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674. Molecular, Biochemical and Biomechanical Analysis of Articular Cartilage Repaired with Genetically Modified Chondrocytes Expressing Insulin-Like Growth Factor-I
GENE THERAPY FOR CONNECTIVE TISSUE in salivary glands after gene transfer to non-obese diabetic (NOD) mice, which develop exocrine gland infiltrates and are a commonly used disease model for SS. We constructed two serotype 2 rAAV vectors, rAAVSPI-6 and rAAVLacZ (encoding β-galactosidase; vector control) using the CMV promoter. rAAVs (1010 particles/gland) were administered directly into both submandibular glands of 8-week old female NOD mice (n = 8) via retroductal delivery. Salivary flow rates were determined before vector delivery and at time of sacrifice (16-weeks old). Serum glucose levels and weight were measured weekly and diabetic mice (serum glucose ≥ 400 mg/dL) were treated with insulin. After sacrifice, submandibular glands were harvested and analyzed for inflammatory infiltrates (focus scores), as well as SPI-6 expression and cytokine profile [mouse interleukins (IL)-2, 4, 6, 12, 18, interferon (IFN)-γ, tumor necrosis factor-α and RANTES] in aqueous gland extracts. At 8 weeks, salivary flow rates were not different between mice assigned to the rAAVSPI-6 and rAAVLacZ groups. In contrast, at 16 weeks flow rates were significantly higher in the rAAVSPI-6treated mice (p = 0.018). Submandibular gland IL-10 levels were significantly increased (p = 0.009), while IFN-γ levels were significantly decreased (p = 0.029), in the rAAVSPI-6 mice compared to the rAAVLacZ group. There were no differences in focus scores, serum glucose levels or animal weights between the two treatment groups at 16 weeks. Overall, these results suggest that SPI-6 gene transfer may be beneficial in limiting the autoimmune exocrinopathy of NOD mice.
673. Plasmid-Based GHRH Supplementation Delivered by Electroporation as a Treatment of Arthritis/Laminitis in Horses Patricia A. Brown,1 David M. Hood,2 Ruxandra Draghia-Akli.1 Research and Clinical Services, ADViSYS, Inc., The Woodlands, TX; 2Hoof Diagnostic and Rehabilitation Clinic, Texas A&M University, College Station, TX. 1
Background. Osteoarthritis is a significant social and economic problem in the elderly and continued research and improvements in therapy are needed. Horses have osteoarthritic conditions that are similar to humans, thus, the horse can be used as a model to investigate gene transfer as a potential therapeutic modality for the treatment of osteoarthritis. In aging mammals, the GHRH-GH-IGF-I axis undergoes considerable decrement with reduced GH secretion and IGF-I production associated with a loss of skeletal muscle mass, osteoporosis, arthritis, increased fat deposition and decreased lean body mass. Stimulation of the GH axis can reverse many of the agerelated declines in muscle mass and decrease symptoms of arthritis. Toxicology study. An initial toxicology study was performed in horses using 2.5mg of myogenic GHRH-expressing plasmid, to determine if the GHRH plasmid delivery by intramuscular injection followed by electroporation was safe and effective. This 180-day study involved six normal adult mares (mean age 4.8±1.06 years). None of the horses used in this study suffered any detectable adverse reactions or complications following treatment. Treated animals exhibited increases in body mass and erythrocyte production as well as decreases in ACTH and cortisol levels. Laminitis/Arthritis cases. A second proof-of-concept study was designed as a randomized study of eight horses with chronic laminitic/ arthritic conditions. In this study, four horses were treated with GHRH-expressing plasmid. Two of the horses had arthritis and laminitis, and two had only arthritic symptoms. Following treatment, subjects were followed for 6 months with clinical evaluations completed at 30-day intervals. An additional four horses with similar clinical disease served as non-treated controls. Evaluation parameters consisted of 1) radiographic evaluation, 2) physical lameness
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assessment, 3) quantitative force plate evaluation defining voluntary limb load and load distribution profile, 4) body condition score, 5) body mass, 6) a complete blood count, and 7) a standardized chemistry panel. GHRH plasmid-treated laminitis/arthritis-affected horses demonstrated a significant improvement in lameness status as detected by both physical and force plate assessment. At 6 months post-treatment, neither subject in the treatment group required systemic analgesics and the horses were rated as pasture sound. Physical and radiographic evaluation of the hooves demonstrated significant improvement. The ability to maintain an adequate body condition and body mass improved significantly following treatment. Non-laminitic arthritic subjects demonstrated an initial improvement in lameness status and body condition. Lameness in the non-treated controls was exacerbated over the 6-month study, and one animal had to be euthanized. Conclusions. Plasmid-mediated GHRH supplementation delivered by intramuscular injection followed by electroporation is a safe, effective therapy which can be used to treat laminitis/arthritis in horses. Further studies are needed to assess its effectiveness in treating arthritis in humans.
