Skip to main content
SpringerLink
Log in
Book cover

Adeno-Associated Virus pp 317–337Cite as

Preclinical Study Design for rAAV

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((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.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Barker, L. F. (1975) Food and Drug Administration regulations and licensure. Fed Proc 34, 1522–1524.

    PubMed  CAS  Google Scholar 

  2. Snyder, R. O., and Francis, J. (2005) Adeno-associated viral vectors for clinical gene transfer studies. Curr Gene Ther 5, 311–321.

    Article  PubMed  CAS  Google Scholar 

  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. 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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  20. Flotte, T. R., and Berns, K. I. (2005) Adeno-associated virus: a ubiquitous commensal of mammals. Hum Gene Ther 16, 401–407.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pediatrics, University of Massachusetts Medical School Gene Therapy Center, Worcester, MA, USA

    Terence R. Flotte

  2. Department of Pediatrics, University of Florida Powell Gene Therapy Center, Gainesville, FL, USA

    Thomas J. Conlon

  3. Department of Pediatrics, University of Massachusetts Medical School, Worcester, MA, USA

    Christian Mueller

Authors
  1. Terence R. Flotte
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas J. Conlon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christian Mueller
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Terence R. Flotte .

Editor information

Editors and Affiliations

  1. Center of Excellence for Regenerative, Health Biotechnology (CERHB), University of Florida, Innovation Drive 13706, Alachua, 32615, Florida, USA

    Richard O. Snyder

  2. INSERM U649, Labo. Thérapie Génique, CHU Hôtel Dieu, bd. Jean Monnet 30, Nantes CX 1, 44035, France

    Philippe Moullier

Appendices

General Guidelines for Real-Time PCR Standard Operating Procedure

Procedure Setup

  1. 1.

    Document on Form Real-Time PCR Procedure Record all information requested.

  2. 2.

    Wipe down entire work surface, 1.5 mL tube racks, and pipettes with 10% bleach.

  3. 3.

    Wipe down work surface area, 1.5 mL tube racks, and pipettes with DNA away.

  4. 4.

    Follow with an ethanol wipe down of work area, 1.5 mL tube racks, and pipettes

    Note: A separate set of pipettes are used for PCR preparation. The pipettes used for gDNA extractions must not be used.

  5. 5.

    Place a clean absorbent pad down on work area and lay all cleaned racks and pipettes on the pad.

    Note: Place and label all tubes in the rack to be used. Make sure to place the tubes used for preparing the standard curves in a separate rack.

  6. 6.

    Document the lot number and expiration date of the universal master mix.

Preliminary Run Set Up

  1. 1.

    Wipe down pipettes with DNA Away.

  2. 2.

    Based on the calculations, pipette water volumes required for gDNA sample wells.

  3. 3.

    Pipette water into the FAM standard wells.

  4. 4.

    Pipette water into a well indicated for FAM no template control (NTC) wells.

  5. 5.

    Prepare the master mix for the FAM-labeled probe.

    Note: Do not pipette master mix to plate at this time.

  6. 6.

    Prepare gDNA samples based on gDNA needed for sample type.

    Note: All genomic DNA dilutions should be made on the day the PCR assay is run. They should not be made in advance.

    Note: Do not pipette gDNA samples at this time.

  7. 7.

    Vortex briefly and pipette master mix Cocktail to FAM standard and NTC wells.

  8. 8.

    Vortex briefly and pipette master mix Cocktail to sample wells.

  9. 9.

    Vortex briefly and pipette diluted gDNA samples to designated wells.

  10. 10.

    Vortex briefly and pipette the required amount of each standard dilution into the appropriate well based on vector length.

  11. 11.

    Vortex briefly and pipette Spike in to designated wells.

    Note: Spike in is calculated to keep a 100 copy/μg genomic DNA ratio. If less than 0.1 μg DNA is loaded, a ten-copy spike in is always used. Example: If 0.7 μg is loaded, then a 70-copy spike in will be used.

  12. 12.

    Cover plate with optical cover according to instructions. The optical cover should not be touched.

  13. 13.

    Settle the samples in the plate by tapping on side. This gets out any bubbles that are sitting at the bottom of the wells.

  14. 14.

    Transfer to isolated room housing the real-time PCR machine and run.

Analysis

  1. 1.

    When the run is complete, analyze the run using the appropriate software.

  2. 2.

    Remove outliers on the standard curve if necessary. GLP standards require at least six standard curve points; therefore up to three outliers may be removed. Reanalyze run if outliers are removed.

  3. 3.

    Start baseline (red flag) should be set at a standard predetermine value or default to be considered acceptable.

  4. 4.

    Stop baseline (red flag) should be set 1 cycle before amplification of the 10E8 standard point begins (rounded to a whole number).

  5. 5.

    After setting the baselines section, the threshold should be set to maximize the R2 using the following criteria:

    • The threshold should be set above the background

    • Below the plateaued and linear regions of the amplification curve

    • Within the geometric phase of the amplification curve

  6. 6.

    The acceptable range for the slope should be between ±0.3 of the study mean. The R2 should be equal or greater than 0.97 for the run to be acceptable.

  7. 7.

    The acceptable range for the efficiency should be 95–105%.

    $$ \text{Efficiency}=-1+{10}^{(-1/\text{slope})}$$
  8. 8.

    Observe the copy count (Ct) for the NTC. The acceptable range for the NTC is less than or equal to 15 copies/μg gDNA.

  9. 9.

    Complete the SOP associated form to determine if the run is acceptable.

  10. 10.

    If the run is unacceptable, discuss with laboratory director before attempting the run again.

