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.

Successful target cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent endocytosis

Abstract

Cell surface targeting of recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to modify AAV's natural tropism. As modification of the capsid surface is likely to affect the mechanism of vector internalization and consequently the vector's intracellular fate, we investigated early steps in cell transduction of rAAV capsid insertion mutants. Mutants displaying peptides with neutral overall charge at position 587 transduced cells independently of AAV2's primary receptor heparan sulfate proteoglycan (HSPG), whereas mutants carrying positively charged insertions were capable of HSPG binding with affinities correlating with their net positive charge. Whereas rAAV2 is internalized via an HSPG- and clathrin-dependent pathway, HSPG-binding mutants used a clathrin- and caveolin-independent mechanism. Surprisingly, although this pathway was as efficient in mediating vector entry as the one used by rAAV2, successful cell transduction was hampered at a post-entry step, presumably caused by inefficient endosomal escape. In contrast, HSPG-independent, clathrin-dependent internalization used by non-HSPG-binding mutants correlated with efficient nuclear delivery of vector genomes and robust transgene expression. These findings indicate that cell surface targeting strategies should direct uptake of rAAV targeting vectors to clathrin-mediated endocytosis, the naturally evolved entry route of AAV, to promote successful intracellular processing and re-targeting of rAAV's tropism.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

References

  1. Büning H, Perabo L, Coutelle O, Quadt-Humme S, Hallek M . Recent developments in adeno-associated virus vector technology. J Gene Med 2008; 10: 717–733.

    Article  PubMed  Google Scholar 

  2. Pien GC, Basner-Tschakarjan E, Hui DJ, Mentlik AN, Finn JD, Hasbrouck NC et al. Capsid antigen presentation flags human hepatocytes for destruction after transduction by adeno-associated viral vectors. J Clin Invest 2009; 119: 1688–1695.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Kwon I, Schaffer DV . Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer. Pharm Res 2007; 25: 489–499.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Wu Z, Asokan A, Samulski RJ . Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol Ther 2006; 14: 316–327.

    CAS  Article  PubMed  Google Scholar 

  5. Summerford C, Samulski RJ . Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J Virol 1998; 72: 1438–1445.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Kern A, Schmidt K, Leder C, Muller OJ, Wobus CE, Bettinger K et al. Identification of a heparin-binding motif on adeno-associated virus type 2 capsids. J Virol 2003; 77: 11072–11081.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Opie SR, Warrington Jr KH, Agbandje-McKenna M, Zolotukhin S, Muzyczka N . Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding. J Virol 2003; 77: 6995–7006.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Boucas J, Lux K, Huber A, Schievenbusch S, von Freyend MJ, Perabo L et al. Engineering adeno-associated virus serotype 2-based targeting vectors using a new insertion site-position 453-and single point mutations. J Gene Med 2009; 11: 1103–1113.

    CAS  Article  PubMed  Google Scholar 

  9. Asokan A, Hamra JB, Govindasamy L, Agbandje-McKenna M, Samulski RJ . Adeno-associated virus type 2 contains an integrin alpha5beta1 binding domain essential for viral cell entry. J Virol 2006; 80: 8961–8969.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Sanlioglu S, Benson PK, Yang J, Atkinson EM, Reynolds T, Engelhardt JF . Endocytosis and nuclear trafficking of adeno-associated virus type 2 are controlled by rac1 and phosphatidylinositol-3 kinase activation. J Virol 2000; 74: 9184–9196.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  12. Perabo L, Goldnau D, White K, Endell J, Boucas J, Humme S et al. Heparan sulfate proteoglycan binding properties of adeno-associated virus retargeting mutants and consequences for their in vivo tropism. J Virol 2006; 80: 7265–7269.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Shi W, Bartlett JS . RGD inclusion in VP3 provides adeno-associated virus type 2 (AAV2)-based vectors with a heparan sulfate-independent cell entry mechanism. Mol Ther 2003; 7: 515–525.

    CAS  Article  PubMed  Google Scholar 

  14. Marsh M, Helenius A . Virus entry: open sesame. Cell 2006; 124: 729–740.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Wang LH, Rothberg KG, Anderson RG . Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J Cell Biol 1993; 123: 1107–1117.

