Simplified retroviral vector GCsap with murine stem cell virus long terminal repeat allows high and continued expression of enhanced green fluorescent protein by human hematopoietic progenitors engrafted in nonobese diabetic/severe combined immunodeficient mice

Despite efforts toward improvements in retrovirus-mediated gene transfer, s table high-level expression of a therapeutic gene in human hematopoietic st em cells remains a great challenge. We have evaluated the efficiency of dif ferent viral long terminal repeats (LTRs) in long-term expression of a tran sgene in vivo, using severe combined immunodeficiency (SCID)-repopulating c ell assays. Vectors used were variants of the simplified retroviral vector GCsap with the different LTRs of Moloney murine leukemia virus (MLV), myelo proliferative sarcoma virus (MPSV), and murine stem cell virus (MSCV). The enhanced green fluorescent protein (EGFP) gene was used as a marker to asse ss levels of transduction efficiency. CD34(+) cells isolated from human cor d blood were transduced by exposure to virus-containing supernatants on fib ronectin fragments and in the presence of stem cell factor, interleukin 6, Flt-3 ligand, and thrombopoietin, and then transplanted into nonobese diabe tic/SCID mice. Engraftment of human cells highly expressing EGFP, with diff erentiation along multiple cell lineages, was demonstrated for up to 18 wee ks posttransplant, although the three different vectors showed different tr ansduction frequencies (MLV, <0.1-33.2%; MPSV, <0.1-22.8%; MSCV, 0.3-51.7%) . Of importance is that high-level transduction frequencies in human progen itor cells were also confirmed by colony-forming cell assays using bone mar row from transplanted mice, in which EGFP-expressing, highly proliferative potential colonies were observed hy fluorescence microscopy. In these mice the vector carrying the MSCV LTR generated more EGFP-expressing human cells than did either of the other two constructs, indicating that GCsap carryin g the MSCV LTR may be an efficient tool for stem cell gene therapy.