We have initiated a Phase 1 safety and gene trial in patients with Type 1 Gaucher's disease. CD34+ stem and progenitor cells were obtained from peripheral blood by apheresis after stimulating patients with G-CSF (NIH) or from bone marrow biopsies (Children's Hospital, Los Angeles). The first recipient was a 21-year-old male with type 1 Gaucher's disease where only bone marrow stromal cells were used in the expansion of his CD34+ cells. Barely detectable and short-lasting marking of granulocytes was observed. The second patient was a 22-year-old female. Autologous marrow stromal cells along with IL-3, IL-6 and SCF were employed to expand the transduced cells. More significant and longer lasting granulocyte marking was observed in this patient. We are developing more efficient procedures to infect CD34+ cells and to segregate the transduced cells. We are also investigating methods to provide a growth advantage to the transduced cells. We have carried out a series of pre-clinical investigations concerning gene therapy for Frabry disease. A recombinant retroviral gene transfer vector was designed that engineers efficient traduction of cells and expression of human alpha-Gal A activity. Enzymatic correction was observed in cultured skin fibroblasts and B cell lines derived from patients with Fabry disease. Corrected cells secreted significant quantities of alpha-Gal A into the culture medium, and the enzyme is taken up by uncorrected bystander cells. We have also achieved enzymatic correction in CD34+ and progenitor colony cells obtained from bone-marrow aspirates from patients with Fabry disease. We are producing a high-titer clinical grade vector under GMP conditions for gene therapy trials in Fabry disease. We shall also explore gene therapy in the transgenic alpha- Gal A knock-out mouse model of Fabry disease that we created. In addition, we have made gene transfer vector constructs based on HIV-1 that are highly effective in the transduction of quiescent cells including rat cerebellar neurons. This investigation has particular relevance to gene therapy since most stem cells in the bone marrow are nondividing cells within the nervous system are in the post-mitotic state. We are initiating studies aimed at correcting the Niemann-Pick C phenotype in vivo using the NPC mouse model.
