene was intravenously administered into neonatal knock‐out mice (n = 5). Treated animals showed increase in their lifespan (p = 0.0081). Since the animals did not develop any evident pathological symptoms, we decided to cull them at 55 days of age. The neuropathology was ameliorated and some of the most affected areas of the brain, like thalamus, brain stem and cerebellum were partially rescued. Histologic analysis, enzymatic assay and blood test revealed improvement in the visceral pathology.

We are currently testing novel and more efficient constructs, with the aim of improving gene expression in the CNS while enhancing the therapeutic effects on the visceral pathology.

PO09 The Chemical Modification of Adenovirus Vectors for Targeted DNA Vaccination and Immune Evasion

Minall Leanne Miss 1

1 Oxford University, Oxford, United Kingdom

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Viruses are powerful vectors for the delivery of nucleic acids, with applications in gene therapy and vaccination. However, major challenges for this technology include mis‐targeting of the vector and neutralization by host immune responses. Here we show that chemical addition of synthetic glycans to adenovirus (Ad) both increased resistance to neutralizing antibody, and switched the naturally board Ad viral tropism to target dendritic cells (DCs). The new glyco‐Ads showed high selectivity for the complementary sugar receptors, thereby enhancing gene delivery. In vaccination studies, DC‐targeted glyco‐Ads enhanced antigen‐specific T cell responses. The glyco‐Ads were also protected from some of the major immune sequestration mechanisms, including binding by blood factor X and anti‐Ad antibodies. Thus, chemical glycosylation of the Ad capsid allows modulation of tropism and shielding from sequestration and neutralizing antibody. Since manipulation of this process is facile, it provides a flexible and potentially universal solution to key obstacles facing the utilization of viral vectors in therapeutic and vaccination contexts.

PO10 Innovative approaches for the manufacture of the starting plasmid DNA vectors: High Quality (HQ) plasmid DNA

Hitchcock Antony 1

1 Cobra Biologics, Keele, United Kingdom

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

The increased demand for the production of AAV and Lenti viral vectors, used in both regenerative and immunotherapy clinical studies, through transient expression has created a need for innovative approaches to be taken for the manufacture of the starting plasmid DNA vectors used in their production. For drug developers cost, time and quality for these vectors are key to the underlying cost of goods of the final product. Plasmids used in virus production have been designated as GMP starting material by the EU regulatory authorities. In response to customer needs and regulatory compliance, Cobra has developed a High Quality (HQ) plasmid DNA service which has streamlined the existing manufacturing GMP platform. With such a strategy HQ plasmid manufacturing can be performed in less than 8 weeks (as opposed to months for GMP) to produce fully compliant plasmid DNA which can be used to support early phase clinical virus production.

PO11 AAV9‐mediated central nervous system‐targeted gene delivery via cisterna magna route in mice

Lukashchuk Vera Dr 1 Lewis Katherine Dr 1 Coldicott Ian Mr 1 Grierson Andrew Dr 1 Azzouz Mimoun Prof 1 2

1 University of Sheffield, Sheffield, United Kingdom 2 King Abdulaziz University, Jeddah, Saudi Arabia

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Current barriers to the use of adeno‐associated virus serotype 9 (AAV9) in clinical trials for treating neurological disorders are its high expression in many off‐target tissues such as liver and heart, and lack of cell specificity within the central nervous system (CNS) when using ubiquitous promoters such as human cytomegalovirus (CMV) or chicken‐β‐actin hybrid (CAG). To enhance targeting the transgene expression in CNS cells, self‐complementary (sc) AAV9 vectors, scAAV9‐GFP vectors carrying neuronal Hb9 and synapsin 1, and non‐specific CMV and CAG promoters were constructed. We demonstrate that synapsin1 and Hb9 promoters exclusively targeted neurons in vitro, although their strengths were up to 10‐fold lower than that of CMV. In vivo analyses of mouse tissue after scAAV9‐GFP vector delivery via the cisterna magna revealed a significant advantage of synapsin1 promoter over both Hb9 variants in targeting neurons throughout the brain, since Hb9 promoters were driving gene expression mainly within the motor‐related areas of the brain stem. In summary, this study demonstrates that cisterna magna administration is a safe alternative to intra‐cranial or intra‐cerebroventricular vector delivery route using scAAV9, and introduces a novel utility of the Hb9 promoter for the targeted gene expression for both in vivo and in vitro applications.

PO12 Production of VSV‐G Envelope Glycoprotein from Saccharomyces cerevisiae for non‐viral gene delivery

Themis Michael Dr 1 Walls Thomas Mr 1 Khonsari Hassan Dr 1 Themis Michael Dr 1

1 Brunel University, Uxbridge, United Kingdom

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Non‐viral gene therapy vectors are amenable to simple, large‐scale production. They provide low immunogenicity compared to their viral counterparts and have little or no genotoxic side effects. However, non‐viral vector gene transfer can be limited by low transfection efficiency.

VSV‐G pseudotyped retroviral vectors are highly infectious to several cell types and free VSV‐G can render non‐enveloped particles infectious. VSV‐G can also be complexed with plasmid vectors to enhance non‐viral vector gene delivery to cell types not previously amenable to gene transfer with traditional transfection agents.

Unfortunately, VSV‐G is cytotoxic as it promotes syncytia formation in mammalian cultures resulting in cell death. Hence, VSV‐G production from mammalian cells, like that of VSV‐G pseudotype viruses, is restricted to transient transfection, which makes the harvesting of this effective conjugate expensive and time consuming. To address this problem, we have generated a strain of the yeast Saccharomyces cerevisiae that can produce the VSV‐G protein from an inducible integrated yeast artificial chromosome vector. Using this construct we show that pure VSV‐G can be continuously and simply produced by Saccharomyces cerevisiae without syncytia formation due to the yeast cell wall. We also show that yeast generated VSV‐G achieves greater transfection efficiency than VSV‐G produced from mammalian cells.

PO13 Systemic L‐DOPA Secretion by AAV Gene Therapy to Improve the Treatment of Parkinson's Disease

Antunes André Saraiva Leão Marcelo Mr 1 Hikima Atsuko Dr 1 Hobbs Carl Mr 1 McDonald Michael Dr 2 Rose Sarah Dr 1 Henckaerts Els Dr 1

1 King's College London, London, United Kingdom 2 McDonaldsPharm Ltd, London, United Kingdom

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Parkinson's disease (PD) is characterised by progressive degeneration of the dopaminergic neurons in the substantia nigra leading to severe motor complications. Standard oral treatment with L‐DOPA provides powerful symptomatic relief, but disabling side effects such as dyskinesia eventually develop due to the pulsatile stimulation of the dopamine receptors.

Our goal is to demonstrate proof‐of‐principle for continuous L‐DOPA synthesis in peripheral organs by AAV gene therapy for the treatment of PD. We propose a combination therapy of oral L‐DOPA and gene delivery of dopamine‐synthetic enzymes to peripheral organs in order to allow for continuous L‐DOPA production outside the CNS. If successful, a one‐time intravenous injection would be sufficient to elevate plasma L‐DOPA levels and diminish the fluctuations inherent to oral treatment.

