Advertisement

Preclinical Study Design for rAAV

  • Terence R. FlotteEmail author
  • Thomas J. Conlon
  • Christian Mueller
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 807)

Abstract

The process of moving a novel drug such as an adeno-associated viral vector from the bench top to bedside is an arduous process requiring coordination and skill from multiple laboratories and regulatory agencies. Proceeding to a phase I safety trial in humans after most of the proof-of-concept data have been acquired may take several years. During this time, agencies including the FDA, NIH Office of Biotechnology Activities (OBA), and Recombinant DNA Advisory Committee (RAC) along with the investigator’s team will develop a series of preclinical toxicology and biodistribution studies in order to develop a safety profile for the intended novel drug. In this chapter, key features of the pharm–tox study design and conduct will be discussed. Highlighted features include choosing a sufficient animal number and species to use in testing, dose determination, typical toxicological assays performed, the use of Standard Operating Procedures in respect to good laboratory practices compliancy, and role of the Quality Assurance Unit.

Key words

AAV gene therapy Preclinical testing AAV toxicology Vector biodistribution Good laboratory practices 

References

  1. 1.
    Barker, L. F. (1975) Food and Drug Administration regulations and licensure. Fed Proc 34, 1522–1524.PubMedGoogle Scholar
  2. 2.
    Snyder, R. O., and Francis, J. (2005) Adeno-associated viral vectors for clinical gene transfer studies. Curr Gene Ther 5, 311–321.PubMedCrossRefGoogle Scholar
  3. 3.
    Francis, J. D., and Snyder, R. O. (2005) Production of Research and Clinical Grade Recombinant Adeno-associated Virus Vectors, in Laboratory Techniques in Biochemistry and Molecular Biology (Berns, K. I., and Flotte, T. R., Eds.) pp 19–56, Elsevier, Amsterdam.Google Scholar
  4. 4.
    Conrad, C. K., Allen, S. S., Afione, S. A., Reynolds, T. C., Beck, S. E., Fee-Maki, M., Barrazza-Ortiz, X., Adams, R., Askin, F. B., Carter, B. J., Guggino, W. B., and Flotte, T. R. (1996) Safety of single-dose administration of an adeno-associated virus (AAV)- CFTR vector in the primate lung. Gene Ther 3, 658–668.PubMedGoogle Scholar
  5. 5.
    Song, S., Scott-Jorgensen, M., Wang, J., Poirier, A., Crawford, J., Campbell-Thompson, M., and Flotte, T. R. (2002) Intramuscular administration of recombinant adeno-associated virus 2 alpha-1 antitrypsin (rAAV-SERPINA1) vectors in a nonhuman primate model: safety and immunologic aspects. Mol Ther 6, 329–335.PubMedCrossRefGoogle Scholar
  6. 6.
    Poirier, A., Campbell-Thompson, M., Tang, Q., Scott-Jorgensen, M., Combee, L., Loiler, S., Crawford, J., Song, S., and Flotte, T. R. (2004) Toxicology and biodistribution studies of a recombinant adeno-associated virus 2-alpha-1 antitrypsin vector. Preclinica 2, 43–51.Google Scholar
  7. 7.
    Flotte, T. R., Conlon, T. J., Poirier, A., Campbell-Thompson, M., and Byrne, B. J. (2007) Preclinical characterization of a recombinant adeno-associated virus type 1-pseudotyped vector demonstrates dose-dependent injection site inflammation and dissemination of vector genomes to distant sites. Hum Gene Ther 18, 245–256.PubMedCrossRefGoogle Scholar
  8. 8.
    Jacobson, S. G., Boye, S. L., Aleman, T. S., Conlon, T. J., Zeiss, C. J., Roman, A. J., Cideciyan, A. V., Schwartz, S. B., Komaromy, A. M., Doobrajh, M., Cheung, A. Y., Sumaroka, A., Pearce-Kelling, S. E., Aguirre, G. D., Kaushal, S., Maguire, A. M., Flotte, T. R., and Hauswirth, W. W. (2006) Safety in nonhuman primates of ocular AAV2-RPE65, a candidate treatment for blindness in Leber congenital amaurosis. Hum Gene Ther 17, 845–858.PubMedCrossRefGoogle Scholar
  9. 9.
