June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Increasing the amount of template favors HDR mediated DSB repair in photoreceptors in vivo following AAV mediated gene transfer
Author Affiliations & Notes
  • Knut Stieger
    Department of Ophthalmology, Justus Liebig Universitat Giessen, Giessen, Hessen, Germany
  • Michelle Knapp
    Department of Ophthalmology, Justus Liebig Universitat Giessen, Giessen, Hessen, Germany
  • David Götzmann
    Department of Ophthalmology, Justus Liebig Universitat Giessen, Giessen, Hessen, Germany
  • Annabella Janise-Libawski
    Department of Ophthalmology, Justus Liebig Universitat Giessen, Giessen, Hessen, Germany
  • Bettina Gill
    Department of Ophthalmology, Justus Liebig Universitat Giessen, Giessen, Hessen, Germany
  • Bärbel Fühler
    Department of Ophthalmology, Justus Liebig Universitat Giessen, Giessen, Hessen, Germany
  • Tobias Wimmer
    Department of Ophthalmology, Justus Liebig Universitat Giessen, Giessen, Hessen, Germany
  • Brigitte Müller
    Department of Ophthalmology, Justus Liebig Universitat Giessen, Giessen, Hessen, Germany
  • Footnotes
    Commercial Relationships   Knut Stieger SpliceBio, CoaveTx, Code C (Consultant/Contractor), CoaveTx, Code F (Financial Support); Michelle Knapp None; David Götzmann None; Annabella Janise-Libawski None; Bettina Gill None; Bärbel Fühler None; Tobias Wimmer None; Brigitte Müller None
  • Footnotes
    Support  DFG SPP2127, DFG STI-597/5
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1116. doi:
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      Knut Stieger, Michelle Knapp, David Götzmann, Annabella Janise-Libawski, Bettina Gill, Bärbel Fühler, Tobias Wimmer, Brigitte Müller; Increasing the amount of template favors HDR mediated DSB repair in photoreceptors in vivo following AAV mediated gene transfer. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1116.

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

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Abstract

Purpose : Genome editing takes center stage in the development of new therapeutic applications for the treatment of inherited diseases. While a lot is known with regard to DNA repair processes and necessary modifications upon induction of a double strand break (DSB) at the target site in dividing cells in vitro, hardly anything is known in nondividing cells of different tissues in vivo, such as neurons. Therefore, the aim of the present study was to analyze the influence of template DNA quantity on the activity of nonhomologous endjoining (NHEJ) and homology directed repair (HDR) of DSBs in photoreceptor cells in vivo.

Methods : Sixteen hemizygous male or homozygous female mice (age: 2 months) of the C57BL/6-RPGR.tm1Sti mouse line were injected subretinally with AAV2/8 vectors harboring the ISce-I homing endonuclease and a GFP frame linked by a T2A linker under the control of the rhodopsin kinase (Rk) promoter alone or together with a template (changing the ISce-I target site into a HindIII site) either in an all-in-one vector, or as two vectors at the ratio of 1:1 or 1:10 (endonuclease : template). Following euthanasia, GFP positive cells were enriched by FACS, and consequences of DNA repair activity studied at the target site by sanger sequencing and TIDE analysis.

Results : Between 10% (ISce-I vector alone), 15 and 17% (ISce-I and template all in one or 1:1 ratio, respectively) and 25% (ISce-I and template at 1:10 ratio) total editing activity was observed in targeted photoreceptor cells. While the NHEJ activity declined slightly from 10% (ISce-I vector alone) to 8% for the all in one vector and 6% for both vector combinations, the HDR activity increased from 8 and 9% for all in one vector and 1:1 ratio to 19% for the 1:10 ratio vector combination. Deletions and substitutions outnumbered insertions in NHEJ repair events and appeared to occur more often at the 5’ site of the DSB.

Conclusions : Targeted DNA editing is possible in photoreceptor cells using the ISce-I homing endonuclease in vivo. The rate of HDR increases significantly with higher amounts of template. This correlation, which is known from in vitro experiments, has important implications in the design of therapeutic applications and may contradict the notion that all in one vectors are more efficient in gene transfer for genome editing applications.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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