Enhancement of Adeno-Associated Virus Infection by Mobilizing Capsids into and Out of the Nucleolus

Gene Therapy Center and Department of Pharmacology, The University of North Carolina at Chapel Hill, 7119 Thurston Bowles, CB 7352, Chapel Hill, North Carolina 27599-7352, USA.
Journal of Virology (Impact Factor: 4.44). 03/2009; 83(6):2632-44. DOI: 10.1128/JVI.02309-08
Source: PubMed


Adeno-associated virus (AAV) serotypes are being tailored for numerous therapeutic applications, but the parameters governing the subcellular fate of even the most highly characterized serotype, AAV2, remain unclear. To understand how cellular conditions control capsid trafficking, we have tracked the subcellular fate of recombinant AAV2 (rAAV2) vectors using confocal immunofluorescence, three-dimensional infection analysis, and subcellular fractionation. Here we report that a population of rAAV2 virions enters the nucleus and accumulates in the nucleolus after infection, whereas empty capsids are excluded from nuclear entry. Remarkably, after subcellular fractionation, virions accumulating in nucleoli were found to retain infectivity in secondary infections. Proteasome inhibitors known to enhance transduction were found to potentiate nucleolar accumulation. In contrast, hydroxyurea, which also increases transduction, mobilized virions into the nucleoplasm, suggesting that two separate pathways influence vector delivery in the nucleus. Using a small interfering RNA (siRNA) approach, we then evaluated whether nucleolar proteins B23/nucleophosmin and nucleolin, previously shown to interact with AAV2 capsids, affect trafficking and transduction efficiency. Similar to effects observed with proteasome inhibition, siRNA-mediated knockdown of nucleophosmin potentiated nucleolar accumulation and increased transduction 5- to 15-fold. Parallel to effects from hydroxyurea, knockdown of nucleolin mobilized capsids to the nucleoplasm and increased transduction 10- to 30-fold. Moreover, affecting both pathways simultaneously using drug and siRNA combinations was synergistic and increased transduction over 50-fold. Taken together, these results support the hypothesis that rAAV2 virions enter the nucleus intact and can be sequestered in the nucleolus in stable form. Mobilization from the nucleolus to nucleoplasmic sites likely permits uncoating and subsequent gene expression or genome degradation. In summary, with these studies we have refined our understanding of AAV2 trafficking dynamics and have identified cellular parameters that mobilize virions in the nucleus and significantly influence AAV infection.

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    • "Cathepsins B and L, which have been found to cleave AAV8 in vitro with higher efficiency than AAV2 (Akache et al., 2007), may also be contributing to capsid dissociation. Finally, there is the possibility that AAV particles that enter the nucleus first localize to nucleoli, necessitating an additional transport step prior to capsid uncoating in the nucleoplasm (Johnson and Samulski, 2009). "
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    ABSTRACT: Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8׳s robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII & IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype.
    Virology 04/2014; s 454–455(1):227–236. DOI:10.1016/j.virol.2014.02.017 · 3.32 Impact Factor
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    • "General IS (taken from treatment of autoimmune disorders or transplantations) (9) and more specific interventions for AAV have been proposed and tested in pre-clinical models, such as proteasomal inhibitors (PI) (e.g., bortezomib, MG 132, carfilzomib). Many groups focus on increasing AAV transduction levels in various tissues using PI (47–50), and other compounds like arsenic trioxide (51), rather than their influence on the immune response. At the same time, another group observed reduced CTL responses as well (42). "
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    ABSTRACT: Transitioning to human trials from preclinical models resulted in the emergence of inhibitory AAV vector immune responses which has become a hurdle for sustained correction. Early animal studies did not predict the full range of host immunity to the AAV vector in human studies. While pre-existing antibody titers against AAV vectors has been a lingering concern, cytotoxic T-cell (CTL) responses against the input capsid can prevent long-term therapy in humans. These discoveries spawned more thorough profiling of immune response to rAAV in pre-clinical models, which have assessed both innate and adaptive immunity, and explored methods for bypassing these responses. Many efforts towards measuring innate immunity have utilized Toll-Like Receptor (TLR) deficient models and have focused on differential responses to viral capsid and genome. From adaptive studies, it is clear that humoral responses are relevant for initial vector transduction efficiency while cellular responses impact long-term outcomes of gene transfer. Measuring humoral responses to AAV vectors has utilized in vitro neutralizing antibody (NAb) assays and transfer of seropositive serum to immunodeficient mice. Overcoming antibodies using CD20 inhibitors, plasmapheresis, altering route of delivery and using different capsids have been explored. CTL responses were measured using in vitro and in vivo models. In in vitro assays expansion of antigen-specific T cells as well as cytotoxicity towards AAV transduced cells can be shown. Many groups have successfully mimicked antigen-specific T cell proliferation, but actual transgene level reduction and parameters of cytotoxicity towards transduced target cells has only been shown in one model. The model utilized adoptive transfer of capsid specific in vitro expanded T-cells isolated from immunized mice with LPS as an adjuvant. Finally, the development of immune tolerance to AAV vectors by enriching regulatory T-cells has also been explored as well as modulating t
    Frontiers in Immunology 02/2014; 5:28. DOI:10.3389/fimmu.2014.00028
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    • "Interestingly, a combination of GeLC-MS and 2DE identified thirteen cellular proteins (Table 1 and 2), including nucleolin [17], [18] and nucleophosm [17], [19] which have been reported to bind to the capsid during AAV packaging or infection process. However, the remaining 11 appear to represent novel cellular AAV capsid-binding proteins as evidenced by one such protein SET. "
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    ABSTRACT: Recombinant adeno-associated vectors (rAAV) are commonly purified by either chromatography or equilibrium CsCl gradient. Nevertheless, even after purification various cellular proteins often associate with rAAV vector capsids. Such co-purifying cellular proteins may raise concern about safety of gene therapy. Here we report identification and characterization of the co-purifying cellular protein in the vector preparations by using a combination of two proteomics approaches, GeLC-MS (gel electrophoresis liquid chromatography-mass spectrometry) and 2DE (two-dimensional gel electrophoresis). Most prominent bands revealed by Coomassie Blue staining were mostly similar to the AAV capsid proteins. Posttranslational modifications of capsid proteins were detected by the proteomics analysis. A total of 13 cellular proteins were identified in the rAAV vectors purified by two rounds of cesium chloride gradient centrifugation, including 9 by the GeLC-MS analysis and 4 by the 2DE analysis. Selected cellular proteins were verified by western blot. Furthermore, the cellular proteins could be consistently found associated with different AAV serotypes and carrying different transgenes. Yet, the proteins were not integral components of the viral capsis since a stringent washing procedure by column purification could remove them. These co-purified proteins in AAV vector preparations may have a role in various stages of the AAV life cycle.
    PLoS ONE 02/2014; 9(2):e86453. DOI:10.1371/journal.pone.0086453 · 3.23 Impact Factor
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