Cellular Binding and Trafficking of AAV Vectors

The ability of a vector particle to interact with a specific receptor molecule on a target cell is critical for successful transduc-tion. Table I lists currently identified primary and secondary receptor molecules for AAV serotypes. The primary receptors identified (heparin sulfate and sialic acid) are commonly found on many cells and are also the receptor molecules used by a large number of viruses besides AAV. This suggests that additional receptors that lend more specificity to attachment and penetration of cells might exist, and several such corecep-tors have been identified (Table I).

Although attachment is a critical first event, it does not necessarily imply that the vector will be able to efficiently transduce the cell. This has been increasingly apparent in recent years as a more detailed understanding of the trafficking and uncoating of AAV vectors has been accumulated (46-50). For example, polarized human airway epithelial cells are transduced with varying efficiencies by AAV2-based vectors, depending on the route of delivery and entry from the basolat-eral surface, results in about a 200-fold increase in gene expression in the cells compared with vector administered from the apical surface (46,156). Surprisingly, the difference in vector attachment to the 2 cell surfaces is only about 5fold. This finding led to the discovery that the vectors traffic differently in these cells, depending on the side of the polarized airway to which they bind (46,156). Vectors administered from the apical surface are modified by ubiquitination. The addition of proteosome or ubiquitin-ligase inhibitors led to an increase in transduction following apical administration of vectors. These comparisons of differences in binding and differences in transduction efficiency have led to an appreciation that cellular trafficking of vectors is important to successful transduction, and that it may differ between cell types and even within individual polarized cells between the apical and basal surfaces.

After binding to the cellular receptor, AAV2 is internalized by an endocytosis mechanism that for heparin binding appears to be mainly via clathrin-coated pits (157,158), although some clathrin-independent uptake may also occur (157,158). Additional studies using fluorescent Cy3-conjugated AAV2 vector particles showed that endocytosis can be mediated by an avp5 integrin/Rac1-dependent mechanism and that subsequent trafficking to the nucleus requires activation of PI3K pathways, as well as functional microtubules and microfilaments (160). Evidence for the involvement of early endosomes in AAV trafficking, as well as the involvement of microtubules and microfilaments, exists mainly from the use of inhibitors (48,49). There is a growing consensus that, in the absence of helper virus, there is a block at the step of import of viral vector genomes into the nucleus of the cell (161-163). However, there is no clear understanding as to how or where un-coating of the viral genome occurs, or how the vector enters the nucleus, although it does not appear to use the nuclear pore complex for this step (163,164).

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