Cytoplasmic Dynein-Dependent Adeno-Associated Virus Interaction with Microtubules

AAV VECTORS: GENOMES AND BIOLOGY 754. Intracellular Trafficking Patterns of rAAV-2 Demonstrate Significant Cell-Type Specificity Wei Ding,1 Neal Zhang,1 Ziying Yan,1,2 Richard Peluso,3 Barrie Carter,3 John F. Engelhardt.1,2 1 Anatomy and Cell Biology, University of Iowa, Iowa City, IA; 2 Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, The University of Iowa, Iowa City, IA; 31100 Olive Way; Suite 100, Targeted Genetics Corporation, Seattle, WA. 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 (10fold). 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: Luciferaseexpressing 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.

755. Cytoplasmic Dynein-Dependent AdenoAssociated Virus Interaction with Microtubules Samir Kelkar,1 Ronald G. Crystal,1 Philip L. Leopold.1 Weill Medical College of Cornell University, New York, NY.

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The importance of adeno-associated virus (AAV)-based gene therapy vectors as therapeutic vehicles is derived in part from efficient Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright  The American Society of Gene Therapy

cell membrane penetration and translocation of the adeno-associated virus capsid to the nucleus where its genetic cargo is delivered. Other viruses have enhanced intracellular trafficking to the nucleus through interaction with the microtubule cytoskeleton with subsequent energy-dependent translocation in a retrograde manner towards the nucleus. The retrograde molecular motor, cytoplasmic dynein, participates in the microtubule-based nuclear-directed translocation of numerous viruses. Based on the hypothesis that the cytoplasmic dynein motor complex mediates a biochemical linkage of the AAV capsid with microtubules, a microtubule binding assay was developed in which binding of fluorophore-conjugated AAV capsids to microtubules was evaluated by mixing fluorophoreconjugated AAV capsid with polymerized microtubules, centrifuging to pellet the microtubules, and measuring the presence of the AAVassociated fluorescence in either the microtubule-free supernatant or microtubule-containing pellet. SDS-polyacrylamide gel electrophoresis of supernatant and pellet fractions showed that fluorophore-labeled AAV capsid was located predominantly in the supernatant when AAV was incubated with taxol-stabilized microtubules. However, when purified bovine microtubuleassociated proteins were combined with microtubules, the majority of AAV was found in the pellet after centrifugation (69 ± 4 % vs 23 ± 4% in the absence of microtubule-associated proteins). The interaction of microtubule-associated proteins and AAV with microtubules did not occur in the presence of 500 mM NaCl. Using freshly isolated A549 lung epithelial cell lysate, the direct involvement of cytoplasmic dynein was evaluated. A549 cell lysate was depleted of endogenous cytoplasmic dynein by immunoprecipitation with a monoclonal antibody against the 74.1 kDa intermediate chain of cytoplasmic dynein. Microtubule-associated proteins isolated from whole A549 cell lysate versus cytoplasmic dynein-depleted A549 cell lysate were compared for the ability to support AAV-microtubule interaction. Whereas AAV capsid pelleted with microtubules when incubated with microtubule-associated proteins from whole A549 cell lysate, AAV capsid was found predominantly in the supernatant when cytoplasmic dynein-depleted cell lysate was used. No effect was observed when an irrelevant primary antibody was used for immunprecipitation. To confirm the involvement of cytoplasmic dynein in AAV-microtubule interaction, the nucleotide-sensitive binding property of cytoplasmic dynein was utilized. Under conditions that are known to disrupt cytoplasmic dynein interaction with microtubules (10 mM ATP), the efficiency of the AAVmicrotubule interaction was reduced. However, the presence of an identical concentration of AMP-PNP, a non-hydrolyzable analog of ATP, had no effect on AAV-microtubule interactions. The results of this study suggest that the AAV capsid interacts with microtubules in a cytoplasmic dynein-dependent manner, providing a potential mechanism for the efficient translocation of the AAV capsid to the nucleus during infection.

756. Distinct Classes of Proteasome-Modulating Agents Cooperatively Augment Recombinant Adeno-Associated Virus Type 2 and Type 5Mediated Transduction from the Apical Surface of Human Airway Epithelia Ziying Yan,1,2 Roman Zak,1,2 Yulong Zhang,1,2 Wei Ding,1,2 Simon Godwin,3 Keith Munson,3 Richard Peluso,3 John F. Engelhardt.1,2 1 Anatomy and Cell Biology, The Univeristy of Iowa, Iowa City, IA; 2 Center for Gene Therapy, The University of Iowa, Iowa City, IA; 3 Target Genetics Corporation, Seattle, WA. Tripeptidyl aldehyde proteasome inhibitors have been shown to effectively increase viral capsid ubiquitination and transduction of recombinant adeno-associated virus (rAAV) type 2 and 5 serotypes. In the present study, we have characterized a second class of proteasome-modulating agents (anthracycline derivatives) for their S287