We show that injection of 1E+12 gcp/mouse of a liver‐specific bicistronic AAV vector, encoding tyrosine hydroxylase (TH) and GTP‐cyclohydrolase (GCH1), results in the transduction of ∼40% of the hepatocytes and a two‐fold increase in the levels of systemic L‐DOPA 28 days post‐injection. These data show that hepatic L‐DOPA production and release can be achieved but that additional organs may need to be targeted in order to achieve plasma L‐DOPA levels that approximate those achieved with oral treatment. We have generated a tricistronic vector encoding TH, GCH1 and 6‐pyrovoyltetrahydopterin synthase (PTPS) under the control of a ubiquitous promoter to achieve L‐DOPA synthesis in skeletal muscle and liver. This vector features a cardiac‐specific microRNA target sequence to exclude cardiac expression. Pre‐clinical studies are currently ongoing to test the potential of this vector.

PO14 Gene Therapy for Familial ALS Using AAV9 Mediated Silencing of Mutant SOD1

Iannitti Tommaso Dr 1 Scarrot Joseph M Mr 1 Coldicott Ian RP Mr 1 Kaspar Brian K Prof. 2 Ferraiuolo Laura Dr 1 Shaw Pamela J Prof. 1 Azzouz Mimoun Prof. 1

1 University of Sheffield, Sheffield Institute for Translational Neuroscience (SITraN), 385 Glossop Road, S10 2HQ, Sheffield, United Kingdom 2 The Research Institute at Nationwide Children's Hospital, Department of Neuroscience, The Ohio State University, 43205, Columbus, USA

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder resulting in death of brain and spinal cord motor neurons. Some familial cases are caused by missense mutations in the gene encoding the Cu/Zn superoxide dismutase 1 (SOD1) conferring a toxic gain of function to this protein. We hypothesised that specifically silencing expression of the mutant form of the human SOD1 gene would alleviate SOD1‐linked ALS symptoms.

Objectives: Evaluate the therapeutic efficacy of AAV9‐shRNA mediated SOD1 silencing in the SOD1G93A mouse model.

Methods: Animals were treated either at postnatal day 1 (P1, pre‐onset) or P40 (onset). scAAV9‐hSOD1si or scrambled control scAAV9‐hSOD1ssi were delivered using 2 routes of delivery, facial vein or cisterna magna. Mice were then tested using behavioural tests including weekly rotarod runs, neurological scoring and CatWalk gait analysis. Weekly body weight was also collected.

Results: We observed an improvement in rotarod performance in mice treated with scAAV9‐hSOD1si vs. scAAV9‐hSOD1ssi and untreated controls in all studies. Survival analysis revealed that scAAV9‐hSOD1si delivery via cisterna magna at P1 and P40 extended SOD1G93A mouse life span by 67 days and 16 days, respectively. The P1 cisterna magna study is still ongoing and scAAV9‐hSOD1si‐treated mice have now reached 225 days. SOD1 silencing at P40 also improved motor function as revealed by CatWalk gait analysis.

Conclusions: AAV9‐shRNA mediated SOD1 silencing through ransdu magna improved motor performance and led to remarkable life span extension in the SOD1G93A mouse model. Our gene therapy approach offers promising strategy for clinical application in SOD1‐linked familial ALS.

PO15 Development of lung and muscle protein factories to deliver therapeutic monoclonal antibodies

Antepowicz Agata Miss 1 2 Tan Tiong Mr 1 2 Pringle Ian Dr 1 2 Hyde Stephen Professor 1 2 Gill Deborah Professor 1 2

1 University of Oxford, Oxford, United Kingdom 2 UK Cystic Fibrosis Gene Therapy Consortium, Oxford, Edinburgh & London, United Kingdom

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Recombinant therapeutic proteins, of which monoclonal antibodies (mAbs) represent the largest market, are used to treat diseases including cancer, blood and immune disorders. The cost and complexity of large‐scale production and purification of mAbs translates to high cost of therapy. We hypothesise that our rSIV.F/HN lentiviral lung gene delivery platform, currently undergoing preclinical evaluation for cystic fibrosis gene therapy, could be used to establish protein factories within patients' lungs. This approach is being evaluated using two model mAbs, infliximab and palivizumab, for delivery to the lung lumen and circulatory system, respectively. The efficiency of mAb‐secreting lung protein factories using rSIV.F/HN lung gene transfer is being compared with conventional muscle protein factories using rAAV2/8. Preliminary in vivo studies using vectors expressing secretory reporter protein Gaussia luciferase (Glux) delivered 1E7 and 1E8 transducing units (TU) of rSIV.F/HN to the mouse lung and 1E10 and 1E11 genome copies (GC) of rAAV2/8 delivered intramuscularly. Each resulted in robust and sustained secretion of Glux into the circulation (p < 0.001 at 9 and 4 months post‐delivery, respectively), supporting progression to experiments using mAb‐encoding vectors. We have developed rSIV.F/HN and rAAV2/8 vectors encoding infliximab and palivizumab, which direct detectable antibody expression in cell culture (46–350 ng/mL), and are currently conducting in vivo studies with these vectors to determine whether therapeutically relevant levels of mAb expression can be achieved in serum and lung lumen. If successful, this approach could translate into reduced patient burden and treatment costs.

PO16 AAV‐mediated gene transfer of FKRP in mice

Gicquel Evelyne Mrs 1 Maizonnier Natacha Mrs 1 Richard Isabelle Dr 1

1 Genethon, INTEGRARE research unit, Evry, France

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Dystroglycanopathies constitute a group of genetic diseases caused by defective glycosylation of alpha‐dystroglycan (aDG), a membrane glycoprotein involved in the cell/matrix anchoring of muscle fibers. The aDG glycosylation, a very complex process, requires many proteins. In particular, mutations in the FKRP gene encoding Fukutin related protein, lead to hypoglycosylation of aDG, resulting in different forms of dystroglycanopathies, among which Limb Girdle Muscular Dystrophy type 2I (LGMD2I).

We generated a knock‐in mouse model, presenting with abnormal glycosylation of aDG, reduction of its binding to the extracellular matrix protein laminin, presence of central nuclei and low resistance to eccentric mechanical stress. Using this mouse model, we performed FKRP gene transfer, by means of a rAAV2/9 vector expressing the murine FKRP under the transcriptional control of the desmin promoter. The rAAV vector was injected intramuscularly or intravenously. Expression of the FKRP transgene was obtained, both at RNA and protein levels. The glycosylation of aDG was restored, as well as binding to laminin. A histological rescue was observed. The AAV vector also improved the muscle function, since it conferred a better resistance to eccentric stress to the injected muscles. In parallel with these promising results, we uncovered that increasing the rAAV‐FKRP dose was leading to a decrease of aDG glycosylation and of its binding to laminin. Moreover, an immune response against the transgene was revealed in some of the injected mice. Altogether, these data support the possibility to use the AAV‐mediated transfer of FKRP but call for a control of gene expression.

PO17 Targeted gene addition strategies for the treatment of X‐linked lymphoproliferative disease

Houghton Benjamin Dr 1 Mussolino Claudio Dr 2 Cathomen Toni Professor 2 Gaspar H Bobby Professor 1 Booth Claire Dr 1

1 Institute of Child Health, UCL, London, United Kingdom 2 Institute for Cell and Gene Therapy & Center for Chronic Immunodeficiency, University of Freiburg, Freiburg, Germany

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

X‐linked lymphoproliferative disease is an immunodeficiency arising from mutations in the SH2D1A gene encoding SAP, a key regulator of immune function expressed in T cells, natural killer, and natural killer T cells. Haemopoietic stem‐cell transplantation is curative, and we have shown proof of concept of haemopoietic stem‐cell gene therapy in a mouse model. Preliminary data suggests that adoptive transfer of T cells corrected using lentiviral gene therapy can correct immune defects. However, targeted gene editing of T cells may allow for physiologically regulated SAP expression.

