GENE-TRANSFER INTO MARROW REPOPULATING CELLS - COMPARISON BETWEEN AMPHOTROPIC AND GIBBON APE LEUKEMIA-VIRUS PSEUDOTYPED RETROVIRAL VECTORS IN A COMPETITIVE REPOPULATION ASSAY IN BABOONS

Many diseases might be treated by gene therapy targeted to the hematop oietic system, but low rates of gene transfer achieved in humans and l arge animals have limited the application of this technique. We have d eveloped a competitive hematopoietic repopulation assay in baboons to evaluate methods for improving gene transfer and have used this method to compare gene transfer rates for retroviral vectors having an envel ope protein (pseudotype) from amphotropic murine retrovirus with simil ar vectors having an envelope protein derived from gibbon ape leukemia virus (GALV). We hypothesized that vectors with a GALV pseudotype mig ht perform better based on our previous work with cultured human hemat opoietic cells. CD34(+) marrow cells from each of four untreated baboo ns were divided into two equal portions that were cocultivated for 48 hours with packaging cells producing equivalent titers of either ampho tropic or GALV pseudotyped vectors containing the neo gene. The vector s contained small sequence differences to allow differentiation of cel ls genetically marked by the different vectors. Nonadherent and adhere nt cells from the cultures were infused into animals after they receiv ed a myeloablative dose of total body irradiation. Polymerase chain re action (PCR) analysis for neo gene-specific sequences in colony-formin g unit-granulocyte-macrophage from cell populations used for transplan t showed gene transfer rates of 2.7%, 7.1%, >15%, and 3.9% with the am photropic vectors and 7.1%, 11.3%, >15%, and 26.4% with the GALV pseud otyped vector, PCR analysis of peripheral blood and marrow cells after engraftment showed the neo gene to be present in all four animals ana lyzed at levels between 0.1% and 5%. Overall gene transfer efficiency was higher with the GALV-pseudotyped vector than with the amphotropic vectors. Southern blot analysis in one animal confirmed a gene transfe r efficiency of between 1% and 5%. The higher gene transfer efficiency with the GALV-pseudotyped vector correlated with higher levels of GAL V receptor RNA compared with the amphotropic receptor in CD34(+) hemat opoietic cells. These results show that GALV-pseudotyped vectors are c apable of transducing baboon marrow repopulating cells and may allow m ore efficient gene transfer rates for human gene therapy directed at h ematopoietic cells. In addition, our data show considerable difference s in gene transfer efficiency between individual baboons, suggesting t hat a competitive repopulation assay will be critical for evaluation o f methods designed to improve gene transfer into hematopoietic stem ce lls. (C) 1997 by The American Society of Hematology.