Gene Therapies for the Treatment of Leber Hereditary Optic Neuropathy

Abstract

Introduction Leber hereditary optic neuropathy (LHON) is a rare, blinding, maternally inherited mitochondrial genetic disease in need of effective treatment. LHON is a nonsyndromic optic neuropathy affecting the retinal ganglion cells (RGCs), whose axons form the optic nerve and extend into the brain via the optic chiasm and optic tracts. The physiopathology of LHON is characterized by selective loss of RGCs and their axons, which leads to rapidly progressive bilateral central vision loss. Molecular Genetics and Disease Mechanisms LHON was the first identified inherited human disease associated with point mutations in mitochondrial DNA (mtDNA)1 and it is considered the most common mitochondrial genetic disorder.2 Three primary point mutations are responsible for LHON in ~90% of subjects: m.3460G>A, m.11778G>A and m.14484T>C, respectively in the MT-ND1, MT-ND4 and MT-ND6 mitochondrial genes. These genes code for 3 subunits of the nicotinamide adenine dinucleotide: ubiquinone oxidoreductase, complex I of the mitochondrial respiratory chain. Mutations in these subunits of complex I ultimately impair ATP synthesis through oxidative phosphorylation and increase the production of reactive oxygen species. This combination may lead vulnerable cells like RGCs, under triggering conditions, to cross the threshold for apoptosis.3 RGCs appear to be selectively vulnerable to mitochondrial dysfunction possibly due to their peculiar neuronal architecture, as their intra-retinal axons remain unmyelinated, and other metabolic factors.4,5 This vulnerability may trigger a catastrophic wave of degeneration that manifests clinically with acute or rapidly progressive painless bilateral central vision loss, either simultaneously or sequentially in the 2 eyes.6 Clinical Features and Natural History The prevalence of LHON has been estimated at between 1 in 30,000 to 1 in 50,000 in Northern Europe.4,7,8 The mtDNA mutations are necessary, but not sufficient to cause vision loss, and there is a well-documented incomplete penetrance. It is estimated that ~25% to 50% of males and up to 10% of females who carry one of the primary point mutations will manifest the clinical disease.9 The mitochondrial genotype is the most significant prognostic factor of visual outcome, followed by age at onset of vision loss. Variable visual recovery rates have been reported depending on the criteria used, ranging from 4% to 25% for the m.11778G>A mutation to 22% to 25% for the m.3460G>A mutation, and 37% to 1% for the m.14484T>C mutation.9,10 The m.11778G>A MT-ND4 mutation accounts for about 75% of LHON in North America and Europe,5,11 and an even higher proportion among Asian countries,12 and it is known to cause a severe clinical form of LHON.13 Furthermore, it is well established that the natural history of the disease is different for young-onset patients, especially if they are below the age of 12 years.10,14,15 In a recent meta-analysis focusing on ND4-LHON patients aged at least 15 years at onset of vision loss, only 11.3% showed some spontaneous visual recovery, although the definitions used for recovery varied among studies.13 Asymptomatic carriers of the m.11778G>A MT-ND4 mutation have normal visual function before the expression of the disease.16,17 When triggered, LHON classically manifests as subacute, bilateral, painless central vision loss. The bilateral vision loss is most often sequential, with 1 eye affected first, followed by the second eye within about 6 to 8 weeks after onset in the first eye.9–11 However, 25% to 50% of patients report bilateral involvement at initial presentation.9,11 This likely reflects both true instances of simultaneous bilateral onset, but also cases in which the patient is unaware of vision loss in the first eye until the fellow eye becomes involved.18 Clinical manifestation in the first eye is essentially predictive of bilateral involvement,10,11 and occurrences of long-term unilateral involvement have been very rarely reported in retrospective studies.10 In 1 report of 53 affected LHON patients, 32 of whom carried the m.11778G>A MT-ND4 mutation, 90.6% of patients showed bilateral involvement 6 months after onset.14 In one of the first analyses of a LHON cohort published in the literature, van Senus19 