LONG-TERM IN-VIVO EXPRESSION OF THE HUMAN GLUCOCEREBROSIDASE GENE IN NONHUMAN-PRIMATES AFTER CD34(-CELL TRANSDUCTION WITH CELL-FREE RETROVIRAL VECTOR PREPARATIONS() HEMATOPOIETIC)

Successful gene transfer into stem cells would provide a potentially u seful therapeutic modality for treatment of inherited and acquired dis orders affecting hematopoietic tissues. Coculture of primate bone marr ow cells with retroviral producer cells, autologous stroma, or an engi neered stromal cell line expressing human stem cell factor has resulte d in a low efficiency of gene transfer as reflected by the presence of 0.1-5% of genetically modified cells in the blood of reconstituted an imals. Our experiments in a nonhuman primate model were designed to ex plore various transduction protocols that did not involve coculture in an effort to define clinically useful conditions and to enhance trans duction efficiency of repopulating cells. We report the presence of ge netically modified cells at levels ranging from 0.1% (granulocytes) to 14% (B lymphocytes) more than 1 year following reconstitution of myel oablated animals with CD34(+) immunoselected cells transduced in suspe nsion culture with cytokines for 4 days with a retrovirus containing t he glucocerebrosidase gene. A period of prestimulation for 7 days in t he presence of autologous stroma separated from the CD34(+) cells by a porous membrane did not appear to enhance transduction efficiency. In fusion of transduced CD34(+) cells into animals without myeloablation resulted in only transient appearance of genetically modified cells in peripheral blood. Our results document that retroviral transduction o f primate repopulating cells can be achieved without coculture with st roma or producer cells and that the proportion of genetically modified cells may be highest in the B-lymphoid lineage under the given transd uction conditions.