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Hong Yu, Tsung-Han Chou, Vittorio Porciatti, John Guy; Mitochondrial Gene Complementation Of The NADH Ubiquinone Oxidoreductase Mediated By A Mitochondrial Targeting Sequence Modification To The VP2 Capsid Of Adeno-associated Virus Rescues Vision In The LHON Mouse. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1413.
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© ARVO (1962-2015); The Authors (2016-present)
Leber Hereditary Optic Neuropathy (LHON) is a disease caused by mutated mitochondrial DNA and has no effective remedy mainly because of lacking adequate delivery system. Previously, we presented a novel system by fusing AAV capsid VP2 with mitochondrial targeting sequence (MTS) and delivered wild type ND4 into mitochondria of LHON cybrid cells to rescue the cell from defective respiration due to the ND4 mutation. Here we use the system delivering human ND4 in vivo to rescue visual loss in the LHON mouse model.
The normal human ND4 gene with a FLAG epitope under control of the mitochondrial promoter (sc-HSP-ND4) was packaged with mito-targeted (COX8) or standard AAV (VP2). The ND4 pseudo virus was injected into eyes of normal mice. Translocation and integration of human ND4 in mouse mitochondria was assessed by PCR, sequencing and immunohistochemistry. Allotopic mutant human R340H ND4 was injected into both eyes of mice with one eye also injected with sc-HSP-ND4 packaged by COX8 or VP2 AAV, and the contralateral eye injected with GFP. Visual function was monitored by pattern electroretinography (PERG).
Immunohistochemistry showed 90% of murine RGCs expressed ND4FLAG that could be detected up to 6 months post-injection. PERGs showed no decrements in RGC function. PCR of mitochondrial and nuclear DNA extracted from retina and optic nerve gave the expected 1.4Kb band. DNA sequencing confirmed that PCR products were indeed human ND4. Quantitative PCR showed that in the retinal mitochondrial fraction, human ND4 relative to endogenous mouse ND4 was approximately 80%. PCR with forward primers flanking the human ND4 and reverse primers flanking mouse mtDNA revealed several bands of 500bp as expected and the corresponding sequence showed that most bands were human ND4. However, some were human/mouse chimeras with human ND4 replacing mouse ND4 and followed by mouse mtDNA starting from 11519. The PERG amplitude for eyes injected with GFP+ allotopic mutant human ND4 decreased by 29% compared to COX8 delivered wild-type human ND4+ allotopic mutant human ND4 (p= 0.0151) and 25% compared to eyes injected with standard AAV for delivery of wild-type ND4 (P=0.0603).
Despite the mismatch, human ND4 integration into mouse mtDNA through homologous recombination together with rescue of visual function suggests that this novel technology may be effective in treatment of patients with LHON.
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