reported that 95.7% of patients were affected bilaterally within 6 months of disease onset. A later study of 107 LHON patients molecularly confirmed to carry one of the 3 primary mtDNA mutations, documented bilateral involvement in 97% of patients within 1 year.10 A more recent prospective natural history study of patients with the m.11778G>A MT-ND4 mutation reported bilateral vision loss within 2 months of onset in 53% of patients, and within 6 months of onset in 80% of patients.20 Vision loss typically progresses to nadir over a median of 6 to 8 weeks.10 In 1 study, 94% of patients reached the nadir within 8 weeks of the onset of vision loss.14 In an earlier study, the average time to visual stabilization was 3.7 months in a sample of 87 eyes.11 Current Management of LHON It is important to establish a molecular diagnosis to help guide genetic counseling. The management of LHON remains largely supportive with the provision of low vision services and in some countries, patients can be registered as legally blind, providing them with access to relevant social services. The first randomized controlled trial for a mitochondrial genetic disorder (RHODOS) was conducted to evaluate the safety and efficacy of idebenone—a synthetic analog of coenzyme Q10—in patients with visual loss from LHON for up to 5 years.21 A total of 85 patients were recruited and randomized in a 2:1 ratio to either idebenone at a dose of 300 mg 3 times per day for 6 months, or placebo. Although the primary endpoint was not met, there was evidence of visual benefit in a subgroup of patients. Idebenone was found to be safe and the visual benefit was maintained following cessation of treatment.22 A concurrent retrospective study of LHON patients treated with variable doses of idebenone also found evidence of benefit, especially in those treated within 1 year of disease onset.23 On the basis of the cumulative data, including an expanded access program, idebenone (Raxone; Chiesi Farmaceutici, Parma, Italy) was approved in June 2015 by the European Medicines Agency under exceptional circumstances for the treatment of LHON.24 Raxone is not approved for the treatment of LHON in North America. In a recently published open-label study of 87 patients affected with one of the 3 major LHON mtDNA mutations and treated with idebenone, a clinically relevant recovery [either a gain of −0.2 logMAR equivalent to 10 letters, or an improvement from off-chart to on-chart best-corrected visual acuity (BCVA) by at least 1 full line on the ETDRS chart (5 letters)] was reported in 46% of patients with an average treatment duration of 25.6 months.25 The current consensus guidelines recommend that all patients with LHON who are within 1 year of disease onset should be offered treatment with idebenone at a dose of 300 mg 3 times per day for a minimum period of 1 year.26 As the treatment response to idebenone is partial and limited to a subgroup of patients, other therapeutic strategies are being explored to improve the visual prognosis in LHON. Gene Therapies for LHON—Preclinical Validation and Clinical Studies The eye is one of the few immune-privileged compartments of the body, and, as such, constitutes an ideal candidate for local gene therapy. Furthermore, RGCs are located on the inner surface of the retina and should be in theory the most easily accessed using drug delivery via standard intravitreal (IVT) injection. These considerations have led researchers to investigate gene therapy approaches for the treatment of vision loss due to LHON. Adenoassociated viruses (AAV) are nonpathogenic viruses that can infect nondividing human cells. Without its Rep gene, the vector DNA persists in the host nucleus as a nonintegrative episome, thereby preventing oncogenesis. AAV of serotype 2 are the most common viral vectors used in clinical trials of gene therapy. Three different research groups have developed AAV2-based viral vectors encoding the human wild-type ND4 gene, with the goal of restoring complex I activity and preventing RGC degeneration in LHON patients. This gene therapy approach is based on the allotopic expression of the therapeutic ND4 transgene, initially developed in yeast27,28 and mammalian cells,29–31 and set as proof of principle for LHON by Guy et al32 in cybrid cells carrying the m.11778G>A mutation (Fig. 1). This approach has