We constructed SAP‐specific TALENs capable of generating a site‐specific double‐stranded break close to the translation start codon, allowing correction of most described SH2D1A mutations. TALEN activity was validated with a surveyor assay in HEK293T cells. To facilitate optimisation of delivery methods, we designed a fluorescent reporter assay that, when stably integrated into a cellular DNA, provides a fluorescence signal when repair of DNA double‐stranded breaks (DSB) created by TALENs occurs.

Using the reporter assay in Jurkat cells we observed reduced toxicity and highly efficient delivery using an mRNA nucleofection platform, with 23% of treated cells showing repair mediated fluorescence. We proceeded to deliver TALEN mRNA to human primary T cells, and demonstrated the generation of double‐stranded breaks in 25% of the bulk population.

To harness the repair of DSB, we constructed homologous recombination donors in both non‐integrating lentiviral vector and adeno‐associated virus format, to mediate donor delivery with low toxicity. Combined with TALEN mRNA nucleofection, homologous recombination‐driven incorporation of donor in T cells was possible.

PO18 Improved survival in a knock‐in model of Rett syndrome after systemic administration of AAV9/MECP2

Gadalla Kamal Dr 1 3 Vudhironarit Thishnapha Ms 1 2 Bahey Noha Dr 1 4 Hector Ralph Dr 1 Bailey Mark Dr 2 Gray Steven Dr 5 6 Cobb Stuart Dr 1

1 Institute of Neuroscience and Psychology, University of Glasgow, UK, GLASGOW, United Kingdom 2 School of Life Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, UK, GLASGOW, United Kingdom 3 Pharmacology Department, Faculty of Medicine, Tanta University, Egypt, Tanta, Egypt 4 Histology Department, Faculty of Medicine, Tanta University, Egypt, Tanta, Egypt 5 University of North Carolina Gene Therapy Center, Chapel Hill, NC, USA, Chapel Hill, USA 6 University of North Carolina Department of Ophthalmology, Chapel Hill, NC, USA, Chapel Hill, USA

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

De novo mutations in the X‐linked gene MECP2 are responsible for ∼95% of typical Rett syndrome (RTT) cases which are characterized by severe motor and cognitive impairments in females. The missense T158M mutation is one of the most common and severe RTT‐causing mutations. It is unknown whether the presence of endogenous mutated protein may impede the therapeutic action of a gene therapy approach. The aim of this study was therefore to investigate the effectiveness of systemic administration of scAAV9/hMECP2 on survival and other phenotypes in the Mecp2T158M/y knock‐in mice.

A myc‐tagged human MECP2_e1 minigene construct packaged in a self‐complementary AAV2/9 vector (10Λ12 vg/mouse) was delivered intravenously into juvenile Mecp2T158M/y mice which were then monitored for survival, body weight and RTT‐like phenotype severity score.

Treated Mecp2T158M/y mice had significantly extended survival (p = 0.001) and increased body weight (p < 0.001) compared to vehicle‐treated mice. However, there was no improvement in the aggregate severity score. Brain transduction efficiency was relatively low (1–3% of DAPI‐positive cells), which likely explains the lack of impact on neurological phenotypes. Similar results were obtained in Mecp2‐/y KO mice. These data support the concept that the action of exogenous MeCP2 is unlikely to be confounded by deleterious interactions with endogenous mutated MeCP2 protein. Currently, we are optimizing the route of administration and vector design to enhance brain transduction and maximize therapeutic efficacy.

PO19 Transitioning RACE‐seq, a mechanism of action confirmation assay for DNA‐directed RNAi gene therapy, from bench to the bedside

Usher Louise 1 Kortschak Chris Moschos Sterghios A. 1

1 Dept. Of Biomedical Sciences, University of Westminster, London, UK,

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

The therapeutic potential of small interfering RNA (siRNA) and antisense molecules as potent mediators of posttranscriptional gene silencing remains of substantial interest, with a number of drugs currently in clinical trials. Among these, TT‐034 is a phase I/Iia‐stage recombinant adeno‐associated virus serotype 8 vector, developed as a single‐dose gene therapy for Hepatitis C virus (HCV). TT‐034 acts via a DNA‐directed RNA interference (ddRNAi) mechanism of action (MOA). The vector expresses three short hairpin RNA (shRNA) pro‐drugs that are processed into effector siRNAs. These target the HCV RNA genome for site specific, silencer mediated, siRNA‐induced silencing complex (siRISC) cleavage. We have previously used Rapid Amplification of cDNA Ends with next generation sequencing (RACE‐seq) on Illumina to investigate Dicer processing of TT‐034 shRNA analogues.

Here we report on an adaptation of RACE‐seq for generating and sequencing these low complexity libraries on the bedside‐relevant Ion Torrent platform. Results with synthetic siRNA and synthetic shRNA are discussed with a focus on read length/depth, quality and MOA interpretation. Furthermore, a simplified, low computational power data analysis pipeline is proposed, in line with a bedside, minimally invasive theranostic assay suitable for demonstrating on‐target MOA in phase II and III clinical trials and beyond. We propose RACE‐seq as a suitable solution for investigating, discriminating and quantifying specific RNA cleavage events and an ideal tool for producing direct evidence for an on‐target MOA of RNA‐ targeting therapeutics.

PO20 Site specific labelling of adeno‐associated virus identifies targets for enhancing viral transduction efficiency

Chandran Jayanth Dr 1 Sharp Paul Dr 2 Collins Mark Dr 3 Azzouz Mimoun Dr 1

1 Department of Neuroscience, Sheffield Institute for Translational Neuroscience (SITraN), The University of Sheffield, 385A Glossop Road, Sheffield S10 2HQ, UK, UK 2 Department of Psychology, The University of Sheffield, Sheffield, UK, UK 3 Department of Biomedical Science, The University of Sheffield, Sheffield, UK, UK

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Adeno‐associated viruses (AAVs) are attractive gene therapy vectors due to their low toxicity, high stability, and ability to ransducer a variety of tissues and cell types. AAVs, however, requires a high vector dosage to achieve a favourable transduction, which increases the risk of inducing a host immune response, and necessitates the identification of molecules capable of improving transduction efficiency. Here, we identify new protein targets for enhancing viral transduction through a site specific modification of the AAV9 viral capsid, enabling us to both track the movement of viral particles, and to isolate proteins forming a complex with the capsid. We identified several different functional pathways associated with the capsid interacting proteins including the DNA damage response, mRNA splicing, epidermal growth factor receptor (EGFR) signalling and intracellular transport, and further validated the screen by identifying two modifiers of AAV9 transduction: histone deacetylase 4 (HDAC4) and splicing factor 2/alternative splicing factor (SF2/ASF). Our data demonstrates a dual utility for a single site‐specific modification in the AAV capsid, and provides a list of proteins targets that can regulate AAV transduction.

