The transfer of therapeutic genes into hematopoietic stem cells (HSC) offers the potential of effective and permanent treatment for a wide spectrum of diseases including metabolic diseases, infections and cancer. However, despite promising results in vitro and in murine models, the results of human gene therapy trials to date have revealed disappointingly low retrovirus-mediated gene transfer frequencies into pluripotential long-term repopulating cells. While the lentivirus vectors may offer one alternative, their inability to integrate in quiescent cells and safety issues make the continued pursuit of alternative approaches for gene delivery to HSC imperative for continued progress. Adeno-associated irus (AAV) vectors are rapidly emerging as gene transfer vectors with unique properties including those of non-pathogenicity and low immunogenicity. We and others have shown the ability of AAV vectors to efficiently transduce a variety of non- dividing cells both in vivo and in vitro. We have also recently demonstrated the capacity of AAV vectors to transduce primitive clonogenic CD34+ and CD34+/CD38- human hematopoietic progenitor cells and to display chromosomal integration. These properties make AAV vectors attractive for further evaluation to for gene delivery to HSC. Here we propose to test the proficiency of AAV vectors to transduce non-dividing and metabolically quiescent CD34+38- hematopoietic progenitor cells. AAV transduction will be tested in vitro in populations defined as being in G0 based upon their 2n DNA and low RNA content and multi-potential clonogenic properties. The full range of hematopoietic potentials of AAV transduced CD34+38- and G0 cells will be tested in a chimeric xenograft model utilizing transplantation of AAV transduced hematopoietic cells into immunodeficient NOD/SCID mice. AAV transduction of a newly defined lin-CD34-38- primitive hematopoietic progenitor cell will also be tested. Experiments directly comparing retroviral and AAV transduction will test the transduction potentials of the two vector systems in vitro and in NOD/SCID mice. Transduction of self-renewing pluripotential stem cells will be tested in a murine serial transplant model. In all three systems to be utilized, the ability of a single transduced progenitor cell to give rise to multi-lineage progeny will be tested using sensitive PCR assays developed for our vectors. Lastly, information obtained from these experiments will be used to design a therapeutic marrow transplant strategy utilizing AAV transduction of the ApoAI Milano gene for the treatment of atherosclerosis. It is expected that knowledge gained from these experiments will provide an accurate assessment of the transduction potential of AAV vectors for hematopoietic stem cell gene therapy.
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