Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles.
The use of Moloney murine leukemia virus (Mo-MLV)-based vectors to deliver therapeutic genes into target cells is limited by their inability to transduce nondividing cells. To test the capacity of HIV-based vectors to deliver genes into nondividing cells, we have generated replication-defective HIV type 1 (HIV-1) reporter vectors carrying neomycin phosphotransferase or mouse heat stable antigen, replacing the HIV-1 sequences encoding gp160. These vectors also harbor inactive vpr, vpu, and nef coding regions. Pseudotyped HIV-1 particles carrying either the ecotropic or the amphotropic Mo-MLV envelope proteins or the vesicular stomatitis virus G protein were released after single or double transfections of either human 293T or monkey COS-7 cells with titers of up to 10(7) colony-forming units per milliliter. A simple ultrafiltration procedure resulted in an additional 10- to 20-fold concentration of the pseudotyped particles. These vectors along with Mo-MLV-based vectors were used to transduce primary human skin fibroblasts and human peripheral blood CD34+ cells. The HIV-1 vector system was significantly more efficient than its Mo-MLV-based counterpart in transducing human skin fibroblasts arrested at the G0/G1 stage of the cell cycle by density-dependent inhibition of growth. Human CD34+ cells were transduced efficiently using HIV-1 pseudotype particles without prior stimulation with cytokines.
Reiser
J
J
Molecular and Medical Genetics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. jreiser@helix.nih.gov
Harmison
G
G
Kluepfel-Stahl
S
S
Brady
R O
RO
Karlsson
S
S
Schubert
M
M
D000818:Animals; D018952:Antigens, CD34; D019556:COS Cells; D002453:Cell Cycle; D002460:Cell Line; D003673:Defective Viruses / Q000502:physiology*; D015754:Genes, nef; D016334:Genes, vpr; D016338:Genes, vpu; D015316:Genetic Therapy; D005822:Genetic Vectors; D019302:HIV Envelope Protein gp160 / Q000096:biosynthesis / Q000235:genetics; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D019868:Kanamycin Kinase; D008214:Lymphocytes; D051379:Mice; D008979:Moloney murine leukemia virus; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D012867:Skin / Q000378:metabolism; D014162:Transfection
D018952:Antigens, CD34; D019302:HIV Envelope Protein gp160; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase