57 Acid alpha Glucosidase Gene Replacement Therapy to the Diaphragm in Ventilator Dependent Pompe Disease One Year Respiratory Motor Outcomes Molecular Therapy Volume 21, Supplement 1, May 2013 Copyri[.]
CLINICAL GENE & CELL THERAPY 55 Preclinical Safety Studies for AAV2-MerTK Gene Therapy Vector for Retinitis Pigmentosa Thomas J Conlon,1 Wen-Tao Deng,2 Renee C Ryals,2 Kirsten E Erger,1 Travis L Cossette,1 Shannon E Boye,2 Isaam McDoom,2 Nathalie Clemente,1 Brian Cleaver,1 Mark Potter,1 Corinne Abernathy,3 Sanford L Boye,2 William W Hauswirth.2 Pediatrics, University of Florida, Gainesville, FL; 2Opthamology, university of Florida, Gainesville, FL; 3Clinical and Translational Science Institute, University of Florida, Gainesville, FL Purpose Efficacy of MERTK gene replacement therapy has been demonstrated using different viral vectors in the Royal College of Surgeons (RCS) rat, a well studied model of recessive retinitis pigmentosa (RP) that contains a mutation in the Mertk gene MERTK is expressed in the RPE cells and plays a key role in renewal of photoreceptor outer segments (OS) by phagocyting shed OS tips Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space and ultimately result in retinal degeneration The results of three separate Phase I clinical trials for Leber’s Congenital Amaurosis type (LCA2) demonstrated the safety of AAV in human patients AAV is well-suited for human gene therapy due to its low immunogenic profile In the present study, we conducted a series of preclinical efficacy and safety evaluations of an AAV2 vector expressing human MERTK cDNA driven by a RPE-specific VMD2 promoter, including a formal GLP toxicology and biodistribution study Methods Efficacy of AAV2-VMD2-hMERTK vector was evaluated by subretinally injected into one eye of RCS rats and retinal function was assayed electroretinographically months post-injection Safety and biodistribution of the vector was assessed in SD rats by subretinal injection of BSS control and two different dosed of AAV vector at a total dose of 4x108 or 4x109 vg The effect of the vector was investigated by electroretinogram, fundoscopic analysis, and gross anatomy in the injected eyes Potential toxicity was studied by organ hispathology and clinical pathology at different time points up to 90 days post-injection Results Efficacy of the vector was demonstrated in RCS rat which showed significant improved ERG responses in treated eyes compared to contralateral untreated eyes Delivery of AAV-MERTK in SD rat eyes at two different doses did not result in a change in ERG magnitude of rod and cone responses relative to BSS control injected eyes indicating that administration of AAV vector did not adversely affect retinal function Fundoscopic analysis and gross anatomy of the injected eyes were normal compared to injected controls There were no gross or microscopic findings or presence of transformed cells in the blood that were related to administration of the test article as assessed by histopathology and blood smear All injected eyes and day blood samples were positive for vector genomes and all peripheral tissues were negative Conclusions Our results demonstrate the efficacy and safety of the AAV2-VMD2hMERTK vector in animal models tested A GMP vector has been manufactured and is presently in clinical trial Supported by The Foundation Fighting Blindness 56 Glaucoma-GT, a Novel Gene Therapy Treatment for Primary Open-Angle Glaucoma Katie Binley,1 Scott Ellis,1 Vicky Scripps,1 Sharifah Iqball,1 Stuart Naylor,1 Kyriacos Mitrophanous.1 Oxford BioMedica (UK) Ltd, Oxford, United Kingdom Glaucoma is the second leading cause of blindness worldwide, affecting around 70 million people, over 2.5 million people in the USA alone It is characterized by irreversible degeneration of the optic nerve, usually associated with an elevated intraocular pressure (IOP) Prostenoid analogues such as Latanoprost are a first-line treatment for glaucoma due to their high level of efficacy and low risk of side-effects, but fail to halt disease progression in many patients due to non-adherence, which can be > 50% Surgery is often Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy required, which is expensive and only partially effective There is therefore a real medical need for a novel drug that overcomes this issue of poor compliance Initial proof-of-concept for this approach has been demonstrated (Barraza et al, 2010), and we are now moving this into a translational gene therapy platform for clinical evaluation Glaucoma-GT is a gene therapy product aimed at lowering IOP via the prostenoid pathway: a single transcorneal administration leads to the expression of human cyclooxygenase-2 (COX-2) and prostaglandin F (FP) receptor in the front of the eye COX-2 is a rate-limiting enzyme in the biosynthesis of prostaglandins, and both COX-2 and the FP receptor are down-regulated in glaucomatous eyes Expression of both genes increases both prostaglandin 2 (PGF2) biosynthesis and signaling, increasing aqueous outflow and reducing longterm IOP Gene transfer following transcorneal administration of EIAV-GFP vector was