Current Moloney Murine Leukemia Virus (MLV) based vector strategies have failed to produce efficient transduction of human stem cells, a population which is quiescent and expresses low levels of virus receptor. A novel vector system in which a lentiviral vector (lenti) is pseudotyped with the vesicular stomatitis virus envelope G protein (VSV) has been developed in order to overcome the innate obstacles of stem cell transduction. We compared the efficiency of transducing CD34+ subpopulations using "lenti/VSV" with MLV pseudotyped with either VSV (MLV/VSV) or with GALV (MLV/GALV) under a variety of conditions and at increasing Multiplicities of Infection (MOI). Each construct contained the enhanced Green Fluorescent Protein (eGFP) as a reporter gene (using the CMV promoter in lenti/VSV and MLV/VSV and the LTR promoter in MLV/GALV). No toxicity was noted with MOI up to 3,000. Transduction of CD34+CD38+cells over three days in IL-3. IL-6 and SF (36S) resulted in high efficiency transduction of short term cultures with all three vectors (eGFP expression 14-58% with lenti/VSV (MOI 300-3,000), 12-39% with MLV/VSV (MOI 300-3,000) and 6-36% with MLV/GALV (MOI 300) (n=4). However, eGFP was only detectable in long term culture of CD34+CD38+ cells transduced with lenti/VSV (12-22% eGFP at 5 weeks). Three day transduction of more primitive progenitors, CD34+CD38- cells, in 36S with lenti/VSV resulted in 15-39% eGFP expression persisting for at least 5 weeks of culture (n=3) In contrast, MLV/VSV and MLV/GALV produced only 0-2% eGFP expression in short or long term cultures of CD34+CD38- cells. We next studied whether progenitors could be transduced by lenti/VSV during brief exposure to virus without growth factor stimulation. Exposure to lenti/VSV for 12 hours in the absence of growth factors resulted in eGFP expression in 7-12% of CD34+CD38+ cells (n=2) and 52% of CD34+CD38- cells (n=l) with stable expression in long term culture No eGFP expression was detectable in short or long term cultures after transduction with MLV/VSV or MLV/GALV in the absence of growth factors. The lenti/VSV system thus significantly improves the efficiency of gene transfer into a quiescent population of primitive progenitors and may provide a method for efficient hematopoietic stem cell transduction.
|Number of pages||1|
|Publication status||Published - 1998|
ASJC Scopus subject areas
- Cancer Research
- Cell Biology