Engineering T cells and molecules for targeting joints and inflammation

Chernajovsky Y, Annenkov A, Dreja H, Adam S, Adams G

Informations

• Publié par: biomed
• Publié le: 01 janvier 2001
• Nombre de lectures: 4
• Langue: English

Extrait

Available onlinehttp://arthritisresearch.com/supplements/3/S1

Meeting abstracts Innovative Rheumatology: Gene and Cell Therapies of Arthritis and Related Autoimmune Disorders Second International Meeting Montpellier, France 17–18 May 2001

Received: 6 April 2001 Published: 25 April 2001

P1 Adenoassociated virus preferentially transduces human compared to mouse synovium K Jennings, S Katakura, H Burstein, G Gao, JM Wilson, R Hirsch 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

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 trans duction of a number of cell types without provoking a significant immune response. These properties make AAV especially attrac tive 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 synoviumin vitroand in vivowas tested. Three human RA synovial fibroblast cell lines and two murine (one DBA/1J and one DBA1J×C3H F1) synovial fibroblast cell lines were used to test AAV transductionin vitro. The 5 4 cell lines (2 × 10 cells) were infected with 10 particles/cell of a murine IL10encoding vector (AAVmIL10) alone or with the addi tion of a low titer (100 particles/cell) of an E1, E3deleted recom binant 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 coin fected 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 synoviumin 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 11 engraftment, tissues were injected with 3.4 × 10 particles of 11 AAVluciferase alone or in combination with 1.0 × 10 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 trans duce human, compared to mouse, synovial tissue and suggest that

Arthritis Res2001,3 (suppl 1):A1–A16 © 2001 BioMed Central Ltd (Print ISSN 14659905; Online ISSN 14659913)

second strand synthesis may be a limiting factor in gene transduc tion. Further studies to elucidate the mechanisms limiting gene transduction in human synovium may allow optimization of this vector for the treatment of arthritis.

P2 Delivery of antisense constructs and ribozymes to inhibit cartilage destruction in the SCID mouse model of RA † † †‡ † § T Pap* , J Schedel , U MüllerLadner , RE Gay , W Zacharias , † S Gay *Department of Experimental Rheumatology, University Hospital † Magdeburg, Germany; WHO Collaborative Center for Molecular Biology and Novel Therapeutic Strategies in Rheumatic Diseases, Department of Rheumatology, University Hospital Zurich, Switzerland; ‡ Department of Internal Medicine, University Regensburg, Germany; § and University of Alabama at Birmingham, USA

Research of the last years has demonstrated clearly the role of rheumatoid arthritis synovial fibroblasts (RASF) in the destruc tion of articular cartilage. It has been understood that RASF not only exhibit features of activation and altered apoptosis, but fol lowing 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 working on specific strategies to interfere with the expression of disease relevant matrix degrading enzymes using the complementary approaches of ribozymes and antisense expression constructs. Ribozymes are short RNA molecules that have catalytic activity and are capable of cleaving mRNA thus inhibiting its translation. We have used retroviral gene transfer of ribozymes against MMP1 as well as cathepsins B and L to inhibit the expression of these enzymes in RASF bothin vitroand when implanted together with normal articular cartilage into severe combined immunodeficient (SCID) mice. As demonstratedin vitro, gene transfer of such ribozymes results in a sustained, up to 60% decrease of enzyme production in RASF over 60 days. Currently, SCID mouse experi ments are underway to study the effect of gene transfer with these ribozymes on cartilage degradationin vivo. To evaluate the potential of antisense constructs as an alternative approach, we have generated antisense constructs against the novel MT1MMP and transduced RASF using a retroviral system. First data indicate a high efficacy of MT1MMP antisense in inhibit ing the production of MT1MMP. However, constantly high levels