754. Intracellular Trafficking Patterns of rAAV-2 Demonstrate Significant Cell-Type Specificity

Abstract

Serotype-dependent differences in recombinant AAV vector transduction have historically been attributed to differences in the distribution of serotype-specific receptors that affect viral binding and uptake by a target cell. Recently, this dogma has been challenged, as intracellular barriers responsible for limiting nuclear accumulation of rAAV have been uncovered. The efficiency of rAAV uptake by a cell is therefore no longer thought to always directly correlate with the efficiency of transgene expression in different target cell types. These intracellular barriers to transduction may also differ for various serotypes of rAAV that utilize distinct receptors' entry pathways. To further investigate the intracellular mechanisms of rAAV-2 transduction that might vary between cell types, we evaluated the subcellular localization of Cy3-rAAV-2 following transduction of HeLa and IB3 cells. Despite the fact that rAAV-2 enters these two cell types with similar efficiency, HeLa cells are much more transducible with rAAV-2 than with IB3 cells. However, tripeptidyl proteasome inhibitors can induce rAAV-2 transduction in IB3 cells (100-fold) with significantly greater efficiency than HeLa cells (10-fold). We hypothesized that these differences in transduction and responsiveness to proteasome inhibitors may be reflected by variations in the intracellular trafficking patterns of rAAV-2 between HeLa and IB3 cells. To test this hypothesis, we used fluorescent microscopy to evaluate the primary vesicular compartments in which Cy3-labeled rAAV-2 accumulate following infection of these two cell types. We have focused our analysis on three major endosomal compartments with different Rab small GTPase markers, including the peri-nuclear recycling endosome (Rab11), late endosome (Rab7), and late endosome to trans-golgi (Rab9). METHODS: Luciferase-expressing rAAV-2 was labeled with Cy3 and purified by column chromatography. EGFP N-terminus fusions with Rab11, Rab7, and Rab9 were generated in expression plasmids as markers for various intracellular compartments. IB3 and HeLa cells were transfected with various EGFP-Rab fusion constructs using lipofectamine for 48 hrs, followed by infection with Cy3-labeled rAAV-2 at 4°C for 30 minutes with an MOI of 10,000 DRP/cell. Cells were then washed and shifted to 37°C for 30 minutes to 2 hours, after which they were fixed and evaluated by fluorescent microscopy. RESULTS: A substantial degree of co-localization of Cy3-AAV-2 and EGFP-Rab11 was observed in HeLa cells from 30 min to 2 hrs postinfection. This pattern, however, was not observed in IB3 cells. In contrast, we found that the Cy3-labeled rAAV-2 was primarily co-localized with EGFP-Rab9 in IB3 cells. In HeLa cells, the degree of co-localization of Cy3-AAV and EGFP-Rab9 was not predominant. A significant amount of Cy3-labeled rAAV-2 was observed to be co-localized with EGFP-Rab7–tagged compartments in both HeLa and IB3 cells. These findings suggest that rAAV-2 traffics through a diversity of intracellular compartments in a cell-type specific manner. The effect of proteasome inhibitor treatment on the localization pattern of rAAV-2 in these two cell types is currently under investigation.