Mitochondrial diseases affect at least 1 in 4,300 people and as a group, it represents the most common form of inherited neuromuscular diseases in the population (1,2). The associated clinical phenotype is extremely heterogeneous, ranging in severity from early-onset, frequently fatal, childhood encephalomyopathies to late-onset, progressive neurodegenerative syndromes that result in significant chronic morbidity and impaired quality of life. Ocular involvement is a prominent feature and over half of all patients with an underlying mitochondrial cytopathy will manifest one or more ophthalmologic manifestations with a particular predilection for the optic nerve, the outer retina, and the extraocular muscles. Vision is the most precious of our senses and unsurprisingly, the risk of blindness is a major cause of concern for patients and their families. The first pathogenic mutations associated with mitochondrial disease were identified in 1988 and the intervening years have witnessed an exponential increase in the number of causative genes and a better understanding of the disease mechanisms that contribute to cell loss and clinical deterioration. Despite these remarkable achievements, effective treatments for patients with mitochondrial disease still remain elusive and the translational gap remains to be bridged. Although one cannot underestimate the scale of the challenges involved, we have now reached a confluence of scientific and technological breakthroughs that could herald the dawn of a new era of personalized genetic medicine. In this review, we will cover potentially transformative therapeutic strategies for patients with mitochondrial eye diseases, including the unknowns and the ethical considerations.
MITOCHONDRIAL DISEASE PARADIGMS
Mitochondrial diseases are categorized into 2 main groups, primary mitochondrial DNA (mtDNA) disorders caused by pathogenic mutations within the mitochondrial genome and nuclear mitochondrial disorders, which are linked with an ever-increasing list of nuclear genes that encode for key structural and functional subunits of the mitochondrial machinery (3). To illustrate the therapeutic potential of genetic manipulation for mitochondrial eye diseases, 3 specific examples will be discussed in this review: Leber hereditary optic neuropathy (LHON); the mitochondrial phenotypes associated with the m.8993T>G (MT-ATP6) mtDNA mutation; and autosomal-dominant optic atrophy (DOA) secondary to pathogenic mutations within the nuclear-encoded OPA1 gene (3q28-q29).
LHON is the most common primary mtDNA disorder and it is the classical paradigm of a mitochondrial optic neuropathy (4,5). Most patients harbor one of the 3 pathogenic mutations that all affect complex I subunits of the mitochondrial respiratory chain: m.3460G>A (MT-ND1), m.11778G>A (MT-ND4), and m.14484T>C (MT-ND6), of which the m.11778G>A mutation is by far the most common mutation (60%–90%) (6). Patients present with bilateral sequential or simultaneous severe visual loss, which is characterized by a dense central or cecocentral scotoma expanding over an acute disease phase lasting about 6 months (4,5). LHON is characterized by incomplete penetrance and a rather intriguing gender bias with the overall attributable risk of visual loss being 50% for a male carrier compared with 10% for a female carrier. Although spontaneous visual recovery can occur, usually within the first year of disease onset, most patients will remain severely visually impaired and legally blind (7).