PO21 Development of gene therapy for Niemann‐Pick Type C disease

Hughes Michael Mr 1 Smith Dave Mr 2 Morris Lauren Ms 2 Tordo Julie Dr 3 Palomar‐Martin Nuria Ms 3 Henckaerts Els Dr 3 Waddington Simon Dr 4 Platt Fran Professor 2 Rahim Ahad Dr 1

1 Department of Pharmacology, UCL School of Pharmacy, London, UK 2 Department of Pharmacology, University of Oxford, Oxford, UK 3 Department of Infectious Diseases, King's College London, London, UK 4 Institute for Women's Health, University College London, London, UK

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Niemann‐Pick type C is a lysosomal storage disorder with neurological and visceral pathology, for which there is currently no major disease modifying treatment. Loss of NPC1 function, a late endosomal transmembrane protein, leads to systemic intracellular lipid accumulation. However, premature death is usually associated with neurological manifestations, such as neurodegeneration and neuroinflammation. This project focuses on the development of an adeno‐associated viral (AAV) vector capable of delivering and expressing human NPC1 in the mouse brain, via perinatal intracranial or intravenous injection, with the ultimate aim of ameliorating brain and visceral pathology in the Npc1^−/− mouse model.

AAV vectors exhibit efficient and widespread gene delivery throughout the brain, however their limited packaging capacity of 4.7 kb can be a constraint for larger genes. Extensive construct modifications were made to incorporate the relatively large 3.8 kb NPC1 into a functional AAV serotype 9 vector, where NPC1 is controlled by the constitutively active neuronal promoter human synapsin I. Initial in vivo studies demonstrated successful NPC1 over‐expression in administered mouse brains, compared to endogenous NPC1 levels in un‐administered controls. A preliminary very low dose study was carried out on the Npc1^−/− model, where newborn Npc1^−/− mice were administered with 4.6×10⁹ vector genomes of AAV9.SYN1.NPC1 via intracranial injections. Treated Npc1^−/− mice exhibited an increased lifespan (n = 8; median survival of 116.5 days; P < 0.0001) compared to untreated Npc1^−/− mice (n = 6; median survival of 75.5 days). Combined with significant behavioural improvements these results demonstrate the potential beneficial use of gene therapy for Niemann‐Pick type C and support higher dose studies.

PO22 The impact of thaw rate on the recovery and proliferation of MSCs and T cells

Baboo Jasmin Dr 1 Delahaye Mike Dr 1 Ugbeyan Reginald Mr 1 Gaddum Nick Dr 1 Blanco Magdalena Dr 1 Nancekievill Alex Mr 2 Morris John Dr 2 Milne Stuart Dr 2 Callens Sarah Miss 1 Ward Stephen Dr 1

1 Cell and Gene Therapy Catapult, London, United Kingdom 2 Asymptote Ltd, Cambridge, United Kingdom

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Understanding the impact of thaw rate on cryopreserved material can be beneficial in achieving the best quality cells in terms of viable cell recovery and functional performance. While a common perception is that faster thawing is best, there is literature both supporting and against this establishment. Furthermore, the cryopreservation medium and cell type are also contributory factors in determining cell quality post thaw. The aim of this study was to therefore elucidate the impact of thaw rate and cryopreservation medium on T cells and Mesenchymal Stem Cells (MSCs).

Cryopreserved MSCs and T cells (>86% CD3 positive cells) in vials (2 ml Nunc cryovials and 6 ml Aseptic Technology crystal vials), were thawed in a water bath supported by a device to hold and agitate the vial. A range of temperatures (10°C, 37°C, 65°C, 95°C) were used to achieve varying thaw rates whereby the end point of thaw was a small ice crystal. Once thawed, cells were processed to determine the viable recovery (%) and proliferative performance. Within this study two cryopreservation medium were used, with and without 10% DMSO.

When formulated with 10% DMSO, the viable cell recovery (%) for both MSCs and T cells was approximately 70% or above, over the tested thaw rate range. DMSO free media was found to be a poor freezing formulation for T cells, but was effective with MSCs. Unexpectedly, the low rates of thawing used in this study were compatible with high viability on thawing for both MSCs and T cells.

PO23 A fragmented AAV dual vector strategy for delivery of over‐sized genes leads to expression of hybrid transcripts

McClements Michelle E Dr 1 Issa Peter Charbel Professor 1 2 3 Blouin Veronique Dr 4 MacLaren Robert E Professor 1 3 5

1 Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom 2 Department of Ophthalmology, University of Bonn, Bonn, Germany 3 Oxford Eye Hospital, The John Radcliffe Hospital, Oxford, United Kingdom 4 Laboratoire de Thórapie Gónique UMR649, Institut de Recherche Thórapeutique – IRT1, Quai Moncousu, BP 70721, 44007 Nantes cedex, France 5 Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Introduction: Fragmented adeno‐associated virus (fAAV) containing truncated single ITR transgenes have been used for gene replacement in cases where the disease gene is too large to be encoded within a single AAV vector (e.g. ABCA4, the gene most commonly associated with recessively inherited retinal degenerations). We explored the mechanism whereby these vectors could lead to successful reformation of an oversized transgene carrying a complete and correct coding sequence (CDS).

Methods: Fragmented AAV5 and AAV8 vectors were generated by packaging an oversized plasmid, containing the human ABCA4 transgene (6.7 kb) flanked by standard AAV2 inverted terminal repeats (ITRs). Transgenes contained in the fAAV vectors were assessed by Southern blot and used to ransducer HEK293T cells. Expression of ABCA4 was evaluated by qPCR, RT‐PCR and western blot.

Results: The fAAV vectors contained an expected heterogeneous population of truncated transgenes containing a single ITR. Transduction with fAAV vectors led to successful ABCA4 expression as determined by mRNA transcript detection and sequencing. Expression was not however identified at the protein level. Further investigation revealed that the fAAV approach led to a mixed population of transcript forms. These were established to be hybrid ABCA4 transcripts generated by the joining of opposing randomly truncated transgenes.

Conclusion: The identification of stable hybrid transcripts suggests there is more refinement required in the fAAV approach if it is to be pursued in clinical trials.

PO24 Investigating cell fusion between the donor and host retina after photoreceptor precursor transplantation

Barnard Alun Dr 1 Balmer Jasmin Dr 1 Singh Mandeep Dr 1 Moralli Daniela Dr 2 Green Catherine Dr 2 MacLaren Robert Prof 1 3 4

1 Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom 2 Wellcome Trust Centre for Human Genetics, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom 3 Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom 4 Moorfields Eye Hospital, NHS Foundation Trust, London, United Kingdom

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Photoreceptor replacement is a potential treatment for inherited retinal degenerations in which all photoreceptors are lost. Laboratory experiments which use non‐degenerate retinas as host eyes for photoreceptor transplantation are not representative of the clinical scenario because they place transplanted cells in direct contact with host photoreceptors. This close association may facilitate membrane fusion and exchange of cellular contents. We used a range of molecular‐genetic techniques to investigate this possibility. We transplanted GFP‐labelled photoreceptor precursors (Nrl‐GFP) into the subretinal space of adult host mice ubiquitously expressing DsRed (CAG‐DsRed) and found that the majority of GFP‐positive cells in the host outer nuclear layer (ONL) also co‐localised DsRed. In a sex‐mismatched experiment, where GFP photoreceptors precursors from female pups were injected subretinally into adult males, cells in the host ONL that were GFP‐positive were also male, as identified by a Y‐positive nucleus using fluorescent in situ hybridisation. Furthermore, we generated a line that combined GFP‐labelled photoreceptor precursors with a Cre‐sensitive red fluorescent reporter allele (CAG‐LSL‐tdTomato, Nrl‐GFP). When photoreceptor precursors from this line were injected subretinally into adult host mice expressing Cre recombinase, we found cells in the ONL of host eyes that were positive for both GFP and tdTomato, indicating Cre recombinase had been taken up by donor cells. These results can be explained by exchange of cellular contents between transplanted photoreceptor precursor donor cells and cells in the host ONL. The most likely mechanism of intercellular exchange might be cell fusion, which could be utilised as future therapeutic strategy.

