Efficient human immunodeficiency virus-based vector transduction of unstimulated human mobilized peripheral blood CD34(+) cells in the SCID-hu Thy/Liv model of human T cell lymphopoiesis

The methods available to efficiently transduce human CD34(+) hematopoietic stem cells (HSCs) derived from mobilized peripheral blood, such that they f ully retain their engraftment potential and maintain high levels of transge ne expression in vivo, have been unsatisfactory. The current murine retrovi rus-based gene transfer systems require dividing cells for efficient transd uction, and therefore the target HSCs must be activated ex vivo by cytokine s to cycle, which may limit their engrafting ability. Lentivirus-based gene transfer systems do not require cell division and, thus, may allow for eff icient gene transfer to human HSCs in the absence of any ex vivo cytokine s timulation. We constructed human immunodeficiency virus (HIV)-based vectors and compared them in vitro and in vivo with MuLV-based vectors in their ab ility to transduce unstimulated human CD34(+) HSCs isolated from mobilized peripheral blood. Both sets of vectors contained the marker gene that expre sses the enhanced green fluorescent protein (EGFP) for evaluating transduct ion efficiency and were pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or the amphotropic murine leukemia virus envelope (A-M ULV Env). The VSV-G-pseudotyped HIV-based vectors containing an internal mo use phosphoglycerate kinase promoter (PGK) were able to transduce up to 48% of the unstimulated CD34(+) cells as measured by EGFP expression. When the se cells were injected into the human fetal thymus implants of irradiated S CID-hu Thy/Liv mice, up to 18% expressed EGFP after 8 weeks in vivo. In con trast, the MULV-based vectors were effective at transducing HSCs only in th e presence of cytokines. Our results demonstrate that the improved HIV-base d gene transfer system can effectively transduce unstimulated human CD34(+) HSCs, which can then differentiate into thymocytes and provide long-term t ransgene expression in vivo.