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416 rAAV Vector Development for Local Treatment of Osteoarthritis Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy S159 MUSCULOSKELETAL CELL[.]
MUSCULOSKELETAL CELL AND GENE THERAPY exercise intolerance, a dropped jaw, and a hoarse bark We recently reported that a single intravascular injection of adeno-associated virus serotype (AAV8) vector expressing myotubularin under the muscle-specific desmin promoter in XLMTM dogs markedly improved muscle weakness and prolonged lifespan To measure clinical response to this therapy, we developed a neuromuscular assessment score, graded on a scale from 10 (normal) to (unable to maintain sternal recumbency) Repeated examinations of wild type dogs by one observer yielded a consistent score in 98% (45/46) of exams Two reviewers scoring 110 neurological examinations agreed on the score in 75% of cases, with agreement within point in 93% of cases Assessment scores positively correlated with isometric hind limb strength, a well-established measure of disease severity We applied this clinical measure to follow response to gene replacement in dogs Four groups were studied in a blinded manner: Wild-type or female carrier controls (N=6); affected dogs given saline only (N=4); affected dogs given AAV8-MTM1 (5e12 vg/kg) (N=3) or (2.5e13 vg/kg) (N=3) AAV8-MTM1 was administered at 10 weeks of age and dogs were followed for months Results: Differences in neuromuscular assessment scores were detected between XLMTM and wild type dogs prior to treatment at 10 weeks of age (p=0.0009) At 17 weeks of age, differences were detected between treatment groups (p=0.0001) At 17 weeks of age, no significant differences were detected between affected and low dose treated dogs or between wild type and high dose treated dogs Together, these data indicate that the neuromuscular assessment score developed for this canine congenital neuromuscular disorder is a reliable and valid measure of clinical response to gene therapy 415 AAV9 Improves Lysosomal Organization and Diaphragmatic Contractile Function in Pompe Disease A Gary Todd,1 Bumsoo Ahn,2 Jessica A McElroy,1 David D Fuller,3 Barry J Byrne,1 Leonardo F Ferreira,2 Darin J Falk.1 Pediatrics, University of Florida, Gainesville, FL; 2Applied Physiology and Kinesiology, University of Florida, Gainesville, FL; 3Physical Therapy, University of Florida, Gainesville, FL Pompe disease is a neuromuscular disorder resulting from lysosomal storage of glycogen in individuals lacking the glycogen degrading enzyme alpha acid glucosidase (GAA) Progressive glycogen accumulation within the lysosome leads to muscle weakness and ultimately respiratory failure The purpose of this study was to determine if AAV9-hGAA can restore myofibrillar diaphragmatic contractile function following intrathoracic administration in a murine model of Pompe disease Myofibers were isolated from wild-type (WT) or Pompe (Gaa-/-) and AAV9-hGAA treated Gaa-/- mouse diaphragms at months of age (5 months post injection) While no significant difference in the rate of tension development was observed between groups the 20% decrease in maximal calcium-activated specific force usually observed in Gaa-/- mice (117.8 ± 8.4 kN/m2) was significantly improved to levels resembling wild-type following AAV9 treatment (AAV9-treated 143.7 ± 5.1 kN/m2; WT 147.9 ± 9.5 kN/m2) Biochemical analysis of diaphragm lysates revealed a significant increase in GAA enzyme levels compared to Gaa-/and WT animals Confocal microscopy confirmed both absence of lysosomal GAA and lysosomal aggregation in Gaa-/- single fibers and subsequent AAV9-mediated lysosomal hGAA targeting in treated animals Our data suggest that AAV9-hGAA remediates the decreased myofibrillar force and restores proper lysosomal organization in a murine model of Pompe disease 416 rAAV Vector Development for Local Treatment of Osteoarthritis Sirkka R M Kyostio-Moore,1 Patricia Berthelette,1 Susan Piraino,1 Nance Moran,2 Joseph Serriello,2 Bindu Nambiar,1 Cathleen Sookdeo,1 Alison Bendele,3 Dinesh Bangari,4 Patty Ewing,4 Jennifer Sullivan,1 Gary White,1 Abraham Scaria,1 Gloria L Matthews,2 Donna Armentano.1 Gene Therapy, Genzyme, a Sanofi Company, Framingham, MA; 2Orthopaedic Research, Genzyme, a Sanofi Company, Framingham, MA; 3Bolder BioPATH, Boulder, CO; 4Pathology, Genzyme, a Sanofi Company, Framingham, MA Localized delivery of rAAV vectors allows therapeutic agent production