• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Oct 15, 1996; 93(21): 11382–11388.

Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.


We describe the construction of a safe, replication-defective and efficient lentiviral vector suitable for in vivo gene delivery. The reverse transcription of the vector was found to be a rate-limiting step; therefore, promoting the reaction inside the vector particles before delivery significantly enhanced the efficiency of gene transfer. After injection into the brain of adult rats, sustained long-term expression of the transgene was obtained in the absence of detectable pathology. A high proportion of the neurons in the areas surrounding the injection sites of the vector expressed the transduced beta-galactosidase gene. This pattern was invariant in animals sacrificed several months after a single administration of the vector. Transduction occurs by integration of the vector genome, as it was abolished by a single amino acid substitution in the catalytic site of the integrase protein incorporated in the vector. Development of clinically acceptable derivatives of the lentiviral vector may thus enable the sustained delivery of significant amounts of a therapeutic gene product in a wide variety of somatic tissues.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.6M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Mulligan RC. The basic science of gene therapy. Science. 1993 May 14;260(5110):926–932. [PubMed]
  • Verma IM. Gene therapy: hopes, hypes, and hurdles. Mol Med. 1994 Nov;1(1):2–3. [PMC free article] [PubMed]
  • Crystal RG. Transfer of genes to humans: early lessons and obstacles to success. Science. 1995 Oct 20;270(5235):404–410. [PubMed]
  • Leiden JM. Gene therapy--promise, pitfalls, and prognosis. N Engl J Med. 1995 Sep 28;333(13):871–873. [PubMed]
  • Yang Y, Nunes FA, Berencsi K, Furth EE, Gönczöl E, Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4407–4411. [PMC free article] [PubMed]
  • Yang Y, Ertl HC, Wilson JM. MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses. Immunity. 1994 Aug;1(5):433–442. [PubMed]
  • Dai Y, Schwarz EM, Gu D, Zhang WW, Sarvetnick N, Verma IM. Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1401–1405. [PMC free article] [PubMed]
  • Knowles MR, Hohneker KW, Zhou Z, Olsen JC, Noah TL, Hu PC, Leigh MW, Engelhardt JF, Edwards LJ, Jones KR, et al. A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis. N Engl J Med. 1995 Sep 28;333(13):823–831. [PubMed]
  • Kremer EJ, Perricaudet M. Adenovirus and adeno-associated virus mediated gene transfer. Br Med Bull. 1995 Jan;51(1):31–44. [PubMed]
  • Miller AD, Miller DG, Garcia JV, Lynch CM. Use of retroviral vectors for gene transfer and expression. Methods Enzymol. 1993;217:581–599. [PubMed]
  • Miller DG, Adam MA, Miller AD. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol. 1990 Aug;10(8):4239–4242. [PMC free article] [PubMed]
  • Dai Y, Roman M, Naviaux RK, Verma IM. Gene therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10892–10895. [PMC free article] [PubMed]
  • Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M, Shearer G, Chang L, Chiang Y, Tolstoshev P, et al. T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years. Science. 1995 Oct 20;270(5235):475–480. [PubMed]
  • Bordignon C, Notarangelo LD, Nobili N, Ferrari G, Casorati G, Panina P, Mazzolari E, Maggioni D, Rossi C, Servida P, et al. Gene therapy in peripheral blood lymphocytes and bone marrow for ADA- immunodeficient patients. Science. 1995 Oct 20;270(5235):470–475. [PubMed]
  • Kohn DB, Weinberg KI, Nolta JA, Heiss LN, Lenarsky C, Crooks GM, Hanley ME, Annett G, Brooks JS, el-Khoureiy A, et al. Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency. Nat Med. 1995 Oct;1(10):1017–1023. [PMC free article] [PubMed]
