Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Nov;70(11):8098–8108. doi: 10.1128/jvi.70.11.8098-8108.1996

Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector.

X Xiao 1, J Li 1, R J Samulski 1
PMCID: PMC190884  PMID: 8892935

Abstract

Muscle-directed gene transfer is being considered for the treatment of several metabolic diseases, including hemophilia and Duchene's muscular dystrophy. Previous efforts to target this tissue for somatic delivery with various vector systems have resulted in transient expression due to silencing of the transgene or to an immune response against the vector-transduced cells. We introduced recombinant adeno-associated virus vector (rAAV) carrying a lacZ reporter into muscle tissue of immunocompetent mice. The lacZ reporter gene was efficiently transduced and expressed with no evidence of a cellular immune response. Moreover, gene expression persisted for more than 1.5 years. Molecular characterization of rAAV vector DNA suggests a mechanism for persistence, since vector episomes convert to high-molecular-weight genomic DNA. These data provide the first report for establishing long-term gene transduction into mammalian muscle cells in vivo without the need for immune modulation of the organism.

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Acsadi G., Jani A., Huard J., Blaschuk K., Massie B., Holland P., Lochmüller H., Karpati G. Cultured human myoblasts and myotubes show markedly different transducibility by replication-defective adenovirus recombinants. Gene Ther. 1994 Sep;1(5):338–340. [PubMed] [Google Scholar]
  2. Acsadi G., Jani A., Massie B., Simoneau M., Holland P., Blaschuk K., Karpati G. A differential efficiency of adenovirus-mediated in vivo gene transfer into skeletal muscle cells of different maturity. Hum Mol Genet. 1994 Apr;3(4):579–584. doi: 10.1093/hmg/3.4.579. [DOI] [PubMed] [Google Scholar]
  3. Acsadi G., Lochmüller H., Jani A., Huard J., Massie B., Prescott S., Simoneau M., Petrof B. J., Karpati G. Dystrophin expression in muscles of mdx mice after adenovirus-mediated in vivo gene transfer. Hum Gene Ther. 1996 Jan 20;7(2):129–140. doi: 10.1089/hum.1996.7.2-129. [DOI] [PubMed] [Google Scholar]
  4. Afione S. A., Conrad C. K., Kearns W. G., Chunduru S., Adams R., Reynolds T. C., Guggino W. B., Cutting G. R., Carter B. J., Flotte T. R. In vivo model of adeno-associated virus vector persistence and rescue. J Virol. 1996 May;70(5):3235–3241. doi: 10.1128/jvi.70.5.3235-3241.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Alexander I. E., Russell D. W., Miller A. D. DNA-damaging agents greatly increase the transduction of nondividing cells by adeno-associated virus vectors. J Virol. 1994 Dec;68(12):8282–8287. doi: 10.1128/jvi.68.12.8282-8287.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berns K. I., Bohenzky R. A. Adeno-associated viruses: an update. Adv Virus Res. 1987;32:243–306. doi: 10.1016/s0065-3527(08)60479-0. [DOI] [PubMed] [Google Scholar]
  7. Berns K. I., Linden R. M. The cryptic life style of adeno-associated virus. Bioessays. 1995 Mar;17(3):237–245. doi: 10.1002/bies.950170310. [DOI] [PubMed] [Google Scholar]
  8. Blau H. M., Springer M. L. Muscle-mediated gene therapy. N Engl J Med. 1995 Dec 7;333(23):1554–1556. doi: 10.1056/NEJM199512073332308. [DOI] [PubMed] [Google Scholar]
  9. Dai Y., Roman M., Naviaux R. K., Verma I. M. 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. doi: 10.1073/pnas.89.22.10892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dai Y., Schwarz E. M., Gu D., Zhang W. W., Sarvetnick N., Verma I. M. 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. doi: 10.1073/pnas.92.5.1401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Davis H. L., Demeneix B. A., Quantin B., Coulombe J., Whalen R. G. Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle. Hum Gene Ther. 1993 Dec;4(6):733–740. doi: 10.1089/hum.1993.4.6-733. [DOI] [PubMed] [Google Scholar]
  12. DeMatteo R. P., Markmann J. F., Kozarsky K. F., Barker C. F., Raper S. E. Prolongation of adenoviral transgene expression in mouse liver by T lymphocyte subset depletion. Gene Ther. 1996 Jan;3(1):4–12. [PubMed] [Google Scholar]
  13. Dunckley M. G., Wells D. J., Walsh F. S., Dickson G. Direct retroviral-mediated transfer of a dystrophin minigene into mdx mouse muscle in vivo. Hum Mol Genet. 1993 Jun;2(6):717–723. doi: 10.1093/hmg/2.6.717. [DOI] [PubMed] [Google Scholar]
  14. Engelhardt J. F., Litzky L., Wilson J. M. Prolonged transgene expression in cotton rat lung with recombinant adenoviruses defective in E2a. Hum Gene Ther. 1994 Oct;5(10):1217–1229. doi: 10.1089/hum.1994.5.10-1217. [DOI] [PubMed] [Google Scholar]
