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1034. Gene Therapy with Decorin Reduces Diabetic Organomegaly in Rats
GENE THERAPY FOR BONE, CONNECTIVE TISSUE AND SKIN TISSUES 1035. Biodistribution and Expression of AAVhuTNFR:Fc after Intra-Articular Injection to Cynomolgus Monkeys Dana B. Martin,1 Tara Allen,1 Ed Kelly,1 Ziv Sandalon,1 Linda Wilson,1 Lauren M. Tatalick,1 Carmel Lynch.1 1 Preclinical Biology, Targeted Genetics, Seattle, WA.
1034. Gene Therapy with Decorin Reduces Diabetic Organomegaly in Rats N. S. Nahman, Jr.,1 A. Bringardner,1 U. Y. Bhatt,1 T. J. Sferra,2 M. Husa,1 T. Verrill,1 J. Vaughn.1 1 Medicine/Nephrology, The Ohio State University, Columbus, OH; 2 Pediatrics, The Ohio State University, Columbus, OH. Decorin is a small glycoprotein known to inhibit the biologic effects of the pro-sclerotic cytokine, TGF-b. Glucose-driven increases in TGF-b are thought to underlie accumulation of matrix protein in diabetes, causing glomerulosclerosis and nephromegaly in the kidney, and organomegaly of the liver and heart. On this basis, we theorized that decorin may retard matrix protein synthesis and organomegaly in diabetes. To test this question in vitro, we assessed the effect of decorin on glucose-driven fibronectin (FN) synthesis in cultured human mesangial cells (HMC). HMC were transduced with an adenovirus generating the human decorin transgene (rAdDec, a generous gift of P. Lowenstein, UCLA) or a control virus (rAd), and exposed to 10 or 50 mM glucose. After 24 hrs, FN gene transcription (quantitative PCR) from cells exposed to 50 mM and treated with rAdDec, was significantly lower than cells exposed to 50mM+rAd (41±0.04 % of 50mM+rAd, mean±SEM, p<0.05). This was similar to cells treated with 10mM+rAd (31±0.04 % of 50mM+rAd p<0.05). To assess the effect of decorin in vivo, 4 SD rats were rendered diabetic with streptozotocin and after 4 weeks, transduced with 109 rAdDec particles (DM-Dec) via carotid injection, as we have described (Kidney Int 58:1500). After 10 wks of diabetes (6 wk post virus injection), the animals were sacrificed and liver, kidney and heart weights determined. When compared to 10 week diabetics (DM) from our previous work (AJKD 23:105), decorin therapy significantly reduced liver, kidney, and heart weights (table). In 4 other rats, increased hepatic expression of the decorin transgene was demonstrated at 5 wks using quantitative PCR (7.4±9.3 fold increase in DM-Dec vs DM). CONCLUSION: Systemic injection of a decorin transgene reduces the organomegaly of diabetes, presumably by antagonizing TGF-b induced matrix protein synthesis. Decorin may be useful in controlling matrix accumulation in diabetes, and thus help reduce the systemic complications of the disease. Effect of decorin on organ weight (g/100g BW) Organ DM DM-Dec Liver 5.4+/-.23 4.3+/-.29* Kidney 0.63+/-.03 0.49+/-.03* Heart 0.59+/-.03 0.35+/-.04* * p < 0.05 vs DM
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
AAV-huTNFR:Fc is an AAV2 vector carrying a soluble TNF receptor. AAV-huTNFR:Fc is for use in the treatment of TNF-αmediated inflammatory arthritides such as rheumatoid arthritis (RA) and psoriatic arthritis (PsA). Intra-articular treatment would allow a high local TNFR:Fc expression level while minimizing systemic levels. The biodistribution and expression of AAV-huTNFR:Fc, was investigated after a single intra-articular injection in cynomolgus monkeys. Vector was administered to the right knee joint at a dose of 2.4 X1011, 3 X1012 and 3X1013 DRP of AAV-huTNFR:Fc. Vector was administered to an additional group of 2 animals at the high dose of 3X1013 IM injection to assess systemic expression. A human TNFR:Fc specific ELISA assessed vector expression in the serum and joint-lavage fluid on days 1 (prior to dosing), 30, 60 and 90. In addition, serum samples were also analyzed for anti-AAV neutralizing antibody titer and anti-huTNFR:Fc antibody titer. Animals were euthanized on day 90 and the injection site, iliac lymph node and spleen were collected for vector biodistribution and transgene expression analysis by vector specific DNA PCR and mRNA-specific RT-PCR, respectively. As expected, all animals developed a neutralizing antibody response to AAV2 by day 30 confirming exposure to vector. On day 30 circulating TNFR:Fc protein was detected in serum and joint lavage fluid samples of both monkeys given tgAAV-huTNFR:Fc by the IM route. However, assessing expression of huTNFR:Fc at day 60 and 90 were inconclusive due to inhibition of the spike recovery in the assay. This inhibition is likely secondary to an anti-huTNFR:Fc antibody response in the non-human primate model. Sporadic huTNFR:Fc transgene expression was detected in the serum and joint lavage fluid of animals dosed by the IA route and detection may be limited by anti-huTNFR:Fc antibody production. Vector was detected in the joint at day 90 in a dose-dependent manner. In conclusion, local delivery of AAV-huTNFR:Fc may be therapeutic in RA and PsA patients with a limited number of remaining painful joints despite current systemic therapy.
