AEGiS-11IAC: Hematopoietic stem cell based gene therapy for AIDS.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

Hematopoietic stem cell based gene therapy for AIDS.

Int Conf AIDS 1996 Jul 7-12; 11:22 (abstract no. LB.A.6002)
Junker U, Plavec I, Bonyhadi M, Baker J, Kaneshima H, Bohnlein E; Systemix, Inc., Palo Alto, CA.

Previously, we and others have demonstrated the anti-HIV efficacy of a dominant-negative Rev mutant (RevM10) in T cell lines and primary T cells. We have further optimized retroviral vectors to maximize expression of the RevM10 gene measured by intracellular FACS analysis and could demonstrate a correlation between RevM10 expression and anti-HIV efficacy. Our data demonstrate that amphotropic retroviral vectors support expression of anti-HIV genes both in human T cell lines and in thymocytes and peripheral blood lymphocytes. Introduction of therapeutic genes into hematopoietic stem cells offers the possibility to persistently suppress replication in all differentiated peripheral blood cells which can be infected by HIV-1. We have developed amphotropic retroviral transduction protocols for hematopoietic stem/progenitor cells enriched from umbilical cord and mobilized peripheral blood. We can reproducibly mark hematopoietic progenitor (CFU-C assay) and more primitive cells (LTC-IC assay) in vitro using cocktails of 3 cytokines (IL-3, IL-6 plus SCF or LIF). We have also developed assay systems to generate macrophages from transduced hematopoietic stem cells. Retroviral-mediated expression of RevM10 is sufficient to suppress HIV-1 JR-FL replication in this lineage. To demonstrate gene delivery into the T cell lineage, we adapted the SCID-hu Thy/Liv model for gene transfer experiments. We could demonstrate engraftment of transduced UCB-derived cells in the Thy/Liv organ after 5 weeks but the marking frequency was below 1%. More recently, we pre-enriched transduced stems cells (MPB, UCB) using a vector-encoded surface antigen (murine Lyt-2) to reconstitute irradiated SCID-hu animals. Using this system, we could demonstrate significantly higher marking frequencies (less than 20% and in some animals, we detected Lyt-2 expression (1-2%) at termination of the study. The percentage of Lyt-2 expressing cells increased significantly after in vitro expansion of these cells and RevM10 expression in these cells is sufficient to suppress HIV-1 replication. These T cells respond to in vitro stimulation similar to control cells (cytokine secretion, CD25, etc.) indicating the safety of the procedure.

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