June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Messenger RNA-mediated augmentation restores mitochondrial function in Opa1-null cells
Author Affiliations & Notes
  • Michael H Farkas
    Ophthalmology, University at Buffalo, Buffalo, New York, United States
    VA Western New York Healthcare System, Buffalo, New York, United States
  • Maria E. Sousa
    Ophthalmology, University at Buffalo, Buffalo, New York, United States
    VA Western New York Healthcare System, Buffalo, New York, United States
  • Tadeusz Kaczynski
    Ophthalmology, University at Buffalo, Buffalo, New York, United States
    VA Western New York Healthcare System, Buffalo, New York, United States
  • Elizabeth D. Au
    Ophthalmology, University at Buffalo, Buffalo, New York, United States
  • Footnotes
    Commercial Relationships   Michael Farkas None; Maria Sousa None; Tadeusz Kaczynski None; Elizabeth Au None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 4451. doi:
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      Michael H Farkas, Maria E. Sousa, Tadeusz Kaczynski, Elizabeth D. Au; Messenger RNA-mediated augmentation restores mitochondrial function in Opa1-null cells. Invest. Ophthalmol. Vis. Sci. 2023;64(8):4451.

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

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Abstract

Purpose : Optic neuropathies due to retinal ganglion cell (RGC) loss are primarily caused by genes involved mitochondrial function. Given the proximity of the RGCs to the vitreal space, they are an attractive target for mRNA-mediated augmentation. Herein, we investigated the ability of OPA1 mRNA transfected into Opa1-null mouse embryonic fibroblasts (MEFs) to restore mitochondrial function.

Methods : OPA1 mRNA was from a linearized plasmid by in vitro transcription (IVT). Following synthesis, polyA tail was added using polyA polymerase. Then a Cap-1 structure was added using the Vaccinia capping system in combination with 2’-O-methyltransferase. The mature OPA1 was transfected into Opa1-null MEFs using lipofectamine MessengerMax. OPA1 production and localization was determined by Western blot and immunofluorescence, respectively. Mitochondrial tubulation was performed by immunofluorescence using MitoTracker Red. Mitochondrial respiration was assayed using a Seahorse XF analyzer.

Results : Initial quality control of OPA1 showed that the full-length (3 Kb) transcript is synthesized by IVT, and the poly A tail was between 200-300 nucleotides in length. Following transfection of mature OPA1, protein levels were restored to similar levels as seen in wild-type MEFs. Immunofluorescence showed that the protein co-localized with the mitochondria. Mitochondrial tubulation in the treated Opa1-null MEFs returned to normal levels, as compared to the wild-type MEFs. Further, a statistically significant improvement of mitochondrial respiration was observed in the treated Opa1-null MEFs, relative to the untreated MEFs. Encouragingly, this improvement in respiration was not significantly different than the wild-type MEFs.

Conclusions : mRNA-mediated augmentation therapies hold great promise given their ease of production and minimal cost. While mRNA stability hurdles still need to be overcome, our studies demonstrate that exogenous mRNA-based treatments can restore function.

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

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