characterised in animal models Various vector genome configurations were assessed for the production of PGF2 and activation of the FP receptor in in vitro assays Further preclinical studies are currently ongoing and data from these will be presented Transcorneal injection of EIAV vector led to the transduction of cells of the trabecular meshwork and corneal endothelium in vivo Different configurations of expression cassette gave a broad range of PGF2 and FP activation, with CMV-COX-2-IRES-FP having the highest activity We have demonstrated significant gene transfer following transcorneal administration of EIAV-GFP vector in animal models, and have optimised the therapeutic expression cassette to produce high levels of prostaglandin 2 and enhanced FP activation in in vitro studies These vector configurations will be assessed for IOP lowering in a relevant animal model Clinical Gene & Cell Therapy 57 Acid alpha-Glucosidase Gene Replacement Therapy to the Diaphragm in Ventilator-Dependent Pompe Disease: One-Year Respiratory Motor Outcomes Barbara K Smith,1 Daniel Martin,1 Lee Ann Lawson,2 Cathryn S Mah,2 Saleem Islam,3 Thomas Conlon J Conlon,2 Dawn Phillips,3 Shelley Collins,3 Barry J Byrne.2 Physical Therapy, University of Florida, Gainesville, FL; Pediatrics and Powell Gene Therapy Center, University of Florida, Gainesville, FL; 3Pediatrics and Surgery, University of Florida, Gainesville, FL Pompe disease is a neuromuscular disease caused by diminished or absent acid alpha-glucosidase (GAA) enzyme, which results in lysosomal glycogen accumulation in striated muscle and motor neurons The early-onset form leads to severe cardiac hypertrophy and respiratory failure The currently approved treatment for Pompe disease, recombinant enzyme replacement therapy, reduces the severity of cardiomyopathy and increases survival However, many surviving patients experience progressive ventilatory insufficiency and may eventually require mechanical ventilation (MV) Preclinical data in the murine model of Pompe disease showed a diaphragmatic AAV-GAA vector corrected defects of phrenic motor function Therefore, our objective was to initiate an open-label, Phase I/ II clinical study of AAV-GAA gene therapy to the diaphragm We hypothesized AAV-GAA gene therapy would correct accumulation of muscle glycogen and promote retrograde transport of AAV-GAA to restore respiratory motor function To date, five children (ages 2-15) with full-time MV dependence on chronic ERT have completed enrollment, received intramuscular rAAV1-CMV-GAA vector delivery into the diaphragm, and concluded follow-up ventilatory testing 365 days after dosing No serious vector-related adverse events occurred and no clinically significant changes in safety labs were noted Respiratory motor assessments showed improvements in maximum tidal volume and maximum voluntary ventilation in S23 CLINICAL GENE & CELL THERAPY the subjects Furthermore, all subjects also were able to generate improved spontaneous breathing without MV assistance (190-2900% improvement over baseline levels) In conclusion, the initial results support the safety and feasibility of AAV-mediated gene therapy for ventilatory failure in early-onset Pompe disease 58 Long Term CD4 Reconstitution in HIV Subjects Receiving ZFN CCR5 Modified CD4 T-Cells (SB-728-T) May Be Attributed to the Sustained Durability of the Central Memory T-Cell Subset Gary K Lee,1 Joumana Zeidan,2 Jay Lalezari,3 Ronald Mitsuyasu,4 Shelley Wang,1 Marty Giedlin,1 Geoff Nichol,1 Winson Tang,1 Dale Ando,1 Rafick-Pierre Sekaly.2 Sangamo BioSciences, Richmond, CA; 2Vector & Gene Therapy Institute, Port St Lucie, FL; 3Quest Clinical Research, San Francisco, CA; 4University of California, Los Angeles, CA Background: HIV subjects Infused with SB-728-T led to sustained increases in CD4 cell counts that peaked at 7-14 days post infusion Assuming normal distribution of input cells, a median 2.1-fold higher CD4 cell number than predicted was observed, suggesting expansion of SB-728-T post infusion At year, CD4 counts were restored and maintained at or above 500 cells/mm3 in of treated subjects, a level commonly used to guide initiation of HAART therapy In this study, we report characterization of CD4 T-cells by naive/memory subtypes, and demonstrate that long term immune reconstitution may be attributed to the durability of the central memory CD4 subset Methods: Aviremic HIV subjects with CD4 counts between 200-500 cells/mm3 received 20 (n=2) or 30 billion (n=3) SB-728-T cells The memory/naive composition of the CD4 compartment was determined by FACS in the SB-728-T products, at baseline (pre-infusion), and at early (14-28 days), and late (6-7 months, and 11-12 months) time points post-infusion Memory CD4 subsets were sorted and CCR5 modification levels were analyzed Results: Peripheral blood CD4 count (mean+SD) increased from 410+94 cells/mm3 at baseline to 956+485 at Day 14 and 680+339 at M12 after treatment A specific ZFN mediated CCR5 modification, as measured by nested PCR and representing 25% of all modifications, ranged from 1.3 to 3.8% of CD4 T-cells at Day 14 and persisted for