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rAAV Vector Product Characterization and Stability Studies

  • Richard O. SnyderEmail author
  • Muriel Audit
  • Joyce D. Francis
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 807)

Abstract

Recombinant adeno-associated viral (rAAV) vectors mediate the safe and long-term correction of genetic diseases following a single administration. Preclinical studies in animal models and human trials have shown rAAV vector persistence and safety. In some trials, sustained or transient transgene expression has been demonstrated in humans treated for alpha-1 antitrypsin deficiency, LPL deficiency, hemophilia B and cystic fibrosis, and sustained correction of inherited blindness has been reported by three groups. For human use, rAAV vectors are manufactured and tested in compliance with current Good Manufacturing Practices as outlined in the Code of Federal Regulations (21CFR) or European Good Manufacturing Practices (Eudralex, Volume 4, GMP Guidelines, 2003/94/CE and 91/356/EEC). Manufacturing control, as well as product quality is evaluated by quality control testing and all manufacturing, facilities, and testing activities are reviewed by the quality assurance department. In-process specifications are set and in-process testing is conducted to confirm that the manufacturing process is controlled, aseptic, and performs consistently. Final product is tested to ensure release specifications are met for identity, safety, purity, potency, and stability.

Key words

Adeno-associated virus Gene therapy Assay Testing Quality control Quality assurance 

Notes

Acknowledgments

We acknowledge Mahuya Bose and Weiyi Ni for insightful discussions. ROS is an inventor on patents related to recombinant AAV technology and owns equity in a gene therapy company that is commercializing AAV for gene therapy applications. To the extent that the work in this manuscript increases the value of these commercial holdings ROS has a conflict of interest.

