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. 1996 Jan;70(1):520–532. doi: 10.1128/jvi.70.1.520-532.1996

Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis.

K J Fisher 1, G P Gao 1, M D Weitzman 1, R DeMatteo 1, J F Burda 1, J M Wilson 1
PMCID: PMC189840  PMID: 8523565

Abstract

Adeno-associated virus is an integrating DNA parvovirus with the potential to be an important vehicle for somatic gene therapy. A potential barrier, however, is the low transduction efficiencies of recombinant adeno-associated virus (rAAV) vectors. We show in this report that adenovirus dramatically enhances rAAV transduction in vitro in a way that is dependent on expression of early region 1 and 4 (E1 and E4, respectively) genes and directly proportional to the appearance of double-stranded replicative forms of the rAAV genome. Expression of the open reading frame 6 protein from E4 in the absence of E1 accomplished a similar but attenuated effect. The helper activity of adenovirus E1 and E4 for rAAV gene transfer was similarly demonstrated in vivo by using murine models of liver- and lung-directed gene therapy. Our data indicate that conversion of a single-stranded rAAV genome to a duplex intermediate limits transduction and usefulness for gene therapy.

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Selected References

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  1. 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]
  2. Babiss L. E., Ginsberg H. S. Adenovirus type 5 early region 1b gene product is required for efficient shutoff of host protein synthesis. J Virol. 1984 Apr;50(1):202–212. doi: 10.1128/jvi.50.1.202-212.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berk A. J. Functions of adenovirus E1A. Cancer Surv. 1986;5(2):367–387. [PubMed] [Google Scholar]
  4. Bridge E., Ketner G. Redundant control of adenovirus late gene expression by early region 4. J Virol. 1989 Feb;63(2):631–638. doi: 10.1128/jvi.63.2.631-638.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bridge E., Medghalchi S., Ubol S., Leesong M., Ketner G. Adenovirus early region 4 and viral DNA synthesis. Virology. 1993 Apr;193(2):794–801. doi: 10.1006/viro.1993.1188. [DOI] [PubMed] [Google Scholar]
  6. Carter B. J., Marcus-Sekura C. J., Laughlin C. A., Ketner G. Properties of an adenovirus type 2 mutant, Ad2dl807, having a deletion near the right-hand genome terminus: failure to help AAV replication. Virology. 1983 Apr 30;126(2):505–516. doi: 10.1016/s0042-6822(83)80008-7. [DOI] [PubMed] [Google Scholar]
  7. Ensinger M. J., Ginsberg H. S. Selection and preliminary characterization of temperature-sensitive mutants of type 5 adenovirus. J Virol. 1972 Sep;10(3):328–339. doi: 10.1128/jvi.10.3.328-339.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fields-Berry S. C., Halliday A. L., Cepko C. L. A recombinant retrovirus encoding alkaline phosphatase confirms clonal boundary assignment in lineage analysis of murine retina. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):693–697. doi: 10.1073/pnas.89.2.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Flotte T. R., Afione S. A., Solow R., Drumm M. L., Markakis D., Guggino W. B., Zeitlin P. L., Carter B. J. Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter. J Biol Chem. 1993 Feb 15;268(5):3781–3790. [PubMed] [Google Scholar]
  10. Halbert D. N., Cutt J. R., Shenk T. Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff. J Virol. 1985 Oct;56(1):250–257. doi: 10.1128/jvi.56.1.250-257.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hermonat P. L., Labow M. A., Wright R., Berns K. I., Muzyczka N. Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants. J Virol. 1984 Aug;51(2):329–339. doi: 10.1128/jvi.51.2.329-339.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hermonat P. L., Muzyczka N. Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6466–6470. doi: 10.1073/pnas.81.20.6466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  14. Janik J. E., Huston M. M., Cho K., Rose J. A. Efficient synthesis of adeno-associated virus structural proteins requires both adenovirus DNA binding protein and VA I RNA. Virology. 1989 Feb;168(2):320–329. doi: 10.1016/0042-6822(89)90272-9. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Kotin R. M. Prospects for the use of adeno-associated virus as a vector for human gene therapy. Hum Gene Ther. 1994 Jul;5(7):793–801. doi: 10.1089/hum.1994.5.7-793. [DOI] [PubMed] [Google Scholar]
  17. Kotin R. M., Siniscalco M., Samulski R. J., Zhu X. D., Hunter L., Laughlin C. A., McLaughlin S., Muzyczka N., Rocchi M., Berns K. I. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2211–2215. doi: 10.1073/pnas.87.6.2211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kozarsky K. F., Wilson J. M. Gene therapy: adenovirus vectors. Curr Opin Genet Dev. 1993 Jun;3(3):499–503. doi: 10.1016/0959-437x(93)90126-a. [DOI] [PubMed] [Google Scholar]
  19. LaFace D., Hermonat P., Wakeland E., Peck A. Gene transfer into hematopoietic progenitor cells mediated by an adeno-associated virus vector. Virology. 1988 Feb;162(2):483–486. doi: 10.1016/0042-6822(88)90491-6. [DOI] [PubMed] [Google Scholar]