the duration of followup After infusion, transitional memory CD4 (TTM) count peaked at Day 14 and the levels at this early time point were positively associated (r=0.9, p=0.083) with the improvement in CD4 count Central memory CD4 (TCM) peaked at Month and levels were positively correlated with late CD4 reconstitution (r=0.9, p=0.083) Unlike TTM, TCM counts remained elevated at Month 12 The level of CCR5 modified cells in the effector memory (TEM) and TTM compartments decreased from the peak over time (median 0.8 to 0.3% and 1.8 to 0.9% respectively) while CCR5 modification level in the TCM compartment was maintained over the same time frame (median 1.4% to 1.7%) Conclusions: SB-728-T infusion is safe and provides sustained improvements in CD4 counts that appear to be mediated by long-term reconstitution of the critical TCM compartment Our preliminary results suggest that increased CD4 counts post infusion of SB-728-T may be due to the durable maintenance of TCM cells that are CCR5 modified and potentially enhanced cell survival of endogenous TCM cells, a surrogate marker of slow disease progression S24 59 Cardiovascular Toxicity and Titin CrossReactivity of Affinity-Enhanced TCR-Engineered T Cells Against HLA-A1 Restricted MAGE-A3 Antigen Marcela V Maus,1 Gerald P Linette,2 Edward A Stadtmauer,1 Aaron P Rapoport,3 Bruce L Levine,1 Lyndsey A Emery,1 Leslie Litzky,1 Gwendolyn K Binder-Scholl,4 Dominic P Smethurst,4,5 Andrew B Gerry,4 Nick J Pumphrey,4 Alan Bennet,4 Joanna Brewer,4 Jane Harper,5 Namir Hassan,5 Bent K Jakobsen,4,5 Michael Kalos,1 Carl H June.1 Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA; 2Siteman Cancer Center, Washington University School of Medicine, St Louis, MO; 3The Greenebaum Cancer Center, University of Maryland, Baltimore, MD; 4Adaptimmune Ltd, Abingdon, OX, United Kingdom; 5Immunocore Limited, Abingdon, OX, United Kingdom We initiated clinical testing of engineered T cells expressing an affinity-enhanced T cell receptor that recognizes the MAGE-A3 HLA-A*0101-EVDPIGHLY antigen complex Pre-clinical studies were highly promising with regard to specificity and efficacy of the affinity-enhanced TCR, and a clinical study in stage III/IV melanoma and relapsed multiple myeloma was initiated The first patient infused with the MAGE TCR had metastatic melanoma, and had received extensive prior therapy The patient died days after T cell infusion from cardiac failure Autopsy revealed severe underlying ASCVD with two prior silent Mis and the initial cause of death was attributed to hemorrhagic MI precipitated by demand ischemia As a result, additional cardiac screening and monitoring was implemented Following FDA review, both studies were reopened The second patient had relapsed multiple myeloma after receiving several prior treatment regimens The patient developed cardiogenic shock and died days after infusion There was evidence of in vivo T cell expansion and T cell activation in both patients The levels of T cell markings were highest in blood, heart, and spleen in the first patient, and in blood and pericardial fluid in the second patient Immunohistochemical evaluation of the heart revealed CD3+ T cell infiltrates in the myocardium of both patients MAGE-A3 or related MAGE proteins were not detected in cardiomyocytes or heart autopsy tissue by quantitative RT-PCR TCR heterodimer formation was not detected using dextramer staining compared to V antibody staining of the MAGE TCR expressing cells in vivo No MAGE TCR T cell reactivity was detected against a panel of over 30 cardiomyocyte cells However, a three-dimensional beating cardiomyocyte culture derived from iPS cells, triggered T cell killing; further systematic investigation of TCR binding and reactivity revealed the TCR recognized an unrelated peptide derived from Titin, which is highly expressed in muscle tissue but was not expressed in most cell lines tested in standard cultures The iPS cardiomyocytes were confirmed to express Titin These results indicate that recognition of similar but unrelated peptides presents the most pertinent safety risk for T cell-based therapies and have highlighted the need for additional preclinical screening assays which may help to mitigate this risk in future clinical trials (see our companion abstract “Identification of an alternative specificity for engineered T cells expressing an enhanced affinity MAGE A3 TCR” , for a detailed discussion on Titin discovery and future safety testing) Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy ... levels) In conclusion, the initial results support the safety and feasibility of AAV-mediated gene therapy for ventilatory failure in early-onset Pompe disease 58 Long Term CD4 Reconstitution in HIV... suggesting expansion of SB-728-T post infusion At year, CD4 counts were restored and maintained at or above 500 cells/mm3 in of treated subjects, a level commonly used to guide initiation of HAART therapy. ..CLINICAL GENE & CELL THERAPY the subjects Furthermore, all subjects also were able to generate improved spontaneous breathing without MV assistance (190-2900% improvement over baseline levels)