The m.8993T>G (MT-ATP6) mtDNA mutation affects one of the 2 mitochondrially encoded subunits of complex V (ATP synthase) and its phenotypic manifestation is largely dictated by the cellular level of the mutation (8,9). The mitochondrial genome has a number of unique characteristics. First, it is strictly maternally inherited due to the selective destruction of paternal mtDNA molecules following fertilization. Second, it is relatively segregated within the mitochondrial matrix compartment, being physically separated from the cytosol by the double-membrane structure of the mitochondrion. Third, compared with the nuclear genome, mtDNA is a very high copy number genome with 2–10 mtDNA molecules per mitochondrion and up to 100,000 copies in metabolically active cells that are particularly reliant on mitochondrial oxidative phosphorylation (OXPHOS). As a result of this high copy number, 2 situations can arise in the presence of a pathogenic mtDNA mutation (10). The latter can exist in the homoplasmic state with all the mtDNA molecules in a cell harboring the mutant variant, or it can be heteroplasmic with a combination of both wild-type and mutant mtDNA molecules. At a heteroplasmy level of less than 70%, the mitochondrial respiratory chain is able to compensate for the m.8993T>G mtDNA mutation and the carrier is clinically unaffected. Above this threshold, however, there is worsening biochemical decompensation, the severity of which translates into the patient's disease burden. When the level of the mutation is between 70% and 90%, patients can develop the NARP phenotype, which is a historical acronym that highlights the main clinical features of neurogenic muscle weakness, ataxia, and retinitis pigmentosa (Fig. 1) (8,9). The pattern of visual loss will vary depending on the extent of peripheral and/or central retinal involvement, and the rate of progression, which is highly variable both within and between families carrying the m.8993T>G mutation (11). Although there are limited pathological data, the observed pigmentary changes are likely to be due to outer retinal degeneration affecting the retinal pigment epithelium (RPE) and the rod and cone photoreceptors (12–14). Mutation carriers with heteroplasmy levels exceeding 90% develop a more generalized, early-onset Leigh or Leigh-like neurodegenerative disease with a subacute necrotizing encephalomyelopathy that is fatal in nearly half of all patients before the age of 3 years (9).
With the advent of next-generation exome and whole-genome sequencing, an increasing number of dominantly inherited nuclear genes have been identified that result in either isolated or syndromic optic atrophy (15–18). Despite this genetic heterogeneity, at least 60% of cases of DOA are caused by pathogenic mutations within the OPA1 gene (19,20), which encodes for an inner mitochondrial membrane protein involved in regulating a host of functions ranging from mitochondrial biogenesis to mtDNA maintenance, apoptosis, and the balance between mitochondrial fusion and fission (21,22). Similar to LHON, retinal ganglion cells (RGCs) within the papillomacular bundle are exquisitely sensitive to the mitochondrial dysfunction precipitated by OPA1 mutations. Patients typically present in early childhood with subnormal vision and although the rate of progression tends to be relatively slow, about half of all patients are registered legally blind by the fifth decade of life (23,24).
Traditional therapeutic interventions, mostly involving orally administered compounds with putative antioxidant or mitochondrial OXPHOS boosting properties, have met with limited success for the above mitochondrial eye diseases, probably reflecting poor local bioavailability and the lack of precise molecular targeting (25). To address these fundamental limitations, more sophisticated strategies have been developed, or are being refined, with the nonmutually exclusive aims of correcting the underlying genetic defect, enhancing neuronal survival, or making use of accessory retinal pathways to stabilize or improve visual function. Inevitably, the disease might be too advanced in some patients or the window of opportunity might prove too narrow to salvage sufficient cells and make a real difference to the patient's vision. With these considerations in mind, an attractive, but controversial approach to eliminate mitochondrial disease is to target the germline directly and prevent the transmission of the genetic defect to the next generation.
STRATEGIES FOR MINIMISING DISEASE
The correction of the underlying genetic defect is the obvious strategy for inherited monogenic diseases that have been characterized at the molecular level and for which, there is a relatively clear understanding of the pathological pathway(s) that contribute to cell loss and tissue dysfunction (26). Although simple in principle, a number of factors need to be considered starting with the mode of inheritance and the functional consequences of the mutation at the protein level. The most straightforward scenario is an autosomal-recessive disease where the lack of the wild-type protein can be countered by inserting another gene copy into the nuclear genome (Fig. 2A). The situation for autosomal-dominant diseases is more complicated depending on whether the cellular phenotype is being driven primarily by haploinsufficiency (Fig. 2B), or a mutant protein exhibiting gain-of-function or a dominant-negative effect (Fig. 2C). For some genes, the expression of both wild-type alleles is needed to produce sufficient amount of protein for normal function. In the context of haploinsufficiency, supplementation with a copy of the wild-type gene should therefore be sufficient to restore the native protein to a level that achieves cell rescue. If the mutation results in an aberrant protein with a direct toxic effect or one that is stable enough to compromise the wild-type protein, for example, through abnormal aggregation, the expression of the mutant allele will first have to be suppressed with the use of antisense oligonucleotides, ribozymes, or RNA interference (Fig. 2D).
