Introduction Therapies for Leber hereditary optic neuropathy (LHON), in common with all disorders caused by mutated mitochondrial DNA, are inadequate. We have developed two gene therapy strategies for the disease: mitochondrial-targeted and allotopic expressed and compared them in a mouse model of LHON. Methods A LHON mouse model was generated by intravitreal injection of a mitochondrialtargeted Adeno-associated virus (AAV) carrying mutant human NADH dehydrogenase 4 gene (hND4/m.11778G>A) to induce retinal ganglion cell (RGC) degeneration and axon loss, the hallmark of the human disease. We then attempted to rescue those mice using a second intravitreal injection of either mitochondrial-targeted or allotopic expressed wildtype human ND4. The rescue of RGCs and their axons were assessed using serial pattern electroretinogram (PERG) and transmission electron microscopy. Results Compared to non-rescued LHON controls where PERG amplitude was much reduced, both strategies significantly preserved PERG amplitude over 15 months. However, the rescue effect was more marked with mitochondrial-targeted therapy than with allotopic therapy (p = 0.0128). Post-mortem analysis showed that mitochondrial-targeted human ND4 better preserved small axons that are preferentially lost in human LHON. Conclusions These results in a pre-clinical mouse model of LHON suggest that mitochondrially-targeted AAV gene therapy, compared to allotopic AAV gene therapy, is more efficient in rescuing the LHON phenotype.

Leber’s hereditary optic neuropathy (LHON) is a common mitochondrial genetic disease, causing irreversible blindness in young individuals. Current treatments are inadequate, and there is no definitive cure. This study evaluates the effectiveness of delivering wildtype human NADH ubiquinone oxidoreductase subunit 4 (hND4) gene using mito-targeted AAV(MTSAAV) to rescue LHOH mice. We observed a declining pattern in electroretinograms amplitudes as mice aged across all groups (p < 0.001), with significant differences among groups (p = 0.023; Control vs. LHON, p = 0.008; Control vs. Rescue, p = 0.228). Inner retinal thickness and intraocular pressure did not change significantly with age or groups. Compared to LHON mice, those rescued with wildtype hND4 exhibited improved retinal visual acuity (0.29 ± 0.1 cy/deg vs. 0.15 ± 0.1 cy/deg) and increased functional hyperemia response (effect of flicker, p < 0.001, effect of Group, p = 0.004; Interaction Flicker × Group, p < 0.001). Postmortem analysis shows a marked reduction in retinal ganglion cell density in the LHON group compared to the other groups (Effect of Group, p < 0.001, Control vs. LHON, p < 0.001, Control vs. Rescue, p = 0.106). These results suggest that MTSAAV-delivered wildtype hND4 gene rescues, at least in part, visual impairment in an LHON mouse model and has the therapeutic potential to treat this disease.

Abstract Background Mutation of the m. 8993 T > G ATP6 subunit of ATP synthase causes a maternally inherited Leigh Syndrome (LS), a rapidly fatal encephalomyelopathy in childhood called and related Neuropathy, Ataxia and Retinitis Pigmentosa (NARP) in adults. There is no cure, and relevant animal models are few. Here we describe a novel ATP6 mouse model of LS and preclinical efficacy of a gene therapy approach to neutralize the ATP6 mutation by competition with a mitochondrial targeted adeno‐associated virus serotype 9 (AAV9) vector containing wild‐type ATP6. Methods LS transgenic mice with an ATP6 mutation were developed by microinjecting AAV2 modified by a COX8 mitochondrial targeting sequence (MTS) into mouse blastocysts to deliver the human ATP6 gene with a T > G mutation at position 8993 of the mitochondrial genome, responsible for clinical LS DNA, protein, and metabolic assays confirmed functional gene delivery and in depth physiological/clinical assessments supported germline transmission of an LS phenotype. Rescue of LS in this model was evaluated after intravenous injection of mito‐targeted AAV9 containing wild‐type ATP6 followed by reevaluation of phenotypes. Results LS transgenic mice expressed mutant ATP6 protein in multiple relevant tissues including brain, eye, liver. Mutant ATP6 incorporated into Complex V and conferred decreased ATP synthase efficiency. Mice exhibited hallmarks of the human disease with one or multiple systemic symptoms including early death, paralysis, hunching, vision loss and seizures over six generations. Necropsy revealed spongiform encephalopathy, retinal degeneration, and cardiomegaly. Rescue by mitochondrially directed AAV9 containing wild type ATP6 after disease onset conferred prolonged survival and reduced paralysis. When administered prior to disease onset all treated ATP6 mice survived with evidence of globally improved function. Conclusions The results are consistent with efficient delivery and expression of foreign genes in mitochondria by AAV vectors directed by MTS and support further development of such strategies via intravenous injection of mitochondrial targeted AAV9 to deliver corrective genes.

