Modulation of adeno-associated virus transduction by the promyelocytic leukemia protein, arsenic trioxide, and proteasome inhibitors Public Deposited

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Last Modified
  • March 22, 2019
Creator
  • Mitchell, Angela Marie
    • Affiliation: School of Medicine, Department of Microbiology and Immunology
Abstract
  • Adeno-associated virus (AAV) has been developed as a gene therapy vector and has been utilized in over 100 clinical trials, which demonstrate increasing efficacy. However, the efficacy of systemic applications is often hampered by low transgene expression at lower doses or loss of transgene expression over time at higher doses. Therefore, mechanisms are needed to increase rAAV transduction efficiency without increasing viral dose. The promyelocytic leukemic protein (PML) is a known cell-intrinsic antiviral factor, which has not been examined in the context of rAAV. Using PML knockout mice, we determined that PML inhibits rAAV transduction up to 50-fold in a serotype-independent manner and at several doses. Mechanistically, this transduction inhibition occurred at the level of second-strand DNA synthesis and not at earlier transduction steps. We further demonstrated human PML inhibited rAAV transduction, mostly through the actions of PML isoform II. This effect was extended to rAAV and wild type AAV production and replication. These data demonstrate PML inhibits rAAV transduction and suggest that PML may be an important target for efforts to enhance rAAV transduction. Moreover, various cell stressors enhance rAAV transduction through diverse mechanisms. We examined the effect of arsenic trioxide (As2O3), a chemotherapeutic agent approved for use in humans, on rAAV transduction. As2O3 treatment caused a dose dependent increase in rAAV transduction in vitro, in cell lines from several cell type and species origins. This transduction increase was due to reaction oxygen species dependent stabilization of rAAV virions at the perinuclear region. As2O3 increased transduction in vivo with several rAAV serotypes. Therefore, As2O3 treatment and the dependent mechanisms are promising avenues to enhancing rAAV transduction. Finally, we investigated whether proteasome inhibition was sufficient to enhance rAAV transduction, as previous work demonstrating proteasome inhibitors enhance rAAV transduction was conducted with non-specific proteasome inhibitors. Using carfilzomib, we determined that proteasome inhibition was sufficient to enhance rAAV transduction and that this was the mechanism through which other proteasome inhibitors act. In addition, we determined that the proteasome inhibitors caused increased efficiency in a late step in rAAV transduction. These data further elucidate the mechanism by which proteasome inhibitors enhance rAAV transduction.
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  • In Copyright
Advisor
  • Samulski, R. Jude
Degree
  • Doctor of Philosophy
Graduation year
  • 2013
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