Abstract
Purpose :
To generate mouse models carrying mutated human G3460A ND1 (hND1G3460A) or T14484C ND6 (hND6T14484C) mtDNA
Methods :
The hND1G3460 gene fused with MYC or hND6T14484C fused with HA epitope tag were cloned into self-complementary AAV backbone , under control of the mitochondrial heavy strand promoter including 3 upstream conserved sequence blocks (HSPCSB). Mitochondrial encoded Cherry (mCherry) was put downstream of the mutant genes. After packaging with mito-targeted (COX8) AAV, rAAV was injected into the adult mouse’s vitreous or was microinjected into fertilized oocytes to generate transgenic mito-mice. The expression and function of the mutant genes was assessed by qPCR, immunostaining, confocal laser-scanning ophthalmoscopy (CLSO), pattern electroretinography (PERG) and spectral domain optical coherence tomography (SD-OCT).
Results :
One month after injection, the mice started to show a statistically significant decrease in PERG amplitude, compared to control mice injected with rAAV carrying only mCherry. The reduction of the amplitude, in mice injected with ND1G3460A, were 26% in one month (p=0.0007) and 27% in three months (p=0.04) postinjection, and the reduction, in mice injected with ND6T14484C, were 26% (p=0.008) and 52% (p=0.0007) in one and three months respectively. SD-OCT showed optic nerve head swelling beginning as early as in 1 month after injection. Postmortem examination 7 months after injection showed severe optic neuropathy in mice been injected with mutant genes with a marked reduction in the diameter compared to control. Using the microinjections, we generated 28 transgenic mitomice carrying hND1G3460A and 20 carrying hND6T14484C. mCHERRY fluorescence was visualized in live mitomice retina and optic nerve head with the CLSO. qPCR of mtDNA and mtRNA, revealed that mutant genes were delivered and expressed in mitomice retina, optic nerve, brain, spinal cord, liver, heart, spleen, kidney and ovary, but the differences in the levels of heteroplasmy of mutant genes relative to host mtDNA between organs was highly variable (from 10-6 to 10%) and significantly different.
Conclusions :
Mutant Human ND1G3460A or ND6T14484C gene injected into the eye resulted in visual loss, optic disc edema and optic atrophy in the mouse. These models may be used for testing of potential treatments for human LHON.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.