Abstract
Lentiviruses infect both dividing and nondividing cells. In this study we characterized a lentiviral vector system consisting of a packaging vector (pHP) and a transducing vector (pTV) derived from a recombinant human immunodeficiency virus type 1 (HIV-1). In pHP, the long terminal repeats (LTRs), the 5′ untranslated leader and portions of the env and nef genes were deleted. The leader sequence of pHP was substituted with a modified Rous sarcoma virus (RSV) 59 bp leader containing a mutated RSV gag AUG and a functional 5′ splice site. The pHP construct was found to direct Gag-Pol synthesis as efficiently as wild-type HIV-1. The pTV construct contains sequences required for RNA packaging, reverse transcription and integration, but lacks viral genes. Co-transfection of pHP, pTV and a vesicular stomatitis virus G (VSV-G) envelope plasmid produced vectors at titers of 105–106 transducing units per milliliter in 48 h. Replication-competent virus (RCV) was not detected when deletions were made in the env gene in pHP. The ability of this vector system to transduce dividing and nondividing cell in vitro and in vivo was also demonstrated. Compared with a Moloney murine leukemia virus (MLV) vector, the HP/TV vectors transduced human muscle-, kidney-, liver-derived cell lines and CD34+ primary hematopoietic progenitor cells more efficiently. Although the levels of the pTV transgene expression were high soon after transduction, the expression tended to decrease with time due either to the loss of proviral DNA or to the inactivation of promoter activity, which was found to be cell type-dependent. Analyses of extrachromosomal DNA showed that the unintegrated proviral DNA of lentiviral vectors survived much longer than that of the retroviral vectors. We demonstrate that the HP/TV vector is capable of high efficiency transduction and that long-term expression of lentiviral vectors is dependent on target cell type, the internal promoter and the transgene itself in the transducing vector.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Gordon EM, Anderson WF . Gene therapy using retroviral vectors Curr Opin Biotechnol 1994 5: 611–616
Miller AD, Miller DG, Garcia JV, Lynch CM . Use of retroviral vectors for gene transfer and expression Meth Enzymol 1993 217: 581–599
Russell D, Miller AD . Foamy virus vector J Virol 1996 70: 217–222
Verma IM, Somia N . Gene therapy – promises, problems and prospects Nature 1997 389: 239–242
Poeschla EM, Wong-Staal F, Looney DJ . Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors Nature Med 1998 4: 354–357
Burns JC et al. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to a very high titer and efficient gene transfer into mammalian and nonmammalian cells Proc Natl Acad Sci USA 1993 90: 8033–8037
Shimada T, Fujii H, Mitsuya H, Nienhuis AW . Targeted and highly efficient gene transfer into CD4+ cells by a recombinant human immunodeficiency virus retroviral vector J Clin Invest 1991 88: 1043–1047
Naldini L et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector Science 1996 272: 263–267
Poeschla E, Corbeau P, Wong-Staal F . Development of HIV vectors for anti-HIV gene therapy Proc Natl Acad Sci USA 1996 93: 11395–11399
Chang L-J, Zhang C . Infection and replication of Tat-minus human immunodeficiency viruses: genetic analyses of LTR and tat mutants in primary and long-term human lymphoid cells Virology 1995 211: 157–169
Chang L-J, McNulty E, Martin M . Human immunodeficiency viruses containing heterologous enhancer/promoters are replication competent and exhibit different lymphocyte tropisms J Virol 1993 67: 743–752
Steffy K, Wong-Staal F . Genetic regulation of human immunodeficiency virus Microb Rev 1991 55: 193–205
Zufferey R et al. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo Nat Biotechnol 1997 15: 871–875
Robinson D, Elliott JF, Chang L-J . Retroviral vector with a CMV-IE/HIV-TAR hybrid LTR gives high basal expression levels and is upregulated by HIV-1 Tat Gene Therapy 1995 2: 269–278
Stevenson M et al. Integration is not necessary for expression of human immunodeficiency virus type 1 protein products J Virol 1990 64: 2421–2425
Sakai H et al. Integration is essential for efficient gene expression of human immunodeficiency virus type 1 J Virol 1993 67: 1169–1174
Boshart M et al. A very strong enhancer is located upstream of an immediate–early gene of human cytomegalovirus Cell 1985 41: 521–530
Baskar JF et al. The enhancer domain of the human cytomegalovirus major immediate–early promoter determines cell type-specific expression in transgenic mice J Virol 1996 70: 3207–3214
Xia H, Fitzgerald J, Bredt DS, Forsayeth JR . Detection of mycoplasma infection of mammalian cells BioTechniques 1997 22: 934–936
Chang LJ, Stoltzfus CM . Cloning and nucleotide sequences of cDNA spanning the splice junctions of Rous sarcoma virus mRNAs J Virol 1985 53: 969–972
Ure DR, Campenot RB, Acheson A . Cholinergic differentiation of rat sympathetic neurons in culture: effects of factors applied to distal neurites Dev Biol 1992 154: 388–395
Kimpton J, Emerman M . Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene J Virol 1992 66: 2232–2239
Motmans K, Thirion S, Raus J, Vandevyver C . Isolation and quantification of episomal expression vectors in human T cells BioTechniques 1997 23: 1044–1046
Maniatis T, Fritsch EF, Sambrook J . Molecular Cloning: a Laboratory Manual Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY 1989
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Chang, LJ., Urlacher, V., Iwakuma, T. et al. Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system. Gene Ther 6, 715–728 (1999). https://doi.org/10.1038/sj.gt.3300895
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.gt.3300895
Keywords
This article is cited by
-
Improved intravenous lentiviral gene therapy based on endothelial-specific promoter-driven factor VIII expression for hemophilia A
Molecular Medicine (2023)
-
Intracerebral lentiviral ABCD1 gene therapy in an early disease onset ALD mouse model
Gene Therapy (2023)
-
Histone deacetylase 1 interacts with HIV-1 Integrase and modulates viral replication
Virology Journal (2019)
-
Inositol phosphates are assembly co-factors for HIV-1
Nature (2018)
-
Bacterially derived synthetic mimetics of mammalian oligomannose prime antibody responses that neutralize HIV infectivity
Nature Communications (2017)