NOVEL RETROVIRAL PACKAGING CELL-LINES - COMPLEMENTARY TROPISMS AND IMPROVED VECTOR PRODUCTION FOR EFFICIENT GENE-TRANSFER

We report increased transduction of human hematopoietic progenitor cel ls through a combination of novel retroviral vector packaging cell lin es, and improved vector supernatant production. The new ProPak packagi ng cell lines produce either murine leukemia virus (MLV) xenotropic (P roPak-X cells) or amphotropic particles (Pro-Pak-A cells), and ProPak- based producer cells were demonstrated to be of replication-competent retrovirus (RCR) by stringent testing. Vector supernatants from ProPak or existing packaging cell lines producing different pseudotyped part icles (amphototropic MLV, xenotropic MLV or gibbon ape leukemia virus) were compared for the ability to transduce clinically relevant human hematopoietic cells. All vector types transduced primary human CD34-po sitive or CD4-positive cells, regardless of tropism However, consisten tly higher transduction of target cells was achieved with ProPak-deriv ed amphotropic vector than with PA317-packaged amphotropic vector. The highest transduction of human hematopoietic progenitor cells was achi eved with vector supernatant generated from a coculture of the ProPak- X and ProPak-A cell lines. This ping-pong amplification yielded supern atant containing vector targeted to two distinct receptor present on h uman cells, and did not result in detectable RCR formation. In additio n, we describe conditions for improved vector supernatant production i n a packaged-bed bioreactor.