July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Exploration of Mitochondrially Targeted Gene Therapies
Author Affiliations & Notes
  • Daniel Manraj Maloney
    Smurfit Institute of Genetics , Trinity College Dublin, Dublin, Dublin, Ireland
  • Matthew Carrigan
    Smurfit Institute of Genetics , Trinity College Dublin, Dublin, Dublin, Ireland
  • Naomi Chadderton
    Smurfit Institute of Genetics , Trinity College Dublin, Dublin, Dublin, Ireland
  • Sophia Millington-Ward
    Smurfit Institute of Genetics , Trinity College Dublin, Dublin, Dublin, Ireland
  • Arpad Palfi
    Smurfit Institute of Genetics , Trinity College Dublin, Dublin, Dublin, Ireland
  • Peter Humphries
    Smurfit Institute of Genetics , Trinity College Dublin, Dublin, Dublin, Ireland
  • Jane G Farrar
    Smurfit Institute of Genetics , Trinity College Dublin, Dublin, Dublin, Ireland
  • Footnotes
    Commercial Relationships   Daniel Maloney, None; Matthew Carrigan, None; Naomi Chadderton, None; Sophia Millington-Ward, None; Arpad Palfi, None; Peter Humphries, None; Jane Farrar, None
  • Footnotes
    Support  SFI 11/P!/1080 and 16/IA/4452, HRB/FBI/MRCG 2012-4, HRB HRA_POR_2015_1140, IRC 505.G30495
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 4539. doi:
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    • Get Citation

      Daniel Manraj Maloney, Matthew Carrigan, Naomi Chadderton, Sophia Millington-Ward, Arpad Palfi, Peter Humphries, Jane G Farrar; Exploration of Mitochondrially Targeted Gene Therapies. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4539.

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

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Abstract

Purpose : Mitochondrial deficiencies have been implicated in a wide array of neurological disorders. In particular a number of retinopathies have Complex I deficiencies. The S. cerevisiae gene Ndi1, a rotenone insensitive NADH oxidoreductase, has shown potential as a therapeutic for Complex I related disorders. Here we investigate the efficacy of an Ndi1-based therapy to provide a potential complex I surrogate and furthermore explore if this therapeutic approach can be optimised for greater efficacy.

Methods : HEK293 cells were grown in culture and transfected with Ndi1, OphNdi or GFP. NADH oxidation activity, measured using a UV VIS spectrophotometer at 340nm, was monitored and normalised to protein content as nmol min-1 mg-1 in extracted mitochondria. Oxygen Consumption Rate (OCR) was measured using a Clark-Type Electrode in plasmid transfected HEK293 cells and using a Seahorse XF24 in virally transduced (AAV2-2) HEK293 cells.

Results :
The Ndi1 transgene was codon optimised (OphNdi; GeneArt). Western Blot and Real-Time PCR analysis showed a significant increase in protein and RNA expression from OphNdi compared to Ndi1 transfected HEK293 cells. Additionally, OphNdi transfection resulted in a significant increase in NADH oxidation activity compared to both endogenous complex I activity (p < .001) and wild type Ndi1 activity (p < .001). OCR measurements using the Clark-Type electrode showed a significant difference in OCR in OphNdi1-transfected cells after addition of rotenone (complex I inhibitor) compared to eGFP transfected cells (p < .001). HEK293 cells transduced with AAV2-2 OphNdi showed a significant increase in the OCR compared to AAV2-2 eGFP (p < .001) and AAV2-2 Ndi1 (p < .001). Co-localisation studies show a strong overlap between HA-tagged OphNdi and mitochondrially targeted ds-Red, indicating no perturbation of mitochondrial localisation post optimisation.

Conclusions : The results demonstrate that Ndi1 can fulfil the NADH oxidation role of endogenous complex I and that there is value to optimising the Ndi1 transgene for human expression. Using this strategy we have significantly increased Ndi1 expression from plasmid or an AAV vector. In principle such optimisations will be of significant value in the design of Ndi1-based therapies enabling efficacy to be achieved with a lower quantity of OphNdi vector, an important consideration when targeting retinopathies.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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