  • Grossman M, Rader DJ, Muller DW, Kolansky DM, Kozarsky K, Clark BJ, 3rd, Stein EA, Lupien PJ, Brewer HB, Jr, Raper SE, et al. A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolaemia. Nat Med. 1995 Nov;1(11):1148–1154. [PubMed]
  • Scharfmann R, Axelrod JH, Verma IM. Long-term in vivo expression of retrovirus-mediated gene transfer in mouse fibroblast implants. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4626–4630. [PMC free article] [PubMed]
  • Palmer TD, Rosman GJ, Osborne WR, Miller AD. Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1330–1334. [PMC free article] [PubMed]
  • Naldini L, Blömer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma IM, Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996 Apr 12;272(5259):263–267. [PubMed]
  • Trono D, Feinberg MB, Baltimore D. HIV-1 Gag mutants can dominantly interfere with the replication of the wild-type virus. Cell. 1989 Oct 6;59(1):113–120. [PubMed]
  • Ratner L, Haseltine W, Patarca R, Livak KJ, Starcich B, Josephs SF, Doran ER, Rafalski JA, Whitehorn EA, Baumeister K, et al. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. [PubMed]
  • Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. [PMC free article] [PubMed]
  • Charneau P, Mirambeau G, Roux P, Paulous S, Buc H, Clavel F. HIV-1 reverse transcription. A termination step at the center of the genome. J Mol Biol. 1994 Sep 2;241(5):651–662. [PubMed]
  • Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8033–8037. [PMC free article] [PubMed]
  • Zhang H, Dornadula G, Pomerantz RJ. Endogenous reverse transcription of human immunodeficiency virus type 1 in physiological microenviroments: an important stage for viral infection of nondividing cells. J Virol. 1996 May;70(5):2809–2824. [PMC free article] [PubMed]
  • Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specific nuclear protein in vertebrates. Development. 1992 Sep;116(1):201–211. [PubMed]
  • Leavitt AD, Robles G, Alesandro N, Varmus HE. Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity yet fail to integrate viral DNA efficiently during infection. J Virol. 1996 Feb;70(2):721–728. [PMC free article] [PubMed]
  • Lever A, Gottlinger H, Haseltine W, Sodroski J. Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions. J Virol. 1989 Sep;63(9):4085–4087. [PMC free article] [PubMed]
  • Aldovini A, Young RA. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol. 1990 May;64(5):1920–1926. [PMC free article] [PubMed]
  • Luban J, Goff SP. Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA. J Virol. 1994 Jun;68(6):3784–3793. [PMC free article] [PubMed]
  • Kim HJ, Lee K, O'Rear JJ. A short sequence upstream of the 5' major splice site is important for encapsidation of HIV-1 genomic RNA. Virology. 1994 Jan;198(1):336–340. [PubMed]
  • Vicenzi E, Dimitrov DS, Engelman A, Migone TS, Purcell DF, Leonard J, Englund G, Martin MA. An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences. J Virol. 1994 Dec;68(12):7879–7890. [PMC free article] [PubMed]
  • Geigenmüller U, Linial ML. Specific binding of human immunodeficiency virus type 1 (HIV-1) Gag-derived proteins to a 5' HIV-1 genomic RNA sequence. J Virol. 1996 Jan;70(1):667–671. [PMC free article] [PubMed]
  • Paillart JC, Skripkin E, Ehresmann B, Ehresmann C, Marquet R. A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5572–5577. [PMC free article] [PubMed]
  • McBride MS, Panganiban AT. The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. J Virol. 1996 May;70(5):2963–2973. [PMC free article] [PubMed]
