Suitability of Bone Marrow from HIV-1-Infected Donors for Retrovirus-Mediated Gene Transfer

Abstract

Bone marrow samples from 21 human immunodeficiency virus type 1 (HIV-1)-infected subjects were evaluated for their suitability for retrovirus-mediated gene transduction with anti-HIV-1 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 methods. All seven donors with CD4 counts >100/mm³ had normal percentages of CD34⁺ cells; of 14 patients with low CD4 cell counts (<100/mm³), 5 had reduced and 9 had normal percentages of CD34⁺ cells. Samples of the marrow were plated in a methylcellulose colony-forming unit (CFU) assay to determine the clonogenic capacity of the progenitor cells. Overall, the marrow samples from HIV-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, when compared to marrow samples from uninfected donors. Isolated CD34⁺ cells were transduced with retroviral vectors containing various anti-HIV-1 genes to determine their susceptibility to gene transfer. Transduction of the clonogenic CD34⁺ cells by retroviral vectors did not differ among marrow samples from 13 HIV-1⁺ donors and 9 uninfected donors. Long-term bone marrow cultures established from the transduced CD34⁺ cells demonstrated equivalent survival of clonogenic progenitor cells from both HIV-1-infected and uninfected marrows. Toxicity from expression of the anti-HIV-1 genes was not observed; the percentages of clonogenic progenitor cells that survived in cultures transduced by vectors carrying anti-HIV-1 genes were similar to those transduced by the control LN vectors. Stromal cells cultured from marrow samples from HIV-1-infected donors showed similar growth kinetics, hematopoietic support function, and enhancement of retrovirus-mediated transduction of CD34⁺ cells as seen with stromal cells cultured from uninfected marrow donors. Semi-quantitative polymerase chain reaction (PCR) was performed before and after ex vivo transduction to determine the frequency of HIV-1-containing cells in the CD34⁺ cell preparations. Although HIV-1⁺ cells were present at low levels in the mononuclear cell fractions of some of the marrow samples, the CD34⁺ cell preparation from only one marrow sample contained detectable HIV-1 positive cells (<1 positive cell/100,000 by PCR) prior to transduction. None of the CD34⁺ cell preparations contained detectable HIV-1 after transduction. These studies demonstrate that HIV-1-infected patients are candidates for retrovirus-mediated transduction of anti-HIV-1 genes in bone marrow gene therapy clinical trials.

Overview summary

Gene therapy for patients with human immunodeficiency virus type 1 (HIV-1) infection may be performed by engineering their bone marrow stem cells to express genes that block the growth of HIV-1 in the mature blood cells which are produced. However, deleterious effects from HIV-1 infection on the bone marrow may limit the use of marrow cells from these patients for gene transfer by retroviral vectors. We studied bone marrow samples from 21 HIV-1-infected donors and found that, in most cases, the bone marrow from these patients had normal numbers and functions of the marrow CD34⁺ progenitor cells that would he targeted hy gene therapy. Some patients with advanced disease did have reduced numbers of CD34⁺ cells in their marrow, which can he identified by performing a screening bone marrow aspirate prior to treatment. The CD34⁺ cells isolated from the marrow of these patients had no detectable HIV-1 after the manipulations performed for ex vivo gene transfer. These results demonstrate that the bone marrow from most patients with HIV-1 infection should be suitable for clinical trials of gene therapy to confer resistance to HIV-1 infection.