Induction of synovial apoptosis by gene transfer and peptide mediated protein transduction

0 WHO Collaborative Center for Molecular Biology and Novel Therapeutic Strategies in Rheumatic Diseases, Department of Rheumatology, University Hospital Zurich , Switzerland 1 Department of Internal Medicine, University Regensburg , Germany 2 Division of Rheumatology, Children's Hospital Medical Center , Cincinnati, OH 45229 , USA; Institute for Human Gene Therapy, University of Pennsylvania Health System , Philadelphia, PA 19104, USA; Targeted Genetics Corp, Seattle, WA 98101 , USA 3 K Jennings , S Katakura, H Burstein, G Gao, JM Wilson, R Hirsch 4 Department of Experimental Rheumatology, University Hospital Magdeburg , Germany 5 University of Alabama at Birmingham , USA 6 Institute of Laboratory Medicine, Sahlgrenska University Hospital , Sweden 7 Gothenburg Medical Center , Goteborg , Sweden 8 Cartilage research Unit, Goteborg University, Department of Orthopaedics, Kungsbacka Hospital , Sweden 9 Laboratorio di Immunologia e Genetica, Istituti Ortopedici Rizzoli , Bologna , Italy; Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, NIH , Bethesda, MD , USA; Fidia Advanced Biopolymers , Abano Terme , Italy 10 Institute for Human Gene Therapy, University of Pennsylvania 11 VA Medical Center , PA 19104 , USA 12 Bone and Joint Research Unit, St Bartholomew's and Royal London School of Medicine, Queen Mary, University of London , Charterhouse Square, London EC1M 6BQ , UK 13 Division of Rheumatology 14 Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine , New York, NY 10029 , USA 15 Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine , Philadelphia, PA 19104 , USA 16 Hebrew University Hadassah Medical and Gene Therapy Center , Jerusalem , Israel 17 Department of Human Genetics 18 Department of Rheumatology, UAB , Birmingham , USA 19 Molecular Host Defense Laboratory, Department of Internal Medicine, UT Southwestern Medical Center , Dallas , USA 20 Center for Experimental Rheumatology , UniversitatsSpital Zurich , Switzerland 21 Department of Rheumatology, UMC St Radboud , 6525 GA Nijmegen, PO box 9101 , The Netherlands 22 Center for Molecular Orthopaedics, Brigham and Women's Hospital, Harvard Medical School , Boston, MA , USA 23 University of Pittsburgh, School of Medicine , Pittsburgh, PA , USA 24 Center for Molecular Orthopaedics, Brigham and Women's Hospital, Harvard Medical School , Boston, MA , USA 25 Center for Molecular Orthopaedics, Brigham and Women's Hospital, Harvard Medical School , Boston, MA , USA 26 Boehringer Ingelheim Pharmaceuticals , Ridgefield, CT , USA 27 Department of Molecular Genetics and Biochemistry, University of Pittsburgh , Pittsburgh, PA , USA 28 Division of Rheumatology, Department of Internal Medicine III, University of Vienna, Austria; Department of Pathology, Amgen, Inc , CA , USA; Department of Molecular Genetics, Hellenic Pasteur Institute , Athens , Greece 29 Division of Rheumatology, Department of Internal Medicine III, University of Vienna, Austria; Department of Molecular Genetics, Hellenic Pasteur Institute , Athens , Greece; Bristol Heart Institute, Bristol, UK; Institute for Biomedical Aging Research, Austrian Academy of Sciences , Innsbruck , Austria 30 Genetix Pharmaceuticals Inc , Cambridge, MA 02139 , USA 31 Center for Molecular Orthopaedics, Harvard Medical School , Boston, MA 02115 , USA 32 Department of Pediatrics, Stanford University, School of Medicine , Stanford, CA , USA 33 Department of Joint disease and Rheumatism, Nippon Medical School , Tokyo , Japan 34 Department of Medicine, Division of Immunology and Rheumatology 35 Department of Pediatrics, Stanford University School of Medicine , Stanford, CA , USA Adeno-associated virus preferentially transduces human compared to mouse synovium second strand synthesis may be a limiting factor in gene transduction. Further studies to elucidate the mechanisms limiting gene transduction in human synovium may allow optimization of this vector for the treatment of arthritis. - There is increasing interest in adeno-associated virus (AAV) vectors for a wide variety of gene therapy applications. AAV is a nonpathogenic human parvovirus that can mediate long-term transduction of a number of cell types without provoking a significant immune response. These properties make AAV especially attractive for use in gene therapy of rheumatoid arthritis (RA), a chronic inflammatory disease. To investigate the potential of AAV in gene therapy of arthritis, the ability of AAV to infect synovium in vitro and in vivo was tested. Three human RA synovial fibroblast cell lines and two murine (one DBA/1J and one DBA1JC3H F1) synovial fibroblast cell lines were used to test AAV transduction in vitro. The cell lines (2 105 cells) were infected with 104 particles/cell of a murine IL-10-encoding vector (AAV-mIL-10) alone or with the addition of a low titer (100 particles/cell) of an E1-, E3-deleted recombinant adenovirus to provide E4orf6 activity to enhance second-strand synthesis. The supernatants were harvested from the wells at various time points and assayed for mIL-10 expression by ELISA. Both human synovial cell lines infected with AAV alone demonstrated low-level transgene expression throughout the course of the study. However, by day 10, all human cultures coinfected with adenovirus showed a 16- to 56-fold increase in mIL-10 compared to cultures infected with AAV-mIL10 alone. By day 30, a 31- to 135-fold increase was observed. No such increase was observed in any of the mouse cell lines. To determine the AAV transduction efficiency for synovium in vivo, human RA synovial tissues obtained from patients undergoing joint-replacement surgery were implanted subcutaneously on the backs of NOD.CB17-Prkdc SCID mice. After allowing a 2-week period for engraftment, tissues were injected with 3.4 1011 particles of AAV-luciferase alone or in combination with 1.0 1011 particles of adenovirus. Two weeks following AAV administration, the tissues were homogenized and assayed for expression of luciferase. Only the tissues coinfected with adenovirus had luciferase levels above background. A similar experiment with AAV-LacZ demonstrated X-gal staining only of synovial tissues coinfected with adenovirus. These findings demonstrate a preferential ability of AAV to transduce human, compared to mouse, synovial tissue and suggest that Delivery of antisense constructs and ribozymes to inhibit cartilage destruction in the SCID mouse model of RA Research of the last years has demonstrated clearly the role of rheumatoid arthritis synovial fibroblasts (RA-SF) in the destruction of articular cartilage. It has been understood that RA-SF not only exhibit features of activation and altered apoptosis, but following attachment to cartilage secrete large amounts of matrix degrading enzymes that mediate the destruction of extracellular matrix. Given recent advances in the field of gene transfer, we have been w (...truncated)