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Huijun Yuan, Hong Yu, John Guy; Rescue of a mutant T8993G ATP6 mouse model of NARP and MILS with Mito-Targeted AAV9 Containing Normal ATP6. Invest. Ophthalmol. Vis. Sci. 2016;57(12):754.
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© ARVO (1962-2015); The Authors (2016-present)
To develop gene therapy for the rescue of neurogenic ataxia & retinitis pigmentosa (NARP) and maternally inherited Leigh syndrome (MILS).
To rescue the NARP & MILS mouse model, the human normal ATP6 gene was fused in frame with a FLAG epitope (ATP6FLAG) followed by the mitochondrial encoded mCherry and under the mitochondrial heavy strand promoter.Visual function was monitored using serial flash and pattern electroretinography (PERG). Motor function was evaluated by Rota-Rod testing. Human ATP6 DNA fragments were amplified and sequenced from A6M mouse mitochondria DNA. To rescue A6M mice, mito-targeted AAV9 was generated by modified VP2 with cox8. 10ul of mito-AAV9/ATP6 (1.85e+12 vg/ml) were respectively injected into 3 month old A6M (n=10) and around 18 month old (later stage) A6M mice (n=10) with a genotype and phenotype of T8993G NARP and MILS, and untreated A6M mice (n=10) of same conditions as controls.
After injection of mito-AAV9ATP6 rescue vector, treated A6M mice (n=10) showed 100% survival for one year compared to 30% survival of untreated A6M mice. PERG amplitudes of treated A6M mice was increased to 19.6 uV after six month compared to 11.3 uV for untreated A6M mice (P=0.0001). Confocal laser scanning ophthalmoscopy (CLSO) showed mCherry of treated A6M mice expressed 5 times higher compared to untreated A6M mouse (P<0.01). DNA sequencing showed 70% human wild type ATP6 gene was delivered to A6M mouse mitochondria compared to mutant T8993G ATP6 gene. SDS-PAGE western blot showed the band intensities of mCherry were more than 10 times stronger compared to untreated A6M. At later stage A6M mice (n=10) with hunched, rota rod testing showed latency-to-fall time was significantly increased from 600 s to 439 s with increasing speed in treated A6M mice compared to untreated A6M mice (180 s to 26 s) after injection for two weeks (P<0.01). Video showed the movements with paralysis hind limbs mice prior to their spontaneous deaths were significantly improved after two days of injection of mito-targeting AAV9/ATP6.
Conclusions: Mito-targeted AAV9-wildtype ATP6 can successfully rescue mutant T8993G ATP6 mice significantly reducing mortality rate, improving defective visual function, ataxia and paralysis suggesting it may be helpful for MILS and NARP patients.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Leigh syndome mouse model
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