1036. Platelet-Derived Growth Factor Gene Delivery Promotes Periodontal Tissue Repair Qi-ming Jin,1 Orasa Anusaksathian,1 Sarah A. Webb,1 William V. Giannobile.1 1 Center for Craniofacial Regeneration and Dept. of Periodontics, Prevention, and Geriatrics, University of Michigan, Ann Arbor, MI. Platelet-derived growth factor (PDGF) has potent effects on the wound repair including regeneration of tooth-supporting (periodontal) structures. Limitations of protein delivery to periodontal defects include transient biological activity of PDGFs and bioavailability of factors at the wound site. The objective of this investigation was to determine the ability of in vivo PDGF-B gene transfer to stimulate periodontal regeneration in large surgicallycreated periodontal alveolar bone wounds. Osseous defects were treated with a 2.6% collagen carrier or carrier containing recombinant adenoviruses encoding a control virus luciferase (Ad-luc; 5 x 108 particles (PN)/wound), or PDGF-B (Ad-PDGF-B at two dose levels (5 x 108 (low dose)/wound or 5 x 109 PN (high dose)/wound). Parameters of periodontal wound repair were measured and included bone length, bone area and length of new cementum using image analysis. The collagen carrier with or without genes was delivered to mandibular first and second molars (n=6 animals/group) in SpragueS399
1034. Gene Therapy with Decorin Reduces Diabetic Organomegaly in Rats N. S. Nahman, Jr.,1 A. Bringardner,1 U. Y. Bhatt,1 T. J. Sferra,2 M. Husa,1 T. Verrill,1 J. Vaughn.1 1 Medicine/Nephrology, The Ohio State University, Columbus, OH; 2 Pediatrics, The Ohio State University, Columbus, OH. Decorin is a small glycoprotein known to inhibit the biologic effects of the pro-sclerotic cytokine, TGF-b. Glucose-driven increases in TGF-b are thought to underlie accumulation of matrix protein in diabetes, causing glomerulosclerosis and nephromegaly in the kidney, and organomegaly of the liver and heart. On this basis, we theorized that decorin may retard matrix protein synthesis and organomegaly in diabetes. To test this question in vitro, we assessed the effect of decorin on glucose-driven fibronectin (FN) synthesis in cultured human mesangial cells (HMC). HMC were transduced with an adenovirus generating the human decorin transgene (rAdDec, a generous gift of P. Lowenstein, UCLA) or a control virus (rAd), and exposed to 10 or 50 mM glucose. After 24 hrs, FN gene transcription (quantitative PCR) from cells exposed to 50 mM and treated with rAdDec, was significantly lower than cells exposed to 50mM+rAd (41±0.04 % of 50mM+rAd, mean±SEM, p<0.05). This was similar to cells treated with 10mM+rAd (31±0.04 % of 50mM+rAd p<0.05). To assess the effect of decorin in vivo, 4 SD rats were rendered diabetic with streptozotocin and after 4 weeks, transduced with 109 rAdDec particles (DM-Dec) via carotid injection, as we have described (Kidney Int 58:1500). After 10 wks of diabetes (6 wk post virus injection), the animals were sacrificed and liver, kidney and heart weights determined. When compared to 10 week diabetics (DM) from our previous work (AJKD 23:105), decorin therapy significantly reduced liver, kidney, and heart weights (table). In 4 other rats, increased hepatic expression of the decorin transgene was demonstrated at 5 wks using quantitative PCR (7.4±9.3 fold increase in DM-Dec vs DM). CONCLUSION: Systemic injection of a decorin transgene reduces the organomegaly of diabetes, presumably by antagonizing TGF-b induced matrix protein synthesis. Decorin may be useful in controlling matrix accumulation in diabetes, and thus help reduce the systemic complications of the disease. Effect of decorin on organ weight (g/100g BW) Organ DM DM-Dec Liver 5.4+/-.23 4.3+/-.29* Kidney 0.63+/-.03 0.49+/-.03* Heart 0.59+/-.03 0.35+/-.04* * p < 0.05 vs DM