Purpose The purpose of this study was to compare the baseline steady-state pattern electroretinogram (SS-PERG) of patients with G11778A Leber hereditary optic neuropathy (LHON) with different stages of visual acuity (VA) loss before allotopic gene therapy (GT). Methods Patients (n = 28) were enrolled into groups (GT I: chronic bilateral VA ≤35 Early Treatment Diabetic Retinopathy Study [ETDRS]; GT II: acute bilateral VA ≤35 ETDRS; GT III: acute unilateral, VA ≤35 ETDRS, and better eye VA ≥70 ETDRS) and tested with SS-PERG together with 210 age-matched normal controls (NCs). SS-PERG amplitude (nV) and latency (ms) of each eye were averaged for groups GT I, GT II, and NC. Symptomatic eyes (GT III-S) and asymptomatic eyes (GT III-A) of group GT III were included separately and accounted for by using generalized estimating equation (GEE) methods. Results Compared to NC, SS-PERG amplitudes were reduced similarly by approximately 50% (P < 0.001) among all GT groups (NC > GT I, GT II, GT III-S, and GT III-A). SS-PERG latencies were shorter by ≥3.5 ms in all LHON groups and differed by disease stage (G III-A < NC, P = 0.002; GT III-S < GT III-A, P = 0.01; GT II < GT III-S, P = 0.03; GT I < NC, P < 0.001, but not different from other GT groups, all P > 0.1). Conclusions Although SS-PERG amplitude reduction did not distinguish between disease stages, SS-PERG latency shortening occurred in asymptomatic eyes and symptomatic eyes and distinguished between disease stages. Translational Relevance SS-PERG latency shortening is consistent with primary damage of smaller/slower axons and sparing of larger/faster axons and may provide an objective staging of LHON, which may be helpful to determine efficacy in LHON trials.

Therapies for genetic disorders caused by mutated mitochondrial DNA are an unmet need, in large part due barriers in delivering DNA to the organelle and the absence of relevant animal models. We injected into mouse eyes a mitochondrially targeted Adeno-Associated-Virus (MTS-AAV) to deliver the mutant human NADH ubiquinone oxidoreductase subunit I (hND1/m.3460 G > A) responsible for Leber’s hereditary optic neuropathy, the most common primary mitochondrial genetic disease. We show that the expression of the mutant hND1 delivered to retinal ganglion cells (RGC) layer colocalizes with the mitochondrial marker PORIN and the assembly of the expressed hND1 protein into host respiration complex I. The hND1-injected eyes exhibit hallmarks of the human disease with progressive loss of RGC function and number, as well as optic nerve degeneration. We also show that gene therapy in the hND1 eyes by means of an injection of a second MTS-AAV vector carrying wild-type human ND1 restores mitochondrial respiratory complex I activity, the rate of ATP synthesis and protects RGCs and their axons from dysfunction and degeneration. These results prove that MTS-AAV is a highly efficient gene delivery approach with the ability to create mito-animal models and has the therapeutic potential to treat mitochondrial genetic diseases.