    CAS  Article  PubMed  Google Scholar 

  16. Duan D, Li Q, Kao AW, Yue Y, Pessin JE, Engelhardt JF . Dynamin is required for recombinant adeno-associated virus type 2 infection. J Virol 1999; 73: 10371–10376.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Pellinen T, Ivaska J . Integrin traffic. J Cell Sci 2006; 119 (Part 18): 3723–3731.

    CAS  Article  PubMed  Google Scholar 

  18. Payne CK, Jones SA, Chen C, Zhuang X . Internalization and trafficking of cell surface proteoglycans and proteoglycan-binding ligands. Traffic 2007; 8: 389–401.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Van Hamme E, Dewerchin HL, Cornelissen E, Verhasselt B, Nauwynck HJ . Clathrin- and caveolae-independent entry of feline infectious peritonitis virus in monocytes depends on dynamin. J Gen Virol 2008; 89 (Part 9): 2147–2156.

    CAS  Article  PubMed  Google Scholar 

  20. Bantel-Schaal U, Braspenning-Wesch I, Kartenbeck J . Adeno-associated virus type 5 exploits two different entry pathways in human embryo fibroblasts. J Gen Virol 2009; 90 (Part 2): 317–322.

    CAS  Article  PubMed  Google Scholar 

  21. Shayakhmetov DM, Eberly AM, Li ZY, Lieber A . Deletion of penton RGD motifs affects the efficiency of both the internalization and the endosome escape of viral particles containing adenovirus serotype 5 or 35 fiber knobs. J Virol 2005; 79: 1053–1061.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Denby L, Nicklin SA, Baker AH . Adeno-associated virus (AAV)-7 and -8 poorly transduce vascular endothelial cells and are sensitive to proteasomal degradation. Gene Therapy 2005; 12: 1534–1538.

    CAS  Article  PubMed  Google Scholar 

  23. Douar AM, Poulard K, Stockholm D, Danos O . Intracellular trafficking of adeno-associated virus vectors: routing to the late endosomal compartment and proteasome degradation. J Virol 2001; 75: 1824–1833.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 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: 1573–1587.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Kopatz I, Remy JS, Behr JP . A model for non-viral gene delivery: through syndecan adhesion molecules and powered by actin. J Gene Med 2004; 6: 769–776.

    CAS  Article  PubMed  Google Scholar 

  26. Ogris M, Wagner E . Targeting tumors with non-viral gene delivery systems. Drug Discov Today 2002; 7: 479–485.

    CAS  Article  PubMed  Google Scholar 

  27. Multhaupt HA, Yoneda A, Whiteford JR, Oh ES, Lee W, Couchman JR . Syndecan signaling: when, where and why? J Physiol Pharmacol 2009; 60 (Suppl 4): 31–38.

    PubMed  Google Scholar 

  28. Liang M, Morizono K, Pariente N, Kamata M, Lee B, Chen IS . Targeted transduction via CD4 by a lentiviral vector uses a clathrin-mediated entry pathway. J Virol 2009; 83: 13026–13031.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Mudhakir D, Akita H, Tan E, Harashima H . A novel IRQ ligand-modified nano-carrier targeted to a unique pathway of caveolar endocytic pathway. J Control Rel 2008; 125: 164–173.

    CAS  Article  Google Scholar 

  30. Dmitriev I, Krasnykh V, Miller CR, Wang M, Kashentseva E, Mikheeva G et al. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol 1998; 72: 9706–9713.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Kircheis R, Kichler A, Wallner G, Kursa M, Ogris M, Felzmann T et al. Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery. Gene Therapy 1997; 4: 409–418.

    CAS  Article  PubMed  Google Scholar 

  32. Kreppel F, Gackowski J, Schmidt E, Kochanek S . Combined genetic and chemical capsid modifications enable flexible and efficient de- and retargeting of adenovirus vectors. Mol Ther 2005; 12: 107–117.

