Lentiviral vectors for enhanced gene expression in human hematopoietic cells

Accumulated data indicate that current generation lentiviral vectors, which generally utilize an internal human cytomegalovirus (CMV) immediate early region enhancer-promoter to transcribe the gene of interest, are not yet op timized for efficient expression in human hematopoietic stem/progenitor cel ls (HSPCs). As a first step toward this goal, we constructed self-inactivat ing derivatives of the HIV-l-based transfer vector pHR' containing the enha nced green fluorescent protein (GFP) gene as reporter and the Woodchuck hep atitis virus posttranscriptional regulatory element (WPRE). GFP expression was driven by a variety of strong viral and cellular promoters, including t he murine stem cell virus (MSCV) long terminal repeat (LTR), a Gibbon ape l eukemia virus (GALV) LTR, the human elongation factor 1 alpha (EF1 alpha) p romoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken beta -actin promoter), and the human phosphoglycer ate kinase 1 (PGK) promoter. In contrast to results obtained in human embry onic kidney 293T cells and fibrosarcoma HT1080 cells, in which the CMV prom oter expressed GFP at the highest levels, significantly higher levels of GF P expression (3- to 5-fold) were achieved with the MSCV LTR, the EF1 alpha promoter, and the CAG promoter in the human HSPC line KG1 alpha. Removal of the WPRE indicated that it stimulated GFP expression from all of the vecto rs in KG1 alpha cells (up to 3-fold), although it only marginally improved the performance of the intron-containing EF1 alpha and CAG promoters (<1.5- fold stimulation). The vectors using the MSCV LTR, the GALV LTR, and the PG K promoter were the most efficient at transducing primary human CD34(+) cor d blood progenitors under the conditions employed. High-level GFP expressio n in the NOD/SCID xenograft model was demonstrated with the pHR' derivative bearing the MSCV LTR. These new lentiviral vector backbones provide a basi s for the rational design of improved delivery vehicles for human HSPC gene transfer applications.