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Emerging Gene Manipulation Strategies for the Treatment of Monogenic Eye Disease

Burgess, Frederick R. FRCOphth*,†; Hall, Hildegard Nikki FRCOphth*,‡; Megaw, Roly FRCOphth, PhD*,‡

Author Information
Asia-Pacific Journal of Ophthalmology: July/August 2022 - Volume 11 - Issue 4 - p 380-391
doi: 10.1097/APO.0000000000000545
  • Open



The Fast-Changing Field of Genetic Eye Disease

Genetic eye disease is one of the leading causes of visual loss in children and working adults, and progress in the field over the past few decades has led to changes in disease investigation, diagnosis, and management that general ophthalmologists may not be fully aware of. There are now 280 known causal genes for genetic eye disease (RetNet, the Retinal Information Network), highlighting the marked genetic heterogeneity of conditions such as retinitis pigmentosa (RP). Increasingly, these conditions are referred to by the causal gene (eg, RPE65-mediated retinal disease) rather than the traditional phenotype-based diagnosis that most clinicians will be familiar with (such as Leber congenital amaurosis, LCA).1,2 Further, novel biomarkers are increasingly being used to track the natural history of disease.3 Finally, and importantly, the past 15 years have seen the emergence of novel therapies for these previously untreatable conditions to the extent that we now have a licensed therapy for one form of genetic eye disease and several more in clinical trial.

The eye is an attractive organ for the development and application of gene therapies due to its relative immune privilege,4 noninvasive imaging and functional testing, and the availability of a contralateral control eye in most patients.5 As a result, several “first-in-man” studies aimed at tackling single gene disorders have focused on eye conditions. Several approaches can be used, and the choice of approach depends on both the nature and stage of each genetic condition. The pace of change of genetic eye disease research further adds to the burden for the general ophthalmologist. This systematic review, therefore, aims to give an up-to-date overview of treatment options available for genetic eye disease, both in the preclinical and clinical setting, with an introduction to the different modalities of gene therapy.

To gain a better understanding of the different treatment approaches, we need to understand the nature of disease-causing mutations.

Nature of Disease-Causing Mutations

Disease-causing deoxyribonucleic acid (DNA) mutations (pathogenic variants) can be broadly grouped into 3 categories: nonsense, missense, and insertions/deletions (indels) (Fig. 1):

  1. With nonsense mutations, a single nucleotide change results in the amino acid being substituted for a premature stop codon, ie, a signal to terminate protein synthesis. The resulting truncated product is usually degraded by the body’s surveillance systems, either at the messenger ribonucleic acid (mRNA) level (nonsense-mediated decay) or at the protein level, as the truncated proteins are typically nonfunctional (loss of function, LOF), or even deleterious.
  2. With missense mutations, a single nucleotide change results in one amino acid being changed to another. This substitution can alter the structure and/or function of the protein, either reducing its activity (LOF), giving it a new function (gain of function, GOF), or interfering with the healthy copy of the protein (dominant-negative).6 An example of the latter is proteins that assemble into homomeric complexes (eg, to form an ion channel), where one mutant subunit perturbs the whole assembly. It should also be noted that many missense mutations do not alter structure or function and enter the population as polymorphisms.
  3. With indel mutations, individual nucleotides or small sections of DNA are inserted into or deleted from a gene. This can disrupt the function of the resulting protein (LOF) or give it a new function (GOF). Most often, however, it causes a “frameshift.” RNA is read in triplet codons and so if the indel is not in a multiple of 3, the reading frame is shifted, often introducing a premature stop codon and subsequent LOF.

Categories of disease-causing DNA mutations. DNA indicates deoxyribonucleic acid; NMD, nonsense-mediated decay.

Frameshift and nonsense mutations are sometimes termed “likely gene-disruptive” mutations. In addition to the above, larger chromosomal deletions can also cause LOF by deleting one copy of a gene (plus or minus neighboring genes).

Other than those on the sex chromosomes, we have 2 copies (alleles) of all genes. LOF mutations can result in 2 forms of Mendelian inheritance of genetic disease. A single LOF mutation reduces gene expression by 50%. If this is not enough for normal function of the gene, then this is known as “haploinsufficiency” and dominantly-inherited disease results. If normal function is possible with only 50% gene expression, then 2 mutations (one on each allele) are required to cause “recessive” disease. Whilst recessive disease tends to be caused by LOF, there are several mechanisms of dominant disease in addition to haploinsufficiency (LOF), particularly GOF and dominant-negative mutations.7

The pathomechanism of disease caused by individual mutations will determine the therapeutic approach (Fig. 2). Diseases resulting from LOF mutations can be targeted by conventional gene replacement, nonsense suppression, and exon skipping8 therapies, whereas diseases resulting from GOF mutations need to be targeted by gene editing,9 antisense oligonucleotide,10 or small interfering RNA (siRNA)11 therapies.

