Gene Therapy with Single-Subunit Yeast NADH-Ubiquinone Oxidoreductase (NDI1) Improves the Visual Function in Experimental Autoimmune Encephalomyelitis (EAE) Mice Model of Multiple Sclerosis (MS).

Mitochondrial dysfunction mediated loss of respiration, oxidative stress, and loss of cellular homeostasis contributes to the neuronal and axonal degenerations permanent loss of function in experimental autoimmune encephalomyelitis model (EAE) of multiple sclerosis (MS). To address the mitochondrial dysfunction mediated visual loss in EAE mice, self-complementary adeno-associated virus (scAAV) containing the NADH-dehydrogenase type-2 (NDI1) complex I gene was intravitreally injected into the mice after the onset of visual defects. Visual function assessed by pattern electroretinogram (PERGs) showed progressive loss of function in EAE mice were improved significantly in NDI1 gene therapy-treated mice. Serial optical coherence tomography (OCT) revealed that progressive thinning of inner retinal layers in EAE mice was prevented upon NDI1 expression. The 45% optic nerve axonal and 33% retinal ganglion cell (RGC) loss contributed to the permanent loss of visual function in EAE mice were ameliorated by NDI1-mediated prevention of mitochondrial cristae dissolution and improved mitochondrial homeostasis. In conclusion, targeting the dysfunctional complex I using NDI1 gene can be an approach to address axonal and neuronal loss responsible for permanent disability in MS that is unaltered by current disease modifying drugs.

Thomas Secker M.D.: Archbishop and man-midwife.

This paper provides a biographical outline of the career of Thomas Secker, MD, who from 1758-68 was Archbishop of Canterbury. Although much has been written on Secker, this study seeks to highlight his training in medicine, which has been largely overlooked hitherto by historians.

Gene Therapy for Leber Hereditary Optic Neuropathy: Initial Results.

PURPOSE: Leber hereditary optic neuropathy (LHON) is a disorder characterized by severe and rapidly progressive visual loss when caused by a mutation in the mitochondrial gene encoding NADH:ubiquinone oxidoreductase subunit 4 (ND4). We have initiated a gene therapy trial to determine the safety and tolerability of escalated doses of an adeno-associated virus vector (AAV) expressing a normal ND4 complementary DNA in patients with a G to A mutation at nucleotide 11778 of the mitochondrial genome. DESIGN: In this prospective open-label trial (NCT02161380), the study drug (self-complementary AAV [scAAV]2(Y444,500,730F)-P1ND4v2) was intravitreally injected unilaterally into the eyes of 5 blind participants with G11778A LHON. Four participants with visual loss for more than 12 months were treated. The fifth participant had visual loss for less than 12 months. The first 3 participants were treated at the low dose of vector (5 × 10(9) vg), and the fourth participant was treated at the medium dose (2.46 × 10(10) vg). The fifth participant with visual loss for less than 12 months received the low dose. Treated participants were followed for 90 to 180 days and underwent ocular and systemic safety assessments along with visual structure and function examinations. PARTICIPANTS: Five legally blind patients with G11778A LHON. MAIN OUTCOME MEASURES: Loss of visual acuity. RESULTS: Visual acuity as measured by the Early Treatment Diabetic Retinopathy Study (ETDRS) eye chart remained unchanged from baseline to 3 months in the first 3 participants. For 2 participants with 90-day follow-up, acuity increased from hand movements to 7 letters in 1 and by 15 letters in 1, representing an improvement equivalent to 3 lines. No one lost vision, and no serious adverse events were observed. Minor adverse events included a transient increase of intraocular pressure (IOP), exposure keratitis, subconjunctival hemorrhage, a sore throat, and a transient increase in neutralizing antibodies (NAbs) against AAV2 in 1 participant. All blood samples were negative for vector DNA. CONCLUSIONS: No serious safety problems were observed in the first 5 participants enrolled in this phase I trial of virus-based gene transfer in this mitochondrial disorder. Additional study follow-up of these and additional participants planned for the next 4 years is needed to confirm these preliminary observations.

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.

Induction of rapid and highly efficient expression of the human ND4 complex I subunit in the mouse visual system by self-complementary adeno-associated virus.

OBJECTIVE: To demonstrate the high efficiency and rapidity of allotopic expression of a normal human ND4 subunit of complex I in the vertebrate retina using a self-complementary adeno-associated virus (scAAV) vector for ocular gene delivery to treat acute visual loss in Leber hereditary optic neuropathy (LHON). METHODS: The nuclear-encoded human ND4 subunit fused to the P1 isoform of subunit C of adenosine triphosphate synthase (ATPc) mitochondrial targeting sequence and FLAG epitope was packaged in scAAV2 capsids or single-stranded (ss) AAV2 capsids. These constructs were injected into the vitreous cavities of mice. The contralateral eyes were injected with scAAV-green fluorescent protein (GFP). One week later, pattern electroretinograms and gene expression of the human ND4 subunit and GFP were evaluated. Quantitative analysis of ND4FLAG-injected eyes was assessed relative to Thy1.2-labeled retinal ganglion cells (RGCs). RESULTS: Pattern electroretinogram amplitudes remained normal in eyes inoculated with scAAV-ND4FLAG, ssAAV-ND4FLAG, and GFP. Confocal microscopy revealed the typical perinuclear mitochondrial expression of scAAV-ND4FLAG in almost the entire retinal flat mount. In contrast, scAAV-GFP expression was cytoplasmic and nuclear. Relative to Thy1.2-positive RGCs, quantification of scAAV-ND4FLAG-positive RGCs was 91% and that of ssAAV-ND4FLAG-positive RGCs was 51%. CONCLUSION: Treatment of acute visual loss due to LHON may be possible with a normal human ND4 subunit gene of complex I, mutated in most cases of LHON, when delivered by an scAAV vector. Clinical Relevance Unlike most retinal degenerations that result in slowly progressive loss of vision over many years, LHON due to mutated mitochondrial DNA results in apoplectic, bilateral severe and usually irreversible visual loss. For rescue of acute visual loss in LHON, a highly efficient and rapid gene expression system is required.