center dot PURPOSE: To assess safety of gene therapy in G11778A Leber hereditary optic neuropathy (LHON). center dot DESIGN: Phase 1 clinical trial. center dot METHODS: Setting : single institution. Participants : Pa-tients with G11778A LHON and chronic bilateral visual loss > 12 months (group 1, n = 11), acute bilateral vi-sual loss < 12 months (group 2, n = 9), or unilateral vi-sual loss (group 3, n = 8). Intervention : unilateral intrav-itreal AAV2(Y444,500,730F)-P1ND4v2 injection with low, medium, high, and higher doses to worse eye for groups 1 and 2 and better eye for group 3. Outcome Measures : Best-corrected visual acuity (BCVA), adverse events, and vector antibody responses. Mean follow-up was 24 months (range, 12-36 months); BCVAs were compared with a published prospective natural history co-hort with designated surrogate study and fellow eyes. center dot RESULTS: Incident uveitis (8 of 28, 29%), the only vector-related adverse event, resulted in no attributable vision sequelae and was related to vector dose: 5 of 7 (71%) higher-dose eyes vs 3 of 21 (14%) low-, medium-center dot or high-dose eyes ( P < .001). Incident uveitis requir-ing treatment was associated with increased serum AAV2 neutralizing antibody titers (p = 0.007) but not serum AAV2 polymerase chain reaction. Improvements of > 15 -letter BCVA occurred in some treated and fellow eyes of groups 1 and 2 and some surrogate study and fellow eyes of natural history subjects. All study eyes (BCVA > 20/40) in group 3 lost > 15 letters within the first year despite treatment. center dot CONCLUSIONS: G11778A LHON gene therapy has a favorable safety profile. Our results suggest that if there is an efficacy effect, it is likely small and not dose related. Demonstration of efficacy requires randomization of pa-tients to a group not receiving vector in either eye. Superscript/Subscript Available

Mutations in mitochondrial genes are primary causes of multiple inherited disorders, especially affecting tissues with high aerobic energy demands such as the eye, brain, heart, and skeletal muscles. Such mitochondrial diseases are untreatable, have poor prognoses, and relevant animal models are few. Here we microinjected into the mouse blastocyst an adeno-associated virus (AAV) that we redirected to mitochondria to deliver the mutant human adenosine triphosphate (ATP) synthase subunit 6 gene (m.8993T>G) responsible for Maternally Inherited Leigh Syndrome, a neurologic disease renowned for causing rapidly fatal encephalomyelopathy in childhood or Neuropathy, Ataxia and Retinitis Pigmentosa in adults. Mice bred over six generations exhibited hallmarks of the human disease with one or multiple systemic symptoms including early death, paralysis, hunching, vision loss and seizures. Necropsy revealed spongiform encephalopathy, retinal degeneration, and cardiomegaly. Intravenous injection of a mitochondrially directed AAV serotype 9 vector containing wild-type ATP6 after disease onset, conferred prolonged survival and reduced paralysis. When administered prior to disease onset, all treated mice survived, and visual and motor function improved. These results support further development of gene therapy strategies involving intravenous delivery of mito-targeted AAV9.

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.

Purpose Leber hereditary optic neuropathy (LHON) is a genetic form of vision loss that occurs primarily owing to mutations in the nicotinamide adenine dinucleotide dehydrogenase (ND) subunits that make up complex I of the electron transport chain. LHON mutations result in the apoptotic death of retinal ganglion cells. We tested the hypothesis that gene therapy with the X-linked inhibitor of apoptosis (XIAP) would prevent retinal ganglion cell apoptosis and reduce disease progression in a vector-induced mouse model of LHON that carries the ND4 mutation. Methods Adeno-associated virus (AAV) encoding full length hemagglutinin-tagged XIAP (AAV2.HA-XIAP) or green fluorescent protein (AAV2.GFP) was injected into the vitreous of DBA/1J mice. Two weeks later, the LHON phenotype was induced by AAV delivery of mutant ND4 (AAV2.mND4FLAG) to the vitreous. Retinal function was assessed by pattern electroretinography. Optic nerves were harvested at 4 months, and the effects of XIAP therapy on nerve fiber layer and optic nerve integrity were evaluated using immunohistochemistry, transmission electron microscopy and magnetic resonance imaging. Results During LHON disease progression, retinal ganglion cell axons are lost. Apoptotic cell bodies are seen in the nuclei of astrocytes or oligodendrocytes in the optic nerve, and there is thinning of the optic nerve and the nerve fiber layer of the retina. At 4 months after disease onset, XIAP gene therapy protects the nerve fiber layer and optic nerve architecture by preserving axon health. XIAP also decreases nuclear fragmentation in resident astrocytes or oligodendrocytes and decreases glial cell infiltration. Conclusions XIAP therapy improves optic nerve health and delays disease progression in LHON.