Gene therapy approaches for eye diseases. AAV indicates adeno-associated virus.

Therapeutic Approaches

Gene Replacement

Loss of function mutations can be treated by “conventional” gene replacement therapy. Here, the full-length human gene of interest is packaged into a viral capsid or attached to a nanoparticle that can then be delivered to the target organ, enter the relevant cell type, and be turned on by the host cell, resulting in full-length, wild-type protein being synthesized.12 Viral vector–based delivery of wild-type complementary DNA (cDNA) has been the most investigated method of gene therapy in genetic eye disease. Adenoviral-associated vectors (AAV) and, latterly, lentiviral vectors have been used to deliver this gene replacement to target cells.13 By this approach to treat retinal disease, patients undergo a pars plana vitrectomy followed by retinotomy and subsequent subretinal delivery of the virus to create a localized serous retinal detachment. A limitation to an AAV approach is that there is a size limit to the size of gene (4.7 kb) that can be packaged within each vector. Thus, mutations in very large genes cannot be targeted by this approach.

Nonsense Suppression

Nonsense mutations that introduce a novel, premature stop codon can be treated with drugs that encourage the RNA machinery to “read through” the premature stop and produce a full-length, albeit slightly altered, protein. If this protein has some degree of function, it could rescue the disease.

Exon Skipping Therapies

Coding DNA consists of exons separated by introns. Messenger RNA (mRNA) has the introns removed through a process termed splicing in order to provide the exon-only template for protein synthesis. This occurs by cutting the mRNA at splice sites. Exon-skipping therapies bind to splice sites that surround exons which harbor common, disease-causing mutations, leading to them being “skipped” and not included in the RNA for protein synthesis. This produces truncated proteins which, if they retain function, can rescue disease. This approach can also be achieved by using small molecules.14 Intravitreal injection of these therapies can be used to treat retinal disease.

Gene Editing

Advances in our understanding of DNA repair mechanisms have led to the identification and exploitation of transcription activator-like effector nucleases (TALENs) and, more latterly, clustered, regulatory interspaced, short palindromic repeat (CRISPR)–associated (Cas) nucleases in efforts to edit the mammalian genome. These “gene scissors” can be targeted with extreme precision by attaching them to guide RNAs to bind and cut a specific part of the genome. If a GOF mutation is specifically targeted on a mutant allele, the subsequent DNA repair can introduce a frameshift mutation (and thus a premature stop), halting production of the mutant protein. Thus, only the wild-type allele produces protein and disease can be rescued.15 To treat retinal disease, patients require subretinal delivery of these products in a similar approach to gene replacement therapy (see above).

Antisense Oligonucleotides and Small Interfering RNA

Antisense oligonucleotides are modified single strands of DNA that bind target mRNA resulting in degradation of the pathogenic (but not wild-type) mRNA sequences.16 Similarly, small interfering RNA (siRNA), comprising a short double-strand of RNA, complements the target mRNA and subsequently degrades it via the RNA-induced silencing complex (RISC).17


This literature search used the Cochrane Handbook for Systematic Reviews of Interventions, using the Ovid database for MEDLINE and EMBASE. We used a PICOS search strategy (Patient/Problem, Intervention, Comparison, Outcome) to identify interventional clinical trials for gene therapy (Intervention) for patients with a particular disease (Patient/Problem) for the purpose of improving the disorder (Outcome) given there are no treatments available (Comparison). Structuring and collection of the relevant studies used the PRISMA checklist process. Further search was performed of the database using the relevant disease name. Preclinical studies are included where relevant.

