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Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2

Abstract

We demonstrate the rapid and reliable quantification of physical AAV-2 (adeno-associated virus type 2) particles via a novel ELISA based on a monoclonal antibody which selectively recognizes assembled AAV-2 capsids. Titration of a variety of recombinant AAV-2 (rAAV) preparations revealed that at least 80% of all particles were empty, compared with a maximum of 50% in wild-type AAV-2 stocks, indicating that the recombinant genomes were less efficiently encapsidated. This finding was confirmed upon titration of CsCl gradient fractions from recombinant and wild-type AAV-2 stocks. ELISA-based measurement of capsid numbers revealed a large number of physical particles with low densities corresponding to empty capsids in the recombinant, but not in the wild-type AAV-2 preparations. Moreover, additional expression of VP proteins during rAAV production was found to result in an excessive capsid formation, whilst yielding only minor increases in DNA-containing or transducing rAAV particles. We conclude that encapsidation of viral genomes rather than capsid assembly can be limiting for rAAV production, provided that a critical level of VP expression is maintained. The feasibility of quantifying AAV-2 capsid numbers via the ELISA allows determination of physical to DNA-containing or infectious particle ratios. These are important parameters which should help to optimize and standardize the production and application of recombinant AAV-2.

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References

  1. Berns KI, Bohensky RA . Adeno-associated viruses: an update Adv Vir Res 1987 32: 243–305

    Article  CAS  Google Scholar 

  2. Alexander IE, Russell DW, Spence AM, Miller AD . Effects of gamma irradiation on the transduction of dividing and nondividing cells in brain and muscle of rats by adeno-associated virus vectors Hum Gene Ther 1996 7: 841–850

    Article  CAS  PubMed  Google Scholar 

  3. Kaplitt MG, Makimura H . Defective viral vectors as agents for gene transfer in the nervous system J Neurosci Meth 1997 71: 125–132

    Article  CAS  Google Scholar 

  4. McCown TJ et al. Differential and persistent expression patterns of CNS gene transfer by an adeno-associated virus (AAV) vector Brain Res 1996 713: 99–107

    Article  CAS  PubMed  Google Scholar 

  5. Podsakoff G, Wong KK, Chatterjee S . Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors J Virol 1994 68: 5656–5666

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Russell DW, Miller AW, Alexander IE . Adeno-associated virus vectors preferentially transduce cells in S phase Proc Natl Acad Sci USA 1994 91: 8915–8919

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 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 

  8. 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 

  9. Balague C, Kalla M, Zhang WW . Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome J Virol 1997 71: 3299–3306

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Kessler PD et al. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein Proc Natl Acad Sci USA 1996 93: 14082–14087

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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 

  12. Flotte TR et al. Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector Proc Natl Acad Sci USA 1993 90: 10613–10617

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kaplitt MG et al. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain Nat Genet 1994 8: 148–154

    Article  CAS  PubMed  Google Scholar 

  14. Klein RL et al. Neuron-specific transduction in the rat septohippocampal or nigrostriatal pathway by recombinant adeno-associated virus vectors Exp Neurol 1998 150: 183–194

    Article  CAS  PubMed  Google Scholar 

  15. Peel AL et al. Efficient transduction of green fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell type-specific promoters Gene Therapy 1997 4: 16–24

    Article  CAS  PubMed  Google Scholar 

  16. Flannery JG et al. Efficient photoreceptor-targeted gene expression in vivo by recombinant adeno-associated virus Proc Natl Acad Sci USA 1997 94: 6916–6921

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Snyder RO et al. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors Nat Genet 1997 16: 270–276

    Article  CAS  PubMed  Google Scholar 

  18. Snyder RO, Xiao X, Samulski RJ . Production of recombinant adeno-associated viral vectors. In: Dracopoli N et al (eds) Current Protocols in Human Genetics John Wiley: New York 1996 pp 12.1.1–12.1.24.

    Google Scholar 

  19. Atkinson EM, Debelak DJ, Hart LA, Reynolds TC . A high-throughput hybridization method for titer determination of viruses and gene therapy vectors Nucleic Acids Res 1998 26: 2821–2823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  21. Flotte TR et al. Gene expression from adeno-associated virus vectors in airway epithelial cells Am J Respir Cell Mol Biol 1992 7: 349–356

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  23. 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 

  24. 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 

  25. Fisher KJ et al. 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 

  26. Wistuba A et al. Subcellular compartmentalization of adeno-associated virus type 2 assembly J Virol 1997 71: 1341–1352

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Steinbach S, Wistuba A, Bock T, Kleinschmidt JA . Assembly of adeno-associated virus type 2 capsids in vitro J Gen Virol 1997 78: 1453–1462

    Article  CAS  PubMed  Google Scholar 

  28. Mittereder N, March KL, Trapnell BC . Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy J Virol 1996 70: 7498–7509

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Chen CA, Okayama H . Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA Biotech 1988 6: 632–638

    CAS  Google Scholar 

  30. Samulski RJ, Berns KI, Tan M, Muzyczka N . Cloning of adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells Proc Natl Acad Sci USA 1982 79: 2077–2081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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 

  32. Heilbronn R, Bürkle A, Stephan S, zur Hausen H . The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification J Virol 1990 64: 3012–3018

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Zolotukhin S et al. A ‘humanized’ green fluorescent protein cDNA adapted for high-level expression in mammalian cells J Virol 1996 70: 4646–4654

    CAS  PubMed  PubMed Central  Google Scholar 

  34. de Wet JR et al. Firefly luciferase gene: structure and expression in mammalian cells Mol Cell Biol 1987 7: 725–737

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. MacGregor GR, Caskey CT . Construction of plasmids that express E. coli beta-galactosidase in mammalian cells Nucleic Acids Res 1989 17: 2365

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Rittner K, Stoppler H, Pawlita M, Sczakiel G . Versatile eucaryotic vectors for strong and constitutive transient and stable gene expression Meth Mol Cell Biol 1991 2: 176–181

    CAS  Google Scholar 

  37. Fisher KJ et al. Recombinant adeno-associated virus for muscle directed gene therapy Nature Med 1997 3: 306–312

    Article  CAS  PubMed  Google Scholar 

  38. Myers MW, Carter BJ . Assembly of adeno-associated virus Virol 1980 102: 71–82

    Article  CAS  Google Scholar 

  39. Xiao X, Li J, Samulski RJ . Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus J Virol 1998 72: 2224–2232

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 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 

  41. 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 

Download references

Acknowledgements

We are grateful to Dr Anna Salvetti for providing the HeLaRC32 cell line and to Dr Michael Chapman for supplying CsCl purified wild-type AAV-2. Andrea Hörster and Birgit Teichmann are thanked for their help with the FACS analyses. Thorsten Belz was involved in initial development of the ELISA. Dirk Grimm was supported by the BMBF grant 01KV9517/6.

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Grimm, D., Kern, A., Pawlita, M. et al. Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2. Gene Ther 6, 1322–1330 (1999). https://doi.org/10.1038/sj.gt.3300946

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