Purpose: To evaluate the long-term effects of mitochondrial gene transfer of mutant human NADH ubiquinone oxidoreductase subunit VI (hND6T14484C) in the mouse eye. Methods: Adult mice were injected intravitreally with mitochondrial-targeted adeno-associated virus carrying either hND6T14484C or mitochondrial encoded mCherry. The delivery and expression of the interest gene were detected by polymerase chain reaction (PCR), quantitative PCR (qPCR), and immunostaining. The pathologic effects of the mutant gene in live mice were assessed with RNA-seq, serial spectral domain optical coherence tomography (SD-OCT), and pattern electroretinogram (PERG). Results: Delivered hND6 was found 30-fold greater than endogenous mouse ND6 in microdissected retinal ganglion cells of hND6-injected mice. Compared to controls injected with mCherry, PERG amplitude of hND6 mice dropped significantly at 3 (P = 0.0023), 6 (P = 0.0058), and 15 (P = 0.031) months after injection. SD-OCT revealed swelling of the optic nerve head followed by the progressive retinal and optic nerve atrophy in hND6 mice. Furthermore, RNA-seq data showed a change in 381 transcripts' expression in these mice compared to mCherry mice. Postmortem analysis showed hND6 mice had marked atrophy of the entire optic nerve, from the globe to the optic chiasm, and a significant loss of retinal ganglion cells compared to age-matched control mice (P = 1.7E-9). Conclusions: Delivered hND6T14484C induces visual loss and optic neuropathy in mice, the hallmarks of human Leber's hereditary optic neuropathy (LHON). Translational Relevance: Results from this study will help establish a novel strategy not only to generate an LHON animal model but also to provide a potential to treat this or any other mitochondrial diseases.

Purpose Assessment of Ocular Perfusion Pressure (OPP) requires estimation of the Mean Central Retinal Artery Pressure (MCRAP) [OPP = MCRAP-IOP]. In a seated position, MCRAP is currently estimated as 2/3 of the Mean Arterial Pressure (MAP) to account for the hydrostatic reduction of MAP at eye level. We tested a surrogate method for direct MCRAP assessment by measuring MAP with Arm-Up and cuff at eye level (AUMAP) at different postures and ages. Methods MAP and AUMAP were assessed in a mixed population of 136 subjects (mean age 44 +/- 17.39 years) including healthy participants (N = 30) and patients with optic neuropathies (Glaucoma suspects, N = 14; Open-Angle Glaucoma, N = 26, LHON, N = 19; MS, N = 47) not expected to alter systemic blood pressure. None of the subjects had history of carotid stenosis or pharmacological treatment to regulate blood pressure. AUMAP was also tested in two subgroups in supine (N = 42) and -10 degrees Head Down body Tilt position (HDT, N = 46). Results In the seated position, both 2/3MAP and AUMAP increased with increasing age, however with steeper (2x) slope for AUMAP (P< .0001). With decreasing angle of body tilt, AUMAP increased while MAP decreased. The mean AUMAP/MAP ratio (posture coefficient) was, seated, 0.73 (SE 0.003); supine, 0.90 (SE 0.005); HDT, 0.97 (SE 0.005). In the seated position only, the AUMAP/MAP ratio significantly increased with age (P< .0001). Mean posture coefficients obtained with AUMAP were in the range of those based on either direct ophthalmodynamometric measurements or hydrostatic estimations. Conclusions Surrogate measurement of MCRAP in individual subjects is feasible using the simple AUMAP approach that provides a straightforward estimation of OPP (OPP = AUMAP - IOP) at different body postures. The standard method OPP = 2/3*MAP-IOP in the seated posture underestimates OPP at older ages. Clinical estimation of OPP would benefit from the use of AUMAP, in particular for head-down postures.

