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1093. Ex Vivo Gene Therapy Approach Using Human Lim Mineralization Protein_3 To Induce Bone Healing in a Rodent Model
MUSCLE AND CONNECTIVE TISSUE: BONE/JOINT blockade. Other patients have disease limited to a few joints, which may not warrant use of systemic TNF-α antagonists. Intra-articular (IA) injection of an adeno-associated virus (AAV) vector containing the cDNA for the human tumor necrosis factor-immunoglobulin (IgG1) Fc fusion (TNFR:Fc) gene (tgAAC94) may be a means to provide local high concentrations of soluble TNFR:Fc for an extended period of time without requiring frequent administration. To evaluate the safety of IA administration of tgAAC94 in inflammatory arthritis patients with and without concurrent TNF-α antagonists, the first clinical study of IA tgAAC94 was completed, and a second is underway. In the first study, 15 subjects not on TNF-α antagonists with persistent moderate or severe inflammation in a target joint due to inflammatory arthritis received a single IA injection of either tgAAC94 at 1x1010 (n=5), 1x1011 (n=6) DNase resistant particles (DRP) per mL joint volume or placebo (n=4). In the second study, 21 adults meeting similar entry criteria, except concurrent TNF-α antagonist use was permitted, were randomized to receive a single IA injection of either tgAAC94 1x1011 DRP/mL (n≈16) or placebo (n≈4), followed by an open-label injection of tgAAC94 1x1011 DRP/ mL after 12 to 36 weeks, depending on when swelling in the target joint meets criteria for re-injection. Similarly, a second cohort of 20 subjects will be randomized to receive tgAAC94 1x1012 DRP/mL (n≈16) or placebo (n≈4), followed by an open-label injection 12 to 36 weeks later. In the first study, 12 females and 2 males with rheumatoid arthritis and 1 male with ankylosing spondylitis received an injection into the knee (n=14) or ankle (n=1) and were followed for 24 weeks. Improvement in a composite tenderness and swelling score was noted in all treatment groups, particularly among subjects who received the higher dose of tgAAC94. IA tgAAC94 was safe and well-tolerated. None of the 50 adverse events reported among 12 of 15 subjects were definitely related to study drug. Two events, nasopharyngitis and sinus congestion, were considered possibly related to study drug. One event, mild knee pruritis, was considered probably related to study drug. One 88-year-old female with RA had four SAEs considered unrelated to study drug: labyrinthitis, myocardial infarction and two episodes of congestive heart failure. In the second study, 21 subjects, 10 of whom were receiving concurrent TNF-α antagonists, received an injection of blinded study drug into the knee (n=12), ankle (n=4), wrist (n=2) or MCPs (n=3). No safety concerns were identified after 8-12 weeks of follow-up. Thus, a single dose of IA tgAAC94 appears to be safe and welltolerated in subjects with and without concurrent TNF-α antagonist use. Enrollment in the ongoing study will continue and determine the safety of higher and repeat doses of tgAAC94, and to evaluate the effect of treatment and its duration. Employee of Targeted Genetics.
1093. Ex Vivo Gene Therapy Approach Using Human Lim Mineralization Protein-3 To Induce Bone Healing in a Rodent Model Enrico Pola,1 Wanda Lattanzi,2 Giandomenico Logroscino,1 Giovanni Pecorini,3 Anna Tampieri,4 Andrea Gambotto,5 Carlo A. Logroscino,1 Paul D. Robbins.5 1 Orthopaedics and Traumatology, A. Gemelli University Hospital, Rome, Italy; 2Anatomy and Cell Biology, A. Gemelli University Hospital, Rome, Italy; 3Internal Medicine, A. Gemelli University Hospital, Rome, Italy; 4Department for Biomaterials, Italian National Council of Research CNR, Faenza, Italy; 5Molecular Genetics and Biochemistry, University of Pittsburgh Medical Center, Pittsburgh, PA. Introduction. Gene therapy research in the field of orthopaedics have evolved during the last decade, leading to possible applications for the treatment of pathological conditions, such as bone fractures S420
and defects. Several gene transfer techniques have been employed so far for inducing bone formation in animal models of bone defects. Cell-based approaches, such as the implantation in animals of ex vivo genetically modified cells, produced promising results. In this study we used autologous skin fibroblasts, which are very simple to harvest and propagate in culture, transduced ex vivo with the osteogenic factor Lim Mineralization Protein-3 (LMP-3). These engineered cells produced successful bone healing when implanted by the use of a scaffold in rats, validating the in vivo osteoinductive properties of hLMP-3. Materials and Methods. Primary dermal fibroblasts cultures were established using a biopsy of shaved skin obtained from the abdomen of each rat. Semi-confluent primary fibroblasts were infected with either AdBMP-2 or AdhLMP-3, using a overall multiplicity of infection (MOI) of 100 viral particles per cell. Cells transduced with Ad-eGFP were used as a viral infection control, while untreated cells served as a negative control. The transduced cells were harvested 24 hours after viral infection, let adsorbed on a Hydroxyapatite/ Collagen scaffold and then implanted in a bone defect surgically performed in the mandible of immunocompetent rats. The animals were divided in 4 groups: rats treated with cells infected with AdLMP-3, rats treated with cells infected with AdBMP-2, rats treated with cells infected with Ad-eGFP and rats treated with untreated cells. Rats from each group were sacrified at 1, 2 and 3 months after the treatment and studied by x-rays, Micro-CT and histology. Results. All the animals treated with LMP-3 showed healing of the bone defect after 3 months, as confirmed histologically and radiographically. On the contrary none of the controls showed bone formation at the latest time point. Discussion. Recently, Lim Mineralization Proteins (LMP) have been identified as regulators of the osteoblast differentiation program. We have previously demonstrated that human LMP-3 contributes actively to bone formation, acting through the BMP-2 signaling pathway, being capable of inducing differentiation of cells of mesenchymal derivation towards the osteoblastic lineage, through the up-regulation of bone-specific genes, along with ectopic bone formation in vivo and mineralization in vitro. In this study we have tested the efficacy of an ex-vivo approach using autologous dermal fibroblasts infected with AdLMP-3. Our results show that it’s possible to induce a complete bone healing using this method, and confirm the in vivo osteoinductive properties of hLMP-3.
1094. Self-Complementary Vectors Significantly Enhance AAV-Mediated Gene Transfer to Joint Tissues Jesse Kay,1 Elvire Gouze,1 Kenneth Warrington, Jr.,2 Irene Zolotukhin,2 Rachael Watson,1 Marsha Bush,1 Jeetpaul Saran,1 Jean-Noel Gouze,1 Douglas McCarty,3 Paul Robbins,4 Christopher Evans,5 Steven Ghivizzani.1 1 Orthopaedics & Rehabilitation, University of Florida, Gainesville, FL; 2Pediatrics, Univesity of Florida, Gainesville, FL; 3Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC; 4Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA; 5Center for Molecular Orthopaedics, Harvard Medical School, Boston, MA. Gene delivery approaches have been proposed as a means to improve treatment of the arthritides. By delivering cDNAs encoding anti-arthritic proteins to the cells in the synovial lining, the gene products may be expressed and secreted locally into the joint space. Vectors based on AAV offer advantages that would favor their use as a vehicles for direct, intra-articular gene transfer; however, reports of the effectiveness of this vector have been widely variable. Much of this inconsistency may be due to limitations in synovial lining/ joint capsular cells to initiate second strand DNA synthesis of the Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
