Leber's Hereditary Optic Neuropathy-Gene Therapy: From Benchtop to Bedside

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disorder caused by point mutations in mitochondrial DNA (mtDNA). Most cases are due to mutations in genes encoding subunits of the NADH-ubiquinone oxidoreductase that is Complex I of the electron transport chain (ETC). These mutations are located at nucleotide positions 3460, 11778, or 14484 in the mitochondrial genome. The disease is characterized by apoplectic, bilateral, and severe visual loss. While the mutated mtDNA impairs generation of ATP by all mitochondria, there is only a selective loss of retinal ganglion cells and degeneration of optic nerve axons. Thus, blindness is typically permanent. Half of the men and 10% of females who harbor the pathogenic mtDNA mutation actually develop the phenotype. This incomplete penetrance and gender bias is not fully understood. Additional mitochondrial and/or nuclear genetic factors may modulate the phenotypic expression of LHON. In a population-based study, the mtDNA background of haplogroup J was associated with an inverse relationship of low-ATP generation and increased production of reactive oxygen species (ROS). Effective therapy for LHON has been elusive. In this paper, we describe the findings of pertinent published studies and discuss the controversies of potential strategies to ameliorate the disease

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

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. 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. 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. 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

Clinical utility gene card for : inherited optic neuropathies including next-generation sequencing-based approaches

Non peer reviewe

Characterization of active miniature inverted-repeat transposable elements in the peanut genome

Miniature inverted-repeat transposable elements (MITEs), some of which are known as active non-autonomous DNA transposons, are found in the genomes of plants and animals. In peanut (Arachis hypogaea), AhMITE1 has been identified in a gene for fatty-acid desaturase, and possessed excision activity. However, the AhMITE1 distribution and frequency of excision have not been determined for the peanut genome. In order to characterize AhMITE1s, their genomic diversity and transposition ability was investigated. Southern blot analysis indicated high AhMITE1 copy number in the genomes of A. hypogaea, A. magna and A. monticola, but not in A. duranensis. A total of 504 AhMITE1s were identified from the MITE-enriched genomic libraries of A. hypogaea. The representative AhMITE1s exhibited a mean length of 205.5 bp and a GC content of 30.1%, with AT-rich, 9 bp target site duplications and 25 bp terminal inverted repeats. PCR analyses were performed using primer pairs designed against both flanking sequences of each AhMITE1. These analyses detected polymorphisms at 169 out of 411 insertional loci in the four peanut lines. In subsequent analyses of 60 gamma-irradiated mutant lines, four AhMITE1 excisions showed footprint mutations at the 109 loci tested. This study characterizes AhMITE1s in peanut and discusses their use as DNA markers and mutagens for the genetics, genomics and breeding of peanut and its relatives

Marker-Assisted Selection for Biotic Stress Resistance in Peanut

Peanut is the second-most important legume grown worldwide. Cultivated peanut is a disomic tetraploid, 2n—4x—40, with limited genetic diversity due to a genetic bottleneck in formation of the polyploid from ancestors A. duranensis and A. ipaensis. Consequently, resistance_to biotic stresses is limited in the cultigen; however, wild species possess strong resistances. Transfer o f these resistances is hindered by differences o f ploidy, but production o f synthetic amphidiploids, coupled with use o f molecular markers, enables efficient gene transfer. Marker maps have been made from interspecific crosses, and SSR-based maps from cultivated parents have been developed recently. At least 410 resistance gene analogues have been identified. The first markers for biotic stress tolerance were for root-knot nematode resistance and introgressed from one A. cardenasii chromosome. These and improved markers have been used for marker-assisted backcrossing, contributing to release of three cultivars. Additional QTLs have been identified since. Early and late leafspots cause significant yield losses worldwide, and resistance depends on multiple genes. Using interspecific populations, five resistance QTLs for early leafspot were identified using greenhouse inoculations, and five QTLs for late leafspot were identified using detached leaf assays. Using cultivated species populations, 28 QTLs were identified for LLS resistance; all but one were minor QTLs; the major QTL was donated by an interspecific introgression line parent. Rust often occurs alongside leafspots, and rust resistance was characterized as one major QTL, plus several smaller QTLs. Marker-assisted backcrossing o f this major QTL has been performed into different populations. QTLs for resistance to other biotic stresses have been identified, namely to groundnut rosette virus, Sclerotinia blight, afiatoxin contamination, aphids, and tomato spotted wilt virus. Marker-assisted breeding is still in early stages, and development o f more rapid and inexpensive markers from transcriptome and genome sequencing is expected to accelerate progress

Review Article Leber's Hereditary Optic Neuropathy-Gene Therapy: From Benchtop to Bedside

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disorder caused by point mutations in mitochondrial DNA (mtDNA). Most cases are due to mutations in genes encoding subunits of the NADH-ubiquinone oxidoreductase that is Complex I of the electron transport chain (ETC). These mutations are located at nucleotide positions 3460, 11778, or 14484 in the mitochondrial genome. The disease is characterized by apoplectic, bilateral, and severe visual loss. While the mutated mtDNA impairs generation of ATP by all mitochondria, there is only a selective loss of retinal ganglion cells and degeneration of optic nerve axons. Thus, blindness is typically permanent. Half of the men and 10% of females who harbor the pathogenic mtDNA mutation actually develop the phenotype. This incomplete penetrance and gender bias is not fully understood. Additional mitochondrial and/or nuclear genetic factors may modulate the phenotypic expression of LHON. In a population-based study, the mtDNA background of haplogroup J was associated with an inverse relationship of low-ATP generation and increased production of reactive oxygen species (ROS). Effective therapy for LHON has been elusive. In this paper, we describe the findings of pertinent published studies and discuss the controversies of potential strategies to ameliorate the disease

Gene Therapy for Leber Hereditary Optic Neuropathy

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