RESUMO
Monocytes and macrophages (Mo/Mphi) contribute to the pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection. A successful hematopoietic stem/progenitor cell (HSPC)-based gene therapy strategy for HIV-1 disease must protect Mo/Mphi as well as T cells from HIV-1-related pathology. In this report, we demonstrate that RevM10-transduced HSPCs isolated from cytokine-mobilized peripheral blood give rise to Mo/Mphi suppressing replication of Mphi-tropic HIV-1 isolates. A Moloney murine leukemia virus (MoMLV)-based retroviral vector encoding a bicistronic mRNA co-expressing RevM10 and the murine CD8alpha' chain (Lyt2) was used to transduce HSPCs. Following transduction, these cells were expanded and differentiated by short-term culture in methylcellulose containing various cytokines. In vitro differentiated Mo/Mphi were enriched by fluorescence activated cell sorting (FACS) for the co-expressed transgene (Lyt2) and myelomonocytic (CD33, CD14) surface markers. HIV-1 replication of two Mphi-tropic isolates (JR-FL, BaL) was inhibited in Mo/Mphi expressing RevM10 and Lyt2 relative to control cells expressing only Lyt2 but no functional RevM10 gene product. Cell proliferation and expression of lineage-specific surface markers was not altered in transduced, in vitro differentiated Mo/Mphi cells. This study supports the feasibility of HSPC-based gene therapy as a future treatment for HIV-1 disease.
Assuntos
Transformação Celular Viral , Vetores Genéticos , HIV-1/fisiologia , Células-Tronco Hematopoéticas/virologia , Vírus da Leucemia Murina de Moloney , Monócitos/virologia , Replicação Viral , Células-Tronco Hematopoéticas/citologia , Humanos , Leucócitos Mononucleares/citologia , Macrófagos/citologia , Macrófagos/virologia , Monócitos/citologiaRESUMO
Gene therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infection using intracellular immunization strategies is currently being tested in clinical trials. With the continuing development of potent antiretroviral drugs (e.g., reverse transcriptase [RT] and protease [PR] inhibitors), it is likely that HIV-1 gene therapy will be applied to humans concurrently receiving such antiretroviral medication. In this study, we assessed the in vitro antiviral efficacy of two gene therapy strategies (trans-dominant RevM10, Gag antisense RNA) in combination with clinically relevant RT (AZT, ddC) or PR (indinavir) inhibitors. Retrovirally transduced, human T cell lines expressing antiviral gene constructs were inoculated with high doses of HIV-1HXB3 in the presence or absence of inhibitors. The combination of RevM10 or Gag antisense RNA with antiviral drugs inhibited HIV-1 replication 10-fold more effectively than the single antiviral drug regimen alone. More importantly, we also addressed whether gene therapy strategies are effective against drug-resistant HIV-1 isolates. Both the RevM10 and Gag antisense RNA strategies showed antiviral efficacy against several RT inhibitor-resistant HIV-1 isolates equivalent to their inhibition of HIV-1HXB3 replication. In summary, our data demonstrate the greater than additive antiviral efficacy of gene therapy strategies and RT or PR inhibitors, and that gene therapy approaches are effective against drug-resistant HIV-1 viral isolates.
Assuntos
Síndrome da Imunodeficiência Adquirida/terapia , Fármacos Anti-HIV/uso terapêutico , Terapia Genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Linhagem Celular , Terapia Combinada , DNA Recombinante , Relação Dose-Resposta a Droga , Produtos do Gene gag/genética , Produtos do Gene gag/fisiologia , Produtos do Gene rev/genética , Produtos do Gene rev/fisiologia , Vetores Genéticos/genética , Genoma Viral , Proteína do Núcleo p24 do HIV/efeitos dos fármacos , Proteína do Núcleo p24 do HIV/metabolismo , HIV-1/crescimento & desenvolvimento , Humanos , Indinavir/uso terapêutico , RNA Antissenso/genética , RNA Antissenso/fisiologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/virologia , Zalcitabina/administração & dosagem , Zalcitabina/uso terapêutico , Zidovudina/administração & dosagem , Zidovudina/uso terapêutico , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
The potential of hematopoietic stem cells (HSCs) from human immunodeficiency virus type-1 (HIV-1)-infected individuals, eg, self-renewal and multilineage differentiative capacity, might be perturbed due to the underlying disease. In this study, we assessed the HSC activity in the CD34+ Thy-1+ cell population of peripheral blood stem cells (PBSCs) of three asymptomatic HIV-1-infected individuals after granulocyte colony-stimulating factor (G-CSF; 10 microg/kg/d) mobilization. On day 4 of G-CSF treatment, 0.8% to 1% of the total blood mononuclear cells were CD34+. Leukapheresis followed by a two-step cell isolation process yielded a CD34+ Thy-1+ cell population of high purity (76% to 92% CD34+ Thy-1+ cells). This cell population showed no evidence of HIV-1-containing cells based on a semiquantitative HIV-1 DNA polymerase chain reaction. Furthermore, the purified cells showed normal hematopoietic potential in in vitro clonogenic assays. Successful gene transfer into committed progenitor cells (colony-forming units-cells) and more primitive stem/progenitor cells (long-term culture colony-forming cells) could be shown after amphotropic retroviral transduction. These data provide evidence that the CD34+ Thy-1+ stem cell compartment can be mobilized and enriched in early stage HIV-1-infected patients. Furthermore, successful transduction of this cell population as a prerequisite for stem cell-based clinical gene therapy protocols was demonstrated.