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
AAV-huTNFR:Fc is an AAV2 vector carrying a soluble TNF receptor. AAV-huTNFR:Fc is for use in the treatment of TNF-αmediated inflammatory arthritides such as rheumatoid arthritis (RA) and psoriatic arthritis (PsA). Intra-articular treatment would allow a high local TNFR:Fc expression level while minimizing systemic levels. The biodistribution and expression of AAV-huTNFR:Fc, was investigated after a single intra-articular injection in cynomolgus monkeys. Vector was administered to the right knee joint at a dose of 2.4 X1011, 3 X1012 and 3X1013 DRP of AAV-huTNFR:Fc. Vector was administered to an additional group of 2 animals at the high dose of 3X1013 IM injection to assess systemic expression. A human TNFR:Fc specific ELISA assessed vector expression in the serum and joint-lavage fluid on days 1 (prior to dosing), 30, 60 and 90. In addition, serum samples were also analyzed for anti-AAV neutralizing antibody titer and anti-huTNFR:Fc antibody titer. Animals were euthanized on day 90 and the injection site, iliac lymph node and spleen were collected for vector biodistribution and transgene expression analysis by vector specific DNA PCR and mRNA-specific RT-PCR, respectively. As expected, all animals developed a neutralizing antibody response to AAV2 by day 30 confirming exposure to vector. On day 30 circulating TNFR:Fc protein was detected in serum and joint lavage fluid samples of both monkeys given tgAAV-huTNFR:Fc by the IM route. However, assessing expression of huTNFR:Fc at day 60 and 90 were inconclusive due to inhibition of the spike recovery in the assay. This inhibition is likely secondary to an anti-huTNFR:Fc antibody response in the non-human primate model. Sporadic huTNFR:Fc transgene expression was detected in the serum and joint lavage fluid of animals dosed by the IA route and detection may be limited by anti-huTNFR:Fc antibody production. Vector was detected in the joint at day 90 in a dose-dependent manner. In conclusion, local delivery of AAV-huTNFR:Fc may be therapeutic in RA and PsA patients with a limited number of remaining painful joints despite current systemic therapy.
1036. Platelet-Derived Growth Factor Gene Delivery Promotes Periodontal Tissue Repair Qi-ming Jin,1 Orasa Anusaksathian,1 Sarah A. Webb,1 William V. Giannobile.1 1 Center for Craniofacial Regeneration and Dept. of Periodontics, Prevention, and Geriatrics, University of Michigan, Ann Arbor, MI. Platelet-derived growth factor (PDGF) has potent effects on the wound repair including regeneration of tooth-supporting (periodontal) structures. Limitations of protein delivery to periodontal defects include transient biological activity of PDGFs and bioavailability of factors at the wound site. The objective of this investigation was to determine the ability of in vivo PDGF-B gene transfer to stimulate periodontal regeneration in large surgicallycreated periodontal alveolar bone wounds. Osseous defects were treated with a 2.6% collagen carrier or carrier containing recombinant adenoviruses encoding a control virus luciferase (Ad-luc; 5 x 108 particles (PN)/wound), or PDGF-B (Ad-PDGF-B at two dose levels (5 x 108 (low dose)/wound or 5 x 109 PN (high dose)/wound). Parameters of periodontal wound repair were measured and included bone length, bone area and length of new cementum using image analysis. The collagen carrier with or without genes was delivered to mandibular first and second molars (n=6 animals/group) in SpragueS399