  15. Fang B., Eisensmith R. C., Wang H., Kay M. A., Cross R. E., Landen C. N., Gordon G., Bellinger D. A., Read M. S., Hu P. C. Gene therapy for hemophilia B: host immunosuppression prolongs the therapeutic effect of adenovirus-mediated factor IX expression. Hum Gene Ther. 1995 Aug;6(8):1039–1044. doi: 10.1089/hum.1995.6.8-1039. [DOI] [PubMed] [Google Scholar]
  16. Ferrari F. K., Samulski T., Shenk T., Samulski R. J. Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J Virol. 1996 May;70(5):3227–3234. doi: 10.1128/jvi.70.5.3227-3234.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fisher K. J., Gao G. P., Weitzman M. D., DeMatteo R., Burda J. F., Wilson J. M. Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis. J Virol. 1996 Jan;70(1):520–532. doi: 10.1128/jvi.70.1.520-532.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Flotte T. R., Afione S. A., Conrad C., McGrath S. A., Solow R., Oka H., Zeitlin P. L., Guggino W. B., Carter B. J. Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10613–10617. doi: 10.1073/pnas.90.22.10613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Flotte T. R., Afione S. A., Zeitlin P. L. Adeno-associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration. Am J Respir Cell Mol Biol. 1994 Nov;11(5):517–521. doi: 10.1165/ajrcmb.11.5.7946381. [DOI] [PubMed] [Google Scholar]
  20. Flotte T. R., Barraza-Ortiz X., Solow R., Afione S. A., Carter B. J., Guggino W. B. An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction. Gene Ther. 1995 Jan;2(1):29–37. [PubMed] [Google Scholar]
  21. Flotte T. R., Carter B. J. Adeno-associated virus vectors for gene therapy. Gene Ther. 1995 Aug;2(6):357–362. [PubMed] [Google Scholar]
  22. Flotte T. R., Solow R., Owens R. A., Afione S., Zeitlin P. L., Carter B. J. Gene expression from adeno-associated virus vectors in airway epithelial cells. Am J Respir Cell Mol Biol. 1992 Sep;7(3):349–356. doi: 10.1165/ajrcmb/7.3.349. [DOI] [PubMed] [Google Scholar]
  23. Goldman M. J., Wilson J. M. Expression of alpha v beta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway. J Virol. 1995 Oct;69(10):5951–5958. doi: 10.1128/jvi.69.10.5951-5958.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Goodman S., Xiao X., Donahue R. E., Moulton A., Miller J., Walsh C., Young N. S., Samulski R. J., Nienhuis A. W. Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells. Blood. 1994 Sep 1;84(5):1492–1500. [PubMed] [Google Scholar]
  25. Halbert C. L., Alexander I. E., Wolgamot G. M., Miller A. D. Adeno-associated virus vectors transduce primary cells much less efficiently than immortalized cells. J Virol. 1995 Mar;69(3):1473–1479. doi: 10.1128/jvi.69.3.1473-1479.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Huard J., Lochmüller H., Acsadi G., Jani A., Holland P., Guérin C., Massie B., Karpati G. Differential short-term transduction efficiency of adult versus newborn mouse tissues by adenoviral recombinants. Exp Mol Pathol. 1995 Apr;62(2):131–143. doi: 10.1006/exmp.1995.1015. [DOI] [PubMed] [Google Scholar]
  27. Kaplitt M. G., Leone P., Samulski R. J., Xiao X., Pfaff D. W., O'Malley K. L., During M. J. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain. Nat Genet. 1994 Oct;8(2):148–154. doi: 10.1038/ng1094-148. [DOI] [PubMed] [Google Scholar]
  28. Kay M. A., Holterman A. X., Meuse L., Gown A., Ochs H. D., Linsley P. S., Wilson C. B. Long-term hepatic adenovirus-mediated gene expression in mice following CTLA4Ig administration. Nat Genet. 1995 Oct;11(2):191–197. doi: 10.1038/ng1095-191. [DOI] [PubMed] [Google Scholar]
  29. Kotin R. M., Berns K. I. Organization of adeno-associated virus DNA in latently infected Detroit 6 cells. Virology. 1989 Jun;170(2):460–467. doi: 10.1016/0042-6822(89)90437-6. [DOI] [PubMed] [Google Scholar]
  30. Levy M. Y., Barron L. G., Meyer K. B., Szoka F. C., Jr Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products into blood. Gene Ther. 1996 Mar;3(3):201–211. [PubMed] [Google Scholar]
  31. Lochmüller H., Jani A., Huard J., Prescott S., Simoneau M., Massie B., Karpati G., Acsadi G. Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (delta E1 + delta E3) during multiple passages in 293 cells. Hum Gene Ther. 1994 Dec;5(12):1485–1491. doi: 10.1089/hum.1994.5.12-1485. [DOI] [PubMed] [Google Scholar]
  32. McCown T. J., Xiao X., Li J., Breese G. R., Samulski R. J. Differential and persistent expression patterns of CNS gene transfer by an adeno-associated virus (AAV) vector. Brain Res. 1996 Mar 25;713(1-2):99–107. doi: 10.1016/0006-8993(95)01488-8. [DOI] [PubMed] [Google Scholar]
