June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
In vivo two-photon excitation-based assessment of intracellular protein transfer to mouse retina
Author Affiliations & Notes
  • Krzysztof Palczewski
    Ophthalmology, University of California Irvine, Irvine, California, United States
    Department of Physiology & Biophysics, University of California Irvine, Irvine, California, United States
  • Samuel W Du
    Ophthalmology, University of California Irvine, Irvine, California, United States
    Department of Physiology & Biophysics, University of California Irvine, Irvine, California, United States
  • Jianye Zhang
    Ophthalmology, University of California Irvine, Irvine, California, United States
  • Roman Smidak
    Ophthalmology, University of California Irvine, Irvine, California, United States
  • Huajun B Yan
    Ophthalmology, University of California Irvine, Irvine, California, United States
  • Rafal Holubowicz
    Ophthalmology, University of California Irvine, Irvine, California, United States
  • Grazyna Palczewska
    Ophthalmology, University of California Irvine, Irvine, California, United States
    Polgenix, Poland
  • Footnotes
    Commercial Relationships   Krzysztof Palczewski None; Samuel Du None; Jianye Zhang None; Roman Smidak None; Huajun Yan None; Rafal Holubowicz None; Grazyna Palczewska None
  • Footnotes
    Support  NIH Grants R01EY009339, R24EY027283 and T32GM008620, the Research to Prevent Blindness Stein Innovation Award and unrestricted grants from Research to Prevent Blindness to the Department of Ophthalmology at the University of California, Irvine
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3171 – F0445. doi:
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      Krzysztof Palczewski, Samuel W Du, Jianye Zhang, Roman Smidak, Huajun B Yan, Rafal Holubowicz, Grazyna Palczewska; In vivo two-photon excitation-based assessment of intracellular protein transfer to mouse retina. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3171 – F0445.

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

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Abstract

Purpose : Inherited eye diseases leading to degeneration of the retina predominantly result from genomic alterations to retinal proteins. Genome editing is especially attractive to correct mutations in genes associated with phototransduction, the visual cycle and other cellular processes essential for the viability of retinal cells. It is crucial to study and quantify the delivery of genome editors to the eye, to optimize editing efficiency and minimize toxicity in animals before clinical application.

Methods : All animal procedures were approved by Animal Care Committees at the University of California, Irvine and conformed to recommendations of the American Veterinary Medical Association Panel on Euthanasia and ARVO. We used mT/mG Cre reporter mice (Jackson #007676), which constitutively express membrane tdTomato in all cell types, and switch to membrane GFP after Cre-mediated loxP excision.
We assessed the efficiency of Cre delivery via intraocular injection to anesthetized mice, and compared numerous delivery strategies of recombinant Cre protein, including peptidic tags, small molecule labeling, and custom nanoparticle formulations.
Two-photon (2P) microscopy was performed in mouse eyes 6-8 days after intraocular injection of Cre. A 950 nm light was used to simultaneously visualize mT- and mG-labelled cells. Signals were directed into mT and mG channels based on the 2P emission spectra. Fraction of transfected RPE and photoreceptor cells was assessed for different delivery modalities.

Results : After injection of Cre with various delivery tags, we observed distinct color switching from tdTomato to GFP in photoreceptors and retinal pigment epithelium (RPE). The characteristic features of these cells were preserved, including the hexagonal shape and undisturbed mosaic of RPE cells, and outer segments, inner segments, nuclear layer and connecting outer fibers; all of which were clearly visible in photoreceptors. Many delivery tags were effective in the RPE, while some compounds were targeted to photoreceptors.

Conclusions : Clear 2P images were obtained from the entire retina ranging from the RPE to the ganglion cell layer, enabling 3D reconstructions and assessment of color switching. Thus, 2P excitation-based imaging in intact mouse eyes is an effective way to compare the relative efficacy of diverse delivery routes of genome editors by measuring transfection efficiency and impact on retinal structure.

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

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