401. Evaluation of Primitive Murine Hematopoietic Stem and Progenitor Cell Transduction In Vitro and In Vivo by Recombinant Adeno-Associated Virus Vectors Based on Serotypes 1 through 5

Top of pageAbstract Hematopoietic stem cells (HSCs) are attractive targets for gene therapy of a number of diseases. Recent studies have demonstrated the therapeutic potential of recombinant retroviral vectors in hematopoietic stem cell gene transfer in children with severe combined immunodeficiency by, but subsequent development of leukemia in three treated children hasunderscored the need to develop alternative vectors. Recombinant AAV vectors have to date not been associated with any malignant disease and have gained attention as potentially useful vectors. Although conflicting data exist with regard to hematopoietic stem cell transduction by AAV serotype 2 (AAV2) vectors, additional serotypes vectors, AAV1-5, have not been evaluated for their efficacy for hematopoietic stem and progenitor cell transduction. In the present studies, we evaluated the efficacy of AAV serotype vectors 1 through 5 containing the CMV promoter-driven EGFP gene in hematopoietic progenitor cell assays in vitro, as well as following bone marrow transplantation in lethally-irradiated recipient syngeneic mice in vivo. Sca1+, c-kit+, lin- hematopoietic cells following either-mock-infection, or infection with serotype vectors were plated in methylcellulose cultures and hematopoietic colonies expressing the transgene were evaluated 7 days-post-infection. Approximately 5% of the colonies in cultures infected with AAV1 were EGFP-positive. All other serotype vectors elicited no transgene expression. Co-infection of AAV1 with self-complementary AAV vectors containing the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increase the transduction efficiency to |[sim]|20%. Sca1+, c-kit+, lin- cells following either-mock-infection, or infection with serotype vectors|[plusmn]|scAAV-TC-PTP vectors were also injected via tail-vein into lethally-irradiated syngeneic recipient mice. Six months post-transplantation, primary recipient mice were sacrificed and transgene expression was assessed in peripheral blood cells. Approximately 8% of cells from mice transplanted with cells co-infected with AAV1+scAAV-TC-PTP vectors were EGFP-positive. Transduction efficiency of all other serotype vectors was low. Bone marrow mononuclear cells from primary recipient mice transplanted with cells co-infected with AAV1+scAAV-TC-PTP vectors were also used to transplant lethally-irradiated syngeneic mice, and 4 months post-transplantation, |[sim]|3% of bone marrow mononuclear cells were EGFP-positive. These results document that AAV1 is by far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Additional studies involving scAAV genomes and cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene transfer as well as in human gene therapy.