PO25 Impact of vital dyes on transduction efficiency of AAV vectors used in retinal gene therapy

Salvetti Anna Paola Dr 1 Patricio Maria Ines Dr 1 Barnard Alun Dr. 1 MacLaren Robert Prof. 1

1 NLO, Nuffield Department of Clinical Neurosciences & Oxford Eye Hospital, Oxford, United Kingdom

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Purpose: Treatment of inherited retinal degenerations (IRD) using adeno‐associated viral (AAV) vectors involves delivery by sub‐retinal injection. In the late stages of IRD, progressive alteration of normal anatomy may cause difficulty in localizing the retinotomy, the retinal detachment (RD) and the AAV vector diffusion. Vital dyes can be useful surgical adjuvants. Their impact on AAV transduction is, however, unknown.

Methods: A range of different dilutions of Sodium Fluorescein (SF, Martindale Pharma) (1:0, 1:50, 1:5.000, 1:50.000 and 1:500.000) and of Membrane Blue Dual (MBD, D.O.R.C.)(1:0, 1:50, 1:125, 1:250 and 1:500) were tested on Human Embryonic Kidney cells (HEK293) in vitro using an AAV2.GFP (green fluorescent protein) reporter. Flow Cytometer (FC) analysis was performed to assess both HEK 293 cell viability and AAV2.GFP transduction efficiency.

Results: Potential toxicity was compared with the negative control. MBD resulted not to be toxic at any dilutions and SF was toxic only when applied undiluted. AAV2.GFP transduction efficiency was tested at 1:5000 for SF and at 1:0, 1:50 and 1: 125 dilutions for MBD. Transduction was not reduced by the presence of the dyes.

Conclusions: Cell viability and transduction efficiency were affected only by the presence of the undiluted SF, but not by the MBD. MBD and diluted SF may therefore be a safe adjuvant during gene therapy surgery to enable a better visualization of the retina and of the vector diffusion.

PO26 Preclinical development of AAV‐NPY gene therapy for epilepsy

Patricio Maria I 1 Barnard Alun R 1 Green Alexander L 2 During Matthew J 3 Sen Arjune 1 MacLaren Robert E 1 4

1 Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK 2 Nuffield Department of Surgery, University of Oxford, Oxford, UK 3 Ohio State University Medical Center, Columbus, USA 4 Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, UK

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Epilepsy is a common condition that is characterised by recurrent, unprovoked seizures, and it is treatable in over 70% of the cases with appropriate medication. A significant proportion of patients however have drug‐resistant epilepsy. Neuropeptide Y (NPY) is an endogenous neurotransmitter found in the brain, including the hippocampus. It acts on G‐protein coupled receptors (GPCR), and rodent studies have shown that overexpression of NPY may protect against seizures.

Here we assessed the effects of AAV‐NPY in a human neuronal cell line. The human NPY coding sequence driven by a chicken β actin (CBA) promoter and augmented by a downstream woodchuck hepatitis virus regulatory element (WPRE) was packaged into recombinant AAV serotype 1 to standards suitable for clinical trial use (rAAV1‐NPY). A GFP reporter vector was used as a negative control (rAAV1‐GFP). Human neuroblastoma cells SH‐SY5Y were first transduced with rAAV1‐GFP at a range of multiplicities of infection (MOI). Abundant green fluorescent cells were detected after 4 days at a MOI of 5,000, with no obvious signs of cellular toxicity. Cells were then exposed to rAAV1‐GFP reporter vector, rAAV1‐NPY therapeutic vector or sham treated with no vector (in triplicate). NPY levels in cell supernatant were assessed by two antibody‐based methods after 4 days. rAAV1‐NPY treated cells showed significantly increased levels of NPY, compared to baseline expression in both the rAAV1‐GFP or sham treated controls.

This work provides useful preclinical data in a relevant human cell line that may facilitate human clinical trials of AAV‐NPY gene therapy in the treatment of epilepsy.

PO27 Experimental modelling of ALS by AAV‐mediated in vivo modulation of the C9ORF72 gene

Herranz‐Martin Saul Dr 1 Lewis Katherine E. Dr 1 Coldicott Ian Mr 1 Chandran Jayanth S. Dr 1 Lukashchuk Vera Dr 1 Iannitti Tommaso Dr 1 Shaw Pamela J. Prof. 1 Azzouz Mimoun Prof. 1

1 University of Sheffield, Sheffield, United Kingdom

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron degeneration. The repeat GGGGCC expansions located in the intronic region of the C9ORF72 gene is the most common mutation linked to ALS, being present in 40% of familial ALS cases. Having more than about 30 repeats can lead to the disease initiation. It is unclear whether the presence of toxic RNA generated after the transcription of these repeats or their further unconventional RAN‐translation to generate toxic dipeptide polypeptides (DPRs) triggers the disease. Here, we assess the use of AAV expressing either RNA or DPRs to model C9ORF72 linked ALS in mice.

Self‐complementary AAV9 (scAAV9) viruses carrying GGGGCC repeats from 10 to 102 or the sequence encoding the DPRs were generated and tested in vitro before using in animals. In vivo experiments were performed by injecting these viral vectors in postnatal day 1 (P1) wild type mice. The phenotype of these animals was evaluated by using behavioural tests and histology analysis.

In comparison to controls, no major differences were detected in animals injected with 10 or 102 repeats. However, mice injected with DPRs showed a time‐dependent decline in rotarod performance, as well as defects in CatWalk gait pattern. Survival was also affected in 34‐injected mice. Histology analysis revealed widespread AAV9‐mediated DPRs expression in the brain and the spinal cord. Together, these findings indicate that DPRs play a key role in the disease initiation. The generated model is being used for gene therapeutic development.

PO28 Investigating the Specificity of RNAi Molecules in Human Gene Therapy for Superoxide Dismutase 1‐linked Familial Amyotrophic Lateral Sclerosis

Scarrott Joseph Mr 1 Ferraiuolo Laura Dr 1 Heath Paul Dr Gelsthorpe Catherine Mrs 1 Kaspar Brian Dr 1 Shaw Dame Pamela Prof. 1 Azzouz Mimoun Prof. 1

1 Sheffield Institute for Translational Neuroscience, SHEFFIELD, United Kingdom

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Aims:

1. Reduce human mutant SOD1 (hSOD1) expression by viral delivered short‐hairpin RNA (shRNA).

2. Investigate and evaluate miRNA‐like sequence specific off‐target effects (OTEs) on gene expression resulting from the viral delivery of a therapeutic shRNA targeting hSOD1.

3. Data will be used to support dossier submission to UK regulatory body in preparation for clinical trials in familial ALS patients.

Methods: Recombinant self‐complementary Adeno‐associated‐virus 9 vector carrying human SOD1‐specific shRNA (scAAV9‐shSOD1) has been validated for SOD1 knockdown in vitro. Gene expression changes were investigated in scAAV9‐shSOD1 transduced HEK293 cells, allowing validation of hSOD1 knockdown by microarray analysis. To investigate potential OTEs in vitro, astrocytes derived from the fibroblasts of ALS patients and healthy controls are being transduced with scAAV9‐shSOD1; seed‐matched non‐SOD1 targeting shRNA and vector only controls. Total RNA extracted from transduced iAstrocytes will be analysed by microarray to elucidate OTEs. Any differential effect of hSOD1 knockdown between sALS, fALS and control iAstrocyte lines will be investigated.