Vector design is an important consideration in any gene therapy program to achieve efficient transgene delivery to the target cell. A number of technical challenges had to be overcome in the early days, the details of which are outside the scope of this article, but there is now sufficient experimental and clinical data to make some general recommendations (27,28). The adeno-associated virus (AAV) has emerged as an effective and safe viral vector for human clinical trials, especially for ocular gene therapy. The AAV serotype 2 (AAV2) in particular has a natural tropism for retinal cell types and it can induce prolonged levels of gene expression to hopefully maximize the intended therapeutic effect. Considerable experience has been gained over the past decade in the field of retinal neurodegenerative diseases and promising results have been obtained in clinical trials for patients with Leber congenital amaurosis (LCA) and choroideremia caused by recessive null mutations in the RPE65 and CHM genes, respectively (29–34). Importantly, these seminal studies have emphasized the advantages of early intervention and the need to achieve more sustained augmentation of the wild-type protein by further optimizing viral vector design if the benefit observed in the first few years of treatment is to be maintained in the long term (35,36).
DOA is an ideal disorder for a proof-of-concept study as most OPA1 mutations result in haploinsufficiency and the relatively slow progression of visual loss affords an adequate window for timely intervention (19,20). Importantly, the transgene is within the size requirement to be packaged into an AAV2 delivery vector and there are well-established mutant Opa1 mouse models to test and optimize the efficiency of RGC transfection (22). Unexpected challenges are to be expected in any groundbreaking research program and one important preliminary finding is the need to carefully titrate the level of murine Opa1 expression as supraphysiological levels are actually detrimental to neuronal survival (Guy Lenaers, PhD, oral communication, June 2016). Ultimately, the positive results obtained in the preclinical phase will need to be validated in affected OPA1 mutation carriers and this endeavor will entail other practical considerations relating to optimal study duration, reliable outcome measures and crucially, funding (37).
Allotopic Gene Expression
Gene therapy to correct a pathogenic mtDNA mutation is technically more challenging because of the need to cross 2 membranes before gaining access to the mitochondrial matrix compartment. An ingenious approach to circumvent these physical barriers is allotopic gene expression where a modified version of the mitochondrial gene to be replaced is inserted into the nuclear genome and the resulting protein carries a specific mitochondrial targeting sequence that allows for its efficient transfer into mitochondria. The seminal in vitro work on allotopic gene expression was based on the rescue of ATP synthesis in cybrid cells carrying the m.8993T>G (MT-ATP6) mutation with an AAV delivery vector (38). Cybrid cells are immortalized cell lines that allow the effects of a particular mtDNA variant to be studied on the same nuclear background, thereby removing an important source of variation. These exciting results were followed very shortly by the validation of this gene delivery system in LHON cybrid cells with the m.11778G>A (MT-ND4) mutation, indicating that this could be a generic approach potentially applicable to all pathogenic mtDNA mutations (39). The application of allotopic gene expression to correct the m.8993T>G mutation to prevent RPE and photoreceptor degeneration has not been attempted yet, but the field is ripe for further exploration, more so now that there are established surgical techniques for the subretinal delivery of the viral vector. In comparison, a significant amount of preclinical work has been conducted over the past 15 years for the m.11778G>A mutation, moving rapidly from cell-based to murine and primate models (40,41), and culminating more recently with the launch of pivotal clinical trials for LHON patients confirmed to harbor this common mtDNA mutation (NCT02652767, NCT02652780 and NCT02161380, https://clinicaltrials.gov/ct2/home, accessed July 28, 2016). There is excitement that we are now at the cusp of a potentially game-changing intervention, but this enthusiasm needs to be tempered by the need to demonstrate clinical efficacy, safety, and acknowledgement of an ongoing debate in the LHON scientific community whether there is stable integration of the wild-type ND4 subunit into complex I of the mitochondrial respiratory chain to produce a biochemically functional unit (42).