References

  1. 1.
    Wagner, J. A., Messner, A. H., Moran, M. L., Daifuku, R., Kouyama, K., Desch, J. K., Manley, S., Norbash, A. M., Conrad, C. K., Friborg, S., Reynolds, T., Guggino, W. B., Moss, R. B., Carter, B. J., Wine, J. J., Flotte, T. R., and Gardner, P. (1999) Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus, Laryngoscope 109, 266–274.PubMedCrossRefGoogle Scholar
  2. 2.
    Flotte, T. R., Zeitlin, P. L., Reynolds, T. C., Heald, A. E., Pedersen, P., Beck, S., Conrad, C. K., Brass-Ernst, L., Humphries, M., Sullivan, K., Wetzel, R., Taylor, G., Carter, B. J., and Guggino, W. B. (2003) Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated virus serotype 2 (rAAV2)-CFTR vector in adult cystic fibrosis patients: a two-part clinical study, Hum Gene Ther 14, 1079–1088.PubMedCrossRefGoogle Scholar
  3. 3.
    Stedman, H., Wilson, J. M., Finke, R., Kleckner, A. L., and Mendell, J. (2000) Phase I clinical trial utilizing gene therapy for limb girdle muscular dystrophy: alpha-, beta-, gamma-, or delta-sarcoglycan gene delivered with intramuscular instillations of adeno-associated vectors, Hum Gene Ther 11, 777–790.PubMedCrossRefGoogle Scholar
  4. 4.
    Manno, C. S., Chew, A. J., Hutchison, S., Larson, P. J., Herzog, R. W., Arruda, V. R., Tai, S. J., Ragni, M. V., Thompson, A., Ozelo, M., Couto, L. B., Leonard, D. G., Johnson, F. A., McClelland, A., Scallan, C., Skarsgard, E., Flake, A. W., Kay, M. A., High, K. A., and Glader, B. (2003) AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B, Blood 101, 2963–2972.PubMedCrossRefGoogle Scholar
  5. 5.
    Janson, C., McPhee, S., Bilaniuk, L., Haselgrove, J., Testaiuti, M., Freese, A., Wang, D. J., Shera, D., Hurh, P., Rupin, J., Saslow, E., Goldfarb, O., Goldberg, M., Larijani, G., Sharrar, W., Liouterman, L., Camp, A., Kolodny, E., Samulski, J., and Leone, P. (2002) Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain, Hum Gene Ther 13, 1391–1412.PubMedCrossRefGoogle Scholar
  6. 6.
    During, M. J., Kaplitt, M. G., Stern, M. B., and Eidelberg, D. (2001) Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation, Hum Gene Ther 12, 1589–1591.PubMedGoogle Scholar
  7. 7.
    Flotte, T. R., Brantly, M. L., Spencer, L. T., Byrne, B. J., Spencer, C. T., Baker, D. J., and Humphries, M. (2004) Phase I trial of intramuscular injection of a recombinant adeno-associated virus alpha 1-antitrypsin (rAAV2-CB-hAAT) gene vector to AAT-deficient adults, Hum Gene Ther 15, 93–128.PubMedCrossRefGoogle Scholar
  8. 8.
    Crystal, R. G., Sondhi, D., Hackett, N. R., Kaminsky, S. M., Worgall, S., Stieg, P., Souweidane, M., Hosain, S., Heier, L., Ballon, D., Dinner, M., Wisniewski, K., Kaplitt, M., Greenwald, B. M., Howell, J. D., Strybing, K., Dyke, J., and Voss, H. (2004) Clinical protocol. Administration of a replication-deficient adeno-associated virus gene transfer vector expressing the human CLN2 cDNA to the brain of children with late infantile neuronal ceroid lipofuscinosis, Hum Gene Ther 15, 1131–1154.PubMedGoogle Scholar
  9. 9.
    Brantly, M. L., Chulay, J. D., Wang, L., Mueller, C., Humphries, M., Spencer, L. T., Rouhani, F., Conlon, T. J., Calcedo, R., Betts, M. R., Spencer, C., Byrne, B. J., Wilson, J. M., and Flotte, T. R. (2009) Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy, Proc Natl Acad Sci U S A 106, 16363–16368.PubMedCrossRefGoogle Scholar
  10. 10.
    Manno, C. S., Pierce, G. F., Arruda, V. R., Glader, B., Ragni, M., Rasko, J. J., Ozelo, M. C., Hoots, K., Blatt, P., Konkle, B., Dake, M., Kaye, R., Razavi, M., Zajko, A., Zehnder, J., Rustagi, P. K., Nakai, H., Chew, A., Leonard, D., Wright, J. F., Lessard, R. R., Sommer, J. M., Tigges, M., Sabatino, D., Luk, A., Jiang, H., Mingozzi, F., Couto, L., Ertl, H. C., High, K. A., and Kay, M. A. (2006) Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response, Nat Med 12, 342–347.PubMedCrossRefGoogle Scholar