  20. Lebkowski J. S., McNally M. M., Okarma T. B., Lerch L. B. Adeno-associated virus: a vector system for efficient introduction and integration of DNA into a variety of mammalian cell types. Mol Cell Biol. 1988 Oct;8(10):3988–3996. doi: 10.1128/mcb.8.10.3988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. McPherson R. A., Ginsberg H. S., Rose J. A. Adeno-associated virus helper activity of adenovirus DNA binding protein. J Virol. 1982 Nov;44(2):666–673. doi: 10.1128/jvi.44.2.666-673.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Myers M. W., Laughlin C. A., Jay F. T., Carter B. J. Adenovirus helper function for growth of adeno-associated virus: effect of temperature-sensitive mutations in adenovirus early gene region 2. J Virol. 1980 Jul;35(1):65–75. doi: 10.1128/jvi.35.1.65-75.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Piña M., Green M. Biochemical studies on adenovirus multiplication. IX. Chemical and base composition analysis of 28 human adenoviruses. Proc Natl Acad Sci U S A. 1965 Aug;54(2):547–551. doi: 10.1073/pnas.54.2.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rice S. A., Klessig D. F. Isolation and analysis of adenovirus type 5 mutants containing deletions in the gene encoding the DNA-binding protein. J Virol. 1985 Dec;56(3):767–778. doi: 10.1128/jvi.56.3.767-778.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Richardson W. D., Westphal H. Adenovirus early gene regulation and the adeno-associated virus helper effect. Curr Top Microbiol Immunol. 1984;109:147–165. doi: 10.1007/978-3-642-69460-8_7. [DOI] [PubMed] [Google Scholar]
  27. Richardson W. D., Westphal H. Requirement for either early region 1a or early region 1b adenovirus gene products in the helper effect for adeno-associated virus. J Virol. 1984 Aug;51(2):404–410. doi: 10.1128/jvi.51.2.404-410.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Samulski R. J., Chang L. S., Shenk T. A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J Virol. 1987 Oct;61(10):3096–3101. doi: 10.1128/jvi.61.10.3096-3101.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Samulski R. J., Shenk T. Adenovirus E1B 55-Mr polypeptide facilitates timely cytoplasmic accumulation of adeno-associated virus mRNAs. J Virol. 1988 Jan;62(1):206–210. doi: 10.1128/jvi.62.1.206-210.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Samulski R. J., Zhu X., Xiao X., Brook J. D., Housman D. E., Epstein N., Hunter L. A. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 1991 Dec;10(12):3941–3950. doi: 10.1002/j.1460-2075.1991.tb04964.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sarasin A. SOS response in mammalian cells. Cancer Invest. 1985;3(2):163–174. doi: 10.3109/07357908509017499. [DOI] [PubMed] [Google Scholar]
  33. Sarnow P., Hearing P., Anderson C. W., Halbert D. N., Shenk T., Levine A. J. Adenovirus early region 1B 58,000-dalton tumor antigen is physically associated with an early region 4 25,000-dalton protein in productively infected cells. J Virol. 1984 Mar;49(3):692–700. doi: 10.1128/jvi.49.3.692-700.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sarnow P., Ho Y. S., Williams J., Levine A. J. Adenovirus E1b-58kd tumor antigen and SV40 large tumor antigen are physically associated with the same 54 kd cellular protein in transformed cells. Cell. 1982 Feb;28(2):387–394. doi: 10.1016/0092-8674(82)90356-7. [DOI] [PubMed] [Google Scholar]
  35. Schlehofer J. R., Ehrbar M., zur Hausen H. Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus. Virology. 1986 Jul 15;152(1):110–117. doi: 10.1016/0042-6822(86)90376-4. [DOI] [PubMed] [Google Scholar]
  36. Shepherd S. E., Howe J. A., Mymryk J. S., Bayley S. T. Induction of the cell cycle in baby rat kidney cells by adenovirus type 5 E1A in the absence of E1B and a possible influence of p53. J Virol. 1993 May;67(5):2944–2949. doi: 10.1128/jvi.67.5.2944-2949.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Straus S. E., Ginsberg H. S., Rose J. A. DNA-minus temperature-sensitive mutants of adenovirus type 5 help adenovirus-associated virus replication. J Virol. 1975 Jan;17(1):140–148. doi: 10.1128/jvi.17.1.140-148.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tratschin J. D., Miller I. L., Carter B. J. Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function. J Virol. 1984 Sep;51(3):611–619. doi: 10.1128/jvi.51.3.611-619.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tratschin J. D., Miller I. L., Smith M. G., Carter B. J. Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells. Mol Cell Biol. 1985 Nov;5(11):3251–3260. doi: 10.1128/mcb.5.11.3251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Weinberg D. H., Ketner G. A cell line that supports the growth of a defective early region 4 deletion mutant of human adenovirus type 2. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5383–5386. doi: 10.1073/pnas.80.17.5383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Weinberg D. H., Ketner G. Adenoviral early region 4 is required for efficient viral DNA replication and for late gene expression. J Virol. 1986 Mar;57(3):833–838. doi: 10.1128/jvi.57.3.833-838.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. West M. H., Trempe J. P., Tratschin J. D., Carter B. J. Gene expression in adeno-associated virus vectors: the effects of chimeric mRNA structure, helper virus, and adenovirus VA1 RNA. Virology. 1987 Sep;160(1):38–47. doi: 10.1016/0042-6822(87)90041-9. [DOI] [PubMed] [Google Scholar]
  43. Wilson J. M., Jefferson D. M., Chowdhury J. R., Novikoff P. M., Johnston D. E., Mulligan R. C. Retrovirus-mediated transduction of adult hepatocytes. Proc Natl Acad Sci U S A. 1988 May;85(9):3014–3018. doi: 10.1073/pnas.85.9.3014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yakobson B., Koch T., Winocour E. Replication of adeno-associated virus in synchronized cells without the addition of a helper virus. J Virol. 1987 Apr;61(4):972–981. doi: 10.1128/jvi.61.4.972-981.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yalkinoglu A. O., Heilbronn R., Bürkle A., Schlehofer J. R., zur Hausen H. DNA amplification of adeno-associated virus as a response to cellular genotoxic stress. Cancer Res. 1988 Jun 1;48(11):3123–3129. [PubMed] [Google Scholar]

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