May 9, 2019April 9, 2019Free AccessREVERSAL OF PARALYSIS AND PREVENTION OF PREMATURE DEATH IN A MOUSE GERMLINE MODEL OF LEIGH SYNDROME CAUSED BY A MUTATION IN MITOCHONDRIAL ATP SYNTHASE (S51.003)John Guy and Huijun YuanAuthors Info & AffiliationsApril 9, 2019 issue92 (15_supplement)https://doi.org/10.1212/WNL.92.15_supplement.S51.003 Letters to the Editor

OBJECTIVE To investigate the pathogenic gene in a child with optic atrophy and analyze the influence of this gene mutation on protein structure. OBJECTIVE We collected the clinical record of the 13-year-old girl and her relatives. The child received examinations of the visual acuity, visual field, fundus, OCT, visual-evoked potential (VEP) and the nerve system, underwent brain MRI and was followed up for 1 year. Genomic DNA was extracted from the peripheral blood of the child and her parents for next-generation sequencing of the whole exon. The pathogenic gene mutation was identified and the resultant changes in the protein structure was analyzed. OBJECTIVE The patient presented with impaired vision and optic nerve atrophy in both eyes with low amplitude of VEP, but did not show dystonia or pyramidal tract symptom. Brain MRI detected no leukodystrophy. Genetic analysis suggested a heterozygous c.53_54delTG mutation in exon 1 in the NDUFV1 gene of complex I, which caused a frameshift starting with the codon valine 18, thus changing the amino acid to an Alanine residue and creating a premature stop codon at position 20 of the new reading frame (p.Val18AlafsX20). A heterozygous for c.1162+4A>C: IVS8 + 4A>C in intron 8 was also found. Protein structure analysis showed the missing of important structure of NDUFV1 subunit in complex I. OBJECTIVE We identified a novel NDUFV1 mutation in a child with optic nerve atrophy. This finding may provide further insight into the genotype-phenotype correlations for NDUFV1 gene.