mainly at the application site thereby providing highest concentration at the site and reducing chance of adverse events at off-target locations As there are currently no disease-modifying agents for osteoarthritis (OA), the osteoarthritic joint represents an ideal target for testing local gene delivery In particular, the synovial tissue surrounding the joint space is an accessible depot for therapeutic agent production We have been evaluating rAAV vectors for synovial transduction using both mouse and rabbit joints as models for intraarticular injections and have shown that rAAV capsid tropism is distinct in these two species In osteoarthritic STR/ort mice, viral vectors with AAV1 capsids demonstrated the highest transduction of various joint cells while little was seen with AAV5 capsids (Osteoarthritis Cartilage 2013, 21:358) However, in normal rabbit joints AAV5 and AAV2 capsid based vectors (encoding BMP7) efficiently transduced synovial tissue as assessed by rAAV vector genome copy numbers Both of these vectors also produced detectable levels of BMP7 in the synovial fluid confirming that transduced synovial tissue expressed and efficiently secreted vector encoded transgene product We also evaluated various agents for efficacy in both the mouse and rabbit OA models In the spontaneous osteoarthritic STR/ort mice, AAV1 Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy S159 MUSCULOSKELETAL CELL AND GENE THERAPY mediated heme-oxygenase (HO-1) gene delivery to joint to test the role of oxidative stress did not reduce disease progression as measured by cartilage degradation and synovial pathology as HO-1 levels were already endogenously elevated In a rabbit surgery-induced OA model, a rAAV2 vector was used to overexpress cystatin C (cysC), an endogenous cathepsin K (catK) inhibitor, to examine the role synovial catK in cartilage degradation Efficient gene transfer into synovial tissues was confirmed by detection of both rAAV vector genomes and cysC mRNA Furthermore, a significantly reduced catK activity in the synovium indicated the production of functional therapeutic agent However, no improvement was observed in the cartilage pathology demonstrating that the synovial catK did not play a significant role in cartilage degradation in this model We are currently generating additional rAAV vectors expressing agents with potential disease-modifying properties in an effort to identify therapeutics for osteoarthritis 417 Correlation between Inflammation Biomarkers and Clinical Scores Following Treatment of TGF-β1 Transduced Chondrocyte, TissueGene-C Bumsup Lee,1 Jungjong Cho,1 Taewon Kim,1 Yeomyeong Park,1 Moonjong Noh,1 Kwan-hee Lee.1 Kolon Life Science, Inc., Gwacheon-si, Gyeonggi-do, Republic of Korea Purpose Osteoarthritis (OA) is a degenerative disease with mild inflammatory response, but is little known about how the level of inflammation is related to severity of OA symptom and tissue destruction The level of circulating acute phase protein, C-reactive protein (CRP) has been widely used as a diagnostic marker in acute inflammatory diseases The matrix metalloproteinase-mediated C-reactive protein (CRPM) is a hallmark of chronic tissue inflammation in the knee joint of OA patients TG-C contains non-transduced (hChonJ) and transduced (hChonJb#7) human allogeneic chondrocytes The hChonJb#7 cells were transduced with TGF-β1 gene-containing retroviral vector The aim of this study was to evaluate correlation between inflammatory biomarkers in OA patients and the changes of clinical scores of IKDC and WOMAC to estimate the prognostic feature for this TG-C therapy Methods Data from clinical scores of IKDC, WOMAC, IKDC6-0(the changed value of IKDC score evaluated months post treatment of TG-C) and WOMAC6-0(the changed value of WOMAC score evaluated months post treatment of TG-C) were collected from patients during TG-C clinical trials, phase IIa (KS-TGC-01-2a) and phase IIb (KS-TGC01-2b) The clinical trials of TG-C included patients with advanced joint damage (K&L grade 3, ICRS grade IV in main cartilage lesion) and various range of knee clinical scores The level of serum high sensitive CRP (hsCRP) and CRPM were detected by enzyme linked immunosorbent assay (ELISA) The baselines of clinical scores in each study were compared by a non-parametric rank test; (WilcoxonMann-Whitney test) The correlation between baseline serum hsCRP and CRPM with IKDC6-0 and WOMAC6-0 were assessed by Spearman correlation