Endocytic processing of adeno-associated virus type 8 vectors for transduction of target cells

Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA, USA.
Gene therapy (Impact Factor: 3.1). 05/2012; 20(3). DOI: 10.1038/gt.2012.41
Source: PubMed


We investigated the transduction of HEK293T cells permissive to adeno-associated virus serotype 8 (AAV8) to understand the mechanisms underlying its endocytic processing. Results showed that AAV8 enters cells through clathrin-mediated endocytosis followed by trafficking through various endosomal compartments. Interestingly, compared to the relatively well-characterized AAV2, a distinct involvement of late endosomes was observed for AAV8 trafficking within the target cell. AAV8 particles were also shown to exploit the cytoskeleton network to facilitate their transport within cells. Moreover, the cellular factors involved during endosomal escape were examined by an in vitro membrane permeabilization assay. Our data demonstrated that an acidic endosomal environment was required for AAV2 penetration through endosomal membranes and that the cellular endoprotease furin could promote AAV2 escape from the early endosomes. In contrast, these factors were not sufficient for AAV8 penetration through endosomal membranes. We further found that the ubiquitin-proteasome system is likely involved in the intracellular transport of AAV8 to nucleus. Taken together, our data have shed some light on the intracellular trafficking pathways of AAV8, which, in turn, could provide insight for potentializing AAV-mediated gene delivery.Gene Therapy advance online publication, 24 May 2012; doi:10.1038/gt.2012.41.

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Available from: Yarong Liu, Sep 30, 2015
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    • "Highly purified stocks of rAAV vectors were generated by the triple-plasmid transfection protocol as previously described (Liu et al., 2012), with modification. Briefly, HEK293 cells were co-transfected with three plasmids using polyethelenimine (linear, MW 25,000; Polysciences, Inc.), and the medium was replaced 6 hr posttransfection. "
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    • "The capsids injected here do not recapitulate some of the conditions encountered during normal cell entry, such as the low pH exposure of receptor-bound capsids, but do show some processes that determine the virus distribution within the cytoplasm. The limited movement of the capsids observed here is likely not a specific barrier to cell infection, as after endosomal transport the incoming particles end up very close to the nucleus (Bantel-Schaal et al., 2009; Harbison et al., 2009; Liu et al., 2013; Mani et al., 2006; Suikkanen et al., 2002), from which position they should readily reach the nuclear pore. This would therefore differ from the infection pathways required for viruses such as herpesvirus and HIV which enter through the plasma membrane and hence need to traverse the entire cytoplasm. "
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    • "The clathrin-mediated endocytic route has been considered as the most common endocytic pathway taken by viruses, such as adeno-associated viruses, vesicular stomatis virus or influenza A viruses [46], [47], [48], [49]. Moreover, alphaviruses, and, in particular, Sindbis viruses and Semliki Forest Virus (SFV) have been known to enter cells through clathrin-mediated endocytosis [47]. "
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