GENE REPLACEMENT THERAPY IN HEMATOPOIETIC STEM CELLS

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(Adapted from the applicant's abstract) The overall goal of this program is to develop a strong experimental foundation for the correction of inherited disease of bone marrow-derived cells by genetic modification of hematopoietic stem cells. Current objectives are focused on the use of recombinant retroviral, lentiviral, and adeno-associated virus (AAV) vectors to achieve efficient transfer of functional genes into primitive hematopoietic stem cells while maintaining their maximal hematopoietic potential. The specific aims are to 1) examine the relationship between cell cycle, fibronectin-mediated adhesion, and cytokine regulators on hematopoietic cell function and viral transduction; 2) determine whether recombinant viral vectors derived from AAV and lentivirus can be used for efficient and stable gene transfer in murine and human hematopoietic cells; 3) identify optimal strategies using viral-mediated gene transfer to correct the phenotype in two inherited blood disorders, X-linked chronic granulomatous disease (X-CGD) and Fanconi anemia group C (FanC); and 4) evaluate non- ablative conditioning regimens and selection of transduced cells using maker proteins or drug resistance genes. Experimental approaches include the use of in vitro culture systems, NOD/SCID mouse-human xenografts, and murine models of X-CGD and FanC previously generated by gene targeting approaches. The implementation of these aims will be shared among 4 projects and 3 core units. This proposal draws from a group of investigators with diverse but complementary experience in stem cell biology, retrovirus-, lentivirus-, and AAV-mediated gene transfer, molecular genetics, virology, bone marrow transplantation, and neonatology. Achievement of these goals will permit the translation of this basic work to the development of clinical protocols for effective viral-mediated gene transfer of genetic blood diseases. On a broader level, these studies should provide insight into the biologic behavior of hematopoietic stem cells and the ability to manipulate them ex vivo.