Gene transfer to repopulating human CD34+cells using amphotropic, GALV or RD114 pseudotyped HIV-1 based vectors from stable producer cells.

A novel, stable human immunodeficiency virus type 1 (HIV-1) vector packaging system, STAR, was tested for its ability to transduce human cord blood CD34+ progenitor cells assayed both in vitro and after transplantation to NOD/SCID mice. Vectors pseudotyped with three different gammaretrovirus envelopes were used; the amphotropic MLV envelope (MLV-A), a modified gibbon ape leukemia virus envelope (GALV+) and a modified feline endogenous virus RD114 envelope (RDpro). Titration of vectors harvested from the stable packaging cells gave the following results: MLV-A: 1–3x106 inf.u/ml, GALV+: 1x106 inf.u/ml and RDpro: 3–10x106 inf.u/ml CB CD34+ cells were transduced on Retronectin® preloaded with vector either in fresh medium (PL alone) or in vector containing medium (PL+VCM). Gene transfer to freshly thawed CD34+ in the absence of cytokines was very low. Addition of cytokines (CK; SCF, TPO and FL) increased gene transfer efficiency significantly and this was further augmented if the cells were prestimulated for 24 hours wit the same cytokines. Concentration of the vectors (15-fold) by low-speed ultracentrifugation increased gene transfer to CD34+ cells in vitro even further. More than 90% of cells were transduced with a single exposure to the RDpro vector as determined by GFP expression using flow cytometry. The two other pseudotypes transduced approximately 70% of the cells under the same conditions. Detailed in vitro transduction data is shown in table as percentage GFP positive cells: | | RDpro | MLV-A | GALV+ | |:------------------------------------- | --------- | --------- | --------- | --------- | | | PL alone | PL+VCM | PL alone | PL+VCM | PL alone | PL+VCM | | No CK | 9.2±2 | 0.9±0.8 | 12.5±3.5 | 6.6±0.1 | 0.9±0.4 | 1.4±0.5 | | With CK | 22.4±8.3 | 3.3±5.3 | 18.2±3.8 | 10.8±3.8 | 4.8±1.7 | 16.7±9.0 | | CK prestim f 24 hours | 57.6±14.9 | 35.3±32.1 | 45.4±14.9 | 36.3±14.8 | 3.4±1.7 | 19.5±12.2 | | Concentrated vector CK prestim f 24 h | 93.4±3.3 | 93.3±3.7 | 73.3±7.6 | 40.3±20.4 | 66.1±7.5 | 77.8±10.9 | Transplantation of CD34+ cells prestimulated for 24 hours and then transduced with a single hit of concentrated vector to irradiated NOD/SCID mice revealed that the RDpro vector transduced 55.1% of NOD/SCID repopulating human cells which was significantly higher than the MLV-A (12.6%) or GALV+ (25.1%) pseudotyped vectors. Thus, the STAR packaging system, especially with the modified RD114 envelope, appears to be a new valuable tool for the genetic modification of primitive human progenitor cells.