The long-term data from the LCA gene therapy trials have shown a regression of the initial improvement of vision and parallel histopathological studies have shown progression of outer retinal degeneration (35,36). The restoration of RPE65 protein levels is not sufficient on its own and additional remedial strategies are needed to counter the ongoing neurodegenerative process affecting the outer retina in LCA. This is not unexpected as we still do not have a complete picture of the complex pathological pathways that lead to cell loss when a specific component is disturbed, and only restoring the protein level might not prove curative when the cell has partially compensated, perhaps in maladaptive fashion, to years of deranged cellular metabolism. The correction of the genetic defect coupled with a more general neuroprotective approach to promote cell survival is a pragmatic dual strategy. A number of promising neurotrophic factors have been identified, such as ciliary neurotrophic factor, glial cell–derived neurotrophic factor and brain-derived neurotrophic factor, and future work is needed to determine whether increasing their levels locally within the retina following AAV2-mediated transfection could prolong the therapeutic effect of replacing the defective gene in LHON, NARP, and DOA (43). Potentially, gene therapy could be used to increase the expression of key mediators involved in the cell's antioxidant defense mechanisms, in particular superoxide dismutase (SOD). In addition to impaired mitochondrial OXPHOS, increased levels of reactive oxygen species is a major factor driving the apoptotic loss of RGCs in mitochondrial optic neuropathies (44). Interestingly, allotopic expression of the SOD2 gene in mutant m.11778G>A cybrids resulted in increased cell survival, clearly attesting to the applicability of these complementary neuroprotective pathways in salvaging RGCs (45).
Optogenetics is a revolutionary concept in neuroscience that involves transfecting neuronal cells with bacterial opsin genes encoding for specific ion channels to make them photosensitive and able to discharge an action potential (46). Outer retinal diseases represent an ideal target for optogenetics as the bipolar cells and RGCs are still structurally and functionally healthy even in the presence of extensive loss of rods and cones (Fig. 3). A particular class of light-gated opsin channels are the channelrhodopsins, which have been used successfully to restore photosensory responses and visually evoked neural activity in mouse models of retinitis pigmentosa, resulting in a major surge of interest from industrial partners (47–49). Although the translational potential of optogenetics for mitochondrial diseases is obvious for patients with visual loss secondary to outer retinal neurodegeneration, a number of technical issues will need to be resolved before this technology can be tested in clinical trials. As for other gene therapy programs, achieving a sufficiently high level of transfection in the retinal cells being targeted is essential and to avoid cumulative light phototoxicity, more sensitive opsin channels are needed that do not require stimulation with the more damaging blue light spectrum.
A conceptually different approach to gene replacement is the actual correction of the mutation at the DNA level. Until recently, this was deemed impractical, but we now have the tools for more sophisticated genome editing with the CRISPR/Cas9 system, which is analogous to a pair of molecular DNA scissors (50–52). These can be individually engineered to locate and excise the target sequence in the immediate vicinity of the mutation, creating a double-strand break that is then repaired with a custom-made DNA template with the desired sequence. The CRISPR/Cas9 system is especially attractive for autosomal-dominant mutations with gain-of-function or dominant-negative effects and proof-of-concept studies have now been published utilizing plasmids or AAV2 delivery vectors for gene editing of retinal cells in vivo (53,54). Research groups are exploiting the CRISPR/Cas9 system to correct the genetic defect in induced pluripotent stem cells (iPSCs) before differentiation into the required cell types (55). Unlike human embryonic stem cells, iPSCs can be established from relatively accessible postmitotic tissues such as a skin biopsy or peripheral blood mononuclear cells. Several reliable protocols for RPE and photoreceptor differentiation have been published, but there is still no universally agreed method for efficiently transforming and maintaining iPSCs into RGCs (56–58). Once these technical issues have been resolved, other major challenges remain relating to the integration of these differentiated RGCs into the inner retina, in addition to ensuring the right axonal projections and connections to the lateral geniculate nucleus. A more realistic work flow would be to establish iPSCs, correct the mutation with CRISPR/Cas9 and then differentiate the established iPSC clones into retinal glial cells, which can promote RGC survival locally through the secretion of various neurotrophic factors (59,60). An even more exciting approach would be to accentuate this effect by transfecting the cells to be transplanted with genes encoding for a cocktail of neurotrophic factors and/or antiapoptotic mediators, as described earlier.