Results: Preliminary microarray analysis of transduced HEK293 cells identified 80 downregulated and 105 upregulated genes (n = 3, p < 0.05, fold change ≥1.5). In silico analysis found 50% of downregulated gene 3′UTR regions contained motifs matching the seed sequence variants of the therapeutic shRNA, putatively identifying their downregulation as a consequence of off‐target gene suppression.

Conclusions: Initial results show that a number of genes in addition to the intended target are downregulated in transduced cells. Further investigation using seed sequence‐matched controls is necessary to validate putative targets of off‐target gene regulation.

PO29 Primary culture of human retinal tissue for pre‐clinical testing of AAV gene therapy

Edwards Thomas Dr 1 2 Hickey Doron Dr 1 2 MacLaren Robert Professor 1 2 3

1 Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom 2 Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Oxford, United Kingdom 3 Moorfields Eye Hospital NHS Foundation Trust, London, United Kingdom

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Introduction: Pre‐clinical evaluation of gene therapy for retinal disease often involves animal models that may not accurately reflect the human condition or its response to treatment. Culture of ex‐vivo human retina provides an opportunity for assessing transduction efficiency and cellular tropism of AAV‐mediated gene therapy in the target tissue and may provide useful information on the effects of capsid modification.

Methods: Patients undergoing retinectomy for retinal detachment at the Oxford Eye Hospital were invited to consent for biopsy of redundant retinal tissue that would be removed during surgery. The explants was cultured in modified neurobasal‐A medium, with AAV transduction after 24 hours and tissue fixation on day 8. AAV vectors expressing green fluorescent protein (GFP) with a ubiquitous promoter (CAG) were packaged into three different capsids: AAV2(QuadY‐F), AAV(7m8) and AAV8(Y773F). Samples were processed for immunohistochemistry and viewed under the confocal laser scanning microscope.

Results: Human retinal explants from three retinectomy cases were successfully cultured ex‐vivo for 8 days. Despite explants generally being biopsied from damaged retina, recognizable inner and outer nuclear layers were visible on DAPI staining. High levels of GFP expression throughout the neurosensory retina were observed with all three vector constructs. No GFP signal was detected in the negative control.

Conclusion: Human retina can be cultured ex‐vivo for at least 8 days, which was sufficient time for AAV transduction and expression of GFP driven by the ubiquitous CAG promoter. Further work is required to fully characterize the cellular tropism of each vector in human retina ex‐vivo.

PO30 Activation of nuclear and cytosolic innate immunity by AAV gene therapy in the retina

Xue Kanmin Dr 1 Fu Howell Mr 1 Caddy Sarah L Dr 2 Barnard Alun R Dr 1 Patricio Maria I Dr 1 James Leo C Dr 2 Rada Cristina Dr 2 MacLaren Robert E Prof 1

1 Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, United Kingdom 2 MRC Laboratory of Molecular Biology, Cambridge, Cambridge, United Kingdom

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Recombinant adeno‐associated viral (AAV) vectors have been shown to be safe and efficacious in a number of gene therapy clinical trials, including the treatment of inherited retinal degenerations. Intracellular immunity against viral DNA can restrict the persistence of several pathogenic viruses (including HIV, hepatitis B and human papilloma virus), but little is known about its role against AAV, which may have a significant impact on the efficacy and durability of gene therapy.

We performed retinal gene therapy on BL/6 mice using recombinant AAV2 encoding green fluorescent protein (GFP) and sham injections to fellow eye. Retinal RNA was purified at baseline, day 3, 7 and 15 post‐treatment. Reverse transcription qPCR was performed to detect the expression of a panel of innate immunity associated genes.

GFP expression was detectable from day 3 onwards in all treated eyes. Delayed induction of Apobec1 and Apobec3 was found on day 7, but other Apobec family members (Apobec2 and Aicda) were not induced. Both APOBEC3 and APOBEC1 have been implicated in targeting viral DNA for mutation within the nucleus through deamination of cytosine to uracil residues, which lead to DNA degradation. We also found AAV‐induced upregulation of cytosolic viral DNA sensors, cGAS and STING, as well as anti‐viral effector cytokines, TNF‐α, IFN‐γ and CXCL10, within the retina.

The data suggest that AAV activates intracellular immune responses within target retinal tissues despite the generally acknowledged low immunogenicity of this target tissue.

PO31 Functional expression of Bestrophin‐1 in vitro following transduction with AAV‐BEST1 vectors

Wood Shaun Mr 1 McClements Michelle E Dr 1 Patricio Maria I Dr 1 Uggenti Carolina Dr 2 Sekaran Sumanthi Dr 1 Manson Forbes D Dr 2 MacLaren Robert E Professor 1 3 4

1 Nuffield Laboratory of Ophthalmology, Department of Clinical Neurosciences, University of Oxford, Oxford, United Kingdom 2 The Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester, Manchester, United Kingdom 3 Oxford Eye Hospital, Oxford University Hospitals NHS Trust and NIHR Biomedical Research Centre, Oxford, United Kingdom 4 Moorfields Eye Hospital NHS Foundation Trust and NIHR Biomedical Research Centre, University College London, Oxford, United Kingdom

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Introduction: Mutations in the human BEST1 gene are responsible for a wide‐range of retinal disorders broadly known as ‘Bestrophinopathies’. The protein product, Bestrophin‐1, is expressed in the retinal pigment epithelium (RPE) where it localises to the basolateral membrane acting as a Ca2+‐activated chloride ion channel. Delivery of Bestrophin‐1 to the RPE cells is a potential treatment option for patients presenting with mutations in BEST1. The purpose of this study was to compare the ability of two recombinant adeno‐associated viral (AAV) vectors to express functional bestrophin‐1 following transduction in Human Embryonic Kidney (HEK) 293 cells.

Methods: HEK293 cells were transduced with AAV2/2 vectors, expressing BEST1 under the control of a ubiquitous chicken beta actin (CAG) promoter, with or without a woodchuck post‐translational regulatory element (WPRE). Bestrophin‐1 expression was assessed by western blot and immunocytochemistry (ICC) against untransduced control cells. Whole‐cell patch clamping was used to measure chloride ion conductance in vitro with extracellular chloride at 146 mM and intracellular chloride at 38.4 mM.

Results: Bestrophin‐1 was detected in cells transduced with both vectors by western blot, with higher expression being obtained from the AAV2/2.CAG.hBEST1.WPRE.pA vector. This result was confirmed by ICC. Increases in chloride ion conductance were seen in HEK293 cells transduced with AAV2/2.CAG.hBEST1.pA and AAV2/2.CAG.hBEST1.WPRE.pA when compared to controls.

Conclusion: The functional expression of bestrophin‐1 from these vectors indicates that both could be used in a gene therapy strategy to treat patients presenting with mutations in BEST1.

PO32 Quantification of Fluorescence in Retinal Sections by Confocal Microscopy following Reporter Gene Delivery

Orlans Harry O Dr 1 2 3 Patricio Maria I Dr 1 Barnard Alun R Dr 1 MacLaren Robert E Prof 1 2 3

1 University of Oxford, Oxford, United Kingdom 2 Moorfields Eye Hospital, London, United Kingdom 3 Oxford Eye Hospital, Oxford, United Kingdom

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Confocal laser scanning microscopy (CM) is frequently used to compare levels of tissue fluorescence following reporter gene transduction. Here we investigated the effect of gain on recorded signal in mouse retinas induced to express green fluorescent protein (GFP), and correlated this with protein expression levels.