1093. Ex Vivo Gene Therapy Approach Using Human Lim Mineralization Protein-3 To Induce Bone Healing in a Rodent Model Enrico Pola,1 Wanda Lattanzi,2 Giandomenico Logroscino,1 Giovanni Pecorini,3 Anna Tampieri,4 Andrea Gambotto,5 Carlo A. Logroscino,1 Paul D. Robbins.5 1 Orthopaedics and Traumatology, A. Gemelli University Hospital, Rome, Italy; 2Anatomy and Cell Biology, A. Gemelli University Hospital, Rome, Italy; 3Internal Medicine, A. Gemelli University Hospital, Rome, Italy; 4Department for Biomaterials, Italian National Council of Research CNR, Faenza, Italy; 5Molecular Genetics and Biochemistry, University of Pittsburgh Medical Center, Pittsburgh, PA. Introduction. Gene therapy research in the field of orthopaedics have evolved during the last decade, leading to possible applications for the treatment of pathological conditions, such as bone fractures S420
and defects. Several gene transfer techniques have been employed so far for inducing bone formation in animal models of bone defects. Cell-based approaches, such as the implantation in animals of ex vivo genetically modified cells, produced promising results. In this study we used autologous skin fibroblasts, which are very simple to harvest and propagate in culture, transduced ex vivo with the osteogenic factor Lim Mineralization Protein-3 (LMP-3). These engineered cells produced successful bone healing when implanted by the use of a scaffold in rats, validating the in vivo osteoinductive properties of hLMP-3. Materials and Methods. Primary dermal fibroblasts cultures were established using a biopsy of shaved skin obtained from the abdomen of each rat. Semi-confluent primary fibroblasts were infected with either AdBMP-2 or AdhLMP-3, using a overall multiplicity of infection (MOI) of 100 viral particles per cell. Cells transduced with Ad-eGFP were used as a viral infection control, while untreated cells served as a negative control. The transduced cells were harvested 24 hours after viral infection, let adsorbed on a Hydroxyapatite/ Collagen scaffold and then implanted in a bone defect surgically performed in the mandible of immunocompetent rats. The animals were divided in 4 groups: rats treated with cells infected with AdLMP-3, rats treated with cells infected with AdBMP-2, rats treated with cells infected with Ad-eGFP and rats treated with untreated cells. Rats from each group were sacrified at 1, 2 and 3 months after the treatment and studied by x-rays, Micro-CT and histology. Results. All the animals treated with LMP-3 showed healing of the bone defect after 3 months, as confirmed histologically and radiographically. On the contrary none of the controls showed bone formation at the latest time point. Discussion. Recently, Lim Mineralization Proteins (LMP) have been identified as regulators of the osteoblast differentiation program. We have previously demonstrated that human LMP-3 contributes actively to bone formation, acting through the BMP-2 signaling pathway, being capable of inducing differentiation of cells of mesenchymal derivation towards the osteoblastic lineage, through the up-regulation of bone-specific genes, along with ectopic bone formation in vivo and mineralization in vitro. In this study we have tested the efficacy of an ex-vivo approach using autologous dermal fibroblasts infected with AdLMP-3. Our results show that it’s possible to induce a complete bone healing using this method, and confirm the in vivo osteoinductive properties of hLMP-3.
1094. Self-Complementary Vectors Significantly Enhance AAV-Mediated Gene Transfer to Joint Tissues Jesse Kay,1 Elvire Gouze,1 Kenneth Warrington, Jr.,2 Irene Zolotukhin,2 Rachael Watson,1 Marsha Bush,1 Jeetpaul Saran,1 Jean-Noel Gouze,1 Douglas McCarty,3 Paul Robbins,4 Christopher Evans,5 Steven Ghivizzani.1 1 Orthopaedics & Rehabilitation, University of Florida, Gainesville, FL; 2Pediatrics, Univesity of Florida, Gainesville, FL; 3Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC; 4Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA; 5Center for Molecular Orthopaedics, Harvard Medical School, Boston, MA. Gene delivery approaches have been proposed as a means to improve treatment of the arthritides. By delivering cDNAs encoding anti-arthritic proteins to the cells in the synovial lining, the gene products may be expressed and secreted locally into the joint space. Vectors based on AAV offer advantages that would favor their use as a vehicles for direct, intra-articular gene transfer; however, reports of the effectiveness of this vector have been widely variable. Much of this inconsistency may be due to limitations in synovial lining/ joint capsular cells to initiate second strand DNA synthesis of the Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy