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.

  • Article
  • Published:

Polarized AAVR expression determines infectivity by AAV gene therapy vectors

Gene Therapy volume 26, pages 240–249 (2019)Cite this article

Subjects

Abstract

Adeno-associated virus (AAV) has been investigated to transfer the cystic fibrosis transmembrane conductance regulator (CFTR) to airways. Inhaled AAV2-CFTR in people with cystic fibrosis (CF) is safe, but inefficient. In vitro, AAV2 transduction of human airway epithelia on the apical (luminal) side is inefficient, but efficient basolaterally. We previously selected AAV2.5T, a novel capsid that apically transduces CF human airway epithelia and efficiently restores CFTR function. We hypothesize the AAV receptor (AAVR) is basolaterally localized, and that AAV2.5T utilizes an alternative apical receptor. We found AAVR in human airway epithelia by western blot and RNA-Seq analyses. Using immunocytochemistry we did not find endogenous AAVR at membranes but overexpression localized AAVR to the basolateral membrane, where it preferentially increased transduction. Anti-AAVR antibodies blocked transduction by AAV2 from the basolateral side but not AAV2.5T from the apical side, suggesting a unique apical receptor. Finally, we found infection by AAV2 but not AAV2.5T was blocked by CRISPR knockout of AAVR in cell lines. Our data suggest the absence of apical AAVR is rate limiting for AAV2, and efficient transduction by AAV2.5T is accomplished using an AAVR independent pathway. Our findings inform the development of gene therapy for CF, and AAV vectors in general.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Griesenbach U, Alton EW. Moving forward: cystic fibrosis gene therapy. Hum Mol Genet. 2013;22(R1):R52–58.

    Article  CAS  PubMed  Google Scholar 

  2. Griesenbach U, Alton EW. Progress in gene and cell therapy for cystic fibrosis lung disease. Curr Pharma Design. 2012;18:642–62.

    Article  CAS  Google Scholar 

  3. Welsh MJ. Gene transfer for cystic fibrosis. J Clin Invest. 1999;104:1165–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Steines B, Dickey DD, Bergen J, Excoffon KJ, Weinstein JR, Li X et al. CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes. JCI Insight. 2016;1:e88728.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Excoffon KJ, Koerber JT, Dickey DD, Murtha M, Keshavjee S, Kaspar BK et al. Directed evolution of adeno-associated virus to an infectious respiratory virus. Proc Natl Acad Sci USA. 2009;106:3865–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Yan Z, Sun X, Feng Z, Li G, Fisher JT, Stewart ZA et al. Optimization of recombinant adeno-associated virus-mediated expression for large transgenes, using a synthetic promoter and tandem array enhancers. Hum Gene Ther. 2015;26:334–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Flotte TR, Afione SA, Zeitlin PL. Adeno-associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration. Am J Resp Cell Mol Biol. 1994;11:517–21.

    Article  CAS  Google Scholar 

  8. Moss RB, Milla C, Colombo J, Accurso F, Zeitlin PL, Clancy JP et al. Repeated aerosolized AAV-CFTR for treatment of cystic fibrosis: a randomized placebo-controlled phase 2B trial. Hum Gene Ther. 2007;18:726–32.

    Article  CAS  PubMed  Google Scholar 

  9. Moss RB, Rodman D, Spencer LT, Aitken ML, Zeitlin PL, Waltz D et al. 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. 2004;125:509–21.

    Article  PubMed  Google Scholar 

  10. Aitken ML, Moss RB, Waltz DA, Dovey ME, Tonelli MR, McNamara SC et al. A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease. Hum Gene Ther. 2001;12:1907–16.

    Article  CAS  PubMed  Google Scholar 

  11. 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;7:842–50.

    Article  CAS  PubMed  Google Scholar 

  12. Guggino WB, Cebotaru L. Adeno-Associated Virus (AAV) gene therapy for cystic fibrosis: current barriers and recent developments. Exp Opin Biol Ther. 2017;17:1265–73.

    Article  CAS  Google Scholar 

  13. Sanders N, Rudolph C, Braeckmans K, De Smedt SC, Demeester J. Extracellular barriers in respiratory gene therapy. Adv Drug Deliv Rev. 2009;61:115–27.

    Article  CAS  PubMed  Google Scholar 

  14. Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther. 2008;16:1073–80.

    Article  CAS  PubMed  Google Scholar 

  15. Mizukami H, Young NS, Brown KE. Adeno-associated virus type 2 binds to a 150-kilodalton cell membrane glycoprotein. Virology. 1996;217:124–30.

    Article  CAS  PubMed  Google Scholar 

  16. Kashiwakura Y, Tamayose K, Iwabuchi K, Hirai Y, Shimada T, Matsumoto K et al. Hepatocyte growth factor receptor is a coreceptor for adeno-associated virus type 2 infection. J Virol. 2005;79:609–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Qing K, Mah C, Hansen J, Zhou S, Dwarki V, Srivastava A. Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2. Nat Med. 1999;5:71–7.

    Article  CAS  PubMed  Google Scholar 

  18. Summerford C, Bartlett JS, Samulski RJ. AlphaVbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection. Nat Med. 1999;5:78–82.

    Article  CAS  PubMed  Google Scholar 

  19. Summerford C, Samulski RJ. Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J Virol. 1998;72:1438–45.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Seiler MP, Miller AD, Zabner J, Halbert CL. Adeno-associated virus types 5 and 6 use distinct receptors for cell entry. Hum Gene Ther. 2006;17:10–19.

    Article  CAS  PubMed  Google Scholar 

  21. Wu Z, Miller E, Agbandje-McKenna M, Samulski RJ. α2,3 and α2,6 N-linked sialic acids facilitate efficient binding and transduction by adeno-associated virus types 1 and 6. J Virol. 2006;80:9093–103.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Walters RW, Yi SM, Keshavjee S, Brown KE, Welsh MJ, Chiorini JA et al. Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer. J Biol Chem. 2001;276:20610–6.

    Article  CAS  PubMed  Google Scholar 

  23. Kaludov N, Brown KE, Walters RW, Zabner J, Chiorini JA. Adeno-associated virus serotype 4 (AAV4) and AAV5 both require sialic acid binding for hemagglutination and efficient transduction but differ in sialic acid linkage specificity. J Virol. 2001;75:6884–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bell CL, Vandenberghe LH, Bell P, Limberis MP, Gao GP, Van Vliet K et al. The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice. J Clin Invest. 2011;121:2427–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Pillay S, Meyer NL, Puschnik AS, Davulcu O, Diep J, Ishikawa Y et al. An essential receptor for adeno-associated virus infection. Nature. 2016;530:108–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Pillay S, Zou W, Cheng F, Puschnik AS, Meyer NL, Ganaie SS et al. AAV serotypes have distinctive interactions with domains of the cellular receptor AAVR. J Virol. 2017;91:00391–17. 

  27. Poon MW, Tsang WH, Waye MM, Chan SO. Distribution of Kiaa0319-like immunoreactivity in the adult mouse brain--a novel protein encoded by the putative dyslexia susceptibility gene KIAA0319-like. Histol Histopathol. 2011;26:953–63.

    CAS  PubMed  Google Scholar 

  28. Platt MP, Adler WT, Mehlhorn AJ, Johnson GC, Wright KA, Choi RT et al. Embryonic disruption of the candidate dyslexia susceptibility gene homolog Kiaa0319-like results in neuronal migration disorders. Neuroscience. 2013;248:585–93.

    Article  CAS  PubMed  Google Scholar 

  29. Bhella D. The role of cellular adhesion molecules in virus attachment and entry. Philos Trans R Soc Lond B Biol Sci. 2015;370:20140035.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Karp PH, Moninger TO, Weber SP, Nesselhauf TS, Launspach JL, Zabner J et al. An in vitro model of differentiated human airway epithelia. Methods for establishing primary cultures. Methods Mol Biol (Clifton, N.J.). 2002;188:115–37.

    Google Scholar 

  31. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protocol. 2013;8:2281–308.

    Article  CAS  Google Scholar 

  32. Flotte TR, Solow R, Owens RA, Afione S, Zeitlin PL, Carter BJ. Gene expression from adeno-associated virus vectors in airway epithelial cells. Am J Resp Cell Mol Biol. 1992;7:349–56.

    Article  CAS  Google Scholar 

  33. Griesenbach U, Alton EW. Current status and future directions of gene and cell therapy for cystic fibrosis. BioDrugs. 2011;25:77–88.

    Article  CAS  PubMed  Google Scholar 

  34. Zabner J, Seiler M, Walters R, Kotin RM, Fulgeras W, Davidson BL et al. Adeno-associated virus type 5 (AAV5) but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer. J Virol. 2000;74:3852–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Walters RW, Duan D, Engelhardt JF, Welsh MJ. Incorporation of adeno-associated virus in a calcium phosphate coprecipitate improves gene transfer to airway epithelia in vitro and in vivo. J Virol. 2000;74:535–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Uhlén MFL, Hallström BM, Lindskog C, Oksvold P, Mardinoglu A, Sivertsson Å et al. The human protein atlas. Mol Cell Proteomics. 2005;4:1920–32.

