HIGH-EFFICIENCY RETROVIRAL-MEDIATED GENE-TRANSFER INTO HUMAN AND NONHUMAN PRIMATE PERIPHERAL-BLOOD LYMPHOCYTES

Peripheral blood lymphocytes (PBLs) are primary targets for gene thera py of inherited and acquired disorders of the immune system, We descri be the development of an optimized transduction system that provides f or high-efficiency retrovirus-mediated gene transfer into primary PBLs . This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low-temperature incubation (32 degrees C), and the use of the packaging cell line PG13. Gene marking studies of human and primat e PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte popu lation. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%, The transduction procedure d oes not alter phenotype, viability, or expansion of the transduced cel ls, Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which o bviates the requirement for selection of transduced cells prior to gen e-therapy procedures, Thus, large quantities of healthy retrovirally t ransduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a si gnificant improvement in the methodology for the transduction of lymph ocytes for gene therapy.