STRATEGIES FOR PREVENTING DISEASE
Germline Genome Editing
Mitochondrial diseases cause significant chronic morbidity and in a proportion of cases, especially children with rapidly progressive encephalomyopathies or neurometabolic complications, the outcome is invariably fatal (10). Family planning for women of childbearing age is a complex area of medical practice that requires a multidisciplinary team of physicians working closely with genetic counselors and fertility experts. For women who are known to carry a nuclear-encoded mitochondrial gene, the risk of disease transmission will be dictated by the mode of inheritance although the situation can be more complicated if there is incomplete or variable penetrance. The CRISP/Cas9 technology has been put forward as a method for correcting specific genetic defects in oocytes, but this is less of a pressing issue for recessive and dominant nuclear mutations as there are already established in vitro fertilization (IVF) protocols based on preimplantation genetic diagnosis (PGD). Genetic counseling is more complicated for women who carry pathogenic mtDNA mutations and 2 scenarios will be discussed to illustrate the potential dilemmas—an asymptomatic female carrier who is 60% heteroplasmic for the m.8993T>G mutation, and a visually unaffected female carrier who is homoplasmic for the m.11778G>A mutation.
A female carrier who harbors 60% of the m.8993T>G mutation will not express the biochemical phenotype. However, her oocytes will contain a broad distribution of heteroplasmy with some being above the disease threshold of 70% or even homoplasmic mutant. These drastic shifts in the level of the mutant mitochondrial allele arise as a result of the “mitochondrial bottleneck,” which operates in the early stages of oocyte development to protect the human species from the irreversible accumulation of deleterious mtDNA mutations through selection (Muller ratchet) (61–63). Although PGD is an option for a woman carrying a heteroplasmic mtDNA mutation, there is limited worldwide experience and a number of issues need to be considered, namely, the inability in some cases to select a viable embryo with a sufficiently low mutational load, and the possibility that the level of the mutation in the biopsied blastomeres or trophectoderm is not representative of other tissues, in particular those that are most at risk (64,65). To address this area of unmet clinical need, a number of research groups have focused on methods to shift the level of mtDNA heteroplasmy in various cell models and more recently, in mouse models of mitochondrial disease with the aim of eventually transferring this technique to human oocytes (66,67). The principle is underpinned by the use of mitochondrial-targeted nucleases that can be designed to detect the altered restriction sites in the region surrounding a particular mtDNA mutation. Two attractive experimental protocols have emerged involving zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), both of which create double-strand DNA breaks to selectively target mutant mtDNA molecules for destruction (68–71). The technology for heteroplasmy shifting has matured over the past few years, but we are still some way off from clinical implementation due to safety concerns about potential off-target effects affecting the nuclear genome and the inability, to carefully titrate the level of the mutation without causing pathological secondary mtDNA depletion. The future looks promising as more efficient delivery methods are being devised to introduce ZFNs and TALENs with more sophisticated targeting designs into the mitochondrial matrix where the mtDNA molecules are packaged within nucleoid structures.
Heteroplasmy shifting is obviously not a viable reproductive option for a female carrier carrying a homoplasmic mtDNA mutation and most LHON carriers (over 90%) are homoplasmic mutant (6). Genetic counseling is further complicated by our inability to accurately predict who is going to lose vision beyond the age-dependent penetrance of the mtDNA mutation and the well-established male predominance. Therefore, what are the possible reproductive options for a woman who carries the m.11778G>A mutation at homoplasmic or near homoplasmic levels? Sex selection for a daughter would reduce, but not completely remove the absolute risk of visual loss or the woman could opt for oocyte donation. A different experimental strategy, known as mitochondrial replacement, is being developed that could potentially allow a female LHON carrier to have her own biological child, but without the latter inhering the pathogenic mtDNA mutation present in her oocytes (72). Two groups in Newcastle upon Tyne (United Kingdom) and Portland, OR have pioneered this field of research with the development of 2 related IVF techniques, pronuclear transfer and metaphase II spindle transfer, respectively. In pronuclear transfer, after the mother's oocyte has been fertilized with the father's sperm, the parental pronuclei are removed from the single-cell embryo before being transferred into a mitochondrial donor zygote harboring only wild-type mtDNA (Fig. 4A) (73). The maternal spindle is the structural unit that packages the mother's nuclear DNA in the unfertilized oocyte. In metaphase II spindle transfer, the mother's metaphase II spindle is transferred into a mitochondrial donor oocyte and this is then followed by intracytoplasmic sperm injection fertilization (Fig. 4B) (74). Both IVF techniques have shown promising results with minimal carryover of mutant mtDNA and crucially, viable embryonic development and the births of healthy offspring in a primate model (75,76).
