doi 10 1016/j ymthe 2004 06 689 ��������� ����� ��������� � �������� �� ��������� ��������� ��������������� ���� ����������������� S287 �� �� ���� ������� ��� !������ 754 Intracellular Trafficking Pa[.]
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 Anatomy and Cell Biology, University of Iowa, Iowa City, IA; 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 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 to 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 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 ± % 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 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 Anatomy and Cell Biology, The Univeristy of Iowa, Iowa City, IA; Center for Gene Therapy, The University of Iowa, Iowa City, IA; 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 and serotypes In the present study, we have characterized a second class of proteasome-modulating agents (anthracycline derivatives) for their S287 AAV VECTORS: GENOMES AND BIOLOGY ability to induce rAAV transduction The anthracycline derivatives doxorubicin and aclarubicin were chosen for analysis because they have been shown to interact with the proteasome through a mechanism distinct from that of tripeptidyl aldehydes Our studies demonstrated that doxorubicin and aclarubicin also significantly augmented rAAV transduction in airway cell lines and polarized human airway epithelia, human bronchial xenografts, and mice lungs Both tripeptidyl aldehyde and anthracycline proteasome-modulating agents similarly augmented nuclear accumulation of rAAV in A549 and IB3 airway cell lines but did not directly enhance the efficiency of second- strand synthesis for AAV genome conversion However, these two cell types demonstrated cell specificity in terms of the ability of LLnL or doxorubicin to augment rAAV transduction Interestingly, the combined administration of LLnL and doxorubicin resulted in substantially increased transduction (>2000 fold) following apical infection of human polarized epithelia with either rAAV-2 or rAAV-5 In summary, the cell-type specificity of LLnL and doxorubicin to induce rAAV transduction, together with the ability of these compounds to synergistically enhance rAAV transduction in polarized airway epithelial induction, suggest that these two classes of compounds likely modulate different proteasome functions that affect rAAV transduction Findings from this study provide new insights into how modulation of the proteasome function can be used to augment rAAV transduction in airway epithelia for gene therapy of cystic fibrosis (This work was supported by NIH RO1 HL58340, Gene Therapy Center DK54759, and Targeted Genetics Corp.) 757 Enhancement of Gene Expression by a Recombinant Adeno-Associated Virus by Various Chemical Treatments Sung Jin Kim,1,3 Young Ran Nam,1 Chul-Hyen Joo,1 Jin Woo Chang,2,3 Yoo-Kyum Kim,1 Heuiran Lee.1 Department of Microbiology, University of Ulsan College of Medicine, Seoul, Korea; 2Department of Neurosurgery2, Yonsei University College of Medicine, Seoul, Republic of Korea; 3The Graduate School, Yonsei University College of Medicine, Seoul, Republic of Korea A recombinant adeno-associated virus (rAAV) has drawn attention as a novel gene delivery system with the advantage of long-term and efficient gene expression, particularly in neuronal and skeletal muscle cells However, transduction efficiency (TE) by rAAV is still low, which may explain its limited application, even in neuronal cells in vitro To overcome this drawback of rAAV system, we attempted to enhance the transduction efficacy using many different kinds of chemicals, mostly modulating DNA synthesis, cell cycle or rAAV receptor expression To investigate the effects of various chemical treatments on TE, we infected several human cancer cells lines (cervical Hela, colon HCT 116, breast MCF-7, hepatocellular carcinoma SK-Hep1, and glioma U 251 cancer cells) with rAAVexpressing b-galactosidase We then examined the degree of x-gal staining 48 h after post-infection Total chemicals were employed to enhance TE by rAAV of Trichostatin A as an inducer of rAAV receptor expression, Aphidicolin and Hydroxyurea as a DNA damaging agent, Etopocide and Camptothecin as a DNA synthesis blocker, MG-132 as a proteosome inhibitor, and Tyrphostin-1 