July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Precise and Safe Gene Editing in Retinal Pigment Epithelial Cells in vivo
Bikash R Pattnaik; Pawan K Shahi; Guojun Chen; Amr A Abdeen; Yuyuan Wang; Krishanu Saha; Shaoqin Gong
Author Affiliations & Notes
  • Bikash R Pattnaik
    Pediatrics Ophthal & Visual Sci, Univ of Wisconsin, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin, Madison, Wisconsin, United States
  • Pawan K Shahi
    Pediatrics, University of Wisconsin, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin, Madison, Wisconsin, United States
  • Guojun Chen
    Material Science and Engineering, University of Wisconsin, Madison, Wisconsin, United States
    Wisconsin Institute of Discovery, University of Wisconsin, Madison, Wisconsin, United States
  • Amr A Abdeen
    Wisconsin Institute of Discovery, University of Wisconsin, Madison, Wisconsin, United States
  • Yuyuan Wang
    Material Science and Engineering, University of Wisconsin, Madison, Wisconsin, United States
    Wisconsin Institute of Discovery, University of Wisconsin, Madison, Wisconsin, United States
  • Krishanu Saha
    Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, United States
    Wisconsin Institute of Discovery, University of Wisconsin, Madison, Wisconsin, United States
  • Shaoqin Gong
    Material Science and Engineering, University of Wisconsin, Madison, Wisconsin, United States
    Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Bikash Pattnaik, None; Pawan Shahi, None; Guojun Chen, None; Amr Abdeen, None; Yuyuan Wang, None; Krishanu Saha, None; Shaoqin Gong, None
  • Footnotes
    Support  NIH Grant EY024995,
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4942. doi:
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      Bikash R Pattnaik, Pawan K Shahi, Guojun Chen, Amr A Abdeen, Yuyuan Wang, Krishanu Saha, Shaoqin Gong; Precise and Safe Gene Editing in Retinal Pigment Epithelial Cells in vivo. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4942.

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Abstract

Purpose : CRISPR-Cas9 genome editing, a revolutionary approach, has the potential to treat inherited retinal degeneration. Its specificity in vivo and the safe profile of viral delivery has always been a concern. Retinal pigment epithelial (RPE) cells play an important role in the normal vision. Mutations in several RPE cell genes cause genetic disorders like Stargardt disease, Best disease, Leber congenital and amaurosis etc. In this study, we sought to test editing efficiency of RPE cells in vivo using a non-viral packaged ribonucleoprotein (RNP) complex of Cas9 nuclease and sgRNA.

Methods : We used a reporter mouse model Ai14 wherein the targeted sgRNA in RNP complex edits the stop cassette leading to the tdTomato expression. We targeted RPE cells using PBS, RNP alone, RNP packaged in Lipofectamine2000 (RNP-Lipo), RNP packaged in polymer nanoparticles (NPs) or all-trans retinoic acid (ATRA) conjugated NPs (NP-ATRA). The RNP was delivered by sub-retinal injection which was confirmed by measurable bleb formation. After 12-days, tdTomato expression was assessed by imaging RPE flat mounts using a Nikon C2 confocal microscope. Electroretinography (ERG) was performed on the mice before and after the subretinal injections to test toxicity of the RNP complexes.

Results : Successful editing of the RPE cells was confirmed by localized observation of tdTomato fluorescence. PBS injected eye (the control) did not show any fluorescence. NP and NP-ATRA showed localized distribution of fluorescence at the injection site. Overall, NP-ATRA treated mice showed wider spread and higher tdTomato intensity than NP-treated mice. ERG values were averaged and normalized from same eye before and after the subretinal injection. On an average there was about 70-80% inhibition of scotopic a-, b- and c-wave amplitudes by RNP and RNP-Lipo. Both NP and NP-ATRA, on the other hand, inhibited scotopic ERG responses by only 20-40%.

Conclusions : RNP can be used for precise and targeted RPE gene editing in vivo. Encapsulated RNP further reduced toxicity and preserved retina function as measured by ERG.

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

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
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Copyright © 2025 Association for Research in Vision and Ophthalmology.
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