19. rAAV-2 Transduction of Primitive Human Hematopoietic Stem Cells Capable of Serial Engraftment in Immune Deficient Mice

Abstract

An rAAV-2 vector encoding antisense RNA targeting sequences in the HIV-1 LTR including the TAR and polyadenylation sequences common to all HIV transcripts was tested for gene transfer to human cord blood CD34+CD3//Q hematopoietic stem/progenitor cells (HSC) following serial engraftment in immune-deficient NOD/SCID mice. Flow cytometrically purified cord blood stem cells were transduced with rAAV overnight at low cytokine concentrations and transplanted into sub-lethally irradiated recipients. Long-term multi-lineage marking of myeloid, lymphoid and CD34 progenitor cells was readily detected in primary and secondary recipients following transplantation with purified human HSC. Notably, the frequency of rAAV marking was found to be higher in secondary recipients, indicating efficient transduction of primitive HSC. Parallel serial analyses performed in vitro with clonogenic progenitor cells derived from the marrow of transplant recipients confirmed stable and efficient rAAV marking of HSC. Transgene function was investigated in the macrophage progeny of transduced CD34 stem/progenitor cells. Antisense and control transduced CD34 cells were cultured with cytokines to induce macrophage differentiation but were not subjected to positive selection. Three weeks later adherent macrophages were challenged with HIV-1 JR-FL, a monocytotropic strain of HIV, at a multiplicity of 0.01. Long-term inhibition of HIV-1 replication was noted in every donor tested relative to either untransduced or control rAAV transduced cultures, indicating stable and functional transgene expression in the progeny of transduced stem/progenitor cells. Previous reports of rAAV transduction of hematopoietic stem cells in short term in vitro studies have shown conflicting results, possibly due to the recently defined limitations to initiation of transgene expression, including nuclear entry, uncoating and second strand synthesis. However, demonstration of long term rAAV transduction of human stem cells capable of serial engraftment and transgene expression in differentiated progeny supports the use of these vectors for genetic modification of stem cells.