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.

Download full-text


Available from: Yarong Liu

Click to see the full-text of:

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

728.29 KB

See full-text
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Current challenges for recombinant adeno-associated virus (rAAV) vector-based cancer treatment include the low-efficiency and the lack of specificity in vivo. rAAV serotype 3 (rAAV3) vectors have previously been shown to be ineffective in normal mouse tissues following systemic administration. In the present study, we report that rAAV3 vectors can efficiently target and transduce various human liver cancer cells in vivo. Elimination of specific surface-exposed serine and threonine residues on rAAV3 capsids results in further augmentation in the transduction efficiency of these vectors, without any change in the viral tropism and cellular receptor interactions. In addition, we have identified a potential chemotherapy drug, shikonin, as a multifunctional compound to inhibit liver tumor growth as well as to significantly enhance the efficacy of rAAV vector-based gene therapy in vivo. Furthermore, we also document that suppression of tumorigenesis in a human liver cancer xenograft model can be achieved through systemic administration of the optimized rAAV3 vectors carrying a therapeutic gene, and shikonin at a dose that does not lead to severe liver damage. Our research provides a novel means to achieve not only targeted delivery, but also the potential for gene therapy of human liver cancer.
    Full-text · Article · Oct 2014 · Human Gene Therapy
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T-antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes.
    Preview · Article · May 2014 · Virology
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Dendritic cells (DCs) are potent antigen-presenting cells and therefore have enormous potential as vaccine targets. We have previously developed an engineered lentiviral vector (LV) that is pseudotyped with a mutated Sindbis virus glycoprotein (SVGmu), which is capable of targeting DCs through Dendritic Cell-specific ICAM3-grabbing Nonintegrin (DC-SIGN), a receptor that is predominantly expressed by DCs. In this study, we aimed to elucidate the internalization and trafficking mechanisms of this viral vector system through direct visualization of GFP-Vpr-tagged viral particles in target DCs, which was further corroborated by drug inhibition and dominant-negative mutants of cellular proteins that regulate the endocytic traffic. We demonstrated that our engineered LVs enter the cell via receptor-mediated clathrin- and dynamin-dependent endocytosis. Microtubule networks were also involved in a productive infection. Viral vector fusion was low-pH-dependent and occurred in the early endosomal stage of the intracellular transport. Autophagy was also examined for its effect on transduction efficiency, and we observed that enhanced autophage activity reduced vector infectivity, while suppressed autophagy boosted transduction efficiency. This study shed some light on the internalization and trafficking mechanisms of DC-directed LVs and offers some strategies to further improve the efficiency of LV-mediated gene therapy.
    Full-text · Article · Jun 2013 · PLoS ONE
Show more