  11. 11.
    High, K. A. (2007) Update on progress and hurdles in novel genetic therapies for hemophilia, Hematology Am Soc Hematol Educ Program, 466–472.Google Scholar
  12. 12.
    Ponder, K. P. (2011) Hemophilia gene therapy: a holy grail found, Mol Ther 19, 427–428.PubMedCrossRefGoogle Scholar
  13. 13.
    Mingozzi, F., Meulenberg, J. J., Hui, D. J., Basner-Tschakarjan, E., Hasbrouck, N. C., Edmonson, S. A., Hutnick, N. A., Betts, M. R., Kastelein, J. J., Stroes, E. S., and High, K. A. (2009) AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells, Blood 114, 2077–2086.PubMedCrossRefGoogle Scholar
  14. 14.
    Bainbridge, J. W., Smith, A. J., Barker, S. S., Robbie, S., Henderson, R., Balaggan, K., Viswanathan, A., Holder, G. E., Stockman, A., Tyler, N., Petersen-Jones, S., Bhattacharya, S. S., Thrasher, A. J., Fitzke, F. W., Carter, B. J., Rubin, G. S., Moore, A. T., and Ali, R. R. (2008) Effect of gene therapy on visual function in Leber’s congenital amaurosis, N Engl J Med 358, 2231–2239.PubMedCrossRefGoogle Scholar
  15. 15.
    Maguire, A. M., Simonelli, F., Pierce, E. A., Pugh, E. N., Jr., Mingozzi, F., Bennicelli, J., Banfi, S., Marshall, K. A., Testa, F., Surace, E. M., Rossi, S., Lyubarsky, A., Arruda, V. R., Konkle, B., Stone, E., Sun, J., Jacobs, J., Dell’Osso, L., Hertle, R., Ma, J. X., Redmond, T. M., Zhu, X., Hauck, B., Zelenaia, O., Shindler, K. S., Maguire, M. G., Wright, J. F., Volpe, N. J., McDonnell, J. W., Auricchio, A., High, K. A., and Bennett, J. (2008) Safety and efficacy of gene transfer for Leber’s congenital amaurosis, N Engl J Med 358, 2240–2248.PubMedCrossRefGoogle Scholar
  16. 16.
    Cideciyan, A. V., Hauswirth, W. W., Aleman, T. S., Kaushal, S., Schwartz, S. B., Boye, S. L., Windsor, E. A., Conlon, T. J., Sumaroka, A., Pang, J. J., Roman, A. J., Byrne, B. J., and Jacobson, S. G. (2009) Human RPE65 gene therapy for Leber congenital amaurosis: persistence of early visual improvements and safety at 1 year, Hum Gene Ther 20, 999–1004.PubMedCrossRefGoogle Scholar
  17. 17.
    Clark, K. R., Voulgaropoulou, F., and Johnson, P. R. (1996) A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors, Gene Ther 3, 1124–1132.PubMedGoogle Scholar
  18. 18.
    Chadeuf, G., Favre, D., Tessier, J., Provost, N., Nony, P., Kleinschmidt, J., Moullier, P., and Salvetti, A. (2000) 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 2, 260–268.PubMedCrossRefGoogle Scholar
  19. 19.
    Knop, D. R., and Harrell, H. (2007) Bioreactor production of recombinant herpes simplex virus vectors, Biotechnol Prog 23, 715–721.PubMedCrossRefGoogle Scholar
  20. 20.
    Merten, O. W. (2007) Attention with virus contaminated cell lines, Cytotechnology 55, 1–2.PubMedCrossRefGoogle Scholar
  21. 21.
    Van Vliet, K., Blouin, V., Agbandje-McKenna, M., and Snyder, R. O. (2006) Proteolytic mapping of the adeno-associated virus capsid, Mol Ther 14, 809–821.PubMedCrossRefGoogle Scholar
  22. 22.
    Van Vliet, K., Mohiuddin, Y., McClung, S., Blouin, V., Rolling, F., Moullier, P., Agbandje-McKenna, M., and Snyder, R. O. (2009) Adeno-associated virus capsid serotype identification: Analytical methods development and application, J Virol Methods 159, 167–177.PubMedCrossRefGoogle Scholar
  23. 23.
    Lock, M., McGorray, S., Auricchio, A., Ayuso, E., Beecham, E. J., Blouin-Tavel, V., Bosch, F., Bose, M., Byrne, B. J., Caton, T., Chiorini, J. A., Chtarto, A., Clark, K. R., Conlon, T., Darmon, C., Doria, M., Douar, A., Flotte, T. R., Francis, J. D., Francois, A., Giacca, M., Korn, M. T., Korytov, I., Leon, X., Leuchs, B., Lux, G., Melas, C., Mizukami, H., Moullier, P., Muller, M., Ozawa, K., Philipsberg, T., Poulard, K., Raupp, C., Riviere, C., Roosendaal, S. D., Samulski, R. J., Soltys, S. M., Surosky, R., Tenenbaum, L., Thomas, D. L., van Montfort, B., Veres, G., Wright, J. F., Xu, Y., Zelenaia, O., Zentilin, L., and Snyder, R. O. (2010) Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material, Hum Gene Ther 21, 1273–1285.PubMedCrossRefGoogle Scholar