674. Molecular, Biochemical and Biomechanical Analysis of Articular Cartilage Repaired with Genetically Modified Chondrocytes Expressing Insulin-Like Growth Factor-I Laurie R. Goodrich,1 Eric Strauss,2 Chris T. Chen,2 Chisa Hidaka,2 Paul D. Robbins,3 Chris H. Evans,4 Alan J. Nixon.1 1 College of Veterinary Medicine, Cornell University, Ithaca, NY; 2 Institute for Sports Medicine Research, Hospital for Special Surgery, New York, NY; 3Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA; 4 Orthopedic Surgery-Brigham & Women’s Hospital, Harvard Medical School, Boston, MA. Insulin-like growth factor-I (IGF-I) has been shown to be important for normal articular chondrocyte function. Gene therapy offers the prospect of extending the time IGF-I is released in cartilage injury. The purpose of this study was to investigate the molecular, biochemical and biomechanical effects of AdIGF-I genetically modified chondrocytes on the repair and surrounding tissue compared to defects implanted with naïve chondrocytes. Sixteen horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint was repaired with AdIGF-I transduced chondrocytes (20 x 106) and the opposite was implanted with the same number of control (uninfected) chondrocytes. Horses were sacrificed 4 weeks, 9 weeks, and 8 months. At necropsy, the gross appearance of repair tissue was evaluated. Repair tissue was removed for RNA and DNA. Histological, immunohistochemical, in situ hybridization, biochemical analysis for collagen Type II and proteoglycan content were performed. Taqman PCR was performed to measure mRNA content of repair tissue to compare IGF-I and collagen type II. Biomechanical analysis was also performed. Gross and histological scores were improved for the genetically modified repair tissue at 4 and 9 weeks and 8 months. Type II collagen content was also increased in the AdIGF-treated group and in immunohistochemical analysis as well as by biochemical analysis. Taqman PCR analysis revealed IGF-I and Collagen Type II expression in the AdIGF-treated group was elevated over control (p<0.05) at 4 weeks (Figure 1 and 2). The biomechanical properties of the treated and control tissues did not differ between treatment groups. The present study evaluated the short and long-term effects of AdIGF-I-transduction of chondrocytes prior to implantation. Treatment of chondrocytes with AdIGF-I increased IGF-I expression in the repaired defect for 4 weeks. This increase correlated with an Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
GENE THERAPY FOR CONNECTIVE TISSUE increase in Collagen Type II expression as well as an increase in the percentage of Collagen Type II for up to 8 months. These findings suggest that chondrocyte-based gene transfer may be a promising strategy of improving cartilage repair.
greater cell survival after repair with AdIGF-I-modified cells when compared with control (P<0.05). At 8 months there was a trend towards greater cell survival in defects repaired with IGF-I expressing chondrocytes (P=.06). Chondrocyte survival in this study was positively influenced by genetic modification with AdIGF-I. While the range of percentage donor cells surviving (0-8%) is low, it is consistent with other studies quantifying perichondrial cell or chondrocyte survival in articular cartilage defects. Previously we have reported an average 4% survival of chondrocytes at 8 months following repair using this equine model and this PCR technique. While in that study, no differences were detected between BMP7-treated and control chondrocytes, the current study clearly shows a correlation between IGF-I treatment and cell survival, especially at the early time point.