coefficient Results Although the basal level of hsCRP in serum of the patients was not different from its reference level (3ug/ml) in healthy adults, the level of serum CRPM was mildly elevated in these patients compared to normal reference level (5ng/ml) There were no statistically significant differences between phase IIa study (n=19) and phase IIb study (n=51) with regard to the baseline values of serum hsCRP and IKDC score However, Serum CRPM (p=0.0007) and WOMAC score (p=0.03) were significantly elevated in the phase IIb study compared with the scores observed in the phase IIa study None of two markers showed a statistically S160 significant correlation with basal knee scores or changes in knee scores in all of the study arms However serum CRPM showed a consistent inverse correlation with change in IKDC score (IIa low; IIa high; IIb TG-C r=-0.67; -0.42; -0.40) and an inverse correlation with change in WOMAC score (r=0.26; 0.34; 0.31) in the treatment arms, but not in the placebo arm Conclusion Although this study was not able to suggest that the level of CRPM could be a prognostic marker for the TG-C therapy in the given patients, we could identify that TG-C treatment is tend to be more effective in the patients with low CRPM over the patients with high CRPM at months of post treatment Based on the results above, we are going to investigate CRPM as a prognostic marker in phase clinical trials of TG-C 418 The Effect of TissueGene-C, TGF-β1 Transduced Chondrocyte, on Pain Improvement and Cartilage Regeneration in a Rat Osteoarthritis Model Bumsup Lee,1 Kyoungbaek Choi,1 Hyeonyoul Lee,1 Heonsik Choi,1 Taewon Kim,1 Hyesun Lee,1 Sujeong Kim.1 Kolon Life Science, Inc., Gwacheon-si, Gyeonggi-do, Republic of Korea Purpose: TissueGene-C (TG-C) has been developed as a cell and gene therapy for patients with osteoarthritis It is a mixture of nontransduced human chondrocytes (hChonJ) and genetically modified chondrocytes expressing TGF-β1 (hChonJb#7) Phase 2b clinical trials have shown that TG-C significantly improved IKDC and 100 mm VAS scores over placebo when administered to the knee joints of osteoarthritic patients In this study, a rat model of osteoarthritis was used to identify the role of individual components of TG-C Methods: A rat model of osteoarthritis was induced by injection of monosodium iodoacetate (MIA) into the left knee joint, which resulted in the degeneration of the joint cartilage and pain The rats showing pain-related behaviors were selected for the evaluation of TG-C for its individual cell type (1 hChonJ; hChonJb#7; or TG-C(hChonJ and hChonJb#7)) for pain and cartilage regeneration At two weeks post MIA administration, the individual cells were injected into the osteoarthritic knee cavity The effect of the cells on the mechanical allodynia was evaluated by von Frey filament test in blinded fashion After completing the test, the knees were harvested for histological analysis Results: In MIA model, the reduction of pain was observed in hChonJ#7 treated and TG-C treated groups from day 15 post injection but not in hChonJ alone or in untreated group The magnitude of pain reduction in hChonJ#7 treated group was comparable to that of TG-C treated group up to day 28 However, TG-C showed longer and greater pain reduction thereafter up to weeks, indicating synergy between hChonJ and hChonJ#7 cells According to the analysis, hChonJ#7 cell treatment improved cartilage severely damaged by MIA and TG-C treatment showed even greater improvement in the regeneration of the cartilage Conclusion: This study showed that TG-C treatment could reduce pain and regenerate damaged cartilage in a rat MIA osteoarthritis model and contributes to the understanding of the effect of TG-C on the improvement of clinical outcomes observed in phase 2b trials Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy ... the knee joints of osteoarthritic patients In this study, a rat model of osteoarthritis was used to identify the role of individual components of TG-C Methods: A rat model of osteoarthritis was... feature for this TG-C therapy Methods Data from clinical scores of IKDC, WOMAC, IKDC6-0(the changed value of IKDC score evaluated months post treatment of TG-C) and WOMAC6-0(the changed value of WOMAC... We are currently generating additional rAAV vectors expressing agents with potential disease-modifying properties in an effort to identify therapeutics for osteoarthritis 417 Correlation between