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S. L. Williams, S. R. Bacman, C. T. Moraes; Manipulation of Mitochondrial DNA Mutant Load in Retina Using Viral Delivery of a Restriction Endonuclease Targeted to Mitochondria. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4602.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal involvement in diseases caused by mitochondrial DNA (mtDNA) point mutations such as Leber hereditary optic neuropathy (LHON) and neuropathy, ataxia and retinitis pigmentosa (NARP) is well documented. Using a heteroplasmic mouse model, we have previously shown that viral delivery of a mitochondria-targeted restriction enzyme can specifically reduce the relative abundance of the target mtDNA haplotype in muscle, brain and liver. We have now extended these studies to the retina.
Heteroplasmic mice harboring both NZB and BALBc mtDNA haplotypes were injected either intravitreally or sub-retinally with an AAV1/2 construct encoding an HA-tagged, mitochondria-targeted restriction endonuclease (ApaL1) that specifically cleaves the BALBc mtDNA haplotype. Eyes were harvested at 3 months post injection and radioactive RFLP was used to determine the relative haplotype abundance in laser-microdissected HA-positive and negative cells.
Intravitreal injection efficiently targeted retinal ganglion cells (RGC) and sub-retinal injection the retinal pigmented epithelium (RPE). Multiple RGC samples from a single animal gave a relative NZB mtDNA abundance of 0.1-0.6% in the uninjected eye and 0.3-5.5% in HA-negative cells from the injected eye. The abundance of NZB mtDNA increased to 45-90% in HA-positive cells from the injected eye. Similarly, multiple RPE samples from a single animal gave an NZB mtDNA abundance of 30-33% in the uninjected eye and 17-25% in HA-negative cells from the injected eye, increasing to 76-80% in HA-positive cells from the injected eye.
These results confirm the potential of mitochondria-targeted restriction endonucleases for reduction of mtDNA mutant load in retina and offer an alternative therapeutic strategy to allotopic expression of wild-type mtDNA genes for retinopathy associated with heteroplasmic mtDNA mutations.
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