as a EGFR inhibitor Hydorxyurea increased TE by 25- or 5-fold in Hela or HCT 116 cells, respectively MG-132 increased TE by 8.5-fold in SK-Hep1 cells Co-treatment of MG-132 and camptothecin enhanced TE by 12-fold in Hela cells The combined treatment of hydroxyurea and tyrphostin-1 also enhanced the transduction efficiency by 12-fold in Hela cells Finally, MTT assay consistently implied that the cytotoxicity was not observed in any concentrations of each chemical used in this study In conclusion, the transduction efficiency of rAAV mediated S288 transgene expression could be significantly enhanced in human cancer cells by various chemical treatment The result acquired in current study might be easily applied to other cells, such as primary neuronal cells 758 A Histone Deacetylase Inhibitor Enhances Recombinant Adeno-Associated Virus-Mediated Gene Expression in Cancer Cells In Vitro and In Vivo Takashi Okada,1 Tatsuya Nomoto,1 Yuhe Liu,1 Mayumi IwataOkada,2 Masafumi Takahashi,3 Kuniko Shimazaki,4 Takashi Matsushita,1 Hiroaki Mizukami,1 Akihiro Kume,1 Keiya Ozawa.1,2 Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan; 2Division of Hematology, Department of Medicine, Jichi Medical School, Tochigi, Japan; Department of Organ Regeneration, Shinshu University, Nagano, Japan; 4Department of Physiology, Jichi Medical School, Tochigi, Japan Background: The transduction of cancer cells using recombinant adeno-associated virus (rAAV) occurs with low efficiency, which limits its utility in cancer gene therapy We have previously sought to enhance rAAV-mediated transduction of cancer cells by applying DNA-damaging stresses An alternative technique to improve the rAAV-mediated transduction of tumor cells is enhancement at a transcription step Treatment with a histone deacetylase (HDAC) inhibitor is known to recover the gene expression from a silenced rAAV genome that has been integrated into the host’s genome However, rAAV exists mostly as an extrachromosomal genome rather than as an integrated genome, and this extrachromosomal form is the primary source of gene expression We examined whether the HDAC inhibitor could contribute to the enhanced transcription before integration occurs Methods: Cell lines U251MG, 9L, HEp-2, HepG-2 and NKO-1 were used to examine HDAC inhibitor-assisted gene transfer Target cells were transduced with the AAV1-5 vectors expressing the eGFP gene at x 10(4) genome copies/cell along with the HDAC inhibitor FR901228 The levels of alpha v integrin and eGFP expression were analyzed with FACS Copy number of the rAAV genome in the infected cells was estimated with real-time quantitative PCR analysis U251MG cells were transduced with AAV2Luciferase at x 10(4) genome copies/cell, and then x 10(6) of the transduced cells were inoculated subcutaneously into the BALB/c mice along with the inraperitoneal injection of the FR901228 at mg/kg Twenty-four hours after the administration of the FR901228, optical bioluminescence imaging was performed using the CCD camera Results: The FR901228 treatment improved the rAAV-mediated gene transfer in a dose-dependent manner, and the highest enhancement was observed in the U-251MG cells with AAV2eGFP Twenty-four hours after the AAV2eGFP infection with ng/ml of the FR901228, 48% of the U-251MG cells were eGFP-positive, whereas very few cells were eGFP-positive in the absence of the FR901228 The enhancement of the integrin level was modest and copy number of the rAAV in the transduced cells was also modestly affected by the FR901228 treatment In the analysis using the optical bioluminescence imaging of the subcutaneous tumors, drastic enhancement of the luciferase gene expression was confirmed in vivo The signal intensity in animals treated with the FR901228 (n=5) was 37.4 fold higher than in control animals (n=3) Conclusion: These results suggest that the superior transduction induced by HDAC inhibitor was due to an enhancement of transgene expression rather than increased viral entry This phenomenon may be related to proposed histone-associated chromatin form of the Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright The American Society of Gene Therapy ... proposed histone-associated chromatin form of the Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright The American Society of Gene Therapy