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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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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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  • DOI: https://doi.org/10.1038/sj.gt.3300946

Keywords

  • AAV-2
  • recombinant AAV-2
  • AAV-2 titration
  • AAV-2 ELISA

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