ABCA4/Stargardt Disease

ABCA4-associated retinopathy, incorporating autosomal recessive Stargardt disease (STDG1), is a clinically-diverse inherited retinal disease (IRD) characterized by maculopathy, although known to cause cone, cone-rod, and global retinal degeneration.18 It is the commonest IRD, with an estimated prevalence of between 1:8000 and 1:10,000,19 and typically presents in the first 2 decades of life.20 Due to the large size of ABCA4 (6.8 kb), a single-gene AAV approach is not feasible to treat this disease with gene replacement. Instead, a lentiviral based–approach has been adopted, due to the increased packaging capacity of technology. Interventional human phase I/II clinical trials of a lentiviral vector–delivered ABCA4 subretinal injection trial for StarGen (Sanofi, Oxford Biomedica, Oxford, UK, NCT01367444) ran from 2011 to 2019. Although the results of this study have yet to be peer reviewed, initial results were presented at international meetings and have been posted on,22 All 27 patients recruited experienced an adverse event, including 2 serious adverse events (raised intraocular pressure and intraocular inflammation), although no patients experienced significant visual loss. Recruits were split into 7 cohorts, with variation between cohorts in terms of disease severity and dose administered via subretinal injection. Sponsorship has now been withdrawn from the study. Another group has recently published 3-year safety data from a dose-escalation phase I/IIa clinical trial (ProgStar, NCT01367444) for subretinal injection of a lentiviral-based vector for ABCA4-associated retinopathy.23 Similarly, all patients experienced an adverse event, although most were mild, and there were no clinically significant changes in visual function.

Another approach to address the issue of AAV packaging of large genes is to adopt a “dual vector” delivery strategy. These systems allow large genes to be split and transduced via 2 vectors before recombination of the full-length gene within the targeted cell type. This strategy has been developed and trialed on ABCA4 mouse models,24 with a reduction in pathogenic A2E accumulation reported. The mechanism through which split genes recombine has been the subject of animal studies, leading to a hybrid approach that improved ABCA4 expression in target tissue.25 In other preclinical work, there have been promising results from animal studies regarding nanoparticle vectors for ABCA4 gene delivery, although this has not moved beyond the preclinical setting.26 There is also the potential for antisense oligonucleotides and gene editing to provide exciting options for the treatment of ABCA4-associated retinopathy, as well as other forms of Stargardt disease such as the dominant forms ELOVL4 (STGD3) or PROM1 (STGD4).27

RPE65- and CEP290-Mediated Leber Congenital Amaurosis

Leber congenital amaurosis (LCA) is a severe congenital or early infant-onset IRD characterized by vision loss, nystagmus, an absence of a normal pupil response, and an almost absent electroretinogram (ERG).28 The prevalence is estimated to be 1:33,000 live births.29 It is another example of an historical, phenotype-based diagnosis that has subsequently been shown to describe a genetically-heterogenous group of conditions.30,31

RPE65, localized to chromosome 1p31, is responsible for approximately 5% to 10% of LCA.32RPE65 presents an attractive option for gene therapy due to the relative delay in the onset of retinal degeneration, despite the early onset of visual loss, thereby providing a large treatment window in which cells are available for “rescue.” Gene replacement therapy via an AAV approach rescued the vision of large animal models harboring RPE65 mutations,33 leading to the world’s first randomized, controlled phase 3 trial for gene therapy for genetic disease (voretigene neparvovec, Luxturna), an AAV gene therapy which now has the approval of both the US Food and Drug Administration (FDA) and the UK's National Institute for Health and Care Excellence (NICE).34

A recent review and meta-analysis of published RPE65 gene therapy trials using several different vectors noted considerable variability in primary and secondary end points assessed, with visual acuity the only outcome measure common to all studies.35 Two studies reported full-field stimulus testing (FST) that were appropriate for meta-analysis.36,37 FST is useful in patients with very poor vision, and these studies demonstrated improvement in retinal sensitivity. Additional studies also reported minimal light mobility testing (mobility maze) as an endpoint;38,39 though clinical significance was not reached, there was a trend for improvement. Similarly, visual acuity showed improvements in case eyes compared to control eyes that did not reach clinical significance on meta-analysis. These studies all involved treating the eye with the poorer vision, which introduces inherent bias that could be overcome with bilateral trials.40,41 Longer-term follow-up, especially of pediatric trial participants, suggests there may be a benefit of earlier treatment.42

Another LCA subtype, LCA10, is due to mutations in the CEP290 gene.43 An antisense oligonucleotide has been developed targeting a splice site mutation common in Europe to target pre-mRNA splicing, delivered by intravitreal injection, with published data from the phase I/II trials and ongoing phase II/III trials.10 All 10 participants were injected at baseline and then again at month 3. Analysis showed statistically significant improvements from baseline in terms of logMAR visual acuity, although follow-up was only to month 4 in 6 of the participants. FST showed reversal of asymmetry (as before, the worse eye was treated) but mobility testing showed no significant improvements. The data were somewhat skewed by the impressive response of 1 participant, although statistical significance for visual acuity and FST were maintained when that 1 participant was removed from analysis. The ongoing phase II/III trials (NCT03913143) include a sham injection arm, an important aspect that has been overlooked in previous trials, especially those involving subretinal injections.

