June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Increasing downstream protein expression of a double Dicer-independent shRNA-containing cassette for multi-targeting gene therapy in neovascular age-related macular degeneration
Author Affiliations & Notes
  • Bjørn Kristensen Fabian-Jessing
    Department of Biomedicine, Aarhus University, Aarhus C, Denmark
    Department of Ophthalmology, Aarhus University Hospital, Aarhus N, Denmark
  • Sidsel Alsing
    Department of Biomedicine, Aarhus University, Aarhus C, Denmark
  • Anne Louise Askou
    Department of Ophthalmology, Aarhus University Hospital, Aarhus N, Denmark
  • Toke Bek
    Department of Ophthalmology, Aarhus University Hospital, Aarhus N, Denmark
    Department of Clinical Medicine, Aarhus University, Aarhus C, Denmark
  • Lars Aagaard
    Department of Biomedicine, Aarhus University, Aarhus C, Denmark
  • Thomas Juhl Corydon
    Department of Biomedicine, Aarhus University, Aarhus C, Denmark
    Department of Ophthalmology, Aarhus University Hospital, Aarhus N, Denmark
  • Footnotes
    Commercial Relationships   Bjørn Fabian-Jessing None; Sidsel Alsing None; Anne Louise Askou None; Toke Bek None; Lars Aagaard None; Thomas Corydon None
  • Footnotes
    Support  Fully financed PhD fellowship from Aarhus University and grant from Alcon.
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 2832 – A0348. doi:
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      Bjørn Kristensen Fabian-Jessing, Sidsel Alsing, Anne Louise Askou, Toke Bek, Lars Aagaard, Thomas Juhl Corydon; Increasing downstream protein expression of a double Dicer-independent shRNA-containing cassette for multi-targeting gene therapy in neovascular age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 2022;63(7):2832 – A0348.

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

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Abstract

Purpose : Repeated intravitreal injections with vascular endothelial growth factor (VEGF) inhibitors constitute current standard treatment for neovascular age-related macular degeneration (nAMD). However, two thirds of patients are either partial or non-responders to standard treatment, and new treatment modalities harbouring the potential to target multiple pathways are needed.
We have designed multigenic vectors incorporating intron-embedded double Dicer-independent short hairpin RNA (agshRNA) with potent VEGF knockdown followed by an antiangiogenic gene. However, analysis has revealed mis-splicing leading to low expression of the downstream gene. By eliminating potential competing splice sites, we hypothesized that downstream gene expression could be significantly increased.
Accordingly, this study aimed to eliminate mis-splicing by designing intron-embedded agshRNAs without potentially competing splice sites and to increase downstream gene expression in vitro.

Methods : Using the NetGene2 server for splice site prediction and mFold for RNA secondary structure prediction, we designed new constructs predicted to eliminate problematic splice sites. For easy validation, green fluorescent protein (GFP) was inserted downstream of the agshRNA-containing cassette, and fluorescent microscopy, flow cytometry and RT-PCR with sequencing of amplicons were used to investigate splicing of the agshRNA construct and GFP expression. All experiments were performed in HEK-293 cell lines.

Results : Fluorescent microscopy and flow cytometry showed a significant increase in downstream GFP expression, and RT-PCR with amplicon sequencing confirmed correct splicing.

Conclusions : By eliminating predicted aberrant splice sites in our agshRNA-containing cassette, we corrected splicing and thereby increased downstream GFP expression, paving the way for inserting therapeutic proteins with the aim of developing efficient and persisting therapy for patients with nAMD.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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