Both pronuclear transfer and metaphase II spindle transfer are being further refined in readiness for clinical application, but looking beyond the technical considerations, several complex ethical, moral, and societal questions need to be considered as part of an open debate about technologies that will irreversibly alter the germline for future generations (26,77). Children born as a result of mitochondrial replacement will inherit all their genetic make-up from their biological parents, except for the 37 mitochondrial genes inherited from the female donor oocyte, which is somewhat at odds with the “3-parent babies” label that has gained traction in the lay press. Mitochondrial replacement does imply germline modification and some experts have proposed that this technique be limited to male embryos to avoid transmitting any unknown adverse effects to subsequent generations. The counterarguments are the limited availability of donor oocytes and the need for further, and potentially deleterious, embryo manipulation to isolate a single cell for sex determination by karyotyping. Another controversy is centered on the risks of nuclear mitochondrial incompatibility and the need, if any, for mtDNA haplogroup selection in reducing the long-term hypothetical consequences on mitochondrial homeostasis, including metabolic adaptation, immunity, and aging (78,79). There can be no fail-safe guarantee with the introduction of a new technique, but the stakes with mitochondrial replacement are clearly magnified by the germline consequences and the fear that unwanted side effects might not become apparent for many decades after the birth of a seemingly healthy child (26,77). After a long period of national consultation involving all the major stakeholders, including the Nuffield Council on Bioethics, both Houses of Parliament in the United Kingdom have voted strongly in favor of mitochondrial donation to prevent the maternal transmission of mitochondrial disease in February 2015 (http://www.parliament.uk/business/news/2015/february/lords-mitochondrial-donation-si/, accessed July 28, 2016). Future clinical applications will still need to be approved first by the Human Fertilisation and Embryology Authority (HFEA, United Kingdom) and the criteria for implementation are currently being discussed, including the regulatory framework for monitoring. Coming back to our scenario of a female carrier carrying a homoplasmic m.11778G>A mtDNA mutation, the balance of risks and benefits also need to be put in the context of the incomplete penetrance and the non–life-threatening nature of the LHON phenotype, although no one can deny the major impact of bilateral severe visual loss in an otherwise young and healthy adult individual.
We now stand at an important crossroad in our fight against mitochondrial diseases and the eye is an ideal, relatively accessible, target organ for proof-of-concept and safety studies. The considerable body of knowledge gained about genetic etiology and the associated disease pathways now need to be translated into tangible benefits for patients, a considerable proportion of whom suffer from debilitating and frequently progressive visual failure. The fields of molecular engineering and gene delivery have matured and scientists now have access to an impressive array of tools that can be used to replace or directly correct genetic defects, or in the case of optogenetics, construct alternative retinal pathways for phototransduction and image formation. In parallel, innovative reproductive strategies are being contemplated to prevent the maternal transmission of pathogenic mtDNA, which controversially, involves manipulating the germline. Progress cannot be stopped and rather than impose counterproductive blanket bans, which will inevitably be flouted in countries with less rigorously regulated research environments, scientists need to engage with the public and politicians to introduce a framework that will allow new genetic technologies to be tested and refined, but without losing sight of patient safety as the ultimate guiding principle before any intervention is deemed suitable to be tested in clinical trials.
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