June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Efficient AAV-mediated prime editing in mouse retina
Author Affiliations & Notes
  • Qin Liu
    Ophthalmology, Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Rossano Butcher
    Ophthalmology, Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Jessie R Davis
    Broad Institute, Cambridge, Massachusetts, United States
  • Caitlin Collin
    Ophthalmology, Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • David R Liu
    Broad Institute, Cambridge, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Qin Liu Entrada Therapeutics, Code C (Consultant/Contractor); Rossano Butcher None; Jessie Davis broad institute, Code P (Patent); Caitlin Collin None; David Liu Prime Medicine, Beam Therapeutics, Pairwise Plants, Chroma Medicine, and Nvelop Therapeutics, Code C (Consultant/Contractor), Broad Institute, Code P (Patent)
  • Footnotes
    Support  NIH 1R01EY033107-01A1, UG3AI150551, U01AI142756, R35GM118062, RM1HG009490; the Bill and Melinda Gates Foundation; and the Howard Hughes Medical Institute.
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3787. doi:
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    • Get Citation

      Qin Liu, Rossano Butcher, Jessie R Davis, Caitlin Collin, David R Liu; Efficient AAV-mediated prime editing in mouse retina. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3787.

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

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Abstract

Purpose : Prime editing (PE) is a genome editing technology which can perform precise base-pair substitutions, deletions or insertions in the genome without creating double-stranded breaks. Previous studies have shown PE could achieve genome editing in mouse retina, however, at very low levels (~1-2%). This study aims to develop AAV-PE systems that can achieve higher delivery and editing efficiency in mouse retinas.

Methods : We first tested several AAV-eGFPs viral vectors with different AAV capsids, promoters and terminators to determine the most efficient delivery system in mouse photoreceptor cells. We then used Dnmt1 gene as a target to test the AAV-PE system by introducing a single base-pair substitution. We cloned the PE components into three AAV vectors with split-intein (a. N-terminus Cas9 nickase; b. C-terminus Cas9 nickase and M-MLV reverse transcriptase; c. epegRNA, nicking gRNA and eGFP). Triple AAV-PE were injected subretinally into two-week-old CD1 or Ai9-Rho-Cre reporter mice; the latter expresses tdTomato in photoreceptor cells. Two or seven weeks post injection, DNA was extracted from whole retinas of CD1 mice or dissociated EGPF+/tdTomato+ retinal cells from Ai9-Rho-Cre mice. PCR amplification of targeted Dnmt1 locus was performed, followed by NGS amplicon sequencing.

Results : We found that the highest transduction efficiency was observed in retinas injected with AAV-PHP.eB Cbh-eGFP-W3bGH vector. For in vivo prime editing with split-intein triple AAV-PE, we observed an average of 17.7 ± 3.1% (n=3) precise editing and 1.3 ± 0.6% indels from the whole retinas that express a high level of GFP. In Ai9-Rho-Cre mice, we observed an average of 23.8 ± 9.1% (n=6) precise editing and 0.75 ± 0.4% indels in sorted eGPF+/tdTomato+ rod photoreceptor cells, with the highest editing efficiency up to 38.6%.

Conclusions : We have demonstrated, for the first time, a split-intein based triple AAV prime editor delivery platform could generate precise editing at therapeutically-relevant efficiencies in the photoreceptor cells following subretinal injection. These findings open the avenue of therapeutic prime editing for inherited retinal degenerations.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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