Abstract
Purpose::
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.
Methods::
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.
Results::
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.
Conclusions::
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.
Keywords: mitochondria • gene transfer/gene therapy • retinal degenerations: hereditary