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.
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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.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on Gene Therapy website
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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
- AAV cell surface targeting
- internalization pathway
- intracellular fate
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