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Replication competent helper functions for recombinant AAV vector generation

Gene Therapy volume 9, pages 1199–1206 (2002)Cite this article

Abstract

Adeno-associated virus (AAV) is a promising gene transfer vector tested in both animal studies and human clinical trials. However, current production methods are generally inefficient and require improvements to meet the increasing clinical need for economical, high titer and high quality rAAV vectors. The inefficiency of the current systems largely arises from the AAV helper function, which contains only the AAV coding region but lacks inverted terminal repeats. The terminal repeats were originally removed to prevent replication competent AAV contamination. Here we designed a novel and highly efficient rAAV helper function containing AAV terminal repeats. The new helper function not only mimics the wild-type AAV growth as it replicates along with the vector plasmid, but also restores the cis regulating function of the AAV terminal repeats. Addition of heterologous introns to the helper genome and use of a mutant AAV terminal repeat defective in packaging effectively controls the contamination of replication competent AAV particles. This new strategy also performs better in AAV producing cell lines than those based on non-replicating AAV rep and cap genome.

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References

  1. Srivastava A, Lusby EW, Berns KI . Nucleotide sequence and organization of the adeno-associated virus 2 genome J Virol 1983 45: 555–564

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Samulski RJ et al. Targeted integration of adeno-associated virus (AAV) into human chromosome 19 EMBO J 1991 10: 3941–3950

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kotin RM et al. Site-specific integration by adeno-associated virus Proc Natl Acad Sci USA 1990 87: 2211–2215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yang CC et al. Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro J Virol 1997 71: 9231–9247

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Rutledge EA, Russell DW . Adeno-associated virus vector integration junctions J Virol 1997 71: 8429–8436

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Muzyczka N . Use of adeno-associated virus as a general transduction vector for mammalian cells Curr Topic Microbiol Immunol 1992 158: 97–129

    CAS  Google Scholar 

  7. Berns KI . Parvoviridae: the viruses and their replication Fields FN, Knipe DM, Howley PM (eds); Fundamental Virology Lippincott-Raven 1995 pp 1007–1041

  8. Brister JR, Muzyczka N . Rep-mediated nicking of the adeno-associated virus origin requires two biochemical activities, DNA helicase activity and transesterification J Virol 1999 73: 9325–9336

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Weitzman MD, Kyostio SR, Kotin RM, Owens RA . Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA Proc Natl Acad Sci USA 1994 91: 5808–5812

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Xiao X, Xiao W, Li J, Samulski RJ . A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle J Virol 1997 71: 941–948

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Wang XS, Qing K, Ponnazhagan S, Srivastava A . Adeno-associated virus type 2 DNA replication in vivo: mutation analyses of the D sequence in viral inverted terminal repeats J Virol 1997 71: 3077–3082

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Xiao W et al. Gene therapy vectors based on adeno-associated virus type 1 J Virol 1999 73: 3994–4003

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Chiorini JA, Kim F, Yang L, Kotin RM . Cloning and characterization of adeno-associated virus type 5 J Virol 1999 73: 1309–1319

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Rutledge EA, Halbert CL, Russell DW . Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2 J Virol 1998 72: 309–319

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Muramatsu S, Mizukami H, Young NS, Brown KE . Nucleotide sequencing and generation of an infectious clone of adeno-associated virus 3 Virology 1996 221: 208–217

    Article  CAS  PubMed  Google Scholar 

  16. Chiorini JA, Afione S, Kotin RM . Adeno-associated virus (AAV) type 5 Rep protein cleaves a unique terminal resolution site compared with other AAV serotypes J Virol 1999 73: 4293–4298

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Samulski RJ . Adeno-associated virus: integration at a specific chromosomal locus Curr Opin Genet Dev 1993 3: 74–80

    Article  CAS  PubMed  Google Scholar 

  18. Xiao X, Li J, Samulski RJ . Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector J Virol 1996 70: 8098–8108

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Jooss K, Yang Y, Fisher KJ, Wilson JM . Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers J Virol 1998 72: 4212–4223

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Herzog RW et al. Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector Nat Med 1999 5: 56–63

    Article  CAS  PubMed  Google Scholar 

  21. Kay MA et al. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector Nat Genet 2000 24: 257–261

    Article  CAS  PubMed  Google Scholar 

  22. During MJ, Leone P . Adeno-associated virus vectors for gene therapy of neurodegenerative disorders Clin Neurosci 1995 3: 292–300

    PubMed  Google Scholar 

  23. During MJ et al. An oral vaccine against NMDAR1 with efficacy in experimental stroke and epilepsy Science 2000 287: 1453–1460

    Article  CAS  PubMed  Google Scholar 

  24. Flotte TR, Carter BJ . Adeno-associated virus vectors for gene therapy Gene Therapy 1995 2: 357–362

    CAS  PubMed  Google Scholar 

  25. Samulski RJ, Chang LS, Shenk T . Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression J Virol 1989 63: 3822–3828

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Li J, Samulski RJ, Xiao X . Role for highly regulated rep gene expression in adeno-associated virus vector production J Virol 1997 71: 5236–5243

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Ferrari FK, Xiao X, McCarty D, Samulski RJ . New developments in the generation of Ad-free, high-titer rAAV gene therapy vectors Nat Med 1997 3: 1295–1297

    Article  CAS  PubMed  Google Scholar 

  28. Vincent KA, Piraino ST, Wadsworth SC . Analysis of recombinant adeno-associated virus packaging and requirements for rep and cap gene products J Virol 1997 71: 1897–1905

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Flotte TR et al. An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction Gene Therapy 1995 2: 29–37

    CAS  PubMed  Google Scholar 

  30. Allen JM, Halbert CL, Miller AD . Improved adeno-associated virus vector production with transfection of a single helper adenovirus gene, E4orf6 Mol Ther 2000 1: 88–95

    Article  CAS  PubMed  Google Scholar 

  31. Wang XS et al. Characterization of wild-type adeno-associated virus type 2-like particles generated during recombinant viral vector production and strategies for their elimination J Virol 1998 72: 5472–5480

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Cao L, Liu Y, During MJ, Xiao W . High-titer, wild-type free recombinant adeno-associated virus vector production using intron-containing helper plasmids J Virol 2000 74: 11456–11463

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Clark KR, Voulgaropoulou F, Fraley DM, Johnson PR . Cell lines for the production of recombinant adeno-associated virus Hum Gene Ther 1995 6: 1329–1341

    Article  CAS  PubMed  Google Scholar 

  34. Clark KR, Voulgaropoulou F, Johnson PR . A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors Gene Therapy 1996 3: 1124–1132

    CAS  PubMed  Google Scholar 

  35. Liu XL, Clark KR, Johnson PR . Production of recombinant adeno-associated virus vectors using a packaging cell line and a hybrid recombinant adenovirus Gene Therapy 1999 6: 293–299

    Article  CAS  PubMed  Google Scholar 

  36. Tamayose K, Hirai Y, Shimada T . A new strategy for large-scale preparation of high-titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography Hum Gene Ther 1996 7: 507–513

    Article  CAS  PubMed  Google Scholar 

  37. Chadeuf G et al. Efficient recombinant adeno-associated virus production by a stable rep-cap HeLa cell line correlates with adenovirus-induced amplification of the integrated rep-cap genome J Gene Med 2000 2: 260–268

    Article  CAS  PubMed  Google Scholar 

  38. Inoue N, Russell DW . Packaging cells based on inducible gene amplification for the production of adeno-associated virus vectors J Virol 1998 72: 7024–7031

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Liu X et al. Selective Rep-Cap gene amplification as a mechanism for high-titer recombinant AAV production from stable cell lines Mol Ther 2000 2: 394–403

    Article  CAS  PubMed  Google Scholar 

  40. Salvetti A et al. Factors influencing recombinant adeno-associated virus production Hum Gene Ther 1998 9: 695–706

    Article  CAS  PubMed  Google Scholar 

  41. Conway JE et al. High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap Gene Therapy 1999 6: 986–993

    Article  CAS  PubMed  Google Scholar 

  42. Conway JE et al. Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap J Virol 1997 71: 8780–8789

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Zhang X et al. High-titer recombinant adeno-associated virus production from replicating amplicons and herpes vectors deleted for glycoprotein H Hum Gene Ther 1999 10: 2527–2537

    Article  CAS  PubMed  Google Scholar 

  44. Wang XS, Ponnazhagan S, Srivastava A . Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions J Virol 1996 70: 1668–1677

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Wang XS, Ponnazhagan S, Srivastava A . Rescue and replication signals of the adeno-associated virus 2 genome J Molec Biol 1995 250: 573–580

    Article  CAS  PubMed  Google Scholar 

  46. Gao GP et al. High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus Hum Gene Ther 1998 9: 2353–2362

    Article  CAS  PubMed  Google Scholar 

  47. Weger S, Wistuba A, Grimm D . Kleinschmidt JA.. Control of adeno-associated virus type 2 cap gene expression: relative influence of helper virus, terminal repeats and Rep proteins J Virol 1997 71: 8437–8447

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Tessier J et al. Characterization of adenovirus-induced inverted terminal repeat-independent amplification of integrated adeno-associated virus rep-cap sequences J Virol 2001 75: 375–383

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Hermonat PL, 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 USA 1984 81: 6466–6470

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Dong JY, Fan PD, Frizzell RA . Quantitative analysis of the packaging capacity of recombinant adeno-associated virus Hum Gene Ther 1996 7: 2101–2112

    Article  CAS  PubMed  Google Scholar 

  51. McLaughlin SK, Collis P, Hermonat PL, Muzyczka N . Adeno-associated virus general transduction vectors: analysis of proviral structures J Virol 1988 62: 1963–1973

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Zhou HA et al. Cre-expressing cell line and an E1/E2a double-deleted virus for preparation of helper-dependent adenovirus vector Mol Ther 2001 3: 613–622

    Article  CAS  PubMed  Google Scholar 

  53. Parks RJ et al. A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal Proc Natl Acad Sci USA 1996 93: 13565–13570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Mitani K, Graham FL, Caskey CT, Kochanek S . Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector Proc Natl Acad Sci USA 1995 92: 3854–3858

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Maxwell IH et al. Recombinant LuIII autonomous parvovirus as a transient transducing vector for human cells Hum Gene Ther 1993 4: 441–450

    Article  CAS  PubMed  Google Scholar 

  56. Brandenburger A, Russell S . A novel packaging system for the generation of helper-free oncolytic MVM vector stocks Gene Therapy 1996 3: 927–931

    CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Neurosurgery, CNS Gene Therapy Center, Thomas Jefferson University, Philadelphia, PA, USA

    L Cao & M During

  2. Division of Hematology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA, USA

    W Xiao

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  1. L Cao
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  2. M During
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  3. W Xiao
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Cao, L., During, M. & Xiao, W. Replication competent helper functions for recombinant AAV vector generation. Gene Ther 9, 1199–1206 (2002). https://doi.org/10.1038/sj.gt.3301710

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