  • Berkowitz RD, Hammarskjöld ML, Helga-Maria C, Rekosh D, Goff SP. 5' regions of HIV-1 RNAs are not sufficient for encapsidation: implications for the HIV-1 packaging signal. Virology. 1995 Oct 1;212(2):718–723. [PubMed]
  • Kaye JF, Richardson JH, Lever AM. cis-acting sequences involved in human immunodeficiency virus type 1 RNA packaging. J Virol. 1995 Oct;69(10):6588–6592. [PMC free article] [PubMed]
  • Buchschacher GL, Jr, Panganiban AT. Human immunodeficiency virus vectors for inducible expression of foreign genes. J Virol. 1992 May;66(5):2731–2739. [PMC free article] [PubMed]
  • Parolin C, Dorfman T, Palú G, Göttlinger H, Sodroski J. Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes. J Virol. 1994 Jun;68(6):3888–3895. [PMC free article] [PubMed]
  • Trono D. Partial reverse transcripts in virions from human immunodeficiency and murine leukemia viruses. J Virol. 1992 Aug;66(8):4893–4900. [PMC free article] [PubMed]
  • Lori F, di Marzo Veronese F, de Vico AL, Lusso P, Reitz MS, Jr, Gallo RC. Viral DNA carried by human immunodeficiency virus type 1 virions. J Virol. 1992 Aug;66(8):5067–5074. [PMC free article] [PubMed]
  • Zhu J, Cunningham JM. Minus-strand DNA is present within murine type C ecotropic retroviruses prior to infection. J Virol. 1993 Apr;67(4):2385–2388. [PMC free article] [PubMed]
  • Zhang H, Zhang Y, Spicer TP, Abbott LZ, Abbott M, Poiesz BJ. Reverse transcription takes place within extracellular HIV-1 virions: potential biological significance. AIDS Res Hum Retroviruses. 1993 Dec;9(12):1287–1296. [PubMed]
  • Zhang H, Zhang Y, Spicer T, Henrard D, Poiesz BJ. Nascent human immunodeficiency virus type 1 reverse transcription occurs within an enveloped particle. J Virol. 1995 Jun;69(6):3675–3682. [PMC free article] [PubMed]
  • Zack JA, Haislip AM, Krogstad P, Chen IS. Incompletely reverse-transcribed human immunodeficiency virus type 1 genomes in quiescent cells can function as intermediates in the retroviral life cycle. J Virol. 1992 Mar;66(3):1717–1725. [PMC free article] [PubMed]
  • Gao WY, Cara A, Gallo RC, Lori F. Low levels of deoxynucleotides in peripheral blood lymphocytes: a strategy to inhibit human immunodeficiency virus type 1 replication. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8925–8928. [PMC free article] [PubMed]
  • Meyerhans A, Vartanian JP, Hultgren C, Plikat U, Karlsson A, Wang L, Eriksson S, Wain-Hobson S. Restriction and enhancement of human immunodeficiency virus type 1 replication by modulation of intracellular deoxynucleoside triphosphate pools. J Virol. 1994 Jan;68(1):535–540. [PMC free article] [PubMed]
  • Zhang H, Duan LX, Dornadula G, Pomerantz RJ. Increasing transduction efficiency of recombinant murine retrovirus vectors by initiation of endogenous reverse transcription: potential utility for genetic therapies. J Virol. 1995 Jun;69(6):3929–3932. [PMC free article] [PubMed]
  • Cohen A, Barankiewicz J, Lederman HM, Gelfand EW. Purine and pyrimidine metabolism in human T lymphocytes. Regulation of deoxyribonucleotide metabolism. J Biol Chem. 1983 Oct 25;258(20):12334–12340. [PubMed]
  • McCormick PJ, Danhauser LL, Rustum YM, Bertram JS. Changes in ribo- and deoxyribonucleoside triphosphate pools within the cell cycle of a synchronized mouse fibroblast cell line. Biochim Biophys Acta. 1983 Mar 15;756(1):36–40. [PubMed]
  • Tripathy SK, Black HB, Goldwasser E, Leiden JM. Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors. Nat Med. 1996 May;2(5):545–550. [PubMed]
  • Baskar JF, Smith PP, Nilaver G, Jupp RA, Hoffmann S, Peffer NJ, Tenney DJ, Colberg-Poley AM, Ghazal P, Nelson JA. The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice. J Virol. 1996 May;70(5):3207–3214. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...