    Jacobson, S. G., Acland, G. M., Aguirre, G. D., Aleman, T. S., Schwartz, S. B., Cideciyan, A. V., Zeiss, C. J., Komaromy, A. M., Kaushal, S., Roman, A. J., Windsor, E. A., Sumaroka, A., Pearce-Kelling, S. E., Conlon, T. J., Chiodo, V. A., Boye, S. L., Flotte, T. R., Maguire, A. M., Bennett, J., and Hauswirth, W. W. (2006) Safety of recombinant adeno-associated virus type 2-RPE65 vector delivered by ocular subretinal injection. Mol Ther 13, 1074–1084.PubMedCrossRefGoogle Scholar
  10. 10.
    Goldman, M. J., Litzky, L. A., Engelhardt, J. F., and Wilson, J. M. (1995) Transfer of the CFTR gene to the lung of nonhuman primates with E1- deleted, E2a-defective recombinant adenoviruses: a preclinical toxicology study. Hum Gene Ther 6, 839–51.PubMedCrossRefGoogle Scholar
  11. 11.
    Ferrari, F. K., Samulski, T., Shenk, T., and Samulski, R. J. (1996) Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J Virol 70, 3227–3334.PubMedGoogle Scholar
  12. 12.
    Fisher, K. J., Gao, G. P., Weitzman, M. D., DeMatteo, R., Burda, J. F., and Wilson, J. M. (1996) Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis. J Virol 70, 520–532.PubMedGoogle Scholar
  13. 13.
    Afione, S. A., Wang, J., Walsh, S., Guggino, W. B., and Flotte, T. R. (1999) Delayed expression of adeno-associated virus vector DNA. Intervirology 42, 213–220.PubMedCrossRefGoogle Scholar
  14. 14.
    Song, S., Morgan, M., Ellis, T., Poirier, A., Chesnut, K., Wang, J., Brantly, M., Muzyczka, N., Byrne, B. J., Atkinson, M., and Flotte, T. R. (1998) Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors. Proc Natl Acad Sci U S A 95, 14384–14388.PubMedCrossRefGoogle Scholar
  15. 15.
    McCarty, D. M., Monahan, P. E., and Samulski, R. J. (2001) Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther 8, 1248–1254.PubMedCrossRefGoogle Scholar
  16. 16.
    McCarty, D. M., Fu, H., Monahan, P. E., Toulson, C. E., Naik, P., and Samulski, R. J. (2003) Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Ther 10, 2112–2118.PubMedCrossRefGoogle Scholar
  17. 17.
    Fu, H., Muenzer, J., Samulski, R. J., Breese, G., Sifford, J., Zeng, X., and McCarty, D. M. (2003) Self-complementary adeno-associated virus serotype 2 vector: global distribution and broad dispersion of AAV-mediated transgene expression in mouse brain. Mol Ther 8, 911–917.PubMedCrossRefGoogle Scholar
  18. 18.
    Zhong, L., Chen, L., Li, Y., Qing, K., Weigel-Kelley, K. A., Chan, R. J., Yoder, M. C., and Srivastava, A. (2004) Self-complementary adeno-associated virus 2 (AAV)-T cell protein tyrosine phoshatase vectors as helper viruses to improve transduction efficiency of conventional single-stranded AAV vectors in vitro and in vivo. Mol Ther 10, 950–957.PubMedCrossRefGoogle Scholar
  19. 19.
    Vandenberghe, L. H., Wang, L., Somanathan, S., Zhi, Y., Figueredo, J., Calcedo, R., Sanmiguel, J., Desai, R. A., Chen, C. S., Johnston, J., Grant, R. L., Gao, G., and Wilson, J. M. (2006) Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid. Nat Med 12, 967–971.PubMedCrossRefGoogle Scholar
  20. 20.
    Flotte, T. R., and Berns, K. I. (2005) Adeno-associated virus: a ubiquitous commensal of mammals. Hum Gene Ther 16, 401–407.PubMedCrossRefGoogle Scholar
  21. 21.
    Summerford, C., and Samulski, R. J. (1998) Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J Virol 72, 1438–1445.PubMedGoogle Scholar
  22. 22.
    Summerford, C., Bartlett, J. S., and Samulski, R. J. (1999) AlphaVbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection. Nat Med 5, 78–82.PubMedCrossRefGoogle Scholar
  23. 23.
    Walters, R. W., Yi, S. M., Keshavjee, S., Brown, K. E., Welsh, M. J., Chiorini, J. A., and Zabner, J. (2001) Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer. J Biol Chem 276, 20610–20616.PubMedCrossRefGoogle Scholar
  24. 24.