  33. McLaughlin S. K., Collis P., Hermonat P. L., Muzyczka N. Adeno-associated virus general transduction vectors: analysis of proviral structures. J Virol. 1988 Jun;62(6):1963–1973. doi: 10.1128/jvi.62.6.1963-1973.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Miller J. B., Boyce F. M. Gene therapy by and for muscle cells. Trends Genet. 1995 May;11(5):163–165. doi: 10.1016/s0168-9525(00)89032-0. [DOI] [PubMed] [Google Scholar]
  35. Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol. 1992;158:97–129. doi: 10.1007/978-3-642-75608-5_5. [DOI] [PubMed] [Google Scholar]
  36. Naldini L., Blömer U., Gallay P., Ory D., Mulligan R., Gage F. H., Verma I. M., Trono D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science. 1996 Apr 12;272(5259):263–267. doi: 10.1126/science.272.5259.263. [DOI] [PubMed] [Google Scholar]
  37. Naviaux R. K., Verma I. M. Retroviral vectors for persistent expression in vivo. Curr Opin Biotechnol. 1992 Oct;3(5):540–547. doi: 10.1016/0958-1669(92)90083-u. [DOI] [PubMed] [Google Scholar]
  38. Podsakoff G., Wong K. K., Jr, Chatterjee S. Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors. J Virol. 1994 Sep;68(9):5656–5666. doi: 10.1128/jvi.68.9.5656-5666.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ragot T., Vincent N., Chafey P., Vigne E., Gilgenkrantz H., Couton D., Cartaud J., Briand P., Kaplan J. C., Perricaudet M. Efficient adenovirus-mediated transfer of a human minidystrophin gene to skeletal muscle of mdx mice. Nature. 1993 Feb 18;361(6413):647–650. doi: 10.1038/361647a0. [DOI] [PubMed] [Google Scholar]
  40. Russell D. W., Alexander I. E., Miller A. D. DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5719–5723. doi: 10.1073/pnas.92.12.5719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Russell D. W., Miller A. D., Alexander I. E. Adeno-associated virus vectors preferentially transduce cells in S phase. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8915–8919. doi: 10.1073/pnas.91.19.8915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Samulski R. J. Adeno-associated virus: integration at a specific chromosomal locus. Curr Opin Genet Dev. 1993 Feb;3(1):74–80. doi: 10.1016/s0959-437x(05)80344-2. [DOI] [PubMed] [Google Scholar]
  43. Samulski R. J., Chang L. S., Shenk T. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol. 1989 Sep;63(9):3822–3828. doi: 10.1128/jvi.63.9.3822-3828.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tripathy S. K., Black H. B., Goldwasser E., Leiden J. M. 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. doi: 10.1038/nm0596-545. [DOI] [PubMed] [Google Scholar]
  45. Vincent N., Ragot T., Gilgenkrantz H., Couton D., Chafey P., Grégoire A., Briand P., Kaplan J. C., Kahn A., Perricaudet M. Long-term correction of mouse dystrophic degeneration by adenovirus-mediated transfer of a minidystrophin gene. Nat Genet. 1993 Oct;5(2):130–134. doi: 10.1038/ng1093-130. [DOI] [PubMed] [Google Scholar]
  46. Walsh C. E., Liu J. M., Xiao X., Young N. S., Nienhuis A. W., Samulski R. J. Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7257–7261. doi: 10.1073/pnas.89.15.7257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wolff J. A., Malone R. W., Williams P., Chong W., Acsadi G., Jani A., Felgner P. L. Direct gene transfer into mouse muscle in vivo. Science. 1990 Mar 23;247(4949 Pt 1):1465–1468. doi: 10.1126/science.1690918. [DOI] [PubMed] [Google Scholar]
  48. Yang Y., Ertl H. C., Wilson J. M. 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. doi: 10.1016/1074-7613(94)90074-4. [DOI] [PubMed] [Google Scholar]
  49. Yang Y., Li Q., Ertl H. C., Wilson J. M. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol. 1995 Apr;69(4):2004–2015. doi: 10.1128/jvi.69.4.2004-2015.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yang Y., Nunes F. A., Berencsi K., Furth E. E., Gönczöl E., Wilson J. M. 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. doi: 10.1073/pnas.91.10.4407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yang Y., Nunes F. A., Berencsi K., Gönczöl E., Engelhardt J. F., Wilson J. M. Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat Genet. 1994 Jul;7(3):362–369. doi: 10.1038/ng0794-362. [DOI] [PubMed] [Google Scholar]
  52. Yang Y., Wilson J. M. Clearance of adenovirus-infected hepatocytes by MHC class I-restricted CD4+ CTLs in vivo. J Immunol. 1995 Sep 1;155(5):2564–2570. [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES