Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review
  • Published:

Gene Therapy Progress and Prospects – Vectorology: design and production of expression cassettes in AAV vectors

Gene Therapy volume 13, pages 805–813 (2006)Cite this article

Abstract

Adeno-associated virus (AAV) derived vectors are considered highly eligible vehicles for human gene therapy. Not only do they possess many great potential for clinical applications due to their wide range of tissue targets but also their excellent preclinical safety profile makes them particularly suitable candidates for treating serious diseases. Initial clinical trials have yielded encouraging results and prompted further improvements in their design and methods of production. Many studies have been performed to modify the tropism of recombinant (r)AAV by capsid modification. However, the precise control of spatial and temporal gene expression, which may be important in determining the safety and efficacy of gene transfer, lies in a rational choice and a subtle combination of various regulatory genetic elements to be inserted into the expression cassette. Moreover, new strategies based on such genetic sequences open new perspectives for enhancing vector genome persistence, disrupting or reducing pathogenic gene expression and even targeting genes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3

Similar content being viewed by others

References

  1. Flotte TR . Gene therapy progress and prospects: recombinant adeno-associated virus (rAAV) vectors. Gene Therapy 2004; 11: 805–810.

    Article  CAS  PubMed  Google Scholar 

  2. Gao G, Vandenberghe LH, Alvira MR, Lu Y, Calcedo R, Zhou X et al. Clades of Adeno-associated viruses are widely disseminated in human tissues. J Virol 2004; 78: 6381–6388.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Choi VW, McCarty DM, Samulski RJ . AAV hybrid serotypes: improved vectors for gene delivery. Curr Gene Ther 2005; 5: 299–310.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Grimm D, Kay MA . From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy. Curr Gene Ther 2003; 3: 281–304.

    Article  CAS  PubMed  Google Scholar 

  5. Ostedgaard LS, Rokhlina T, Karp PH, Lashmit P, Afione S, Schmidt M et al. A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia. Proc Natl Acad Sci USA 2005; 102: 2952–2957.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Everett RS, Evans HK, Hodges BL, Ding EY, Serra DM, Amalfitano A . Strain-specific rate of shutdown of CMV enhancer activity in murine liver confirmed by use of persistent [E1(−), E2b(−)] adenoviral vectors. Virology 2004; 325: 96–105.

    Article  CAS  PubMed  Google Scholar 

  7. Park J, Murray GJ, Limaye A, Quirk JM, Gelderman MP, Brady RO et al. Long-term correction of globotriaosylceramide storage in Fabry mice by recombinant adeno-associated virus-mediated gene transfer. Proc Natl Acad Sci USA 2003; 100: 3450–3454.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sirninger J, Muller C, Braag S, Tang Q, Yue H, Detrisac C et al. Functional characterization of a recombinant adeno-associated virus 5-pseudotyped cystic fibrosis transmembrane conductance regulator vector. Hum Gene Ther 2004; 15: 832–841.

    CAS  PubMed  Google Scholar 

  9. Shevtsova Z, Malik JM, Michel U, Bahr M, Kugler S . Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo. Exp Physiol 2005; 90: 53–59.

    Article  CAS  PubMed  Google Scholar 

  10. Liu YL, Mingozzi F, Rodriguez-Colon SM, Joseph S, Dobrzynski E, Suzuki T et al. Therapeutic levels of factor IX expression using a muscle-specific promoter and adeno-associated virus serotype 1 vector. Hum Gene Ther 2004; 15: 783–792.

    Article  CAS  PubMed  Google Scholar 

  11. Yoshimura M, Sakamoto M, Ikemoto M, Mochizuki Y, Yuasa K, Miyagoe-Suzuki Y et al. AAV vector-mediated microdystrophin expression in a relatively small percentage of mdx myofibers improved the mdx phenotype. Mol Ther 2004; 10: 821–828.

    Article  CAS  PubMed  Google Scholar 

  12. Zhang G, Ludtke JJ, Thioudellet C, Kleinpeter P, Antoniou M, Herweijer H et al. Intraarterial delivery of naked plasmid DNA expressing full-length mouse dystrophin in the mdx mouse model of duchenne muscular dystrophy. Hum Gene Ther 2004; 15: 770–782.