    Qing, K., Mah, C., Hansen, J., Zhou, S., Dwarki, V., and Srivastava, A. (1999) Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2. Nat Med 5, 71–77.PubMedCrossRefGoogle Scholar
  25. 25.
    Chen, S., Kapturczak, M., Loiler, S. A., Zolotukhin, S., Glushakova, O. Y., Madsen, K. M., Samulski, R. J., Hauswirth, W. W., Campbell-Thompson, M., Berns, K. I., Flotte, T. R., Atkinson, M. A., Tisher, C. C., and Agarwal, A. (2005) Efficient transduction of vascular endothelial cells with recombinant adeno-associated virus serotype 1 and 5 vectors. Hum Gene Ther 16, 235–247.PubMedCrossRefGoogle Scholar
  26. 26.
    Duan, D., Yue, Y., Yan, Z., Yang, J., and Engelhardt, J. F. (2000) Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus. J Clin Invest 105, 1573–1587.PubMedCrossRefGoogle Scholar
  27. 27.
    Sanlioglu, S., Benson, P. K., Yang, J., Atkinson, E. M., Reynolds, T., and Engelhardt, J. F. (2000) Endocytosis and nuclear trafficking of adeno-associated virus type 2 are controlled by rac1 and phosphatidylinositol-3 kinase activation. J Virol 74, 9184–9196.PubMedCrossRefGoogle Scholar
  28. 28.
    Yan, Z., Zak, R., Luxton, G. W., Ritchie, T. C., Bantel-Schaal, U., and Engelhardt, J. F. (2002) Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors. J Virol 76, 2043–2053.PubMedCrossRefGoogle Scholar
  29. 29.
    Yan, Z., Zak, R., Zhang, Y., Ding, W., Godwin, S., Munson, K., Peluso, R., and Engelhardt, J. F. (2004) Distinct classes of proteasome-modulating agents cooperatively augment recombinant adeno-associated virus type 2 and type 5-mediated transduction from the apical surfaces of human airway epithelia. J Virol 78, 2863–74.PubMedCrossRefGoogle Scholar
  30. 30.
    Weitzman, M. D., Fisher, K. J., and Wilson, J. M. (1996) Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers. J Virol 70, 1845–1854.PubMedGoogle Scholar
  31. 31.
    Qing, K., Wang, X. S., Kube, D. M., Ponnazhagan, S., Bajpai, A., and Srivastava, A. (1997) Role of tyrosine phosphorylation of a cellular protein in adeno- associated virus 2-mediated transgene expression. Proc Natl Acad Sci U S A 94, 10879–10884.PubMedCrossRefGoogle Scholar
  32. 32.
    Qing, K., Hansen, J., Weigel-Kelley, K. A., Tan, M., Zhou, S., and Srivastava, A. (2001) Adeno-associated virus type 2-mediated gene transfer: role of cellular FKBP52 protein in transgene expression. J Virol 75, 8968–8976.PubMedCrossRefGoogle Scholar
  33. 33.
    Flotte, T. R., Afione, S. A., and Zeitlin, P. L. (1994) Adeno-associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration. Am J Respir Cell Mol Biol 11, 517–521.PubMedGoogle Scholar
  34. 34.
    Afione, S. A., Conrad, C. K., Kearns, W. G., Chunduru, S., Adams, R., Reynolds, T. C., Guggino, W. B., Cutting, G. R., Carter, B. J., and Flotte, T. R. (1996) In vivo model of adeno-associated virus vector persistence and rescue. J Virol 70, 3235–3241.PubMedGoogle Scholar
  35. 35.
    Kearns, W. G., Afione, S. A., Fulmer, S. B., Pang, M. C., Erikson, D., Egan, M., Landrum, M. J., Flotte, T. R., and Cutting, G. R. (1996) Recombinant adeno-associated virus (AAV-CFTR) vectors do not integrate in a site-specific fashion in an immortalized epithelial cell line. Gene Ther 3, 748–755.PubMedGoogle Scholar
  36. 36.
    Song, S., Laipis, P. J., Berns, K. I., and Flotte, T. R. (2001) Effect of DNA-dependent protein kinase on the molecular fate of the rAAV2 genome in skeletal muscle. Proceedings of the National Academy of Sciences USA 98, 4084–4088.CrossRefGoogle Scholar
  37. 37.
    Nakai, H., Yant, S. R., Storm, T. A., Fuess, S., Meuse, L., and Kay, M. A. (2001) Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo. J Virol 75, 6969–76.PubMedCrossRefGoogle Scholar
  38. 38.
