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 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 CD3+ 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.
Kearns
K
K
Division of Research Immunology/Bone Marrow Transplantation, Childrens Hospital Los Angeles, University of Southern California School of Medicine 90027, USA.
Bahner
I
I
Bauer
G
G
Wei
S F
SF
Valdez
P
P
Wheeler
S
S
Woods
L
L
Miller
R
R
Casciato
D
D
Galpin
J
J
Church
J
J
Kohn
D B
DB
D000293:Adolescent; D000328:Adult; D018952:Antigens, CD34 / Q000737:chemistry; D001853:Bone Marrow / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D015704:CD4 Antigens / Q000737:chemistry; D018791:CD4 Lymphocyte Count; D002455:Cell Division; D002460:Cell Line; D002469:Cell Separation; D002648:Child; D002675:Child, Preschool; D018628:Gene Dosage; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D015658:HIV Infections / Q000235:genetics* / Q000276:immunology* / Q000473:pathology; D015497:HIV-1; D006801:Humans; D007223:Infant; D008213:Lymphocyte Activation; D008979:Moloney murine leukemia virus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017154:Stromal Cells / Q000276:immunology / Q000473:pathology / Q000821:virology
D018952:Antigens, CD34; D015704:CD4 Antigens