Mutant NADH dehydrogenase subunit 4 gene delivery to mitochondria by targeting sequence-modified adeno-associated virus induces visual loss and optic atrophy in mice.

PURPOSE: Although mutated G11778A NADH ubiquinone oxidoreductase subunit 4 (ND4) mitochondrial DNA (mtDNA) is firmly linked to the blindness of Leber hereditary optic neuropathy (LHON), a bona fide animal model system with mutated mtDNA complex I subunits that would enable probing the pathogenesis of optic neuropathy and testing potential avenues for therapy has yet to be developed. METHODS: The mutant human ND4 gene with a guanine to adenine transition at position 11778 with an attached FLAG epitope under control of the mitochondrial heavy strand promoter (HSP) was inserted into a modified self-complementary (sc) adeno-associated virus (AAV) backbone. The HSP-ND4FLAG was directed toward the mitochondria by adding the 23 amino acid cytochrome oxidase subunit 8 (COX8) presequence fused in frame to the N-terminus of green fluorescent protein (GFP) into the AAV2 capsid open reading frame. The packaged scAAV-HSP mutant ND4 was injected into the vitreous cavity of normal mice (OD). Contralateral eyes received scAAV-GFP (OS). Translocation and integration of mutant human ND4 in mouse mitochondria were assessed with PCR, reverse transcription-polymerase chain reaction (RT-PCR), sequencing, immunoblotting, and immunohistochemistry. Visual function was monitored with serial pattern electroretinography (PERG) and in vivo structure with spectral domain optical coherence tomography (OCT). Animals were euthanized at 1 year and processed for light and transmission electron microscopy. RESULTS: The PCR products of the mitochondrial and nuclear DNA extracted from infected retinas and optic nerves gave the expected 500 base pair bands. RT-PCR confirmed transcription of the mutant human ND4 DNA in mice. DNA sequencing confirmed that the PCR and RT-PCR products were mutant human ND4 (OD only). Immunoblotting revealed the expression of mutant ND4FLAG (OD only). Pattern electroretinograms showed a significant decrement in retinal ganglion cell function OD relative to OS at 1 month and 6 months after AAV injections. Spectral domain optical coherence tomography showed optic disc edema starting at 1 month post injection followed by optic nerve head atrophy with marked thinning of the inner retina at 1 year. Histopathology of optic nerve cross sections revealed reductions in the optic nerve diameters of OD versus OS where transmission electron microscopy revealed significant loss of optic nerve axons in mutant ND4 injected eyes where some remaining axons were still in various stages of irreversible degeneration with electron dense aggregation. Electron lucent mitochondria accumulated in swollen axons where fusion of mitochondria was also evident. CONCLUSIONS: Due to the UGA codon at amino acid 16, mutant G11778A ND4 was translated only in the mitochondria where its expression led to significant loss of visual function, loss of retinal ganglion cells, and optic nerve degeneration recapitulating the hallmarks of human LHON.

Gene therapy using self-complementary Y733F capsid mutant AAV2/8 restores vision in a model of early onset Leber congenital amaurosis.

Defects in the photoreceptor-specific gene aryl hydrocarbon receptor interacting protein-like 1 (Aipl1) are associated with Leber congenital amaurosis (LCA), a childhood blinding disease with early-onset retinal degeneration and vision loss. Furthermore, Aipl1 defects are characterized at the most severe end of the LCA spectrum. The rapid photoreceptor degeneration and vision loss observed in the LCA patient population are mimicked in a mouse model lacking AIPL1. Using this model, we evaluated if gene replacement therapy using recent advancements in adeno-associated viral vectors (AAV) provides advantages in preventing rapid retinal degeneration. Specifically, we demonstrated that the novel self-complementary Y733F capsid mutant AAV2/8 (sc-Y733F-AAV) provided greater preservation of photoreceptors and functional vision in Aipl1 null mice compared with single-stranded AAV2/8. The benefits of sc-Y733F-AAV were evident following viral administration during the active phase of retinal degeneration, where only sc-Y733F-AAV treatment achieved functional vision rescue. This result was likely due to higher and earlier onset of Aipl1 expression. Based on our studies, we conclude that the sc-Y733F-AAV2/8 viral vector, to date, achieves the best rescue for rapid retinal degeneration in Aipl1 null mice. Our results provide important considerations for viral vectors to be used in future gene therapy clinical trials targeting a wider severity spectrum of inherited retinal dystrophies.