Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy

  1. Hironori Uehara  Is a corresponding author
  2. Xiaohui Zhang
  3. Felipe Pereira
  4. Siddharth Narendran
  5. Susie Choi
  6. Sai Bhuvanagiri
  7. Jinlu Liu
  8. Sangeetha Ravi Kumar
  9. Austin Bohner
  10. Lara Carroll
  11. Bonnie Archer
  12. Yue Zhang
  13. Wei Liu
  14. Guangping Gao
  15. Jayakrishna Ambati
  16. Albert S Jun
  17. Balamurali K Ambati  Is a corresponding author
  1. University of Oregon, United States
  2. University of Utah, United States
  3. University of Virginia, United States
  4. Loma Linda University, United States
  5. University of Massachusetts, United States
  6. Wilmer Eye Institute, Johns Hopkins School of Medicine, United States
  7. Moran Eye Center, University of Utah, United States

Abstract

A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early onset Fuchs' endothelial corneal dystrophy (FECD), which progressively impairs vision through loss of corneal endothelial cells. We demonstrate that CRISPR/Cas9-based postnatal gene editing achieves structural and functional rescue in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA), efficiently knocked down mutant COL8A2 expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. There were no adverse sequelae on histology or electroretinography. Col8a2 start codon disruption represents a non-surgical strategy to prevent vision loss in early-onset FECD. As this demonstrates the ability of Ad-Cas9-gRNA to restore phenotype in adult post-mitotic cells, this method may be widely applicable to adult-onset diseases, even in tissues affected with disorders of non-reproducing cells.

Data availability

High-throughput Sequencing data have been deposited in GEO under accession codes GSE146999. Source data files have been provided as excel files.

The following data sets were generated

Article and author information

Author details

  1. Hironori Uehara

    Phil and Penny Knight Campus for Accelerating Scientific Impact, University of Oregon, Eugene, United States
    For correspondence
    uhironori0916@gmail.com
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6133-4918
  2. Xiaohui Zhang

    Moran Eye Center, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Felipe Pereira

    Ophthalmology, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Siddharth Narendran

    Ophthalmology, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Susie Choi

    Department of Ophthalmology and Visual Sciences, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Sai Bhuvanagiri

    Department of Ophthalmology and Visual Sciences, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Jinlu Liu

    Department of Ophthalmology and Visual Sciences, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Sangeetha Ravi Kumar

    Department of Ophthalmology, Loma Linda University, Loma Linda, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Austin Bohner

    Department of Ophthalmology and Visual Sciences, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  10. Lara Carroll

    Department of Ophthalmology and Visual Sciences, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Bonnie Archer

    Moran eye center, University of Utah, Salt lake city, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Yue Zhang

    Division of Epidemiology, Department of Internal Medicine, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Wei Liu

    Division of Epidemiology, Department of Internal Medicine, University of Utah, Salt Lake City, United States
    Competing interests
    The authors declare that no competing interests exist.
  14. Guangping Gao

    Horae Gene Therapy Center, University of Massachusetts, Worcester, United States
    Competing interests
    The authors declare that no competing interests exist.
  15. Jayakrishna Ambati

    Ophthalmology, University of Virginia, Charlottesville, United States
    Competing interests
    The authors declare that no competing interests exist.
  16. Albert S Jun

    Ophthalmology, Wilmer Eye Institute, Johns Hopkins School of Medicine, Baltimore, United States
    Competing interests
    The authors declare that no competing interests exist.
  17. Balamurali K Ambati

    Ophthalmology, Moran Eye Center, University of Utah, Salt Lake City, United States
    For correspondence
    bambati@gmail.com
    Competing interests
    The authors declare that no competing interests exist.

Funding

National Eye Institute (R01EY017950)

  • Balamurali K Ambati

Research to Prevent Blindness

  • Balamurali K Ambati

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#15-11024 and #18-10016) of the University of Utah.

Reviewing Editor

  1. Constance L Cepko, Harvard Medical School, United States

Publication history

  1. Received: January 31, 2020
  2. Accepted: June 7, 2021
  3. Accepted Manuscript published: June 8, 2021 (version 1)
  4. Version of Record published: June 21, 2021 (version 2)

Copyright

© 2021, Uehara et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Hironori Uehara
  2. Xiaohui Zhang
  3. Felipe Pereira
  4. Siddharth Narendran
  5. Susie Choi
  6. Sai Bhuvanagiri
  7. Jinlu Liu
  8. Sangeetha Ravi Kumar
  9. Austin Bohner
  10. Lara Carroll
  11. Bonnie Archer
  12. Yue Zhang
  13. Wei Liu
  14. Guangping Gao
  15. Jayakrishna Ambati
  16. Albert S Jun
  17. Balamurali K Ambati
(2021)
Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy
eLife 10:e55637.
https://doi.org/10.7554/eLife.55637

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