A gene-editing approach utilizing AAV5-packaged CRISPR/Cas9 technology (EDIT-101) has been adopted to target CEP290 mutations by subretinal deliver.44 This study importantly showed that, in both mouse and nonhuman primate models, delivery of EDIT-101 produced on-target correction of the CEP290 splicing defect (IVS26) responsible for a large proportion of LCA10. The rate of successful editing seen in this study exceeded the hypothesized therapeutic requirement of editing at least 10% of foveal photoreceptors. Another group similarly demonstrated high efficiency of CRISPR/Cas-mediated editing to remove the same IVS26 intronic splice mutation in vitro and carried out proof of principle experiments that demonstrated the efficacy of a dual-AAV approach for CRISPR/Cas-mediated CEP290 editing in vivo.45 Subsequently, CEP290 is the subject of a gene-editing trial utilizing CRISPR technology (BRILLIANCE, NCT03872479). Initial safety profile and preliminary results were presented by Pennesi et al at the XIXth International Symposium on Retinal Degeneration (RD2021) in September 2021, showing no Cas-nuclease specific antibody or T-cell response in all 6 participants. No peer-reviewed papers have yet been published from this trial.


Achromatopsia is an autosomal recessive cone dysfunction presenting in early life with photophobia and pendular nystagmus, associated with reduced visual acuity, impaired or absent color vision, and central scotomata.46 There are 6 genes responsible for approximately 90% of Achromatopsia cases,47 with the total prevalence estimated at between 1:30,000 and 1:50,000.48 Nine adult patients were included in a phase I/II trial using AAV8-packaged gene replacement therapy delivered by subretinal injection to target the worse eye affected by a CNGA3 biallelic variant causing complete achromatopsia.49 There was evidence of immune recognition of the AAV8 vector, with 2 potential study drug-related adverse events, although the safety profile was felt to be acceptable. The participants were split into 3 arms, with different doses of study drug injected in each arm, and follow-up was to 12 months. There was tentative, but statistically significant, improvements in visual acuity, contrast sensitivity, and color vision in those eyes receiving study drugs, but the same potential sources of bias existed as for the other AAV gene therapy trials. When this study reported 3-year outcomes, whilst the study eye still maintained statistically significant cone function outcome improvements, this did not reach significance when compared to the nonstudy eye.50 The safety profile was maintained at 3 years. Phase I/II trials are also ongoing for an AAV2-based, subretinal injection targeting CNGA3 Achromatopsia (NCT02935517, NCT02599922), although only preliminary data have been presented so far, with no peer-reviewed published data as yet.

RPGR/X-linked Retinitis Pigmentosa

X-linked retinitis pigmentosa (XLRP), caused by RPGR mutations, represents a large population of those with retinitis pigmentosa,51 affecting between 1:25,000 and 1:50,000 males.52 Patients typically start with night blindness in childhood, going on to develop visual field restriction and subsequent blindness typically in the fourth or fifth decade of life, hence presenting another wide treatment window of opportunity. The first gene therapy trial results for RPGR-associated XLRP were of a phase I/II study, published in 2020, reporting outcomes for 18 patients (split into 6 cohorts based on vector concentration) following subretinal injection of AAV8-packaged RPGR (Cotoretigene toliparvovec; BIIB112).53 Participants injected with higher vector concentrations showed subretinal inflammation, despite all participants taking a prophylactic 21-day course of oral prednisolone (1 mg/kg), but otherwise the study drug was well tolerated. Follow-up was reported at 6 months postinjection, with visual outcomes measured using ETDRS acuity and retinal sensitivity as measured by microperimetry across the central 10 degrees of the macula. The intention of the intervention was to stabilize visual decline, following no improvement in visual function seen in prior animal studies.54 These initial 6-month outcomes showed no significant visual decline, with 7 participants showing an improvement in microperimetry, although this was not clinically significant. In May 2021, Biogen reported the subsequent Phase 2/3 XIRIUS study did not meet its primary endpoint of demonstrating ≥7 dB improvement from baseline at ≥5 of the 16 central loci on microperimetry.55

Another group presented the 6-month outcomes of their 29 participants who received subretinal injection of a recombinant AAV2 RPGR at the Association of Research in Vision and Ophthalmology (ARVO) annual conference in 2021.56 The participants in this phase I/II trial were split into 5 cohorts based on vector concentration, and again ETDRS acuity and microperimetry were used as visual function measures as secondary outcomes. Twenty-one participants were treated centrally (submacular injections) and 8 peripherally. When compared to the fellow (untreated) eye, 8 participants showed a positive response in terms of microperimetry (7+dB improvement in at least 5 central macular loci), and 11 showed an improvement in best-corrected visual acuity (BCVA) (5+ ETDRS letter improvement). A phase II/III trial of this AAV2 subretinal injection, comparing untreated and treated participants, is currently in the prerecruitment phase (NCT04850118).

