Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells

Abstract

The ability of lentiviral vectors to transfer genes into human hematopoietic stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)–derived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G protein of vesicular stomatitis virus (VSV). High-efficiency transduction of human cord blood CD34+cells was achieved after overnight incubation with vector particles. Sixteen to 28 percent of individual colony-forming units granulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34+ cells were positive by polymerase chain reaction (PCR) for the GFP gene. The transduction efficiency of SCID-repopulating cells (SRC) within the cord blood CD34+population was assessed by serial transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. When 400 000 cord blood CD34+ cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondary recipients contained the vector on average as determined by PCR. Transplantation of transduced cells in limiting dilution generated GFP+ lymphoid and myeloid progeny cells that may have arisen from a single SRC. Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. These results show that lentiviral vectors can efficiently transduce very primitive human hematopoietic progenitor and stem cells.

High Levels of Transgene Expression Following Transduction of Long‐Term NOD/SCID‐Repopulating Human Cells with a Modified Lentiviral Vector

“…In particular, during the preparation of this manuscript, Woods et al reported that sustained transgene expression was achieved in transduced human cells engrafted in primary and second NOD/SCID mice using an improved LV and a short transduction protocol similar to ours [42]. Collectively, these studies provide proof-of-principle that LV vectors have the potential to become important delivery vehicles for HSCbased gene therapy.…”

“…of LV containing internal viral and cellular promoters for enhanced transgene expression in hematopoietic cells [39][40][41][42].…”

Development of gene therapy for blood disorders by gene transfer into haematopoietic stem cells

“…Lentiviral vectors can transduce quiescent haematopoietic progenitor cells [11,12,14]. However, it has also been demonstrated that although human CD34+ cells in G0 can be transduced, CD34+ cells in G 1 or G 2 /S/M are transduced more efficiently [16].…”

“…However, it has also been demonstrated that although human CD34+ cells in G0 can be transduced, CD34+ cells in G 1 or G 2 /S/M are transduced more efficiently [16]. It has been shown that umbilical cord blood CD34+ cells, which are mostly in G 0 and G 1 , can be grafted efficiently in immunocompromised NOD/SCID mice, even after serial transplantation [12], and that lentiviral vectors can transduce these SCID repopulating cells (SRCs) with 50-60% efficiency [11,12,14]. The umbilical cord blood (CB) cells that have been transduced are mostly in G 1 (65-70%) and to a lesser extent in G 0 (20-25%) [12].…”

“…While optimizations of the lentiviral vector and lentiviral transduction conditions have allowed for improved transduction of SCID repopulating cells, advances have also been achieved with expression of the transgenes. Because lentiviral vectors were first developed for gene therapy applications, the transgene expression is driven from ubiquitously expressing internal promoters such as the cytomegalovirus (CMV) early promoter, or the murine phosphoglycerate kinase (PGK) gene promoter [11,12]. While these constructs were hailed a success, as they showed persistent expression in serially transplanted NOD/SCID mice, the level of transgene expression was low.…”