June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Towards A DNA-Directed RNA Interference (ddRNAi) Therapy Delivered Intravitreally Using a Novel AAV Capsid to Treat Choroidal Neovascularization (CNV)
Author Affiliations & Notes
  • David Suhy
    Benitec Biopharma, Hayward, California, United States
  • Shih-chu Kao
    Benitec Biopharma, Hayward, California, United States
  • Melissa Ann Kotterman
    4D Molecular Therapeutics, Emeryville, California, United States
  • Albert Doan
    Benitec Biopharma, Hayward, California, United States
  • Vanessa Ufombah-Strings
    Benitec Biopharma, Hayward, California, United States
  • Claudia Kloth
    Benitec Biopharma, Hayward, California, United States
  • Natalie Suhy
    Benitec Biopharma, Hayward, California, United States
  • Peter Roelvink
    Benitec Biopharma, Hayward, California, United States
  • David Kirn
    4D Molecular Therapeutics, Emeryville, California, United States
  • David Schaffer
    4D Molecular Therapeutics, Emeryville, California, United States
  • Footnotes
    Commercial Relationships   David Suhy, Benitec Biopharma (E); Shih-chu Kao, Benitec Biopharma (E); Melissa Kotterman, 4D Molecular Therapeutics (E); Albert Doan, Benitec Biopharma (E); Vanessa Ufombah-Strings, Benitec Biopharma (E); Claudia Kloth, Benitec Biopharma (E); Natalie Suhy, Benitec Biopharma (E); Peter Roelvink, Benitec Biopharma (E); David Kirn, 4D Molecular Therapeutics (E); David Schaffer, 4D Molecular Therapeutics (E)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4121. doi:
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      David Suhy, Shih-chu Kao, Melissa Ann Kotterman, Albert Doan, Vanessa Ufombah-Strings, Claudia Kloth, Natalie Suhy, Peter Roelvink, David Kirn, David Schaffer; Towards A DNA-Directed RNA Interference (ddRNAi) Therapy Delivered Intravitreally Using a Novel AAV Capsid to Treat Choroidal Neovascularization (CNV). Invest. Ophthalmol. Vis. Sci. 2017;58(8):4121.

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

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Abstract

Purpose : Inappropriate expression of VEGF growth factors in ocular tissues is implicated in CNV and the development of age-related macular degeneration (AMD). We describe a ddRNAi therapeutic to silence VEGF-a, VEGF-b and PlGF using a single treatment, gene therapy approach. Because effective RNAi requires large numbers of cells to be transduced, therapeutic evolution of an AAV library in non-human primates (NHP) was applied to identify and select novel viral capsids showing broad transduction in retina following intravitreal injection.

Methods : The anti-VEGF-a, -VEGF-b and -PlGF shRNAs expressed from the recombinant AAV cassette were validated using ARPE19 and JEG3 cells. Therapeutic evolution was performed in NHPs to ensure that novel capsids, delivered via intravitreal injection, can traverse the inner limiting membrane, a dense tissue generally impermissive to passage of viral particles. Initial screening introduced a library of up to 108 unique AAV capsid variants into the vitreous. Capsids transducing retinal layers were recovered and used in subsequent rounds. Multiple rounds resulted in an enrichment of the pool into capsid variants with the desired properties. The tolerance and biodistribution of 2 novel capsid variants harboring a GFP cassette were tested following the intravitreal administration into NHPs. Fluorescence fundus imaging assessed the spatial distribution of transduction.

Results : The recombinant shRNA construct inhibited expression efficiently in a dose dependent manor at levels up to 95% as assessed by both protein and mRNA knockdown and inhibition was concomitant with shRNA expression levels. Completion of six rounds of screening has revealed several candidate AAV capsids that transduce deep retinal layers including the RPE and photoreceptors. Initial biodistribution data, collected thru Day 14 of an ongoing study, demonstrated that wildtype AAV2 transduced retina sparsely with very faint expression at the peri-optic nerve head. In contrast, even at early time points, one of the novel capsid variants already showed strong, discernible expression in perifovea, perivascular, optic nerve head region as well as broad distribution across large areas of the mid- and far-periphery of retina.

Conclusions : Collectively, these data provide compelling motivation for the continued development of a ddRNAi based therapeutic to treat AMD.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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