We read with interest the article by Guy et al1Guy J. Feuer W.J. Davis J.L. et al.Gene therapy for Leber hereditary optic neuropathy: low-and medium-dose visual results.Ophthalmology. 2017; 124: 1621-1634Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar about gene therapy by intravitreous, monocular injection of the AAV2-P1ND4v2 vector carrying the wild-type ND4 gene in 14 patients with Leber hereditary optic neuropathy (LHON) owing to the variant m.11778G>A in the ND4 gene. We have the following comments and concerns. The main disadvantage of the study is the assumption that LHON only affects the optic nerve and the retinal ganglion cells. Although LHON often presents as a single-organ disease, it is in fact a multi-organ disease, which becomes evident only after years or after prospective investigations of organs other than the optic nerve and the retina. It is meanwhile well-established that LHON not only manifests in the eye and optic nerve but may affect other organs and tissues as well, resulting in a more widespread clinical presentation. Other organs and tissues affected in LHON include the central nervous system (brain [multiple sclerosis–like white matter lesions], the spinal cord [diffuse demyelinating lesions of the thoracic spine]), the ears (hypoacusis), the endocrine organs (diabetes, hyperthyroidism), the heart (noncompaction),2Finsterer J. Stöllberger C. Prainer C. et al.Lone noncompaction in Leber's hereditary optic neuropathy.Acta Cardiol. 2004; 59: 187-190Crossref PubMed Scopus (13) Google Scholar the kidneys (renal insufficiency), the peripheral nervous system (neuropathy),3Finsterer J. Zarrouk-Mahjoub S. Leber's hereditary optic neuropathy is multiorgan not mono-organ.Clin Ophthalmol. 2016; 10: 2187-2190Crossref PubMed Scopus (27) Google Scholar the bone marrow (anemia), the vascular system (arterial hypertension, hyperlipidemia), or the spinal column (kyphosis). How to explain that injection of the vector into a single eye should have a beneficial effect on these more distant tissues? Is it conceivable that tingling in legs, numbness of feet, and headache were in fact manifestations of organ involvement other than the eyes or the genetic effect? Did patients with sensory disturbances undergo nerve conduction studies? What were the results? A further objection is that spontaneous remission of visual acuity in LHON patients carrying any of the 3 primary variants has been reported.4Leo-Kottler B. Jacobi F. Christ-Adler M. Leber optic neuropathy with clinical improvement.Ophthalmologe. 2000; 97: 849-854Crossref PubMed Scopus (9) Google Scholar Among the primary LHON mutations, it is particularly the m.11778G>A variant that is associated with a favorable outcome if no further secondary mutations are present.4Leo-Kottler B. Jacobi F. Christ-Adler M. Leber optic neuropathy with clinical improvement.Ophthalmologe. 2000; 97: 849-854Crossref PubMed Scopus (9) Google Scholar In the study from Leo-Kottler et al,4Leo-Kottler B. Jacobi F. Christ-Adler M. Leber optic neuropathy with clinical improvement.Ophthalmologe. 2000; 97: 849-854Crossref PubMed Scopus (9) Google Scholar vision improved spontaneously in all 12 included patients carrying the m.11778G>A variant. Did the authors look for secondary LHON mutations in their 14 patients? How many of them had secondary LHON mutations and which mutations were found? How can the authors exclude the possibility that the presumed therapeutic effect was in fact attributable to spontaneous partial recovery? Did they observe improvement of vision among those who were not injected in either of the 2 eyes, or in the fellow eye among those who were injected? Spontaneous recovery seems to have occurred at least in patients 14 (4>63), 9 (6>9), 5 (20>39), and patient 4 (hand movements >4). Of the 14 included patients 4 were females and 10 males.1Guy J. Feuer W.J. Davis J.L. et al.Gene therapy for Leber hereditary optic neuropathy: low-and medium-dose visual results.Ophthalmology. 2017; 124: 1621-1634Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar Because the course of LHON may be at variance between men and women, we should be informed if course and outcome were different between the genders in the injected eye and noninjected eye. There is hardly spontaneous recovery in female patients carrying any of the 3 primary LHON mutations] and female patients have a higher risk of white matter lesions than male patients.5Matthews L. Enzinger C. Fazekas F. et al.MAGNIMS NetworkMRI in Leber's hereditary optic neuropathy: the relationship to multiple sclerosis.J Neurol Neurosurg Psychiatry. 2015; 86: 537-542Crossref PubMed Scopus (56) Google Scholar Overall, this interesting therapeutic study should consider that LHON is in fact a multi-organ disease, that the course may be different between the genders, that it is unclear if gene therapy could reach organs distal to the eyes, and that spontaneous remission, particularly in male m.11778G>A carriers, should be considered as cause of the improvement. Gene Therapy for Leber Hereditary Optic Neuropathy: Low- and Medium-Dose Visual ResultsOphthalmologyVol. 124Issue 11PreviewTo determine the effects of AAV2(Y444,500,730F)-P1ND4v2 in patients with Leber hereditary optic neuropathy (LHON). Full-Text PDF ReplyOphthalmologyVol. 125Issue 2PreviewPatients entered into our gene therapy study underwent complete physical and neurologic examinations before and after treatment, in addition to the visual studies. They also underwent many laboratory studies, which included hematology, coagulation, renal, and liver profiles. We did not detect any abnormalities other than visual loss. We had replied to this same query previously.1 Newman et al2 described normal electrocardiographs, lumbar punctures, computed tomography scans, and magnetic resonance imaging in all 72 of their 11 778 patients. Full-Text PDF