    CAS  Article  PubMed  Google Scholar 

  33. Hall A . Rho GTPases and the control of cell behaviour. Biochem Soc Trans 2005; 33 (Part 5): 891–895.

    CAS  Article  PubMed  Google Scholar 

  34. Kronenberg S, Bottcher B, von der Lieth CW, Bleker S, Kleinschmidt JA . A conformational change in the adeno-associated virus type 2 capsid leads to the exposure of hidden VP1 N termini. J Virol 2005; 79: 5296–5303.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Sonntag F, Bleker S, Leuchs B, Fischer R, Kleinschmidt JA . Adeno-associated virus type 2 capsids with externalized VP1/VP2 trafficking domains are generated prior to passage through the cytoplasm and are maintained until uncoating occurs in the nucleus. J Virol 2006; 80: 11040–11054.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Stahnke S, Lux K, Uhrig S, Kreppel F, Hosel M, Coutelle O et al. Intrinsic phospholipase A2 activity of adeno-associated virus is involved in endosomal escape of incoming particles. Virology 2010; 409: 77–83.

    Article  PubMed  Google Scholar 

  37. Ding W, Zhang L, Yan Z, Engelhardt JF . Intracellular trafficking of adeno-associated viral vectors. Gene Therapy 2005; 12: 873–880.

    CAS  Article  PubMed  Google Scholar 

  38. Shayakhmetov DM, Li ZY, Ternovoi V, Gaggar H, Gharwan H, Lieber A . The interaction between the fiber knob domain and the cellular attachment receptor determines the intracellular trafficking route of adenoviruses. J Virol 2003; 77: 3712–3723.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Perabo L, Büning H, Kofler DM, Ried MU, Girod A, Wendtner CM et al. In vitro selection of viral vectors with modified tropism: the adeno-associated virus display. Mol Ther 2003; 8: 151–157.

    CAS  Article  PubMed  Google Scholar 

  40. Nicklin SA, Buening H, Dishart KL, de Alwis M, Girod A, Hacker U et al. Efficient and selective AAV2-mediated gene transfer directed to human vascular endothelial cells. Mol Ther 2001; 4: 174–181.

    CAS  Article  PubMed  Google Scholar 

  41. Xiao X, Li J, Samulski RJ . Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 1998; 72: 2224–2232.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Hacker UT, Gerner FM, Büning H, Hutter M, Reichenspurner H, Stangl M et al. Standard heparin, low molecular weight heparin, low molecular weight heparinoid, and recombinant hirudin differ in their ability to inhibit transduction by recombinant adeno-associated virus type 2 vectors. Gene Therapy 2001; 8: 966–968.

    CAS  Article  PubMed  Google Scholar 

  43. Girod A, Ried M, Wobus C, Lahm H, Leike K, Kleinschmidt J et al. Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2. Nat Med 1999; 5: 1052–1056.

    CAS  Article  PubMed  Google Scholar 

  44. Theiss HD, Kofler DM, Büning H, Aldenhoff AL, Kaess B, Decker T et al. Enhancement of gene transfer with recombinant adeno-associated virus (rAAV) vectors into primary B-cell chronic lymphocytic leukemia cells by CpG-oligodeoxynucleotides. Exp Hematol 2003; 31: 1223–1229.

    CAS  Article  PubMed  Google Scholar 

  45. Mizukami H, Young NS, Brown KE . Adeno-associated virus type 2 binds to a 150-kilodalton cell membrane glycoprotein. Virology 1996; 217: 124–130.

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by Deutsche Forschungsgemeinschaft (SPP1230) (HB, MH) and the Center for Molecular Medicine Cologne (ZMMK) (HB). Furthermore, we thank Richard Jude Samulski (University of North Carolina at Chapel Hill, Chapel Hill, NC, USA) and Jürgen Kleinschmidt (DKFZ, Heidelberg, Germany) for kindly providing pXX6 and the anti-capsid antibody A20, respectively, Hanna Janicki for excellent technical assistance and Patrick Schmidt for kind assistance in flow cytometry and microscopy.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to H Büning.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on Gene Therapy website

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Uhrig, S., Coutelle, O., Wiehe, T. et al. Successful target cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent endocytosis. Gene Ther 19, 210–218 (2012). https://doi.org/10.1038/gt.2011.78

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2011.78

Keywords

  • AAV cell surface targeting
  • internalization pathway
  • intracellular fate

Further reading

Search

Quick links