    Article  CAS  PubMed  Google Scholar 

  13. Zhong S, Sun S, Teng BB . The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression. Genet Vaccines Ther 2004; 2: 5.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Toniatti C, Bujard H, Cortese R, Ciliberto G . Gene therapy progress and prospects: transcription regulatory systems. Gene Therapy 2004; 11: 649–657.

    Article  CAS  PubMed  Google Scholar 

  15. Goverdhana S, Puntel M, Xiong W, Zirger JM, Barcia C, Curtin JF et al. Regulatable gene expression systems for gene therapy applications: progress and future challenges. Mol Ther 2005; 12: 189–211.

    Article  CAS  PubMed  Google Scholar 

  16. Jiang L, Rampalli S, George D, Press C, Bremer EG, O'Gorman MR et al. Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR. Gene Therapy 2004; 11: 1057–1067.

    Article  CAS  PubMed  Google Scholar 

  17. Chenuaud P, Larcher T, Rabinowitz JE, Provost N, Joussemet B, Bujard H et al. Optimal design of a single recombinant adeno-associated virus derived from serotypes 1 and 2 to achieve more tightly regulated transgene expression from nonhuman primate muscle. Mol Ther 2004; 9: 410–418.

    Article  CAS  PubMed  Google Scholar 

  18. Chtarto A, Bender HU, Hanemann CO, Kemp T, Lehtonen E, Levivier M et al. Tetracycline-inducible transgene expression mediated by a single AAV vector. Gene Therapy 2003; 10: 84–94.

    Article  CAS  PubMed  Google Scholar 

  19. Sanftner LM, Rivera VM, Suzuki BM, Feng L, Berk L, Zhou S et al. Dimerizer regulation of AADC expression and behavioral response in AAV-transduced 6-OHDA lesioned rats. Mol Ther 2006; 13: 167–174.

    Article  CAS  PubMed  Google Scholar 

  20. Lebherz C, Auricchio A, Maguire AM, Rivera VM, Tang W, Grant RL et al. Long-term inducible gene expression in the eye via adeno-associated virus gene transfer in nonhuman primates. Hum Gene Ther 2005; 16: 178–186.

    Article  CAS  PubMed  Google Scholar 

  21. Rivera VM, Gao GP, Grant RL, Schnell MA, Zoltick PW, Rozamus LW et al. Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer. Blood 2005; 105: 1424–1430.

    Article  CAS  PubMed  Google Scholar 

  22. Flotte TR, Zeitlin PL, Reynolds TC, Heald AE, Pedersen P, Beck S et al. 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 2003; 14: 1079–1088.

    Article  CAS  PubMed  Google Scholar 

  23. Flotte TR, Schwiebert EM, Zeitlin PL, Carter BJ, Guggino WB . Correlation between DNA transfer and cystic fibrosis airway epithelial cell correction after recombinant adeno-associated virus serotype 2 gene therapy. Hum Gene Ther 2005; 16: 921–928.

    Article  CAS  PubMed  Google Scholar 

  24. Virella-Lowell I, Zusman B, Foust K, Loiler S, Conlon T, Song S et al. Enhancing rAAV vector expression in the lung. J Gene Med 2005; 7: 842–850.

    Article  CAS  PubMed  Google Scholar 

  25. Hildinger M, Auricchio A, Gao G, Wang L, Chirmule N, Wilson JM . Hybrid vectors based on adeno-associated virus serotypes 2 and 5 for muscle-directed gene transfer. J Virol 2001; 75: 6199–6203.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Davidoff AM, Gray JT, Ng CY, Zhang Y, Zhou J, Spence Y et al. Comparison of the ability of adeno-associated viral vectors pseudotyped with serotype 2, 5, and 8 capsid proteins to mediate efficient transduction of the liver in murine and nonhuman primate models. Mol Ther 2005; 11: 875–888.

    Article  CAS  PubMed  Google Scholar 

  27. Ngoi SM, Chien AC, Lee CG . Exploiting internal ribosome entry sites in gene therapy vector design. Curr Gene Ther 2004; 4: 15–31.

    Article  CAS  PubMed  Google Scholar 

  28. Ito H, Goater JJ, Tiyapatanaputi P, Rubery PT, O'Keefe RJ, Schwarz EM . Light-activated gene transduction of recombinant adeno-associated virus in human mesenchymal stem cells. Gene Therapy 2004; 11: 34–41.