been refined in terms of efficiency in mitochondrial import and further developed at the preclinical level by Drs Corral-Debrinski, Sahel and colleagues at the Institut de la Vision, Paris, France,34,37,38 and by Dr Guy’s group.39Figure 1: Key breakthroughs in the development of allotopic gene expression to treat mitochondrial defects. This timeline highlights the key breakthroughs in the development of allotopic gene therapies for the treatment of mitochondrial diseases. Leber hereditary optic neuropathy is the first mitochondrial disease for which a gene therapy was assessed in clinical trials and a market authorization was requested from the European Medicines Agency (EMA). Wallace et al.1; Roucou et al.27; Manfredi et al.29; Corral-Debrinski et al.33; Ellouze et al.34; ClinicalTrials.gov.35; GenSight Biologics website.36The IVT injection of viral vectors containing the therapeutic wild-type ND4 gene allows for the preferential targeting of macular RGCs. In targeted cells, the mutated ND4 gene encoded by the multicopy mitochondrial genome is expressed in the mitochondrial matrix; and its translation coupled with protein folding and assemblage within complex I occurs in the mitochondrial inner membrane. Upon cell transfection, the AAV2 gene therapy vector carries the therapeutic ND4 transgene into the cell nucleus where it is episomically transcribed into a messenger RNA (mRNA), which is later translated by ribosomes associated with the external mitochondrial membrane. The 2 mitochondrial targeting sequences flanking the therapeutic ND4 transcript are necessary not only to address the mRNA directly to the external mitochondrial membrane, but also to optimize the translocation of the newly translated ND4 protein through the inner mitochondrial membrane into the mitochondrial matrix,40 where it can finally integrate within complex I and restore its function, competing with the endogenous mutant ND4 protein. Preclinical studies have demonstrated that recombinant adeno-associated virus 2 (rAAV2)/2-ND4 could effectively rescue ATP production in cultured fibroblasts isolated from ND4-LHON patients,37 and that the therapeutic ND4 protein could successfully integrate into complex I in induced LHON murine models, preventing RGC apoptosis and optic nerve atrophy.37,41,42 A summary of the clinical trials of ocular gene therapy for LHON is presented in Table 1, based on the records listed in the database ClinicalTrials.gov. Table 1 - Clinical Trials of Ocular Gene Therapy for the Treatment of ND4-LHON Sponsor Gene Therapy Product Clinical Phase—Study Design—Study Population Huazhong University of Science and Technology, China rAAV2-ND4 with mitochondrial targeting sequence Phase 1 Open-label study 9 ND4-LHON patients who did not experience spontaneous recovery during 12 mo of observation before treatment administration in their worse-seeing eye Phase 2/3 Open-label study 149 ND4-LHON patients treated in their worse-seeing eye with 5E11 vg/μL Bascom Palmer Eye Institute, University of Miami, USA scAAV2-P1ND4v2 Phase 1 Open-label, dose escalation study 12 ND4-LHON patients treated in their worse-seeing eye: 6 with <1 y of vision loss, 6 with >1 y of vision loss 2 ND4-LHON patients with unilateral vision loss GenSight Biologics, France rAAV2/2-ND4 with mitochondrial targeting sequence Phase 1/2 Open-label, dose escalation study 15 ND4-LHON patients treated in their worse-seeing eye Phase 3 (2 separate trials) Double-masked, randomized, sham-controlled studies RESCUE: 39 ND4-LHON patients at least 15 y old at onset, with 0-6 mo of vision loss REVERSE: 37 ND4-LHON patients at least 15 y old at onset, with 6-12 mo of vision loss Right eye randomized to either 9E10 vg or a sham IVT injection, left eye received treatment not allocated to right eye Phase 3 Double-masked, randomized, placebo-controlled study REFLECT: 98 ND4-LHON patients at least 15 y old at onset, with 0-12 mo of vision loss 1:1 patient randomization in 2 treatment arms: Bilateral IVT injection of 9E10 vg or First-affected eye received 9E10 vg, second-affected eye received a placebo IVT injection IVT indicates intravitreal; LHON, Leber hereditary optic neuropathy; rAAV2, recombinant adeno-associated virus 2 vg, viral genomes. In 2011, a phase 1 clinical trial of rAAV2-ND4 for the treatment of ND4-LHON was initiated by the Huazhong