Phase I/II trial data from an AAV5 RPGR subretinal injection intervention were presented at AAO in November 2020.57 Ten participants were split into 3 cohorts (low, medium, high dose), with 2 of the 3 in the high-dose cohort experiencing an inflammatory response. Mobility scores and retinal sensitivity were shown to be improved compared to the nonintervention eye at 12 months follow-up with no dose-limiting adverse events. This group are currently recruiting to a phase III trial (NCT04671433).

Initial data from 8 participants recruited to an AAV (4D-125, utilizing an R100, proprietary vector) phase I/II intravitreal gene therapy trial for XL-RP were presented in October 2021 at ASRS (NCT04517149), showing the intervention was well tolerated.

X-linked Retinoschisis

X-linked retinoschisis (XLRS) is a monogenic disease caused by mutations in the RS1 gene with characteristic features of central retinoschisis and reduced b-wave amplitude on ERG,58 affecting between 1:5000 and 1:25,000 males worldwide.59 A phase I/IIa trial of intravitreal delivery of AAV8-RS1 vector published results of 9 participants in 2018.60 Follow-up was to 18 months and showed a dose-dependent ocular inflammatory response, as for other previously-mentioned AAV gene therapy trials. There was no significant improvement in visual function, as measured by ERG, visual acuity, and retinal sensitivity, but tolerability of the study drug was shown. The group went on to investigate the immune response to the study drug, showing a systemic immune response in terms of lymphocytes, macrophages, and proinflammatory cytokines.61 This could have important ramifications in terms of duration of treatment effect in AAV gene therapy, due to potential immune clearance of the drug, although there is the possibility of an underlying, abnormal immune state in XLRS patients. A further phase I/II AAV2-RS1 vector trial for XLRS has yet to publish findings but has stopped recruiting (NCT02416622).


Choroideremia is an X-linked condition presenting initially as childhood nyctalopia, leading to visual field constriction and eventually blindness in adulthood.62 It is caused by mutation or deletion of the CHM gene and subsequently a lack of the gene product, REP1.63 The estimated prevalence is between 1:50,000 and 1:100,000.64

The first gene therapy trial results for choroideremia were published in 2014, reporting phase I data from 6 participants undergoing subretinal injection of AAV2-REP1.65 Six-month outcomes of low dose showed some visual acuity improvement in those with low baseline visual acuity and stabile vision in the other 4 participants, maintained at 3.5-year follow-up.66 Two-year follow-up of higher-dose AAV2-REP1 showed no significant improvement in visual acuity of the study eye compared to control eye, with an intraretinal immune response seen in 1 patient (potentially due to an intraoperative hemorrhage and subretinal air injection).67 A subsequent phase I/II study using high-dose AAV2-REP1 showed significant visual improvement in 14 treated eyes compared to untreated controls at 2 years, despite vector delivery–related adverse outcomes in 2 patients.68 A further study of the same vector, at the higher dose, using an intraoperative optical coherence tomography (OCT) to improve injection technique69 showed maintenance of BCVA at 2 years with no serious adverse events, whilst a further high-dose study showed no serious adverse events at 2 years.70 Biogen subsequently acquired the therapy (timrepigene emparvovec; BIIB111/AAV2-REP1) and recently reported that their Phase 3 Gene Therapy Study had missed its 12-month primary endpoint of a ≥15 letter improvement in BCVA in the study eye.71 There is ongoing recruitment to a phase I study of intravitreal injection of R100 (proprietary AAV) study drug (NCT04483440).


Mutations in USH2A account for 7% to 23% of autosomal recessive retinitis pigmentosa (RP) cases, with an estimated prevalence of 1:30,000,72 and can either result in nonsyndromic RP or in Usher syndrome (a combination of RP and hearing impairment).73 The large size of the usherin-encoding gene has led to an antisense oligonucleotide approach in gene therapy for visual loss associated with USH2A pathological variants. A human cellular model, a zebrafish model, and a mouse model treated with intravitreal injection of an antisense oligonucleotide (QR-421a) showed selective skipping of the USH2A pathogenic exon.74 Twenty adults were subsequently recruited into a phase I/II randomized trial of QR-421a (Stellar, NCT03780257) in which they received either an intravitreal injection of study drug (at 1 of 3 doses) or a sham injection into the worse-affected eye. Initial reports showed no significant adverse events, leading to the ongoing recruitment of patients to 1 of 2 double-masked, randomized, sham-controlled, 24-month, multiple-dose phase II/III trials, dependent on their disease severity (Celeste, NCT05176717; Sirius, NCT05158296).

