Characterization of anti-CCR5 ribozyme-transduced CD34+ hematopoietic progenitor cells in vitro and in a SCID-hu mouse model in vivo.
The cellular entry of HIV is mediated by the specific interaction of viral envelope glycoproteins with the cell-surface marker CD4 and a chemokine receptor (CCR5 or CXCR4). Individuals with a 32-base-pair (bp) deletion in the CCR5 coding region, which results in a truncated peptide, show resistance to HIV-1 infection. This suggests that the downregulation of CCR5 expression on target cells may prevent HIV infection. Therefore, ribozymes that inhibit the CCR5 expression offer a novel approach for anti-HIV gene therapy. To assess the effect of an anti-CCR5 ribozyme (R5Rbz) on macrophage differentiation, CD34+ hematopoietic progenitor cells were transduced with a retroviral vector carrying RSRbz and allowed to differentiate in the presence of appropriate cytokines. R5Rbz-transduced CD34+ cells differentiated normally into mature macrophages that carried CD14 and CD4 surface markers, expressed the anti-CCR5 ribozyme, and showed significant resistance to viral infection upon challenge with the HIV-1 BaL strain. Using an in vivo thymopoiesis model, the effect of RSRbz on stem cell differentiation into thymocytes was evaluated by reconstituting SCID-hu mice thymic grafts with ribozyme-transduced CD34+ cells. FACS analysis of cell biopsies at 4 and 6 weeks postengraftment for HLA, CD4, and CD8 markers showed comparable levels of reconstitution and similar percentages of subpopulations of thymocytes between grafts receiving R5Rbz-transduced and control CD34+ cells. RT-PCR assays demonstrated the expression of the anti-CCR5 ribozyme in CD4+, CD8+, and CD4+/CD8+ thymocyte subsets derived from RSRbz-transduced CD34+ cells. These results indicate that anti-CCR5 ribozyme can be introduced into hematopoietic stem cells without adverse effects on their subsequent lineage-specific differentiation and maturation. The expression of anti-CCR5 ribozymes in HIV-1 target cells offers a novel gene therapy strategy to control HIV infection.
Department of Pathology, Colorado State University, Fort Collins 80523, USA.
J
J
Bai
S
S
Gorantla
N
N
Banda
L
L
Cagnon
J
J
Rossi
R
R
Akkina
D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism*; D065100:CCR5 Receptor Antagonists; D003114:Colony-Forming Units Assay; D015536:Down-Regulation; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D015658:HIV Infections / Q000517:prevention & control*; D015497:HIV-1; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D016130:Immunophenotyping; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D019713:Receptors, CCR5 / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000378:metabolism
D018952:Antigens, CD34; D065100:CCR5 Receptor Antagonists; D016337:RNA, Catalytic; D012333:RNA, Messenger; D019713:Receptors, CCR5