This application seeks to develop novel retroviral gene transfer systems for the delivery of genes to cells that occupy synovial tissues. Specifically, the investigator proposes to deliver dendritic cells (DC) genes that encode a secreted form of antibody to Tumor Necrosis Factor-alpha (TNF-a) with the goal of inhibiting the effects of this cytokine in sites of pathologic inflammation, such as inflamed arthritic joints. DC have been chosen as target cells because they normally occupy synovial and mucosal sites, and because the investigator and his group have developed efficient methods for their isolation and characterization. They have developed methods for producing retroviruses in extremely high titer and for regulating the expression of retrovirally transferred genes through the inclusion of inducible promoters in the viral genome, and have designed methodologies for retargeting retroviruses to specific cell types by incorporating ligands into the retroviral envelope. The investigator now plans to combine these technologies for the purpose of therapeutic gene transfer to synovial tissues.
The specific aims i nclude: 1) in vitro delivery of genes, including a gene that encodes an anti-TNF-a single chain antibody, under the control of inducible self-inactivating retroviruses, to proliferating DC and their precursors; 2) creation of retroviruses that specifically target and infect CD86+ (B7.2+) cells by generating chimeric retroviral envelopes that contain ligands (e.g., CTLA-4) that specifically bind CD86; 3) creation of retroviruses capable of infecting nondividing cells, such as mature DC, by modification of the gag proteins or use of novel retroviral species; 4) generation of retroviruses that combine the features of the vectors produced in Specific Aims 1-3 such that they specifically target and infect non-dividing CD86+ cells; 5) delivery of transduced DC or CD86 targeted retroviruses, in vivo, to synovium and adjacent lymph nodes in normal mice; and 6) administration of transduced DC or CD86 targeted retroviruses to mice with collagen induced arthritis (CIA) with the goal of ameliorating disease. Although the focus of these studies is delivery of genes to DC, the retroviral constructs developed should provide the means to target and deliver other inducible genes to virtually any cell type.
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