    Article  PubMed  Google Scholar 

  37. Pezzulo AA, Tudas RA, Stewart CG, Buonfiglio LGV, Lindsay BD, Taft PJ et al. HSP90 inhibitor geldanamycin reverts IL-13- and IL-17-induced airway goblet cell metaplasia. J Clin Invest. 2019;129:744-758.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Duan D, Yue Y, Yan Z, McCray PB Jr., Engelhardt JF. Polarity influences the efficiency of recombinant adenoassociated virus infection in differentiated airway epithelia. Hum Gene Ther. 1998;9:2761–76.

    Article  CAS  PubMed  Google Scholar 

  39. Duan D, Yue Y, Yan Z, Yang J, Engelhardt JF. Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus. J Clin Invest. 2000;105:1573–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Boyle MP, Enke RA, Reynolds JB, Mogayzel PJ Jr., Guggino WB, Zeitlin PL. Membrane-associated heparan sulfate is not required for rAAV-2 infection of human respiratory epithelia. Virol J. 2006;3:29.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Walters RW, Grunst T, Bergelson JM, Finberg RW, Welsh MJ, Zabner J. Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia. J Biol Chem. 1999;274:10219–26.

    Article  CAS  PubMed  Google Scholar 

  42. Dudek AM, Pillay S, Puschnik AS, Nagamine CM, Cheng F, Qiu J et al. An alternate route for adeno-associated virus entry independent of AAVR. J Virol. 2018;92:1–15.

  43. Lee H, Lotery A. Gene therapy for RPE65-mediated inherited retinal dystrophy completes phase 3. Lancet (London, England). 2017;390:823–4.

    Article  Google Scholar 

  44. Carroll J. FDA experts offer a unanimous endorsement for pioneering gene therapy for blindness. Science Magazine: Science; 13 October, 2017.

  45. Qing K, Bachelot T, Mukherjee P, Wang XS, Peng L, Yoder MC et al. Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells. J Virol. 1997;71:5663–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Wickham TJ, Roelvink PW, Brough DE, Kovesdi I. Adenovirus targeted to heparan-containing receptors increases its gene delivery efficiency to multiple cell types. Nat Biotechnol. 1996;14:1570–3.

    Article  CAS  PubMed  Google Scholar 

  47. Bartlett JS, Kleinschmidt J, Boucher RC, Samulski RJ. Targeted adeno-associated virus vector transduction of nonpermissive cells mediated by a bispecific F(ab’gamma)2 antibody. Nat Biotechnol. 1999;17:181–6.

    Article  CAS  PubMed  Google Scholar 

  48. Dickey DD, Excoffon KJ, Koerber JT, Bergen J, Steines B, Klesney-Tait J et al. Enhanced sialic acid-dependent endocytosis explains the increased efficiency of infection of airway epithelia by a novel adeno-associated virus. J Virol. 2011;85:9023–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the NIH (H678200-G) and the University of Iowa Center for Gene Therapy (DK054759).

Author information

Authors and Affiliations

  1. Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, Pappajohn Biomedical Institute, The University of Iowa, Iowa City, IA, USA

    Bradley A. Hamilton, Xiaopeng Li, Alejandro A. Pezzulo, Mahmoud H. Abou Alaiwa & Joseph Zabner

  2. Molecular Medicine Program, The University of Iowa, Iowa City, IA, USA

    Bradley A. Hamilton & Joseph Zabner

  3. Department of Biomedical Engineering, The University of Iowa, Iowa City, IA, USA

    Mahmoud H. Abou Alaiwa

Authors
  1. Bradley A. Hamilton
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaopeng Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alejandro A. Pezzulo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mahmoud H. Abou Alaiwa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joseph Zabner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joseph Zabner.

Ethics declarations

Conflict of interest

Dr. Zabner is a founder and holds equity in TaleeBio. The other authors declare that they have no conflict of interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hamilton, B.A., Li, X., Pezzulo, A.A. et al. Polarized AAVR expression determines infectivity by AAV gene therapy vectors. Gene Ther 26, 240–249 (2019). https://doi.org/10.1038/s41434-019-0078-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41434-019-0078-3

This article is cited by

Search

Advanced search

Quick links