    Chen, Z. Y., Yant, S. R., He, C. Y., Meuse, L., Shen, S., and Kay, M. A. (2001) Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver. Mol Ther 3, 403–410.PubMedCrossRefGoogle Scholar
  39. 39.
    Song, S., Lu, Y., Choi, Y. K., Han, Y., Tang, Q., Zhao, G., Berns, K. I., and Flotte, T. R. (2004) DNA-dependent PK inhibits adeno-associated virus DNA integration. Proc Natl Acad Sci USA 101, 2112–2116.PubMedCrossRefGoogle Scholar
  40. 40.
    Duan, D., Yue, Y., and Engelhardt, J. F. (2003) Consequences of DNA-dependent protein kinase catalytic subunit deficiency on recombinant adeno-associated virus genome circularization and heterodimerization in muscle tissue. J Virol 77, 4751–4759.PubMedCrossRefGoogle Scholar
  41. 41.
    Yan, Z., Zhang, Y., Duan, D., and Engelhardt, J. F. (2000) Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy. Proc Natl Acad Sci USA 97, 6716–6721.PubMedCrossRefGoogle Scholar
  42. 42.
    Moss, R. B., Rodman, D., Spencer, L. T., Aitken, M. L., Zeitlin, P. L., Waltz, D., Milla, C., Brody, A. S., Clancy, J. P., Ramsey, B., Hamblett, N., and Heald, A. E. (2004) Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebo-controlled trial. Chest 125, 509–521.PubMedCrossRefGoogle Scholar
  43. 43.
    Beck, S. E., Jones, L. A., Chesnut, K., Walsh, S. M., Reynolds, T. C., Carter, B. J., Askin, F. B., Flotte, T. R., and Guggino, W. B. (1999) Repeated delivery of adeno-associated virus vectors to the rabbit airway. J Virol 73, 9446–9455.PubMedGoogle Scholar
  44. 44.
    Halbert, C. L., Rutledge, E. A., Allen, J. M., Russell, D. W., and Miller, A. D. (2000) Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes. J Virol 74, 1524–1532.PubMedCrossRefGoogle Scholar
  45. 45.
    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
  46. 46.
    Mingozzi, F., Maus, M. V., Hui, D. J., Sabatino, D. E., Murphy, S. L., Rasko, J. E., Ragni, M. V., Manno, C. S., Sommer, J., Jiang, H., Pierce, G. F., Ertl, H. C., and High, K. A. (2007) CD8(+) T-cell responses to adeno-associated virus capsid in humans. Nat Med 13, 419–422.PubMedCrossRefGoogle Scholar
  47. 47.
    Hernandez, Y. J., Wang, J., Kearns, W. G., Loiler, S., Poirier, A., and Flotte, T. R. (1999) Latent adeno-associated virus infection elicits humoral but not cell- mediated immune responses in a nonhuman primate model. J Virol 73, 8549–8558.PubMedGoogle Scholar
  48. 48.
    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
  49. 49.
    Hauswirth, W. W., Aleman, T. S., Kaushal, S., Cideciyan, A. V., Schwartz, S. B., Wang, L., Conlon, T. J., Boye, S. L., Flotte, T. R., Byrne, B. J., and Jacobson, S. G. (2008) Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial. Hum Gene Ther 19, 979–990.PubMedCrossRefGoogle Scholar
  50. 50.
    Cideciyan, A. V., Aleman, T. S., Boye, S. L., Schwartz, S. B., Kaushal, S., Roman, A. J., Pang, J. J., Sumaroka, A., Windsor, E. A., Wilson, J. M., Flotte, T. R., Fishman, G. A., Heon, E., Stone, E. M., Byrne, B. J., Jacobson, S. G., and Hauswirth, W. W. (2008) Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. Proc Natl Acad Sci U S A 105, 15112–15117.PubMedCrossRefGoogle Scholar
  51. 51.
    Flotte, T. R., Fischer, A. C., Goetzmann, J., Mueller, C., Cebotaru, L., Yan, Z., Wang, L., Wilson, J. M., Guggino, W. B., and Engelhardt, J. F. (2009) Dual reporter comparative indexing of rAAV pseudotyped vectors in chimpanzee airway. Mol Ther 18, 594–600.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Terence R. Flotte
    • 1
    Email author
  • Thomas J. Conlon
    • 2
  • Christian Mueller
    • 3
  1. 1.Department of PediatricsUniversity of Massachusetts Medical School Gene Therapy CenterWorcesterUSA
  2. 2.Department of PediatricsUniversity of Florida Powell Gene Therapy CenterGainesvilleUSA
  3. 3.Department of PediatricsUniversity of Massachusetts Medical SchoolWorcesterUSA

Personalised recommendations