679. Gene Transfer to Hematopoietic Stem/ Progenitor Cells Using SV40-Derived Vectors
Authors: Alena A. Chekmasova, Mahender Singh, David S. Strayer
Journal: Molecular Therapy (2006)

Stem cell-based gene therapy of human immunodeficiency virus (HIV) infection aims at inhibiting HIV replication and progression of infection to acquired immunodeficiency syndrome (AIDS) by introducing antiviral genes in primitive hematopoietic stem cells (HSC). Ideally, after differentiation into mature blood cells, these antiviral genes should create a host-cell population that is resistant to HIV infection.We approached this goal using recombinant SV40 (rSV40)-derived vectors because of their high efficiency in transducing primitive hematopoietic progenitors. Two methods of gene delivery were used. Rat and rabbit hematopoietic progenitor cells were prepared using magnetic bead fractionation of whole bone marrow, then transduced in culture. Also, based on previous demonstrations of effective rSV40 transduction of bone marrow (BM) by direct in vivo injection in rat femurs, we tested direct femoral intramarrow injection for in vivo gene delivery in rabbits. Two rSV40s were used. SV(Nef-FLAG), carries cDNA for HIV Nef protein with a C-terminal FLAG epitope tag. A bifunctional vector, SV(RNAiR5#5/ RevM10) carries RevM10 with a carboxyl terminus AU1 epitope tag, plus RNAiR#5, an interfering RNA directed against CCR5 mRNA and driven by the adenovirus VA1 promoter, which utilizes RNA polymerase III. Expression of Nef-FLAG and RevM10-AU1 fusion proteins was driven by CMV immediate early promoter. Transgene expression was tested in bone marrow and peripheral blood by flow cytometry (FACS) or fluorescence microscopy after intracellular immunostaining.About 42% of CD34+ hematopoietic progenitor cells in rabbit femoral bone marrow expressed AU1 after in vitro transduction with SV(RNAiR5#5/RevM10) at MOI =100. Levels of FLAG expressing bone marrow cells and lineage marker expression among the several hematopoietic subpopulations were tested 7 days after intramarrow injection of rats. The highest percentages of FLAG- expressing cells were detected in the granulocyte series and cells that expressed stem cell antigen (SCA). Also, about 35% of rabbit bone marrow cells expressed AU1 7 days after either in vitro or in vivo transduction. Ficoll-separated blood mononuclear cells (pbmc) were strongly positive for transgene expression 14 days after intramarrow injection. Follow-up is continuing to determine the longevity of transgene expression and the specific pbmc populations involved.Thus, hematopoietic stem/progenitor cells are attractive candidates for single- and multi-agent genetic therapy. Our data suggest that, especially combining transgenes targeting CCR5 and delivery by rSV40 vectors directly to the bone marrow, bone marrow-directed genetic therapy for treatment of AIDS may be possible without selection, transplantation or application of complete or partial bone marrow ablation regimens. Stem cell-based gene therapy of human immunodeficiency virus (HIV) infection aims at inhibiting HIV replication and progression of infection to acquired immunodeficiency syndrome (AIDS) by introducing antiviral genes in primitive hematopoietic stem cells (HSC). Ideally, after differentiation into mature blood cells, these antiviral genes should create a host-cell population that is resistant to HIV infection. We approached this goal using recombinant SV40 (rSV40)-derived vectors because of their high efficiency in transducing primitive hematopoietic progenitors. Two methods of gene delivery were used. Rat and rabbit hematopoietic progenitor cells were prepared using magnetic bead fractionation of whole bone marrow, then transduced in culture. Also, based on previous demonstrations of effective rSV40 transduction of bone marrow (BM) by direct in vivo injection in rat femurs, we tested direct femoral intramarrow injection for in vivo gene delivery in rabbits. Two rSV40s were used. SV(Nef-FLAG), carries cDNA for HIV Nef protein with a C-terminal FLAG epitope tag. A bifunctional vector, SV(RNAiR5#5/ RevM10) carries RevM10 with a carboxyl terminus AU1 epitope tag, plus RNAiR#5, an interfering RNA directed against CCR5 mRNA and driven by the adenovirus VA1 promoter, which utilizes RNA polymerase III. Expression of Nef-FLAG and RevM10-AU1 fusion proteins was driven by CMV immediate early promoter. Transgene expression was tested in bone marrow and peripheral blood by flow cytometry (FACS) or fluorescence microscopy after intracellular immunostaining. About 42% of CD34+ hematopoietic progenitor cells in rabbit femoral bone marrow expressed AU1 after in vitro transduction with SV(RNAiR5#5/RevM10) at MOI =100. Levels of FLAG expressing bone marrow cells and lineage marker expression among the several hematopoietic subpopulations were tested 7 days after intramarrow injection of rats. The highest percentages of FLAG- expressing cells were detected in the granulocyte series and cells that expressed stem cell antigen (SCA). Also, about 35% of rabbit bone marrow cells expressed AU1 7 days after either in vitro or in vivo transduction. Ficoll-separated blood mononuclear cells (pbmc) were strongly positive for transgene expression 14 days after intramarrow injection. Follow-up is continuing to determine the longevity of transgene expression and the specific pbmc populations involved. Thus, hematopoietic stem/progenitor cells are attractive candidates for single- and multi-agent genetic therapy. Our data suggest that, especially combining transgenes targeting CCR5 and delivery by rSV40 vectors directly to the bone marrow, bone marrow-directed genetic therapy for treatment of AIDS may be possible without selection, transplantation or application of complete or partial bone marrow ablation regimens.