Gene Transfer of Hematopoietic Stem Cells

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DESCRIPTION(adapted from applicant's abstract): In order for efficient gene transfer of hematopoietic stem cells to be achieved we believe that the testing of new vectors and better defined stem cell populations are required. We have chosen Fanconi anemia (FA) as a model disease to facilitate improved gene transfer protocols of hematopoietic cells. FA is a rare autosomal recessive disorder characterized principally by bone marrow failure and the development of leukemia. The hematologic manifestations of FA are due to a disorder of stem cell function. The functions of the FANC proteins (FANC complementation groups A-H) are not understood but all FANC cells exhibit hypersensitivity to DNA crosslinkers and suggest a role in maintaining DNA stability. Although we demonstrated that a selective growth advantage exists in gene-corrected FA hematopoietic cells in a knockout model, the corrected stem cell population requires further characterization. Here we propose to identify and test gene transfer on isolated fractions of primitive hematopoietic stem cells based on physiologic rather than immunologic methods from both mouse and human. This purification scheme isolates the previously described novel side population fraction (SP) in both mouse and human hematopoietic cells. Our strategy is to isolate and transduce Fanconi anemia complementation group A (FANCA) and C (FANCC) knockout mice primitive stem cells. Recipient animals will be examined for their response to DNA damaging agents and cytokines and their ability to reconstitute hematopoiesis following gene transfer using moloney-murine retroviral and HIV, equine and feline-base lentiviral vectors. Isolation of human CD34+, CD34+/CD38- and SP fractions from FA patients will be tested for transduction by retroviral vectors using the NOD/scid immunodeficient mouse system. Currently we have an ongoing trial for FANCA patients. Information obtained from the planned studies will provide a better understanding of abnormal hematopoiesis in FA and better define therapeutic strategies important for designing future human clinical trials.