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.

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

“…Moreover, position 587 is embedded in the natural HSPG receptor binding motif 32,33 . The presence of positively charged residues as those of the His-tag at this position can reconstitute HSPG binding and natural receptor usage for cell entry 34,35 . Owing to these potential risks and the absence of a functional proof that a His-tag in position 587 indeed efficiently depletes DARPin-deficient particles 30 , we decided to place the His-tag N-terminally of the DARPin, thus avoiding both of the above mentioned risks.…”

The N-Terminal Domain of NLRC5 Confers Transcriptional Activity for MHC Class I and II Gene Expression

“…Forty-eight hours posttransfection, cells were harvested, lysed, and purified by iodixanol density step gradient centrifugation (27). Genomic particle titers were determined by quantitative PCR (qPCR) (LightCycler System; Roche Diagnostics, Mannheim, Germany) using transgene specific primers (28).…”

Structure and Dynamics of Adeno-Associated Virus Serotype 1 VP1-Unique N-Terminal Domain and Its Role in Capsid Trafficking

“…This is followed by interactions with membrane-bound coreceptors, for example, fibroblast growth factor receptor and ␣V␤5 and ␣5␤1 integrin receptors for AAV2 (36)(37)(38), platelet-derived growth factor receptor for AAV5 (39), and the 37-/67-kDa laminin receptor for AAV8 (40). Following attachment, the endocytosis of the capsid has been reported to occur by means of clathrin-coated pits (41), though recent studies have described a clathrin-independent mechanism (42). After endocytosis, the virus traffics through the endocytic pathway and accumulates in the perinuclear region (43) before delivering its infective genome to the nucleus.…”