    Article  CAS  PubMed  Google Scholar 

  29. Ulrich-Vinther M, Stengaard C, Schwarz EM, Goldring MB, Soballe K . Adeno-associated vector mediated gene transfer of transforming growth factor-beta1 to normal and osteoarthritic human chondrocytes stimulates cartilage anabolism. Eur Cell Mater 2005; 10: 40–59.

    Article  CAS  PubMed  Google Scholar 

  30. Kapturczak M, Zolotukhin S, Cross J, Pileggi A, Molano RD, Jorgensen M et al. Transduction of human and mouse pancreatic islet cells using a bicistronic recombinant adeno-associated viral vector. Mol Ther 2002; 5: 154–160.

    Article  CAS  PubMed  Google Scholar 

  31. Fang J, Qian JJ, Yi S, Harding TC, Tu GH, VanRoey M et al. Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol 2005; 23: 584–590.

    Article  CAS  PubMed  Google Scholar 

  32. Marasco WA . Therapeutic antibody gene transfer. Nat Biotechnol 2005; 23: 551–552.

    Article  CAS  PubMed  Google Scholar 

  33. Lipshutz GS, Titre D, Brindle M, Bisconte AR, Contag CH, Gaensler KM . Comparison of gene expression after intraperitoneal delivery of AAV2 or AAV5 in utero. Mol Ther 2003; 8: 90–98.

    Article  CAS  PubMed  Google Scholar 

  34. Xu Z, Yue Y, Lai Y, Ye C, Qiu J, Pintel DJ et al. Trans-splicing adeno-associated viral vector-mediated gene therapy is limited by the accumulation of spliced mRNA but not by dual vector coinfection efficiency. Hum Gene Ther 2004; 15: 896–905.

    Article  CAS  PubMed  Google Scholar 

  35. Reich SJ, Auricchio A, Hildinger M, Glover E, Maguire AM, Wilson JM et al. Efficient trans-splicing in the retina expands the utility of adeno-associated virus as a vector for gene therapy. Hum Gene Ther 2003; 14: 37–44.

    Article  CAS  PubMed  Google Scholar 

  36. Liu X, Luo M, Zhang LN, Yan Z, Zak R, Ding W et al. Spliceosome-mediated RNA trans-splicing with recombinant adeno-associated virus partially restores cystic fibrosis transmembrane conductance regulator function to polarized human cystic fibrosis airway epithelial cells. Hum Gene Ther 2005; 16: 1116–1123.

    Article  CAS  PubMed  Google Scholar 

  37. Lai Y, Yue Y, Liu M, Ghosh A, Engelhardt JF, Chamberlain JS et al. Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors. Nat Biotechnol 2005; 23: 1435–1439.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Yan Z, Zak R, Zhang Y, Engelhardt JF . Inverted terminal repeat sequences are important for intermolecular recombination and circularization of adeno-associated virus genomes. J Virol 2005; 79: 364–379.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yen L, Svendsen J, Lee JS, Gray JT, Magnier M, Baba T et al. Exogenous control of mammalian gene expression through modulation of RNA self-cleavage. Nature 2004; 431: 471–476.

    Article  CAS  PubMed  Google Scholar 

  40. Tomar RS, Matta H, Chaudhary PM . Use of adeno-associated viral vector for delivery of small interfering RNA. Oncogene 2003; 22: 5712–5715.

    Article  CAS  PubMed  Google Scholar 

  41. Pinkenburg O, Platz J, Beisswenger C, Vogelmeier C, Bals R . Inhibition of NF-kappaB mediated inflammation by siRNA expressed by recombinant adeno-associated virus. J Virol Methods 2004; 120: 119–122.

    Article  CAS  PubMed  Google Scholar 

  42. Zhang W, Singam R, Hellermann G, Kong X, Juan HS, Lockey RF et al. Attenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery. Genet Vaccines Ther 2004; 2: 8.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Babcock AM, Standing D, Bullshields K, Schwartz E, Paden CM, Poulsen DJ . In vivo inhibition of hippocampal Ca2+/calmodulin-dependent protein kinase II by RNA interference. Mol Ther 2005; 11: 899–905.

    Article  CAS  PubMed  Google Scholar 

  44. Xia H, Mao Q, Eliason SL, Harper SQ, Martins IH, Orr HT et al. RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat Med 2004; 10: 816–820.

    Article  CAS  PubMed  Google Scholar 

  45. Goyenvalle A, Vulin A, Fougerousse F, Leturcq F, Kaplan JC, Garcia L et al. Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science 2004; 306: 1796–1799.