675. Enhanced Survival of Chondrocytes over Expressing Insulin like Growth Factor I in Equine Cartilage Repair Laurie R. Goodrich,1 Chisa Hidaka,2 Mary L. Strassheim,1 Paul D. Robbins,3 Chris H. Evans,4 Alan J. Nixon.1 1 Clinical Sciences, Cornell University College of Veterinary Medicine, Ithaca, NY; 2Institute for Sports Medicine Research, Hospital for Special Surgery, New York, NY; 3Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA; 4Orthopedic Surgery-Brigham & Women’s Hospital, Harvard Medical School, Boston, MA. Recent studies have shown that insulin-like growth factor (IGFI) plays a powerful role in cell survival and prevention of programmed cell death. When recombinant IGF-I is combined with chondrocyte transplants, local upregulation of matrix production occurs in experimentally induced full-thickness cartilage defects resulting in morphological improvement of cartilage at the site of cartilage injury. This study was designed to test whether genetic modification of chondrocytes to over express IGF-I could enhance cartilage repair by improving chondrocyte survival. Sixteen horses arthroscopically had a 15 mm cartilage defect made in both femoropatellar joints. The defects were repaired with AdIGFI transduced chondrocytes in one limb and naïve chondrocytes in the opposite limb. Four horses were necropsied at 4 and 9 weeks and 8 horses at 8 months postoperative. Ten 10 mg of DNA was harvested from the repair tissue and Taqman™ quantitative realtime polymerase chain reaction (PCR) was used to assess the number of copies of the Y-chromosome marker SRY. A standard curve for the SRY was constructed using genomic DNA from male chondrocytes. A conversion factor of 6.6pg of DNA per diploid cell was used to calculate percentage of cell survival. A paired t test was used to compare cell survival between IGF-I-treated and control groups at each time point. Significance was set at P<0.05. Average percentage of SRY-positive (male, donor) cells present in the defects at 4 weeks, 9 weeks and 8 months was 4.8%, 0%, and 0.9% respectively (Figure 1). At 4 weeks there was a significantly Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
676. Local Stimulation of Articular Cartilage Repair by Transplantation of Encapsulated Chondrocytes Overexpressing Human FGF-2 In Vivo Gunter Kaul,1 Magali Cucchiarini,1 David Zurakowski,2 Stephen B. Trippel,3 Dieter Kohn,1 Henning Madry.1 1 Laboratory for Experimental Orthopaedics, Saarland University, Homburg, Saarland, Germany; 2Department of Biostatistics and Medicine, Harvard Medical School, Boston, MA; 3Department of Orthopaedic Surgery, Indiana University, Indianapolis, IN. Background Defects of articular cartilage are an unsolved problem in orthopaedics. Application of fibroblast growth factor-2 (FGF-2) protein has been demonstrated to modulate articular cartilage repair in vivo. We previously reported that overexpression of FGF-2 selectively stimulated cell proliferation in an ex vivo model of chondrocyte transplantation and that transgene expression remains present in vivo for at least 30 days when transfected chondrocytes are implanted in osteochondral cartilage defects. In the present study, we tested the hypothesis that gene transfer of human fibroblast growth factor 2 (FGF-2) via transplantation of encapsulated genetically modified articular chondrocytes stimulates chondrogenesis in cartilage defects in vivo. Methods Lapine articular chondrocytes overexpressing a lacZ or a human FGF-2 gene sequence were encapsulated in alginate and further characterized. The resulting lacZ or FGF-2 spheres were applied to cylindrical osteochondral defects (3.2-mm in diameter) in each patellar groove in the knee joints of rabbits. Cartilage repair was assessed based on Safranin O-stained sections using a previously published histological grading system developed to quantitate the repair of cartilage defects at three and fourteen weeks after implantation. This grading system ranges from 0 points (normal
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673. Plasmid-Based GHRH Supplementation Delivered by Electroporation as a Treatment of Arthritis/Laminitis in Horses Patricia A. Brown,1 David M. Hood,2 Ruxandra Draghia-Akli.1 Research and Clinical Services, ADViSYS, Inc., The Woodlands, TX; 2Hoof Diagnostic and Rehabilitation Clinic, Texas A&M University, College Station, TX. 1
Background. Osteoarthritis is a significant social and economic problem in the elderly and continued research and improvements in therapy are needed. Horses have osteoarthritic conditions that are similar to humans, thus, the horse can be used as a model to investigate gene transfer as a potential therapeutic modality for the treatment of osteoarthritis. In aging mammals, the GHRH-GH-IGF-I axis undergoes considerable decrement with reduced GH secretion and IGF-I production associated with a loss of skeletal muscle mass, osteoporosis, arthritis, increased fat deposition and decreased lean body mass. Stimulation of the GH axis can reverse many of the agerelated declines in muscle mass and decrease symptoms of arthritis. Toxicology study. An initial toxicology study was performed in horses using 2.5mg of myogenic GHRH-expressing plasmid, to determine if the GHRH plasmid delivery by intramuscular injection followed by electroporation was safe and effective. This 180-day study involved six normal adult mares (mean age 4.8±1.06 years). None of the horses used in this study suffered any detectable adverse reactions or complications following treatment. Treated animals exhibited increases in body mass and erythrocyte production as well as decreases in ACTH and cortisol levels. Laminitis/Arthritis cases. A second proof-of-concept study was designed as a randomized study of eight horses with chronic laminitic/ arthritic conditions. In this study, four horses were treated with GHRH-expressing plasmid. Two of the horses had arthritis and laminitis, and two had only arthritic symptoms. Following treatment, subjects were followed for 6 months with clinical evaluations completed at 30-day intervals. An additional four horses with similar clinical disease served as non-treated controls. Evaluation parameters consisted of 1) radiographic evaluation, 2) physical lameness