RHO-Mediated Autosomal Dominant Retinitis Pigmentosa

RHO-mediated autosomal dominant retinitis pigmentosa (adRP) presents with nyctalopia due to rod degeneration with subsequent cone degeneration resulting in legal severe sight impairment, although there is a spectrum of disease severity and speed of onset.75 RP has a prevalence of approximately 1:4000, and approximately 25% of RP is adRP.76 The rhodopsin gene (RHO) was the first gene to cause RP identified, and it is implicated in approximately 25% of all adRP cases.77

Due to the gain of function (GOF) in RHO-mediated adRP, the gene replacement therapeutic strategies are not appropriate, hence the goal of treatment is to reduce the expression of the pathogenic RHO protein or to increase the proportion of wild-type RHO compared to the pathogenic variant.78 RNA-targeted therapies for adRP have included antisense oligonucleotide intravitreal injections, shown to reduced pathogenic RHO mRNA expression in murine adRP models with associated retinal function improvements.79 This has led to a phase I/II clinical trial (AURORA, NCT04123626) that is currently recruiting patients with P23H-adRP (P23H being a pathogenic RHO variant). RNA interference (siRNA and short hairpin RNA) was felt to have promise as a strategy for adRP following an AAV-RHO murine study by an Irish team;80 however, since acquisition of the technology in 2019,(RhoNova, Roche) there have been no further updates. adRP also presents the opportunity for genome editing via CRISPR technology,81 although there are concerns regarding “off-target” effects from CRISPR due to the permanent disruption of both pathogenic and wild-type DNA.82 No CRISPR therapeutics for adRP have progressed to clinial trials in humans at this point in time.


Aniridia is a dominantly-inherited, bilateral, panocular developmental disease of the eye, overwhelmingly associated with PAX6 loss of function. It is characterized by iris and foveal hypoplasia, with secondary complications including early cataracts, glaucoma, and progressive keratopathy. The estimated prevalence of aniridia is between 1:64,000 and 1:96,000.83 Approximately 90% of aniridia is attributable to PAX6 haploinsufficiency,84 with a small number of other causative genes identified including FOXC1, PITX2, and MAB21L1.85,86 The transcription factor PAX6 is crucial to eye and brain development, and also importantly has a role in corneal maintenance in adulthood.87 The development of the eye (including iris and fovea) continues postnatally and thus genetic therapies for aniridia may be of benefit in the early postnatal period, as well as later in life from a corneal perspective.

Ataluren is a nonsense suppressor that targets in-frame nonsense mutations and showed promise in an aniridia mouse model (Sey) with a nonsense mutation.88,89 It has recently completed phase II clinical trials as an oral preparation vs placebo for aniridia patients with a nonsense mutation (STAR trial, NCT02647359). Whilst trial data including secondary endpoints have not been published, a press release from PTC Therapeutics (March 2, 2020) confirmed that it failed to reach the primary endpoint of a change in maximum reading speed using MNREAD acuity charts. Ataluren previously failed to meet the primary endpoint in phase III trials for cystic fibrosis90 and initially Duchenne muscular dystrophy (DMD),91 albeit showing benefit in a subgroup of the latter. A further phase III trial was conducted for DMD, and subsequent meta-analysis of the 2 trials showed some evidence that it delayed disease DMD progression;92 it has now been conditionally approved from use in DMD by the European Medicines Agency.

Employing an alternative strategy, it was recently shown that the antidepressant duloxetine enhances the expression of the PAX6 protein in human limbal stem cells (LSCs)93 via the ERK/MEK pathway. The authors tested the effects of the repurposed drug in vitro in PAX6 gene–edited LSCs, with duloxetine enhancing PAX6 expression and normalizing the expression of the target genes normally repressed by PAX6 in mutant cells. In contrast to nonsense suppression, which risks producing some truncated or abnormal protein, in this study only the healthy or “wild-type” PAX6 protein was enhanced. Additionally, this approach is not limited to those with nonsense mutations. Using a similar strategy and target pathway in vivo in a mouse aniridia model, a MEK inhibitor given topically or systemically in the early postnatal period enhanced PAX6 expression in the cornea, improving corneal clarity and visual function.94 These suggest exciting avenues for clinical translation that could apply to other diseases involving genes that, such as PAX6, are highly dose-sensitive.

