Abstract
Purpose:
To describe vertical gene transfer of the mutant human ND4 (hmutND4) bred from founder mice generated by mitochondria targeting sequence AAV infection of mouse embryonic stem cells.
Methods:
hmutND4 with a FLAG epitope and mitochondrial encoded mCherry under control of a mitochondrial promoter was packaged into mito-targeted AAV2 containing the COX8 leader sequence inserted into the VP2 capsid, then microinjected into the mouse blastocyst. Retina, brain, optic nerve, heart, liver and skeletal muscle were assessed by PCR, sequencing, two-dimensional blue native polyacrylamide gel electrophoresis (2D BN-PAGE), complex I activity and histopathology. Visual function was monitored by serial pattern electroretinography (PERG), retinal structure by spectral domain optical coherence tomography(SD-OCT) and mitochondrial gene expression by confocal laser scanning ophthalmoscopy (CLSO) of mCherry fluorescence.
Results:
60 transgenic mice were generated that contained varying expression of in vivo mCherry fluorescence. Three females with the highest levels of mCherry expression in the retina were mated with males of the same strain and produced a total of 137 pups over 4 generations. Red fluorescent particles were seen in the RGC layer and optic nerve head in 77% of mice, and cells with fluorescence increased as mice aged suggesting replication of the transgene. hmutND4 was detected by PCR in all tissues from the F0 founder mice to their F1 to F4 progeny. Characteristic of mitochondrial heteroplasmy, transgene levels differed significantly between tissues (p<0.001) with the highest levels found in the optic nerves. Expression of hmutND4 was confirmed by immunostaining and it assembled into mouse complex I. Activity of complex I decreased 10% to 60% and was inversely correlated to the amount of hmutND4. PERGs showed a progressive decline in amplitudes from 3 months after birth to noise levels by 11 months of age. SD-OCT confirmed progressive thinning of the RGC+IPL(p=0.00089). Ultrastructural analysis showed severe loss of axons in the optic nerve, demyelination and mitochondrial abnormalities in the brain and degeneration of few fibers in skeletal muscle, but none in the heart.
Conclusions:
Vertical transmission of mutant human ND4 into subsequent generations of transgenic mito-mice results in the characteristic hallmarks of human LHON affecting predominantly the optic nerve despite its presence in all mouse tissues.
Keywords: 538 gene transfer/gene therapy •
600 mitochondria •
740 transgenics/knock-outs