Bilateral subretinal injections of an adeno‐associated vector (AAV2/2) delivering GFP driven by a ubiquitous promoter were administered to 7‐week old C57Bl/6J mice (n = 12). Five weeks post‐injection, 12 eyes were sectioned for CM imaging. Serial images were acquired at multiple gains, and mean grey values (GV) for individual retinal layers were calculated using ImageJ software. For the remaining eyes, GFP levels normalised to β‐actin were quantified for retina and remaining eye cup separately by Western blot.

On CM, GFP was confined to the retinal pigment epithelium (RPE) and photoreceptors (PH), being more highly expressed in the former. The effect of gain on GV followed an exponential relationship for all retinal layers (logarithmic transformation R² = 0.9998, p = 8E‐17 for RPE; R² = 0.9911, p = 1.54E‐10 for PH). Mean ratio of GFP eye cups:retinas by Western blot was 6.13 ± 2.0. Mean GV ratio RPE:PH on confocal sections was 5.31 ± 0.84 for low gain value, 6.40 ± 1.16 for moderate gain, and 6.70 ± 1.35 for high gain.

Quantification of fluorescence in CM images reflects expression levels of fluorescent proteins within ocular tissues. Given the exponential relationship between acquisition gain and measured fluorescence, GV ratios between retinal layers within a single image are likely to be underestimated at lower levels of gain and overestimated at higher levels.

PO33 Adeno‐associated virus gene therapy for the treatment of epidermal growth factor receptor positive (EGFR) lung cancer

Zaki Kam Dr 1 4 Agundez Leticia Dr 2 Sanchez Belinda Dr 3 Linden Michael Prof 2 Henckaerts Els Dr 2 Takeuchi Yasu Dr 4 Collins Mary Prof 1 4

1 Division of Advanced Therapies, National Institute for Biological Sciences & Control, Potters Bar, United Kingdom 2 Department of Infectious Diseases, King's College London School of Medicine, London, United Kingdom 3 Cuban Centre of Molecular Immunology, Havana, Cuba 4 Division of Infection and Immunity, University College London, London, United Kingdom

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Introduction: Gene therapy using recombinant adeno‐associated virus (rAAV) encoding monoclonal antibody (mAb) sequence injected into mice muscle has been shown to produce sustained serum mAb level capable of protecting animals from infectious diseases in mouse models. We aim to develop a protocol to achieve a therapeutic serum level of 7A7, an anti‐epidermal growth factor receptor (EGFR) mAb in mouse models for the treatment of EGFR‐positive lung cancer.

Materials and Methods: rAAV encoding 7A7 light and heavy chains driven by tMCK, a muscle specific promoter (AAV8‐7A7) was produced. Two doses of AAV8‐7A7 vector was injected into the muscle of healthy C57Bl/6 mice. Serial bleeds were performed at appropriate intervals. Detection of serum 7A7 was done using ELISA with human EGFR (hEGFR) antigen. Luciferase clones of murine 3LL‐D122 and human A431 cell lines were used to establish subcutaneous and metastatic mouse tumour models. Tumour monitoring was done by caliper measurements and in vivo imaging system (IVIS).

Results: ELISA using hEGFR was able to detect serum 7A7 in mice following AAV8‐7A7 administration. Higher dose of viral vector results in higher dose of serum mAb. Subcutaneous tumour models with luciferase clones of 3LL‐D122 and A431 cell lines have been established and detectable on IVIS.

Conclusions: Administration of AAV8‐7A7 into mice muscle resulted in a dose‐dependent detectable level of serum 7A7 mAb using ELISA. Establishment of metastatic mouse tumour models and the effect of AAV8‐7A7 on these tumours are currently on‐going. Updated results will be presented.

PO34 Preparation of seed stocks of Pluripotent Stem Cells for human application: “A stitch in time saves nine”!

Stacey Glyn Dr 1 Hunt Charles Dr 1 Man Jennifer Dr 1 Taylor Hannah Dr 1 Iriajen Angela 1 Abranches Elsa Dr 1 O'Shea Orla Dr. 1 Barbato Michela 1 Chapman Charlotte 1 Shephard Matthew 1 Nowell Craig Dr. 1 Collins Mary Dr. 1

1 UK Stem Cell Bank, Division of Advanced Therapies, National Institute for Biological Standards and Control, Medicines and Health‐care Regulatory Agency, South Mimms, United Kingdom

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

The raw‐ and starting materials such as culture media components and production cell lines require careful consideration in order to enable a reliable and safe cell therapy product. Regulators will often say “it is not possible to test safety into a product” and early and robust risk assessment and traceability of these materials is crucial to minimise risk to patients and investors. The UK Stem Cell Bank (UKSCB) is a key component of the UK regenerative medicine infrastructure charged with procuring, processing (banking and testing) and distributing seed stocks of human pluripotent stem cell (hPSC) lines for research and human application and is equipped with laboratories designed to meet EU GMP. In its latest phase of development, the UKSCB is focused on 41 hPSC lines established in the UK, under appropriate conditions and traceability. Key elements of the UKSCB process are ethics review, “due diligence”, processing, storage and distribution; all of which are designed to meet the requirements of the UKSCB HTA license, which has been maintained successfully for more than ten years. All cell lines are subject to ethical scrutiny by a national ethics committee, following which the UKSCB performs a due diligence protocol which establishes whether each cell line could meet European requirements (i.e. EUTCD) and provide a potential starting material for clinical trials. The first of these cell lines has now been banked, representing the start of a pipeline of hPSC lines that will be made available for research or commercial development internationally, under a non‐exclusive license.

PO35 Pre‐existing immunity to human parainfluenza virus (hPIV) does not affect rSIV.F/HN‐mediated transduction efficiency.

Pytel Kamila M. Dr 1 4 Chan Mario MSc 1 4 Meng Cuixiang BSc 1 4 Martinez Andrea Rodriguez MRes 1 Boyd Christopher A. Dr 2 4 Davies Jane C. Prof 1 4 Gill Deborah R. Prof 3 4 Hyde Stephen C. Prof 3 4 Innes Alastair J. Dr 2 4 Alton Eric WFW. Prof 1 4 Griesenbach Uta Prof 1 4

1 Imperial College London, London, United Kingdom 2 University of Edinburgh, UK, Edinburgh, United Kingdom 3 Gene Medicine Research Group, University of Oxford, Oxford, United Kingdom 4 UK Cystic Fibrosis Gene Therapy Consortium, United Kingdom

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

Lentiviral vector pseudotyped with the Sendai virus envelope proteins F and HN (rSIV.F/HN) transduces lungs efficiently, and can be repeatedly administered. These features make the virus a suitable vector for a range of diseases including cystic fibrosis (CF). Sendai virus has significant sequence homology with hPIV1 and ∼80% of adults carry anti‐hPIV1 antibodies. In preparation for a first‐in‐man CF trial we assessed whether hPIV1 antibodies inhibit rSIV.F/HN transduction efficiency. We passively immunised mice with human immunoglobulins (IVIg) administered either intraperitoneally (400 μl) or intranasally (100 μl) and showed that, using these doses, the concentration of hPIV1 antibodies in mouse serum and broncho‐alveolar lavage fluid (BALF) was as high as or higher than in human serum and BALF. Next we transduced mice with rSIV.F/HN‐lux intranasally 24 hrs after immunisation and showed that transduction efficiency was not affected by the presence of hPIV1 antibodies. To confirm results in human samples we performed in vitro transduction inhibition assays in serum positive or negative (n = 4/group) for hPIV1 antibodies and showed that both groups inhibited rSIV.F/HN transduction, whereas rSIV.VSVG control virus was significantly (p < 0.01) less affected. Subsequently we quantified hPIV1 IgG and IgA antibodies in BALF, grouped them into positive and negative cohorts and performed a transduction inhibition assay to address whether hPIV1 antibodies in epithelial lining fluid (ELF) inhibit rSIV.F/HN efficiency. There was no significant difference between the groups, suggesting that hPIV1 antibodies in ELF do not inhibit rSIV‐F/HN transduction. The data suggest that hPIV1 antibodies will not inhibit rSIV.F/HN transduction in human lung.