Leber Hereditary Optic Neuropathy

Leber hereditary optic neuropathy (LHON) is a mitochondrial disease that primarily targets retinal ganglion cells, typically presenting with severe, rapidly sequential, bilateral visual loss.95 Three-point mutations (m.11778G>A, m.3460G>A, m.14484T>C) in the group of mitochondrial DNA (mtDNA) that encodes the subunits of complex I of the respiratory chain, the MT-ND genes, are responsible for approximately 90% of LHON cases.96 The m.11778G>A mutation in mtND4 gene is the most prevalent amongst LHON cases and has been the subject of human trials for gene therapy. The prevalence of LHON in Europe is estimated at between 1:27,000 and 1:54,000.97

Phase III trials assessing the efficacy of the intravitreal injection of AAV2-ND4 gene therapy for those carrying the m.11778G>A mutation have been undertaken by groups in France and China.98,99 The French group undertook 2 phase III trials, one in subjects within 6 months of disease onset (RESCUE, n=39, NCT02652767), and another in those more than 6 months from disease onset (REVERSE, n=37, NCT02652780), with subsequent recruitment of 86% of participants from both studies into a 5-year follow-up study (RESTORE, NCT03406104). Both RESCUE and REVERSE participants were randomized with one eye receiving the intravitreal study drug and the other receiving a sham intravitreal injection (a blunt syringe-end placed against the sclera but no entry into the vitreous cavity). There was no control arm where no study drug was delivered in either study. The Chinese group recruited 149 patients into a phase III trial whereby the eye with the worse BCVA received the study drug with no intervention for the other eye (NCT03153293). All studies showed a sustained, clinically significant improvement in BCVA in both treated and untreated eyes beyond that reported in the natural history of LHON.99–101 Subsequently the French group recruited 98 participants within 12 months of disease onset to a new, phase III, randomized, double-masked, placebo-controlled trial (REFLECT, NCT03293524), in which the first-affected eye receives study drug and the second eye is randomized to either study drug or placebo/sham. The results of this study have yet to be published. The previous phase I/II studies had shown that the intravitreal injection of AAV2-ND4 study drug had been well tolerated with minimal adverse events (some cases of raise intraocular pressure or uveitis, but all treatable and localized), although the primary endpoint in terms of a difference in BCVA between the 2 treatment groups in the REVERSE trial was not met.102–105

The potential for a contralateral effect that is suggested by the bilateral BCVA improvements was investigated by the group behind the REVERSE trial. They performed quantitative polymerase chain reaction (PCR) analysis on nonhuman primates 3 months after unilateral intravitreal AAV2-ND4 injection and found detectable vector in the contralateral optic nerve, optic chiasm, optic tract, and anterior segment.101 The mechanism for this effect remains unexplained, although there is the possibility of synaptic AAV spread and axonal or astrocytic transfer of mitochondria.106–108 To confirm the mechanism of these findings, further studies are required. Ideally, a natural history study of the disease that adopts the same stringent psychophysical tests at the same timepoints as those used in the interventional studies, or a deferred treatment arm of an interventional study, would rule out the possibility that the improved vision seen in the contralateral eye was due to fluctuations in visual processing during the disease process. In the absence of this, however, it is hoped the REFLECT study will shed some light on the phenomenon.


The sheer number of genetic eye diseases undergoing gene therapy clinical trials (Fig. 3 and Table 1) shows the significant level of interest in the potential for translation of these therapies from bench to bedside. Further, the use of stem cell–derived cell replacement products to treat genetic eye disease, though not covered in this review, could offer another therapeutic approach. The breadth of therapy design is encouraging, providing multiple possible therapeutic mechanisms, thereby providing the greatest chance of eventual success in terms of appreciable clinical benefit to patients. That complex retinal disease, such as age-related macular degeneration, is now being targeted by gene replacement strategies, highlights the progress made in the field (comprehensively reviewed here107). Some fundamental questions regarding gene therapy for genetic eye diseases remain, such as optimal dosing, the relative benefits of AAV-packaging vs the alternatives and the potential for a significant inflammatory response to the therapy itself. As a result, despite the promise of the eye as a target, it has proven difficult to deliver clinically effective gene therapies to the eye, as described in this review. Despite setbacks, the licensing of Luxturna (voretigene neparvovec, Novartis) for the treatment of RPE65-mediated LCA is a major advance in our efforts to treat these rare, but devastating, causes of visual loss. Improved study design, increased awareness of ongoing clinical trials, and improved clinical genetic diagnostics should provide greater scope for future clinical trials to deliver results for these patient groups.