  24. 24.
    Zhen, Z., Espinoza, Y., Bleu, T., Sommer, J. M., and Wright, J. F. (2004) Infectious titer assay for adeno-associated virus vectors with sensitivity sufficient to detect single infectious events, Hum Gene Ther 15, 709–715.CrossRefGoogle Scholar
  25. 25.
    Mohiuddin, I., Loiler, S., Zolotukhin, I., Byrne, B. J., Flotte, T. R., and Snyder, R. O. (2005) Herpesvirus-based infectious titering of recombinant adeno-associated viral vectors, Mol Ther 11, 320–326.PubMedCrossRefGoogle Scholar
  26. 26.
    Snyder, R. O., Xiao, X., and Samulski, R. J. (1996) Production of Recombinant Adeno-Associated Viral Vectors, in Current Protocols in Human Genetics (Dracopoli, N., Haines, J., Krof, B., Moir, D., Morton, C., Seidman, C., Seidman, J., and Smith, D., Eds.), pp 12.11.11-24, John Wiley and Sons, New York.Google Scholar
  27. 27.
    Nony, P., Chadeuf, G., Tessier, J., Moullier, P., and Salvetti, A. (2003) Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles, J Virol 77, 776–781.PubMedCrossRefGoogle Scholar
  28. 28.
    Gao, G. P., Qu, G., Faust, L. Z., Engdahl, R. K., Xiao, W., Hughes, J. V., Zoltick, P. W., and Wilson, J. M. (1998) High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus (In Process Citation), Hum Gene Ther 9, 2353–2362.PubMedCrossRefGoogle Scholar
  29. 29.
    Grimm, D., Kern, A., Pawlita, M., Ferrari, F., Samulski, R., and Kleinschmidt, J. (1999) 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.PubMedCrossRefGoogle Scholar
  30. 30.
    Wistuba, A., Kern, A., Weger, S., Grimm, D., and Kleinschmidt, J. A. (1997) Subcellular compartmentalization of adeno-associated virus type 2 assembly, J Virol 71, 1341–1352.PubMedGoogle Scholar
  31. 31.
    Grimm, D., Kay, M. A., and Kleinschmidt, J. A. (2003) Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6, Mol Ther 7, 839–850.PubMedCrossRefGoogle Scholar
  32. 32.
    Sommer, J. J., Smith, P. H., Parthasarathy, S., Isaacs, J., Vijay, S., Kieran, J., Powell, S. K., McClelland, A., and Wright, J. F. (2003) Quantification of adeno-associated virus particles and empty capsids by optical density measurement, Mol Ther 7, 122–128.PubMedCrossRefGoogle Scholar
  33. 33.
    Clark, K. R., Liu, X., McGrath, J. P., and Johnson, P. R. (1999) Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses, Hum Gene Ther 10, 1031–1039.PubMedCrossRefGoogle Scholar
  34. 34.
    Drittanti, L., Rivet, C., Manceau, P., Danos, O., and Vega, M. (2000) High throughput production, screening and analysis of adeno-associated viral vectors, Gene Ther 7, 924–929.PubMedCrossRefGoogle Scholar
  35. 35.
    Veldwijk, M. R., Topaly, J., Laufs, S., Hengge, U. R., Wenz, F., Zeller, W. J., and Fruehauf, S. (2002) Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks, Mol Ther 6, 272–278.PubMedCrossRefGoogle Scholar
  36. 36.
    Flotte, T. R., Burd, P., and Snyder, R. O. (2002) Utility of a Recombinant Adeno-Associated Viral Vector Reference Standard, BioProcessing 1, 75–77.Google Scholar
  37. 37.
    Moullier, P., and Snyder, R. O. (2008) International efforts for recombinant adeno-associated viral vector reference standards, Mol Ther 16, 1185–1188.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Richard O. Snyder
    • 1
    • 3
    Email author
  • Muriel Audit
    • 2
  • Joyce D. Francis
    • 3
  1. 1.Department of Molecular Genetics and MicrobiologyUniversity of Florida College of MedicineGainesvilleUSA
  2. 2.GenoSafe SASEVRY CedexFrance
  3. 3.Center of Excellence for Regenerative Health BiotechnologyUniversity of FloridaAlachuaUSA

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