Non-stable viral and stable non-viral gene transfer to hematopoietic stem cells

Mature blood cells have a limited life-span, and are continuously replaced by proliferation and differentiation of pluripotent hematopoietic stem cells (HSC) during hematopoiesis. Although HSC are an extremely attractive target for gene therapy to treat diseases of the blood there are still few methods that achieve gene delivery to these cells safely and efficiently. In these studies, we have explored novel methods for gene delivery to HSC. We developed Non-integrating Lentiviral (NIL) vectors for use in situations where transient gene expression is desired. NIL vectors harbor mutations designed to disable vector integration without affecting entry into cells. We followed the levels of eGFP expression from NIL vectors in human Jurkat cells and primary human CD34+ cells. Using NIL vectors carrying the neomycin phosphotransferase gene, we determined the relative frequency of G418-resistant HT29-cell colonies as a measure of stable integration and we characterized the nature of the integrants. Independently, we investigated two non-viral strategies for stable gene transfer in HSC. A gene delivery system based on the Sleeping Beauty (SB) transposon exists where a plasmid transiently expressing the SB transposase mediates the stable integration of a second plasmid containing an expression unit flanked by the transposon-derived inverted repeats (IR). In K562 cells we observed eGFP expression in >60% of cells 2 months after electroporation. In CD34+ cells, we observed stable gene transfer at levels up to 6%, but stable gene transfer to more primitive NOD/SCID repopulating cells or CD34+/CD38- cells was low. A system based on the streptomyces phage integrase phiC31 was also used to promote site-restricted, stable integration of a plasmid containing an expression unit and an attI3 site into endogenous pseudo attP-sites in mammalian genomes. In the K562 cell line, we observed eGFP expression in >10% of cells one month after electroporation, however we have been unable to achieve persistent reporter gene expression in human CD34+ progenitor cells thus far. Although further improvements in gene transfer efficacy are needed, the stable gene expression observed by these systems highlights their potential for ameliorating hematopoietic diseases.