Fanconi Anemia (FA) is an autosomal recessive syndrome characterized by progressive pancytopenia, diverse congenital abnormalities, and increased predisposition to malignancy. The only cure for FA has been HLA-matched bone marrow, and more recently umbilical cord blood, transplantation. The cloning of cDNAs for FA by functional complementation has opened up the possibility for correction of the disease by gene therapy. Our hypothesis is that hematopoietic stem and progenitor cells present in cord blood and bone marrow of patients with FA can be efficiently and stably transduced with retroviral- and adeno associated virus (AAV)- vectors containing the FA complementation C gene with correction of the growth patterns of these cells, and the enhanced sensitivity of FA cells to chromosome-breakage and kill by drugs. To this end the following aims are proposed: 1) Construct high titer retroviral- and AAV-vectors containing the FA complementation C gene. 2) Compare vectors for high efficiency stable transduction of stem and progenitor cells in normal cord blood and later in normal bone marrow cells using first relatively unseparated cells present in a low-density fraction, then more highly purified cells. These comparisons will then be done in marrow and cord blood cells from patients with complementation C-FA to see if their growth characteristics, chromosomal fragility, and hypersensitivity to drugs can be corrected. 3) Evaluate stable integration of genes into the earliest subsets of hematopoietic stem/progenitor cells and compare this with integration into later more mature subsets of the cells by using a variety of in vitro and in vivo assays. In vitro assays include those for LTC-IC, HPP-CFC, CFU-GEMM, BFU-E and CFU-GM. Replating capacity of single HPP-CFC- and CFU-GEMM-colonies into secondary and subsequent plates will be used as an estimate of self-renewal capacity, and gene integration of sequentially replated colonies determined. For in vivo analysis of stable integration we will use human cell-inoculated SCID mice. 4) Compare stable gene integration, using retroviral and AAV- vectors pre- and post-cryopreservation of cord blood and before and after expansion of the stem and progenitor cells of these tissues sources. The information obtained could lead to a gene therapy approach to cure the FA syndrome by autologous stem/progenitor cell transplantation.
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