Eye diseases undergoing gene therapy trials. LHON indicates Leber hereditary optic neuropathy; LCA, Leber congenital amaurosis; RP, retinitis pigmentosa.
TABLE 1 - Registered Gene Therapy Clinical Trials for Eye Disease
Disease/Gene Phase Drug Design Drug Delivery NCT No Study Status
Stargardt/ABCA4 I/IIA AAV Subretinal 01367444 Terminated
II/III AAV Subretinal 01736592 Active, not recruiting
LCA/RPE65 I AAV Subretinal 00516477 Completed
I AAV Subretinal 00821340 Completed
I AAV Subretinal 00481546 Active, not recruiting
I/II AAV Subretinal 01208389 Active, not recruiting
I/II AAV Subretinal 02781480 Completed
I/II AAV Subretinal 00643747 Completed
I/II AAV Subretinal 01496040 Completed
I/II AAV Subretinal 00749957 Completed
III AAV Subretinal 00999609 Active, not recruiting
III AAV Subretinal 04516369 Active, not recruiting
LCA/CEP290 I/II AON Intravitreal 03140969 Completed
I/II AON Intravitreal 03913130 Active, not recruiting
I/II AAV/CRISPR Subretinal 03872479 Recruiting
II/III AON Intravitreal 04855045 Recruiting
II/III AON Intravitreal 03913143 Active, not recruiting
Achromatopsia/CNGA3 I/II AAV Subretinal 02610582 Recruiting
I/II AAV Subretinal 02935517 Recruiting
I/II AAV Subretinal 03758404 Completed
Achromatopsia/CNGB3 I/II AAV Subretinal 02599922 Recruiting
I/II AAV Subretinal 03001310 Completed
Achromatopsia/CNGA3 & CNGB3 I/II AAV Subretinal 03278873 Active, not recruiting
X-linked RP/RPGR I/II AAV Subretinal 03116113 Completed
I/II AAV Subretinal 03252847 Completed
I/II AAV Subretinal 04517149 Recruiting
I/II AAV Subretinal 03316560 Recruiting
II/III AAV Subretinal 04850118 Not yet recruiting
III AAV Subretinal 03584165 Enrolling by invitation
III AAV Subretinal 04794101 Recruiting
III AAV Subretinal 04671433 Recruiting
X-linked Retinoschisis/RS1 I/II AAV Intravitreal 02416622 Active, not recruiting
I/II AAV Intravitreal 02317887 Recruiting
Choroideremia/CHM I AAV Intravitreal 04483440 Recruiting
I/II AAV Subretinal 01461213 Completed
I/II AAV Subretinal 02341807 Active, not recruiting
I/II AAV Subretinal 02077361 Active, not recruiting
II AAV Subretinal 02553135 Completed
II AAV Subretinal 02407678 Completed
II AAV Subretinal 02671539 Completed
II AAV Subretinal 03507686 Active, not recruiting
III AAV Intravitreal 03496012 Completed
III AAV Subretinal 03584165 Enrolling by invitation
USH2A I/II AON Intravitreal 03780257 Active, not recruiting
II AON Intravitreal 05085964 Enrolling by invitation
II/III AON Intravitreal 05176717 Recruiting
II/III AON Intravitreal 05158296 Recruiting
adRP/RHO I/II AON Intravitreal 04123626 Recruiting
Aniridia/PAX6 II Small Mol Oral 02647359 Completed
II Small Mol Oral 04117880 Withdrawn
LHON/ND4 I/II AAV Intravitreal 02064569 Completed
I/II AAV Intravitreal 01267422 Completed
I/II AAV Intravitreal 02161380 Active, not recruiting
I/II AAV Intravitreal 04912843 Recruiting
II/III AAV Intravitreal 03153293 Active, not recruiting
III AAV Intravitreal 02652780 Completed
III AAV Intravitreal 02652767 Completed
III AAV Intravitreal 03406104 Active, not recruiting
III AAV Intravitreal 03428178 Active, not recruiting
III AAV Intravitreal 03293524 Active, not recruiting
AAV indicates adenoviral-associated vectors; AON, antisense oligonucleotides; CRISPR, clustered, regulatory interspaced, short palindromic repeat; LHON, Leber hereditary optic neuropathy; LCA, Leber congenital amaurosis; RHO, rhodopsin gene; RP, retinitis pigmentosa.


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gene therapy; Luxturna; retinitis pigmentosa; RPE65; Leber

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