July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Adapting CRISPR towards Mitochondrial DNA Diseases
Author Affiliations & Notes
  • Anh H Pham
    Ophthalmology, UCLA Stein Eye Institute, Los Angeles, California, United States
  • John C Burnett
    Center for Gene Therapy, City of Hope, Duarte, California, United States
  • Footnotes
    Commercial Relationships   Anh Pham, PCT/US2017/036821 (P); John Burnett, PCT/US2017/036821 (P)
  • Footnotes
    Support  none
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 3589. doi:
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      Anh H Pham, John C Burnett; Adapting CRISPR towards Mitochondrial DNA Diseases. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3589.

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

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Purpose : Mutations in mitochondrial DNA (mtDNA) lead to devastating neurological and musculoskeletal disorders that have no effective therapy. Curative strategies towards mtDNA diseases have been successful using mitochondrially targeted engineered nucleases (TALE nucleases and zincfinger nucleases) that recognize and cleave mutant mtDNA molecules, resulting in rapid clearance of linearized mtDNA. In this proposal, we aim to redirect the CRISPR genome editing technology, which requires a Cas9 endonuclease and a RNA guide (gRNA), towards the mitochondria by overcoming the challenges of importing gRNA into the mitochondria.

Methods : We have developed two strategies for the import of gRNA into the mitochondria and confirmed the targeted depletion of mtDNA in tissue culture cell lines. One strategy employs RNA motifs that have previously been identified to import exogenous RNAs into the mitochondria. A second strategy employs a synthetic compound attached to a modified gRNA to permit membrane-potential dependent import of gRNA into the mitochondria.

Results : Using the genetically encoded RNA motifs to deliver gRNA into the mitochondria, we identified two motifs which showed small enrichment in mitoplast subfractions and demonstrated mild depletions in the targeted mtDNA when measured by Taqman qRT-PCR. We achieved more efficient import of gRNA into the mitochondrial matrix when a synthetic compound is covalently attached to the modified gRNA. The gRNA was modified to improve RNA stability inside the cell and the pattern of modification did not interfere with the Cas9 recognition and function in an in vitro cutting assay. Fluorescence confocal microscopy showed >80% localization of the modified gRNA and TEM studies verified matrix localization of the RNA. We obtained moderate depletion of the targeted mtDNA using the synthetic modality for gRNA design.

Conclusions : We have achieved two methods for targeting gRNA into the mitochondria with different efficiencies in RNA import and mtDNA depletion. The modality for delivering gRNA into the mitochondria may largely depend on the extent of mutant mtDNA load inside the cell. We hope to expand the tools for genome engineering of mtDNA by overcoming the primary obstacle in the development of CRISPR therapeutics for mtDNA diseases.

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


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