SUITABILITY OF BONE-MARROW FROM HIV-1-INFECTED DONORS FOR RETROVIRUS-MEDIATED GENE-TRANSFER

Bone marrow samples from 21 human immunodeficiency virus type 1 (HIV-1 )-infected subjects were evaluated for their suitability for retroviru s-mediated gene transduction with anti-HIV-l genes. The percentages of CD34(+) cells that could be isolated from the mononuclear fraction of bone marrow samples were determined. Fifteen of the 21 marrow samples had normal percentages of CD34(+) cells isolated by immunomagnetic me thods. All seven donors with CD4 counts >100/mm(3) had normal percenta ges of CD34(+) cells; of 14 patients with low CD4 cell counts (<100/mm (3)), 5 had reduced and 9 had normal percentages of CD34(+) cells. Sam ples of the marrow were plated in a methylcellulose colony-forming uni t (CFU) assay to determine the clonogenic capacity of the progenitor c ells. Overall, the marrow samples from I-W-infected donors showed a 44 % reduction in CFU derived from the mononuclear cell fraction and a 75 % reduction in CFU derived from the isolated CD34(+) cell fraction, wh en compared to marrow samples from uninfected donors. Isolated CD34(+) cells were transduced with retroviral vectors containing various anti -HIV-l genes to determine their susceptibility to gene transfer. Trans duction of the clonogenic CD34(+) cells by retroviral vectors did not differ among marrow samples from 13 HIV-1(+) donors and 9 uninfected d onors. Long-term bone marrow cultures established from the transduced CD34(+) cells demonstrated equivalent survival of clonogenic progenito r cells from both HN-l-infected and uninfected marrows. Toxicity from expression of the anti-HIV-1 genes was not observed; the percentages o f clonogenic progenitor cells that survived in cultures transduced by vectors carrying anti-HN-l genes were similar to those transduced by t he control LN vectors. Stromal cells cultured from marrow samples from HIV-l-infected donors showed similar growth kinetics, hematopoietic s upport function, and enhancement of retrovirus-mediated transduction o f CD34(+) cells as seen with stromal cells cultured from uninfected ma rrow donors. Semi-quantitative polymerase chain reaction (PCR) was per formed before and after ex vivo transduction to determine the frequenc y of HIV-l-containing cells in the CD34(+) cell preparations. Although HIV-1(+) cells were present at low levels in the mononuclear cell fra ctions of some of the marrow samples, the CD34(+) cell preparation fro m only one marrow sample contained detectable HIV-1 positive cells (<1 positive cell/100,000 by PCR) prior to transduction. None of the CD34 (+) ceh preparations contained detectable HIV-1 after transduction. Th ese studies demonstrate that HIV-l-infected patients are candidates fo r retrovirus-mediated transduction of anti-HIV-l genes in bone marrow gene therapy clinical trials.