IMPROVED GENE-TRANSFER INTO BABOON MARROW REPOPULATING CELLS USING RECOMBINANT HUMAN FIBRONECTIN FRAGMENT CH-296 IN COMBINATION WITH INTERLEUKIN-6, STEM-CELL FACTOR, FLT-3 LIGAND, AND MEGAKARYOCYTE GROWTH AND DEVELOPMENT FACTOR

We have used a competitive repopulation assay in baboons to develop im proved methods for hematopoietic stem cell transduction and have previ ously shown increased gene transfer into baboon marrow repopulating ce lls using a gibbon ape leukemia virus (GALV)-pseudotype retroviral vec tor (Kiem et al, Blood 90:4638, 1997). In this study using GALV-pseudo type vectors, we examined additional variables that have been reported to increase gene transfer into hematopoietic progenitor cells in cult ure for their ability to increase gene transfer into baboon hematopoie tic repopulating cells. Baboon marrow was harvested after in vivo admi nistration (priming) of stem cell factor (SCF) and granulocyte colony- stimulating factor (G-CSF). CD34-enriched marrow cells were divided in to two equal fractions to directly compare transduction efficiencies u nder different gene transfer conditions. Transduction by either incuba tion with retroviral vectors on CH-296-coated flasks or by cocultivati on on vector-producing cells was studied in five animals; in one anima l, transduction on CH-296 was compared with transduction on bovine ser um albumin (BSA)-coated flasks. The highest level of gene transfer was obtained after 24 hours of prestimulation followed by 48 hours of inc ubation on CH-296 in vector-containing medium in the presence of multi ple hematopoietic growth factors (interleukin-6. stem cell factor, FLT -3 ligand, and megakaryocyte growth and development factor). Using the se conditions, up to 20% of peripheral blood and marrow cells containe d vector sequences for more than 20 weeks, as determined by both polym erase chain reaction and Southern blot analysis. Gene transfer rates w ere higher for cells transduced on CH-296 as compared with BSA or cocu ltivation. In one animal, we have used a vector expressing a cell surf ace protein (human placental alkaline phosphatase) and have detected 1 0% and 5% of peripheral blood cells expressing the transduced gene 2 a nd 4 weeks after transplantation as measured by flow cytometry. In con clusion, the conditions described here have resulted in gene transfer rates that will allow detection of transduced cells by flow cytometry to facilitate the evaluation of gene expression. The levels of gene tr ansfer obtained with these conditions suggest the potential for therap eutic efficacy in diseases affecting the hematopoietic system. (C) 199 8 by The American Society of Hematogy.