IMPROVED METHODS OF HIV VECTOR-MEDIATED GENE-TRANSFER

HIV vectors are capable of targeted gene transfer into CD4(+) cells. H owever, extensive testing of HIV vectors in gene therapy applications is hampered by the low titer of current HIV vector preparations. We at tempted to increase the efficiency of HIV vector mediated gene transfe r using recently developed techniques. HIV vectors could be concentrat ed by approximately 20 times by sulfonated cellulose column chromatogr aphy. No replication competent cytopathic HIV was detected in the conc entrated vector preparation. When the vector preparation and the targe t cells were centrifuged at transduction, about a five-fold increase i n the apparent titer was achieved. Accordingly, by combining these two techniques the overall titer was increased by approximately two order s of magnitude. Using this high efficiency strategy, we transduced hum an primary lymphocytes that are refractory to transduction with curren tly available viral vectors. Amplification and sequencing of the integ ration sites showed that HIV vectors could stably integrate into the c hromosomes of CD4 enriched human peripheral blood mononuclear cells. T hese findings indicate that HIV vectors are useful for the development of gene therapy targeting lymphocytes. (C) 1998 Elsevier Science Irel and Ltd. All rights reserved.