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assessment, 3) quantitative force plate evaluation defining voluntary limb load and load distribution profile, 4) body condition score, 5) body mass, 6) a complete blood count, and 7) a standardized chemistry panel. GHRH plasmid-treated laminitis/arthritis-affected horses demonstrated a significant improvement in lameness status as detected by both physical and force plate assessment. At 6 months post-treatment, neither subject in the treatment group required systemic analgesics and the horses were rated as pasture sound. Physical and radiographic evaluation of the hooves demonstrated significant improvement. The ability to maintain an adequate body condition and body mass improved significantly following treatment. Non-laminitic arthritic subjects demonstrated an initial improvement in lameness status and body condition. Lameness in the non-treated controls was exacerbated over the 6-month study, and one animal had to be euthanized. Conclusions. Plasmid-mediated GHRH supplementation delivered by intramuscular injection followed by electroporation is a safe, effective therapy which can be used to treat laminitis/arthritis in horses. Further studies are needed to assess its effectiveness in treating arthritis in humans.
674. Molecular, Biochemical and Biomechanical Analysis of Articular Cartilage Repaired with Genetically Modified Chondrocytes Expressing Insulin-Like Growth Factor-I Laurie R. Goodrich,1 Eric Strauss,2 Chris T. Chen,2 Chisa Hidaka,2 Paul D. Robbins,3 Chris H. Evans,4 Alan J. Nixon.1 1 College of Veterinary Medicine, Cornell University, Ithaca, NY; 2 Institute for Sports Medicine Research, Hospital for Special Surgery, New York, NY; 3Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA; 4 Orthopedic Surgery-Brigham & Women’s Hospital, Harvard Medical School, Boston, MA. Insulin-like growth factor-I (IGF-I) has been shown to be important for normal articular chondrocyte function. Gene therapy offers the prospect of extending the time IGF-I is released in cartilage injury. The purpose of this study was to investigate the molecular, biochemical and biomechanical effects of AdIGF-I genetically modified chondrocytes on the repair and surrounding tissue compared to defects implanted with naïve chondrocytes. Sixteen horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint was repaired with AdIGF-I transduced chondrocytes (20 x 106) and the opposite was implanted with the same number of control (uninfected) chondrocytes. Horses were sacrificed 4 weeks, 9 weeks, and 8 months. At necropsy, the gross appearance of repair tissue was evaluated. Repair tissue was removed for RNA and DNA. Histological, immunohistochemical, in situ hybridization, biochemical analysis for collagen Type II and proteoglycan content were performed. Taqman PCR was performed to measure mRNA content of repair tissue to compare IGF-I and collagen type II. Biomechanical analysis was also performed. Gross and histological scores were improved for the genetically modified repair tissue at 4 and 9 weeks and 8 months. Type II collagen content was also increased in the AdIGF-treated group and in immunohistochemical analysis as well as by biochemical analysis. Taqman PCR analysis revealed IGF-I and Collagen Type II expression in the AdIGF-treated group was elevated over control (p<0.05) at 4 weeks (Figure 1 and 2). The biomechanical properties of the treated and control tissues did not differ between treatment groups. The present study evaluated the short and long-term effects of AdIGF-I-transduction of chondrocytes prior to implantation. Treatment of chondrocytes with AdIGF-I increased IGF-I expression in the repaired defect for 4 weeks. This increase correlated with an Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
GENE THERAPY FOR CONNECTIVE TISSUE increase in Collagen Type II expression as well as an increase in the percentage of Collagen Type II for up to 8 months. These findings suggest that chondrocyte-based gene transfer may be a promising strategy of improving cartilage repair.
greater cell survival after repair with AdIGF-I-modified cells when compared with control (P<0.05). At 8 months there was a trend towards greater cell survival in defects repaired with IGF-I expressing chondrocytes (P=.06). Chondrocyte survival in this study was positively influenced by genetic modification with AdIGF-I. While the range of percentage donor cells surviving (0-8%) is low, it is consistent with other studies quantifying perichondrial cell or chondrocyte survival in articular cartilage defects. Previously we have reported an average 4% survival of chondrocytes at 8 months following repair using this equine model and this PCR technique. While in that study, no differences were detected between BMP7-treated and control chondrocytes, the current study clearly shows a correlation between IGF-I treatment and cell survival, especially at the early time point.
