July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Gene Therapy for eAMD by Lentiviral Delivery of Dicer-Independent shRNAs Providing Increased Specificity and Safety
Author Affiliations & Notes
  • Sidsel Alsing
    Department of Biomedicine, Aarhus University, Aarhus C, Denmark
  • Tobias Bjerg
    Department of Biomedicine, Aarhus University, Aarhus C, Denmark
  • Lars Aagaard
    Department of Biomedicine, Aarhus University, Aarhus C, Denmark
  • Thomas Corydon
    Department of Biomedicine, Aarhus University, Aarhus C, Denmark
    Department of Ophthalmology, Aarhus University Hospital, Denmark
  • Footnotes
    Commercial Relationships   Sidsel Alsing, None; Tobias Bjerg, None; Lars Aagaard, None; Thomas Corydon, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 3385. doi:
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      Sidsel Alsing, Tobias Bjerg, Lars Aagaard, Thomas Corydon; Gene Therapy for eAMD by Lentiviral Delivery of Dicer-Independent shRNAs Providing Increased Specificity and Safety. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3385.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Current treatments of exudative AMD (eAMD) demand repeated administration while gene therapy offers the possibility of achieving lifelong effects with only a single treatment. The expression of Dicer-independent short hairpin RNAs (DIshRNAs) from tissue-specific pol II promoters has the potential to increase the specificity and safety of gene therapy for eAMD. The purpose of this project is to investigate the potency, specificity, and safety of tissue-specific expression of DIshRNAs targeting VEGF for use in gene therapy applications for eAMD, using appropriate in vitro experiments.

Methods : All studies have been performed in HEK-293 cell lines. The features of the DIshRNAs have been investigated using the dual luciferase reporter assay and Northern blotting. The knockdown efficacy was confirmed by Western blotting. Lentiviral expression of the DIshRNAs has been investigated with the dual luciferase reporter assay.

Results : We have designed tissue-specific (pol II) expressed DIshRNAs targeting VEGF. We have found that the on-target knockdown efficacy of DIshRNAs is comparable to a canonical shRNA, while the passenger strand off-target effect is avoided. A knockdown efficacy of approximately 90 % was achieved using pol II-driven DIshRNAs. Additionally, we have confirmed that the knockdown efficacy of the DIshRNAs is unaltered in the absence of Dicer. Using Northern blotting we have investigated the processing of DIshRNA species, and identified a guide species of approximately 25 nucleotides, which is close to the expected size range. Lastly, we have shown that the DIshRNAs may also be expressed using a lentiviral vector system.

Conclusions : Our results demonstrate that the DIshRNAs potently reduce the expression of a selected target while exhibiting no passenger strand off target effects, and that they may be expressed from a tissue specific promoter. Additionally, we have shown that lentiviral RPE-specific expression of DIshRNAs is feasible, making pol II-driven DIshRNAs a valuable tool for gene therapy purposes.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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