University of Science and Technology (ClinicalTrials.gov identifier: NCT01267422). This open-label study included 9 patients with ND4-LHON who were first followed for 12 months, then received a single IVT injection of gene therapy in their worse-seeing eye at a dose of 5E9 viral genomes (vg) if they were below the age of 12, and 1E10 vg if they were older. Unexpectedly, 4 of 9 patients showed a clinically meaningful improvement of BCVA (ie, a change better than −0.3 logMAR, equivalent to at least +15 ETDRS letters) in both eyes. In patients who had <2 years of vision loss at treatment administration, the mean change in BCVA from baseline to 36 months was −0.3 logMAR in the injected eyes and −0.35 logMAR in the uninjected eyes, respectively, equivalent to gains of +15 and +18 ETDRS letters.43 In patients who had >2 years of vision loss at treatment administration, the mean change in BCVA from baseline to 36 months was −0.4 logMAR in the injected eyes and −0.25 logMAR in the uninjected eyes, respectively, equivalent to gains of +20 and +13 ETDRS letters. These results were sustained up to 7 years after treatment administration.44 Of note, 7 of 9 (78%) patients treated in this study were children below the age of 15 at onset of vision loss (from 7 to 14 y of age), and the 2 patients who were above the age of 15 years at onset did not show a sustained improvement in BCVA. In 2014, a phase 1/2a dose-finding clinical trial was initiated by GenSight Biologics to assess the safety and efficacy of rAAV2/2-ND4 gene therapy for the treatment of ND4-LHON (ClinicalTrials.gov identifier: NCT02064569). Fifteen adult patients with a molecularly confirmed diagnosis of ND4-LHON were enrolled in 4 cohorts treated at increasing doses: 3 patients received a dose of 9E9 vg, 3 patients received a dose of 3E10 vg, 6 patients received a dose of 9E10 vg, and 3 patients received a dose of 18E11 vg.45 Two years after treatment administration, the unilateral injection of rAAV2/2-ND4 gene therapy was well tolerated. The dose 9E10 vg/eye was defined as the dose level with the best benefit/risk ratio for the subjects and was therefore chosen for the ensuing pivotal studies. Also in 2014, a phase 1 clinical trial of scAAV2-P1ND4v2 for the treatment of ND4-LHON was initiated at the Bascom Palmer Eye Institute of the University of Miami (ClinicalTrials.gov identifier: NCT02161380). The open-label dose escalation study included 3 groups of patients: 6 patients with chronic LHON (>1 y of vision loss) treated at 2 different doses [low (1.18E9 vg) and medium (5.81E9 vg)]; 6 patients with acute LHON (<1 y of vision loss) treated at 2 different doses [low (1.18E9 vg) and medium (5.81E9 vg)]; and 2 patients with unilateral vision loss treated at the low dose (1.18E9 vg). Each patient was administered a single IVT injection of gene therapy in their worse-seeing eye. Preliminary results reported signs of efficacy in 2 patients of the chronic group, and 4 patients of the acute group, with follow-up periods varying from 3 to 24 months post treatment administration.46 Changes in BCVA compared with baseline ranged from −0.08 to −0.45 logMAR, equivalent to gains between +4 and +23 ETDRS letters. bilateral improvement of BCVA was reported in 4 of the 6 patients who showed signs of dose response was in the study In GenSight Biologics initiated 2 phase randomized, sham-controlled pivotal and that the efficacy and safety of a unilateral IVT injection of rAAV2/2-ND4 vg) in recently affected ND4-LHON patients to 12 mo of vision loss) with a follow-up of 2 had to be at least 15 years old at the time of onset of vision loss. The right eye of was allocated to either rAAV2/2-ND4 or a sham treatment in a 1:1 The fellow eye received the treatment not allocated to the right eye. rAAV2/2-ND4 was administered via a single IVT injection. IVT injection was by to the eye at the of a using the of a a The 2 studies had an study the only being the vision loss duration at with the study (ClinicalTrials.gov identifier: including patients who had a duration of vision loss months in the and the study (ClinicalTrials.gov identifier: including patients who had a duration of vision loss between 6 and 12 months at in both eyes. and included 39 and 37 patients, The primary defined as a in the change in BCVA from baseline to between the of eyes that received