675. Enhanced Survival of Chondrocytes over Expressing Insulin like Growth Factor I in Equine Cartilage Repair Laurie R. Goodrich,1 Chisa Hidaka,2 Mary L. Strassheim,1 Paul D. Robbins,3 Chris H. Evans,4 Alan J. Nixon.1 1 Clinical Sciences, Cornell University College of Veterinary Medicine, Ithaca, NY; 2Institute for Sports Medicine Research, Hospital for Special Surgery, New York, NY; 3Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA; 4Orthopedic Surgery-Brigham & Women’s Hospital, Harvard Medical School, Boston, MA. Recent studies have shown that insulin-like growth factor (IGFI) plays a powerful role in cell survival and prevention of programmed cell death. When recombinant IGF-I is combined with chondrocyte transplants, local upregulation of matrix production occurs in experimentally induced full-thickness cartilage defects resulting in morphological improvement of cartilage at the site of cartilage injury. This study was designed to test whether genetic modification of chondrocytes to over express IGF-I could enhance cartilage repair by improving chondrocyte survival. Sixteen horses arthroscopically had a 15 mm cartilage defect made in both femoropatellar joints. The defects were repaired with AdIGFI transduced chondrocytes in one limb and naïve chondrocytes in the opposite limb. Four horses were necropsied at 4 and 9 weeks and 8 horses at 8 months postoperative. Ten 10 mg of DNA was harvested from the repair tissue and Taqman™ quantitative realtime polymerase chain reaction (PCR) was used to assess the number of copies of the Y-chromosome marker SRY. A standard curve for the SRY was constructed using genomic DNA from male chondrocytes. A conversion factor of 6.6pg of DNA per diploid cell was used to calculate percentage of cell survival. A paired t test was used to compare cell survival between IGF-I-treated and control groups at each time point. Significance was set at P<0.05. Average percentage of SRY-positive (male, donor) cells present in the defects at 4 weeks, 9 weeks and 8 months was 4.8%, 0%, and 0.9% respectively (Figure 1). At 4 weeks there was a significantly Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
676. Local Stimulation of Articular Cartilage Repair by Transplantation of Encapsulated Chondrocytes Overexpressing Human FGF-2 In Vivo Gunter Kaul,1 Magali Cucchiarini,1 David Zurakowski,2 Stephen B. Trippel,3 Dieter Kohn,1 Henning Madry.1 1 Laboratory for Experimental Orthopaedics, Saarland University, Homburg, Saarland, Germany; 2Department of Biostatistics and Medicine, Harvard Medical School, Boston, MA; 3Department of Orthopaedic Surgery, Indiana University, Indianapolis, IN. Background Defects of articular cartilage are an unsolved problem in orthopaedics. Application of fibroblast growth factor-2 (FGF-2) protein has been demonstrated to modulate articular cartilage repair in vivo. We previously reported that overexpression of FGF-2 selectively stimulated cell proliferation in an ex vivo model of chondrocyte transplantation and that transgene expression remains present in vivo for at least 30 days when transfected chondrocytes are implanted in osteochondral cartilage defects. In the present study, we tested the hypothesis that gene transfer of human fibroblast growth factor 2 (FGF-2) via transplantation of encapsulated genetically modified articular chondrocytes stimulates chondrogenesis in cartilage defects in vivo. Methods Lapine articular chondrocytes overexpressing a lacZ or a human FGF-2 gene sequence were encapsulated in alginate and further characterized. The resulting lacZ or FGF-2 spheres were applied to cylindrical osteochondral defects (3.2-mm in diameter) in each patellar groove in the knee joints of rabbits. Cartilage repair was assessed based on Safranin O-stained sections using a previously published histological grading system developed to quantitate the repair of cartilage defects at three and fourteen weeks after implantation. This grading system ranges from 0 points (normal
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