July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
Micro-homology-mediated end joining allows precise excision-insertion genome editing by a single adeno-associated virus vector to restore visual function in blind mice
Author Affiliations & Notes
  • Koji M Nishiguchi
    Ophthalmology, Tohoku University, Sendai, Japan
  • Kosuke Fujita
    Ophthalmology, Tohoku University, Sendai, Japan
  • Shota Katayama
    Ophthalmology, Tohoku University, Sendai, Japan
  • Toru Nakazawa
    Ophthalmology, Tohoku University, Sendai, Japan
  • Footnotes
    Commercial Relationships   Koji Nishiguchi, Tohoku University (P); Kosuke Fujita, Tohoku University (P); Shota Katayama, None; Toru Nakazawa, Tohoku University (P)
  • Footnotes
    Support  Funding sources: This work was supported in part by the Japan Agency for Medical Research and Development (KMN, JP18lk1403004)
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4225. doi:
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      Koji M Nishiguchi, Kosuke Fujita, Shota Katayama, Toru Nakazawa; Micro-homology-mediated end joining allows precise excision-insertion genome editing by a single adeno-associated virus vector to restore visual function in blind mice. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4225.

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

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Abstract

Purpose : To restore visual function in mice defective of both rod and cone function through precise excision-insertion genome editing with a single adeno-associated virus (AAV) vector by adopting micro-homology-mediated end joining (MMEJ).

Methods : Blind mice with genetic defects in both rod-specific Gnat1 (Gnat1IRD2/IRD2) and cone-specific Pde6c (Pde6ccpfl1/cpfl1) were injected with a single AAV2/8 vector that carried CRISPR/SaCas9 system targeting the pathogenic mutation in Gnat1 in one eye. We designed two guide RNAs flanking the Gnat1 mutation as well as the wildtype donor DNA to correct the mutation through MMEJ. The other untreated eye served as the control. After the treatment, genome editing efficacy was analyzed by immunohistochemistry and visual function was measured using electroretinogram (ERG) and visually evoked potential (VEP).

Results : In the blind mutant mice (Gnat1IRD2/IRDPde6ccpfl1/cpfl1 mice), 3.1 ± 1.2% of rods were found to express Gnat1 by immunohistochemistry after the excision-insertion genome editing treatment. As a result, we observed emergence of small ERG responses to 6Hz stimulus (~ 10 µV to 0 logcds/m2) and substantially increased light sensitivity by up to 4 log units by VEP compared to the severely desensitized untreated contralateral eye (P < 0.01) .

Conclusions : Optimal promoter selection and application of MMEJ allow precise excision-insertion genome editing by a single AAV vector for treating inherited retinal dystrophies. The platform described is versatile and could be applied to treat other ocular conditions.

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

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