rAAV2/2-ND4 and the of eyes that received a sham injection, was not in either trial due to an and sustained improvement in the Two years after treatment administration, the mean improvement from nadir BCVA of visual in and studies was respectively and ETDRS equivalent in eyes, and and +23 ETDRS equivalent in eyes. a bilateral improvement in BCVA was in study patients who had been injected with This therapeutic on eyes was sustained and clinically the improvement in eyes treated with and patients are followed in an study up to 5 years after injection In this study, the bilateral treatment of rAAV2/2-ND4 was sustained up to 3 years after gene therapy administration In an open-label clinical trial of rAAV2-ND4 for the treatment of ND4-LHON was initiated by the Huazhong University of Science and Technology (ClinicalTrials.gov identifier: A total of 149 patients with ND4-LHON received a single dose of gene therapy vg) in their worse-seeing eye. age for this cohort was and average disease duration at time of treatment administration was months, varying from 1 to months A and significant improvement of BCVA at least −0.3 was reported within 3 in at least 1 eye of of patients and in both eyes in of Although patient age at onset is not the reported mean age at study of years that a proportion of patients treated in this open-label clinical trial were In GenSight Biologics initiated a phase 3 pivotal clinical study, (ClinicalTrials.gov identifier: a randomized, placebo-controlled The study the efficacy and safety of bilateral IVT injection of rAAV2/2-ND4 in ND4-LHON patients with bilateral vision loss within 1 patients received an IVT injection of rAAV2/2-ND4 in the eye and were allocated to an IVT injection of either rAAV2/2-ND4 or placebo in the second-affected eye. The study primary endpoint will be the in change from baseline of BCVA between the of second-affected eyes that received the drug and the of second-affected eyes that received a at years after treatment The results of the primary endpoint will be these pivotal studies have confirmed that ocular gene therapy for LHON via IVT injection has an safety with and ocular to The most common were and and with treatment for and for the IVT injection of rAAV2/2-ND4 showed limited with the vector DNA in the visual and were and did not with the local ocular of the of Gene Therapy in LHON 3 research groups the efficacy and safety of unilateral IVT gene therapy in ND4-LHON patients reported an and clinically meaningful improvement of BCVA in the eyes not with the reported natural history of the for the with rAAV2/2-ND4 gene therapy was in a from that study demonstrated the of viral vector DNA in not only the injected but also in of the uninjected eye in DNA was in uninjected the bilateral treatment reported in LHON patients in the phase 3 and trials and those of the other 2 rAAV2/2-ND4 DNA was and in the optic chiasm of injected the by the viral vector DNA to from the treated eye to the eye is to be via the optic nerve and chiasm and the optic A of rAAV2/2-ND4 DNA be but is likely that studies have limited and of rAAV2/2-ND4 DNA in could also to the of ocular gene therapy in LHON. of mitochondrial mRNA or be at this especially the on this Furthermore, a recent study of an induced of in reported of from a eye to a eye via the optic brain could for the improvement of visual function in the as reported in some subjects with a retinal LHON is a mitochondrial disease with an need for Gene therapies have been to for the mitochondrial by cells of the inner retina with the wild-type gene, thereby restoring the activity of the respiratory and RGCs. Three research groups have developed gene therapy for the treatment of the most and severe genotype of LHON. trials and clinical published studies report a sustained and clinically meaningful bilateral benefit the natural history of the disease in a proportion of ND4 patients who were A study the of viral vector DNA from the injected to the eye into the of this therapeutic IVT administration targeting the RGCs constitutes a safe and to the the of Furthermore, and and have been to be Gene therapy has an safety with and ocular to The results of clinical trials are and could have major for gene therapy clinical trial and the to this treatment to the other LHON

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