    Article  CAS  PubMed  Google Scholar 

  46. Yu JY, Taylor J, DeRuiter SL, Vojtek AB, Turner DL . Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors. Mol Ther 2003; 7: 228–236.

    Article  CAS  PubMed  Google Scholar 

  47. McCarty DM, Fu H, Monahan PE, Toulson CE, Naik P, Samulski RJ . Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Therapy 2003; 10: 2112–2118.

    Article  CAS  PubMed  Google Scholar 

  48. Wang Z, Ma HI, Li J, Sun L, Zhang J, Xiao X . Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo. Gene Therapy 2003; 10: 2105–2111.

    Article  CAS  PubMed  Google Scholar 

  49. Ren C, Kumar S, Shaw DR, Ponnazhagan S . Genomic stability of self-complementary adeno-associated virus 2 during early stages of transduction in mouse muscle in vivo. Hum Gene Ther 2005; 16: 1047–1057.

    Article  CAS  PubMed  Google Scholar 

  50. Xu D, McCarty D, Fernandes A, Fisher M, Samulski RJ, Juliano RL . Delivery of MDR1 small interfering RNA by self-complementary recombinant adeno-associated virus vector. Mol Ther 2005; 11: 523–530.

    Article  PubMed  Google Scholar 

  51. Nakai H, Wu X, Fuess S, Storm TA, Munroe D, Montini E et al. Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver. J Virol 2005; 79: 3606–3614.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Schnepp BC, Clark KR, Klemanski DL, Pacak CA, Johnson PR . Genetic fate of recombinant adeno-associated virus vector genomes in muscle. J Virol 2003; 77: 3495–3504.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Miller DG, Petek LM, Russell DW . Adeno-associated virus vectors integrate at chromosome breakage sites. Nat Genet 2004; 36: 767–773.

    Article  CAS  PubMed  Google Scholar 

  54. Miller DG, Trobridge GD, Petek LM, Jacobs MA, Kaul R, Russell DW . Large-scale analysis of adeno-associated virus vector integration sites in normal human cells. J Virol 2005; 79: 11434–11442.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. McCarty DM, Young Jr SM, Samulski RJ . Integration of adeno-associated virus (AAV) and recombinant AAV vectors. Annu Rev Genet 2004; 38: 819–845.

    Article  CAS  PubMed  Google Scholar 

  56. Huttner NA, Girod A, Schnittger S, Schoch C, Hallek M, Buning H . Analysis of site-specific transgene integration following cotransduction with recombinant adeno-associated virus and a rep encodingplasmid. J Gene Med 2003; 5: 120–129.

    Article  CAS  PubMed  Google Scholar 

  57. Goncalves MA . Adeno-associated virus: from defective virus to effective vector. Virol J 2005; 2: 43.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Recchia A, Perani L, Sartori D, Olgiati C, Mavilio F . Site-specific integration of functional transgenes into the human genome by adeno/AAV hybrid vectors. Mol Ther 2004; 10: 660–670.

    Article  CAS  PubMed  Google Scholar 

  59. Hendrie PC, Russell DW . Gene targeting with viral vectors. Mol Ther 2005; 12: 9–17.

    Article  CAS  PubMed  Google Scholar 

  60. Kohli M, Rago C, Lengauer C, Kinzler KW, Vogelstein B . Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses. Nucleic Acids Res 2004; 32: e3.

    Article  PubMed  PubMed Central  Google Scholar 

  61. Miller DG, Petek LM, Russell DW . Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks. Mol Cell Biol 2003; 23: 3550–3557.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Chamberlain JR, Schwarze U, Wang PR, Hirata RK, Hankenson KD, Pace JM et al. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 2004; 303: 1198–1201.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We wish to thank Susan Cure for final reading of the manuscript.

Author information

Authors and Affiliations

  1. CNRS UMR 8115, Généthon, Evry, France

    C Le Bec & A M Douar

Authors
  1. C Le Bec
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A M Douar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A M Douar.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Le Bec, C., Douar, A. Gene Therapy Progress and Prospects – Vectorology: design and production of expression cassettes in AAV vectors. Gene Ther 13, 805–813 (2006). https://doi.org/10.1038/sj.gt.3302724

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3302724

Keywords

This article is cited by

Search

Advanced search

Quick links