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Near-perfect infectivity of wild-type AAV as benchmark for infectivity of recombinant AAV vectors

Abstract

Viral vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo. The increasing use of AAV as a gene transfer vector, as well as recently shown immunological complications in clinical trials, highlight the necessity to define the specific activity of vector preparations beyond current standards. In this report, we determined the infectious, physical and genome-containing particle titers of several wild-type AAV type 2 (wtAAV2) and recombinant AAV type 2 (rAAV2) preparations that were produced and purified by standard methods. We found that the infectivity of wtAAV2 approaches a physical-to-infectious particle ratio of one. This near-perfect physical-to-infectious particle ratio defines a ‘ceiling’ for the theoretically achievable quality of recombinant AAV vectors. In comparison, for rAAV2, only approximately 50 out of 100 viral particles contained a genome and, more strikingly, only approximately 1 of the 100 viral particles was infectious. Our findings suggest that current strategies for rAAV vector design, production and/or purification should be amenable to improvements. Ultimately, this could result in the generation of near-perfect vector particles, a prospect with significant implications for gene therapy.

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References

  1. Buch PK, Bainbridge JW, Ali RR . AAV-mediated gene therapy for retinal disorders: from mouse to man. Gene Ther 2008; 15: 849–857.

    Article  CAS  PubMed  Google Scholar 

  2. Cecchini S, Negrete A, Kotin RM . Toward exascale production of recombinant adeno-associated virus for gene transfer applications. Gene Ther 2008; 15: 823–830.

    Article  CAS  PubMed  Google Scholar 

  3. Mingozzi F, Maus MV, Hui DJ, Sabatino DE, Murphy SL, Rasko JE et al. CD8(+) T-cell responses to adeno-associated virus capsid in humans. Nat Med 2007; 13: 419–422.

    Article  CAS  PubMed  Google Scholar 

  4. Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 2006; 12: 342–347.

    Article  CAS  PubMed  Google Scholar 

  5. Hauck B, Murphy SL, Smith PH, Qu G, Liu X, Zelenaia O et al. Undetectable transcription of cap in a clinical AAV vector: implications for preformed capsid in immune responses. Mol Ther 2009; 17: 144–152.

    Article  CAS  PubMed  Google Scholar 

  6. Salvetti A, Oreve S, Chadeuf G, Favre D, Cherel Y, Champion-Arnaud P et al. Factors influencing recombinant adeno-associated virus production. Hum Gene Ther 1998; 9: 695–706.

    Article  CAS  PubMed  Google Scholar 

  7. Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chesnut K et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 1999; 6: 973–985.

    Article  CAS  PubMed  Google Scholar 

  8. Debelak D, Fisher J, Iuliano S, Sesholtz D, Sloane DL, Atkinson EM . Cation-exchange high-performance liquid chromatography of recombinant adeno-associated virus type 2. J Chromatogr B Biomed Sci Appl 2000; 740: 195–202.

    Article  CAS  PubMed  Google Scholar 

  9. Zen Z, Espinoza Y, Bleu T, Sommer JM, Wright JF . Infectious titer assay for adeno-associated virus vectors with sensitivity sufficient to detect single infectious events. Hum Gene Ther 2004; 15: 709–715.

    Article  PubMed  Google Scholar 

  10. Sommer JM, Smith PH, Parthasarathy S, Isaacs J, Vijay S, Kieran J et al. Quantification of adeno-associated virus particles and empty capsids by optical density measurement. Mol Ther 2003; 7: 122–128.

    Article  CAS  PubMed  Google Scholar 

  11. Qu G, Bahr-Davidson J, Prado J, Tai A, Cataniag F, McDonnell J et al. Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography. J Virol Methods 2007; 140: 183–192.

    Article  CAS  PubMed  Google Scholar 

  12. Grimm D, Kern A, Pawlita M, Ferrari F, Samulski R, Kleinschmidt J . Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2. Gene Ther 1999; 6: 1322–1330.

    Article  CAS  PubMed  Google Scholar 

  13. Grimm D, Kern A, Rittner K, Kleinschmidt JA . Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum Gene Ther 1998; 9: 2745–2760.

    Article  CAS  PubMed  Google Scholar 

  14. Grieger JC, Choi VW, Samulski RJ . Production and characterization of adeno-associated viral vectors. Nat Protoc 2006; 1: 1412–1428.

    Article  CAS  PubMed  Google Scholar 

  15. 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 

  16. Ferrari FK, Samulski T, Shenk T, Samulski RJ . Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J Virol 1996; 70: 3227–3234.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Fisher KJ, Gao GP, Weitzman MD, DeMatteo R, Burda JF, Wilson JM . Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis. J Virol 1996; 70: 520–532.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Rohr UP, Wulf MA, Stahn S, Steidl U, Haas R, Kronenwett R . Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR. J Virol Methods 2002; 106: 81–88.

    Article  CAS  PubMed  Google Scholar 

  19. Xie Q, Bu W, Bhatia S, Hare J, Somasundaram T, Azzi A et al. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci USA 2002; 99: 10405–10410.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Urabe M, Ding C, Kotin RM . Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther 2002; 13: 1935–1943.

    Article  CAS  PubMed  Google Scholar 

  21. Tullis GE, Shenk T . Efficient replication of adeno-associated virus type 2 vectors: a cis-acting element outside of the terminal repeats and a minimal size. J Virol 2000; 74: 11511–11521.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ward P, Clement N, Linden RM . cis effects in adeno-associated virus type 2 replication. J Virol 2007; 81: 9976–9989.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Nony P, Tessier J, Chadeuf G, Ward P, Giraud A, Dugast M et al. Novel cis-acting replication element in the adeno-associated virus type 2 genome is involved in amplification of integrated rep-cap sequences. J Virol 2001; 75: 9991–9994.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Francois A, Guilbaud M, Awedikian R, Chadeuf G, Moullier P, Salvetti A . The cellular TATA binding protein is required for rep-dependent replication of a minimal adeno-associated virus type 2 p5 element. J Virol 2005; 79: 11082–11094.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Zhou X, Muzyczka N . In vitro packaging of adeno-associated virus DNA. J Virol 1998; 72: 3241–3247.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Myers MW, Carter BJ . Assembly of adeno-associated virus. Virology 1980; 102: 71–82.

    Article  CAS  PubMed  Google Scholar 

  27. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY . Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 2004; 22: 1567–1572.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Ronald Gordon from the Mount Sinai School of Medicine Pathology Core Electron Microscopy Facility for his help with the EM pictures and Jorge Mansilla-Soto for helpful discussions and critical reading of the manuscript. We gratefully acknowledge Roger Tsien (University of California at San Diego, CA, USA) for providing us with pRSET-B-mCherry and Robert Kotin (National Institutes of Health, Bethesda, MD, USA) for giving us empty viral particles. This work was supported by US National Institutes of Health Grants GM071023, GM075019 and DK062345 (to RML) and HL077322 (to TW).

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Correspondence to T Weber.

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Supplementary Information accompanies the paper on Gene Therapy website

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Zeltner, N., Kohlbrenner, E., Clément, N. et al. Near-perfect infectivity of wild-type AAV as benchmark for infectivity of recombinant AAV vectors. Gene Ther 17, 872–879 (2010). https://doi.org/10.1038/gt.2010.27

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