Transcriptiona silencing of transgene expression from Moloney murine leukemia (MoMLV) retroviral vectors has been a hurdle in bringing effective gene therapy to the clinic. The present invention used an optimized transduction protocol for human hematopoietic stem cells (HSC) from mobilized peripheral blood (MPB) to compare MoMLV and mouse stem cell virus (MSCV) vectors, with or without addition of a scaffold attachment region (SAR) from the human interferon- beta gene. To estimate retroviral vector supernatant quality, transgene delivery to CD34<+> cells was quantitated 72 hours after transduction using real-time PCR. To estimate the impact of vector backbone and SAR on transgene expression, the percentage of HSC progeny expressing retroviral transgene was compared 72 hours after transduction, and following 5 week stromal culture, or 6-8 week <i>in vivo</i> HSC repopulation assays (SCID-hu bone and NOD/SCID). The predominant effect of SAR, observed following long term assays, was to increase the mean fluorescence intensity (MFI) of transgene expression among HSC progeny in both <i>in vivo</i> bone repopulation models (3-4 fold), and 2 fold following long term stromal cultures. Using MSCV-SAR vector and the optimized transduction protocol, transgene expression was observed among a mean of 10 % of donor HSC progeny in the SCID-hu bone (range 0.6 - 43 %), and among 3-5 % of human HSC progeny in bone marrow and peripheral blood of NOD/SCID mice.
INCREASED TRANSGENE EXPRESSION IN RETROVIRAL VECTORS HAVING SCAFFOLD ATTACHMENT REGION