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Huijun Yuan, Hong Yu, John Guy; Gene Delivery of ATP6 by A Mitochondrial Targeting Sequence Modification of AAV Capsid VP2 Rescues Cells with Mutated T8993G MtDNA Responsible for Neuropathy Ataxia and Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2724.
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© ARVO (1962-2015); The Authors (2016-present)
Two mitochondria diseases, neuropathy, ataxia, retinitis pigmentosa (NARP) and maternally inherited Leigh's syndrome (MILS) are caused by a T8993G mutation in mtDNA encoding subunit 6 of ATP synthase that is complex V. Our goal is to rescue the consequences of this mutation that result in blindness and death.
The human ATP6 gene was packaged into AAV serotype 2 or AAV serotype 9 to which the cytochrome oxidase subunit 8 (COX8) leader sequence was inserted into the VP2 capsid. Expression of ATP6 was under the control of the human mitochondrial heavy strand promoter (HSP). NARP cybrid cells with 100% mutated T8993G mitochondrial DNA were infected at different MOI with self-complementary (sc) AAV9 or AAV2 containing scAAV-HSP-ATP6 with FLAG. After 48 hrs, high glucose media was exchanged for glucose free galactose media for 5 days that forced the cells to rely on oxidative phosphorylation to produce ATP. Cell survival was assessed by the MTT assay. ATP synthesis was measured using a luciferin-luciferase assay. Expression of mRNA and protein were respectively assessed by qRT-PCR, immunofluorescence and immunoblotting.
After infection of rAAV with scAAV-HSP-ATP6, cell survival increased in a dose-dependent matte with an absorbance 0.305, 0.372, and 0.4055 for MOI of 0, 500, and 5,000 respectively. Relative to no infection, differences were statistically significant with P value of 0.007 and 0.0009.Relative to uninfected NARP cells, the rate of ATP synthesis increased 41% (p< 0.01). Compared to uninfected, infected cybrids ATP6 RNA transcripts were elevated 9 fold and 34 fold with AAV2 and AAV9 infection (p<0.05). Flag tagged ATP6 surrounded DAPI stained nuclei and exhibited the punctate perinuclear distribution of mitochondrial expression with colocalization to MitoTracker Green. Western blotting showed that FLAG tagged ATP6 was detected at day 8. Blue native PAGE revealed wild-type ATP6 integrated into complex V.
MTS modified AAV2 or AAV9 restores ATP synthesis and prevents cell death. Without an animal model it is unclear whether intraocular injection (AAV2) may save the visual loss of adults with NARP or with systemic injection (AAV9) the lives of children with MILS, half of whom die by age 3.
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