PO36 An evaluation of lentiviral mediated gene delivery to Schwann cells for the purpose of tissue engineering

Busuttil Francesca Ms 1 Hughes Michael Mr 1 Bhangra Kulraj Singh Mr 2 Phillips James Dr 2 Rahim Ahad Dr 1

1 Department of Pharmacology, School of Pharmacy, University College London, 29–39 Brunswick Square, London WC1N 1AX, UK., London, United Kingdom 2 Department of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, University College London, 256 Gray's Inn Road, London WC1X 8LD, UK, London, United Kingdom

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Peripheral nerve injury (PNI) affects up to 5% of trauma patients and is associated with motor, sensory and autonomic impairment in the affected area. Following PNI, axonal regeneration is dependent on Schwann cells, which express regeneration‐assisting genes. However, this pro‐regenerative phenotype is not maintained long enough to ensure functional recovery. Tissue engineered three dimensional (3D) constructs containing therapeutic cells that maintain the appropriate ‘repair Schwann cell’ phenotype may help enhance nerve regeneration.

This in vitro proof‐of‐concept study delivered luciferase and green fluorescent protein genes to Schwann cells and assessed whether the transduced cells were still viable for construction of a 3D collagen scaffold used to enhance nerve repair. Schwann cells were successfully transduced using the lentiviral vector produced. The highest transduction efficiency was obtained at a multiplicity of infection of 100. This was confirmed by fluorescence microscopy, bioluminescence imaging and flow cytometry. Following incorporation into the collagen constructs, stereoscopic fluorescence microscopy and confocal microscopy showed that the transduced cells had distributed evenly. Bioluminescence imaging confirmed that the F7 cells were still viable after seeding into the constructs. No fluorescence or bioluminescence was observed in the untransduced cells or in the control constructs seeded with the untransduced cells.

These results suggest that gene therapy augmented Schwann cells could be used to engineer scaffolds that can provide an enhanced environment for nerve repair. We are currently investigating a number of regeneration‐assisting genes which can be delivered to Schwann cells to enhance nerve regeneration following PNI as part of a tissue engineering approach.

PO37 Calcineurin Abeta1 overexpression using an adeno‐associated virus vector (AAV9) demonstrates decreased pathological remodelling and decreased expression of heart failure markers in ischemic and non‐ischemic heart disease.

Clinton Enda Mr 1 Villalba Maria Dr. 1 Salinero Jesus M Gómez Dr. 1 López‐Olañeta Marina Miss 1 Cruz Francisco Miguel Mr. 1 Bernal Juan Antonio Dr. 1 Sanz‐Rosa David Dr. 2 Ibáñez Borja Dr. 1 Lara‐Pezzi Enrique Dr. 1

1 Fundación Centro Nacional De Investigaciones Cardiovasculares Carlos III, Madrid, Spain 2 European University of Madrid, Madrid, Spain

Poster Presentations (odd numbers), Exhibition, poster and catering areas, April 15, 2016, 12:45 – 13:15

In the adult heart, calcineurin activates the transcription factor NFAT, which leads to cardiac hypertrophy and eventually to heart failure. Alternative splicing of the calcineurin mRNA produces the CnAbeta1 isoform, which has a unique C‐terminal domain that activates AKT instead of NFAT. In mice, CnAbeta1 overexpression in the heart as late as 1 week post‐infarction results in improved cardiac function and reduced infarct size. To investigate its potential as a gene therapy, we developed an adeno‐associated virus 9 (AAV9) vector in which CnAbeta1 and luciferase expression are controlled by the cardiac‐specific cTnT promoter. Mice that received a systemic injection of AAV9‐CnAbeta1 showed tissue specific luciferase expression up to 21 days post injection. In a mouse model of aortic stenosis in which pathological hypertrophy is induced by pressure overload, treatment with AAV9‐CnAbeta1 reduced cardiac hypertrophy, improved function and reduced adverse remodelling. Decreased expression of heart failure markers and fibrosis was also observed. We also tested the potential of AAV9‐CnAbeta1 for the treatment of ischemic heart disease in a mouse model of myocardial infarction. Systemic administration of AAV9‐CnAbeta1 resulted in decreased expression of heart failure markers, reduced adverse remodelling and improved function. These results highlight the therapeutic potential of transient CnAbeta1 overexpression post onset of ischemic and non‐ischemic heart disease using an AAV9 vector.

PO38 Vectors for the chemogenetic dissection of the central noradrenergic system allow dissociation of affective actions

Hirschberg Stefan Mr 1 Li Yong PhD 1 Randall Andrew PhD 3 Kremer Eric PhD 2 Pickering Anthony PhD 1 1 University of Bristol, Bristol, United Kingdom 2 Institut de Gónótique Molóculaire de Montpellier, Montpellier, France 3 University of Exeter, Exeter, United Kingdom

Poster Presentations (even numbers), Exhibition, poster and catering areas, April 15, 2016, 12:15 – 12:45

Noradrenaline reuptake inhibitors are amongst the frontline treatments against neuropathic pain, and believed to act by restoring noradrenergic tone in the spinal dorsal horn (SDH). However, their use is commonly associated with CNS side‐effects limiting their therapeutic utility. The Locus coeruleus (LC) is the principle noradrenergic nucleus in the brain and the main source of spinal noradrenaline. Using retrograde viral vectors we have shown that LC neurons projecting to the SDH are distinct from those innervating the medial prefrontal cortex (mPFC) (DOI: 10.1016/j.brainres.2016.02.023). Here we explore the hypothesis that some adverse effects are caused by tonic increased noradrenergic activity in the mPFC.

We developed lentiviral vectors to locally transduce the LC and canine adeno virus type 2 (CAV2) based vectors to retrogradely target specific subpopulations. Both restrict the expression of EGFP and PSAM to noradrenergic neurons by a specific promotor (PRSx8). PSAM is an engineered excitatory receptor‐ionophore specifically activated by a selective agonist PSEM308.

After transduction EGFP immunofluorescence was restricted to neurons that were positive for noradrenergic markers (98.5+/−0.7%). In vivo neuronal recordings from identified LC units showed time locked and dose dependent excitations to PSEM308 application, indicating functional transduction. Conditioned place aversion to 10 mg/Kg PSEM308 intraperitoneally was seen after direct LC transduction and after retrograde transduction from the mPFC. The same drug had no significant effect on rats expressing PSAM in the spinally projecting subpopulation or control animals.

These findings suggest that by using a selective therapeutic strategy to activate pontospinal noradrenergic neurons it may be possible to dissociate the analgesic benefits from the confounding CNS side effects.