General Guidelines for Necropsy Standard Operating Procedure

Gross Necropsy

  1. 1.

    A complete necropsy will be conducted on all animals dying spontaneously, euthanized in extremis or at the scheduled necropsy dates. Necropsy will include examination of:

    • The external body surface

    • All orifices

    • The cranial, thoracic, abdominal, and pelvic cavities and their contents.

  2. 2.

    Prior to sampling, the pathologist will perform a gross necropsy.

  3. 3.

    All body surfaces and orifices are observed carefully and external surfaces palpated gently. All abnormalities will be recorded in the appropriate sections of the necropsy forms.

  4. 4.

    After dissection and sampling, carefully examine all body cavities. The condition of the blood should be noted.

  5. 5.

    The remainder of the esophagus and GI tract should be opened and the mucosa and contents examined.

  6. 6.

    If required by protocol, a licensed pathologist will then examine the previously removed organ tissues in wet tissue containers for abnormalities and lesions found will be fixed in NBF for further examination.

  7. 7.

    If required by protocol, a licensed pathologist will verify the identification of the animal, verify tissue accountability, assess lesions, and check for completeness and accuracy of the entries on the necropsy form and sign and date the necropsy form.

  8. 8.

    An audio recording may be taken during necropsy and the audio file will be sent to the Study Director for archiving. At the end of the study, this audio file will be copied to CD for archiving.

  9. 9.

    Depending on the analyses that need to be performed on these tissues (e.g., histopathology, PCR, or both), multiple samples may be required.

  10. 10.

    Pathologist refers to the person ultimately responsible for the necropsy.

  11. 11.

    Depending on the tissues to be collected employ specific teams (e.g., one team will remove the head and harvest the brain, a second team will harvest the heart and lungs, and a third team will collect the remaining organs).

  12. 12.

    During the course of the dissection, change gloves and instruments between each organ. Instruments used for obtaining study samples should not have been used previously for any other tissue (e.g., use a separate set of instruments for cutting away connective tissue to free an organ and cutting the actual sample from that organ). All cutting boards, containers, and necropsy surfaces will be either disposable or washed with DNA Away, 10% bleach, and 70% ethanol between animals to prevent contamination of DNA/mRNA. Individuals handling the tissue should not touch bare human skin while handling the tissues in order to prevent contamination with RNAase. Razor blades should be discarded between organs to prevent contamination.

  13. 13.

    All gross macroscopic lesions should be recorded on a necropsy form, using the approved terminology and format, and preserved in 10% neutral buffered formalin until consultation with the Study Director or Sponsor for possible histopathologic evaluation. If any questions regarding the identification or description of an organ/lesion arise, the pathologist should be immediately consulted. At each of the steps outlined, it is understood that careful visual observation and gentle palpation (as needed) are essential. Lesion descriptions will include organ, morphology, and where appropriate, color, site, size in millimeters or milliliters, and distribution.

  14. 14.

    Take note of the condition of the blood (e.g., normal, thin, thick, and clotted). In anemic animals, blood can look thin while in lipemic animals it can appear a homogenous pale pink color with a thick consistency.

  15. 15.

    If a bone marrow smear is required, it should be prepared using a portion of the rib (or the bone specified in the ­protocol). The rib or bone specified for bone marrow smear should be submitted as early in the necropsy procedure as possible.

Organ and Tissue Collection

  1. 1.

    After removing the organs from the body, as much extraneous tissue as possible is removed and the organ is rinsed in a container with iced saline to remove blood and debris.

  2. 2.

    Required organs are weighed and the weight is recorded on the study-specific necropsy form.

  3. 3.

    Clean the weighing pan by brushing with a soft brush, or ­wiping the pan with a clean, lint-free cloth or Kimwipe®.

  4. 4.

    Place a disposable bench pad in the weighing pan and tare the scale.

  5. 5.

    Place the sample on the disposable bench pad and weigh them together in the weighing pan. Record the results on the study-specific necropsy form.

  6. 6.

    Pat the organ dry with the disposable bench pad, remove the organ, and weigh only the pad. Record the pad weight on the study-specific necropsy form.

  7. 7.

    Paired organs will be weighed together. Each organ may be placed back into the container containing ice-cold saline before the tissue area to be collected is harvested and cut into small blocks with dedicated cutting boards and instruments. The tissues may be rinsed in containers (appropriate tubs, buckets, pans, etc.) and saline squeeze bottles should also be available. Saline will be chilled overnight and dispensed immediately before use. It may be kept cold, for example, by refrigeration, using frozen bags of saline or saline ice cubes, or setting the saline container in ice.

  8. 8.

    After trimming, the remaining organ will be examined by the necropsy team for gross abnormalities and samples obtained for fixation if required.

  9. 9.

    If a quick frozen sample is needed for analyses such as PCR, place tissue into the appropriate prelabeled cryogenic containers, snap freeze in liquid nitrogen, and transfer to a −70°C freezer. The sample size should be no more than two thirds of the vial in order to allow for expansion of the tissue during freezing. If trimming occurs on a dissection board, use a separate dissection board for each organ.

  10. 10.

    Tissues for histopathology are generally fixed in neutral buffered formalin (NBF). Tissues will be fixed at a thickness not to exceed 5 mm except as stated.

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Flotte, T.R., Conlon, T.J., Mueller, C. (2012). Preclinical Study Design for rAAV. In: Snyder, R., Moullier, P. (eds) Adeno-Associated Virus. Methods in Molecular Biology, vol 807. Humana Press. https://doi.org/10.1007/978-1-61779-370-7_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-370-7_14

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-369-1

  • Online ISBN: 978-1-61779-370-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation