Emerging Gene Therapy Technologies for Retinal Ganglion Cell Neuroprotection

Optic neuropathies encompass various pathologies with the common endpoint of impaired optic nerve function, due to damage/loss of retinal ganglion cells (RGCs) or their axons. Etiologies of these diseases vary in their primary insult which can be ischemic, inflammatory, traumatic, compressive, genetic or other processes,^1–4 and clinical presentation can be variable. Vision deficits range from decreases in visual acuity, restriction of visual fields, dyschromatopsia, and worsened contrast sensitivity,^3–6 with ancillary testing used to distinguish different etiologies.³ These impairments lead to visual disability which, when combined with the clinically distinct but by far most common optic nerve degenerative disease, glaucoma, ranks among the top ten disabilities in the US, and lead to further profound effects on physical and mental health.⁷ Thus, implementing effective therapeutic approaches to treat these diseases would provide substantial impact.

Currently, most optic neuropathies have limited or no effective therapies, despite various treatments attempted to modify distinct insults, target modifiable risk factors for specific conditions, or promote cell-protective/survival mechanisms independent of the initial cause of injury. In optic neuritis, corticosteroids are used to reduce optic nerve inflammation,^8,9 but despite hastening visual recovery, treatment fails to improve visual outcomes.¹⁰ Corticosteroids are also used to treat arteritic ischemic optic neuropathy, with the major goal of preventing vision loss in an unaffected eye.¹¹ For non-arteritic anterior ischemic optic neuropathy, multiple potential medical and surgical treatments studied have failed to provide significant benefit, or studies have been inconclusive.¹² Traumatic optic neuropathy is still treated with corticosteroids or surgery by some clinicians,¹³ but treatment remains controversial as controlled clinical trials are lacking and overall data fail to show definitive benefits.¹⁴ A rare example of an approved neuroprotective therapy for an optic neuropathy is treatment of deficits in cellular respiration seen in Leber’s hereditary optic neuropathy (LHON) with a benzoquinone analog (Idebenone) to restore electron-carrying capacity.¹⁵ Though this increases RGC survival, it is limited by the constraints of daily medication adherence and medication penetration to the site of action,¹⁶ and treatment has been approved in Europe but not in the US. Glaucoma is recognized as distinct from other optic neuropathies with a commonality of an insult terminating with RGC damage. Its management provides an example of addressing a modifiable risk factor through the use of medication or surgery to lower intraocular pressure (IOP).¹⁷ While these strategies often successfully slow or halt RGC and visual loss, many patients continue to develop progressive damage and dysfunction.^18,19 Ocular and systemic side effects associated with corticosteroids²⁰, high frequency of dosing of medications¹⁶, complications of surgery^21,22, and overall failure to impact RGC survival in most optic neuropathies, highlight the urgent need to develop new, effective neuroprotective or neurorestorative treatments.

Numerous promising neuroprotective therapies have been tested in preclinical optic neuropathy models during the past few decades. However, the probability of success of those therapies in the clinic is reduced by limitations of the available preclinical tools needed to recapitulate the human diseases, optic nerve and retinal differences between humans and animal models, and translatability of experimental endpoints. Consequently, many preclinical therapies have failed in clinical trials.^23,24 A novel promising avenue has come with experimentation with adeno-associated virus-based gene therapy, and identification of potential therapeutic gene targets that play a role in multiple optic neuropathies, such as modifying mitochondrial-related RGC dysfunction. Examples include silent information regulator 1 (SIRT1), PPAR-γ coactivator 1 alpha (PGC1ɑ), nuclear factor erythroid 2–related factor 2 (NRF2), mammalian target of rapamycin (mTOR), and optic atrophy-1 (OPA1). Evidence suggests these molecules’ function in apoptotic pathways, mitochondrial biogenesis, and maintenance of mitochondrial DNA.^25–29 The discovery of molecular players opens the door for targeting these systems for therapeutic advantage using gene therapy.

History of Gene Therapy

Gene therapy introduces genetic material to target cells via viral or non-viral delivery methods to either increase, modify, or suppress expression of the gene of interest. Figure 1 depicts a timeline of salient events ushering gene therapy to its current state. In 1968, Rogers et al. demonstrated proof-of-concept for virus mediated gene transfer.³⁰ Not until several decades later, in 1990, did the US Food and Drug Administration (FDA) approve the first human gene therapy clinical trial. Children with severe combined immunodeficiency (SCID) were treated using retroviral-mediated transfer of the adenosine deaminase gene into T-cells, with one demonstrating some degree of long-term response.³¹ In 1999, gene therapy trials encountered a major setback as a result of the tragic death of an 18-year-old, who developed an acute inflammatory response resulting in multi-organ failure after receiving an adenovirus vector (AV) for ornithine transcarbamylase deficiency, calling into question ethical implications of safety and proper patient selection for gene therapy trials.³² What ensued included statements by the Recombinant DNA Advisory Committee and the Human Genome Meeting ethics committee, which led the FDA in 2000 to halt all clinical trials at the institute where the study was conducted.³³ This highlighted risks associated with AV and led to recognition of the differential risk profile per viral vectors. By 2002, there were two additional patient deaths, associated with a lentivirus (LTV) gene therapy to treat SCID causing oncogene activation.³⁴ Numerous ethical studies on the future of gene therapy ensued, as the field was ushered into a state of quiescence with no new clinical trials being initiated.³³ That changed in 2005 when development of a gene therapy for lipoprotein lipase deficiency used the novel adeno-associated virus (AAV) vector and instituted formal safety assessments in preparation for clinical trial testing under conditions stated in the Good Laboratory Practices.³⁵ Use of this safer viral vector, along with the regulatory changes, allowed clinical trials to proceed, leading to approval of the same gene therapy, Alipogene tiparvovec (Glybera) in 2012, the first gene therapy product approved in Europe.³⁶ In 2017, the FDA approved the first gene therapy-based product in the US, tisagenlecleucel (Kymriah), a chimeric antigen receptor T-cell therapy designed to treat B-cell acute lymphoblastic leukemia. This was a graduated step forward, as it did not require direct injection of the gene into patients. Rather, the patient’s cells were removed first, then a therapeutic gene was transferred into cells cultured in vitro prior to replacing the treated cells back into the patient.³⁷ Although cancer remains one of the most studied targets for gene therapy,³⁸ ocular pathology became a favorable candidate due to the eye’s immune-privileged status, easy accessibility, and relatively contained area due the blood-retinal barrier. In December 2017, the FDA approved voretigene neparvovec (Luxturna) to treat Leber’s Congenital Amaurosis type 2 (LCA2), a bi-allelic RPE65 mutation-associated retinal dystrophy.³⁹ The success of Luxturna provided proof of concept that gene therapy could be safely and effectively delivered directly into patients. Building upon this success, there are currently 27 cell and gene therapies licensed by the FDA, and 13 of these are gene therapy-based products.⁴⁰ There currently are 49 registered clinical trials investigating unique gene therapies in ocular pathologies,⁴¹ including therapy for LHON, and the list is expected to grow exponentially.

Figure 1.

A graphical timeline of gene therapy. ADA, Adenosine Deaminase Deficiency. SCID, Severe Combined Immunodeficiency. OTC, Ornithine Transcarbamylase Deficiency. AAV, Adeno-associated Virus. LPLD, Lipoprotein Lipase Deficiency. AAV, adeno-associated virus. ALL, Acute Lymphoblastic Leukemia. LTV, Lentivirus. rAAV2, recombinant adeno-associated virus serotype 2. LHON, Leber’s Hereditary Optic Neuropathy.

Gene Delivery

Delivery of genetic material typically occurs through molecular carriers called vectors, which are viral or non-viral material into which a gene is inserted to facilitate penetration into cells. The ideal vector demonstrates a high carrying capacity, ability to infect a large portion of target cells (transduction efficiency), and high cell specificity with minimal immune response or toxicity. Non-viral gene delivery systems include lipid-based vectors, polymer-based vectors, gold particles, and plasmid electroporation, among others.⁴² Figure 2 summarizes many of these gene delivery methods, and highlights which may be best suited for clinical use in optic neuropathies. The most established biological delivery systems for ocular diseases are viral vectors, including AAV, AV, and LTV. As the predominant vector used in clinical trials, AAVs are small, non-pathogenic viruses with high transduction efficiency and low immunogenicity, but are limited by their smaller carrying capacity of <5 kb^43,44, which limits the size of a gene that can be delivered. Furthermore, AAVs are typically non-integrating viruses and thus have non-replicating, episomal expression. The longevity of AAVs is currently under investigation, although promising preclinical data suggest therapeutic effects can be long lasting. For example, dogs receiving gene therapy for LCA demonstrated photoreceptor survival up to 10 years after treatment.⁴⁵ AAVs are further classified into serotypes based on the composition of the protein shell that encapsulates the viral genetic material (capsid), which impacts the manner of transport, transduction efficiency, and allows for more precise cell targeting. In particular, AAV serotype 2 (AAV2) is commonly used to target RGCs and other retinal cells due to its efficiency and heavily studied safety profile in large animal models and humans.^46–48

Figure 2.

Modes of gene therapy delivery. Several potential modes of delivering foreign DNA into cells are demonstrated using retinal ganglion cells (RGCs) as the target cell. **viral vectors represent a leading clinical candidate delivery method for targeting RGCs. *Packaging in lipid or inert nanoparticles also has potential to translate to the clinic, whereas other delivery methods are currently better suited for research studies or other target cell populations. dsRNA, double-stranded RNA. ER, endoplasmic reticulum.

In comparison, AVs have a higher packaging capacity, but they can generate significant immune/inflammatory responses. Unlike AVs and AAVs, LTVs, a subtype of retroviruses, allow for longer transgene expression through integration inside the host genome.⁴⁹ However, this function comes with the associated risk of higher immunogenicity and unpredictable insertional mutagenesis. Although retroviruses successfully treated X-linked SCID in nine patients, four developed vector-mediated T-cell leukemia 31 to 68 months after treatment.³⁴ Rates of insertional mutagenesis have been lowered with the use of modified LTVs, which offer lower oncogenic potential and genotoxicity compared to γ-retroviral vectors.⁵⁰ Other safety precautions have included viruses designed with self-inactivating long terminal repeats that further decrease the rates of aberrant splicing.⁵¹

After consideration of which viral vector to use, route of administration needs to be considered. In the eye, these modes can include intravascular, intravitreal (IV), subretinal (SR), and retrobulbar (RB), as well as directed into the ciliary muscle via electrotransvection⁵². For retinal pathologies, the most widely used route is SR injection because it delivers the vector to the cells of interest, namely photoreceptors and retinal pigmented epithelium.⁵³ While useful, SR delivery involves invasive surgery performed by highly trained physicians, and risks side effects such as retinal detachment, choroidal effusions, or retinal tears. IV delivery of AAV has emerged as a more likely strategy to treat RGCs, by delivering therapies closer to the outer layers of the retina.^54–56 While viral vectors have emerged as the most clinically relevant examples, other delivery methods continue to be explored. For example, in a pre-clinical effort to deliver naked plasmid to tissues, ciliary muscle electrotransfection has shown promise for local therapy, but has not yet been used in the clinical setting.^57,58 Finally, many publications suggest that to achieve uniform and more extensive transduction of retinal ganglion cells in primate retina, injections underneath the inner limiting membrane (ILM), or subILM injections will enhance transduction [PMID 30783978].

Gene Therapy Design

Due to packaging size constraints, particularly for AAVs, thoughtful design of components of the genetic material is required. Genes of interest are coupled with all necessary genetic elements for expression of the gene in a product called a plasmid (Figure 3). Essential elements include a promoter, gene-of-interest, and terminator. Additionally, elements to increase plasmid stability such as introns can be added to improve expression. The entire plasmid with all of its elements mentioned is referred to as an expression cassette.⁵⁹ Constitutive promoters, such as cytomegalovirus (CMV) and chicken β-actin, can be used to promote gene overexpression in different cell types if desired; whereas, cassettes can also use tissue-specific promoters to increase target specificity, although this may come at the cost of larger size or lower transgene expression.⁶⁰ Examples of RGC-specific promoters include Neurofilament Heavy (NEFH),^61,62 γ-synuclein,⁶³ and syanpsin-1,⁶⁴ which help limit off-target effects. Stabilizing elements, including viral post-transcriptional regulatory elements and introns, can be used as well to boost expression. The presence of an enhancer may increase transgene expression even when paired with a cell-specific promoter, reducing the necessary amount of genetic material needed for adequate expression levels. There are other elements downstream of the gene-of-interest that can increase transcriptional readthrough and overall transcript stability. Examples include woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and poly (A) tail. Together, growing data on various elements that can be used to increase levels of gene expression, and restrict expression to specific cell types, provide key tools for ongoing development of gene therapies for treatment of optic neuropathies.

Figure 3.

Complete expression cassette. Within the plasmid itself the genetic elements needed for replication start with the inverted terminal repeat (ITR), which allows the complementary strand to also be replicated. Starting from the 5’ end, the enhancer serves the role of binding transcription factors to increase transcription efficiency. The promoter binds the RNA polymerase and starts synthesizing the transcript to be translated. The intron is a genetic element that serves to stabilize the mRNA without coding for functional portions of the protein. The gene-of-interest is the actual protein product being coded for. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) also increases transcriptional stability. The bGH poly (A) signal provides a sequence of adenosines that also stabilize the transcript. Replication (REP), capsid (CAP), and “helper” virus function genes are coded for separately from the plasmid but are required for the replication and amplification of the plasmid when preparing it in a biological system before it is delivered into the target cell. AAV, adeno-associated virus.

Strategies for Gene Therapy

There are several ways to alter expression of a gene of interest. Gene silencing is one strategy, targeted to diseases where gain-of-function or dominant negative gene mutations result in abnormally functioning proteins. Gene silencing via RNA interference can potentially be used to suppress production of these proteins by administering small interfering RNAs (siRNAs) that mediate the rapid and specific degradation of their complementary mRNA. A similar strategy can also be used to suppress normal proteins in order to reduce disease pathology. For example, SYL040012 (Bamosiran), an siRNA targeting β2-adrenergic receptor mRNA, is noninferior to timolol for management of glaucoma in patients with basal IOP > 25.⁶⁵

Other gene therapy strategies involve gene-editing, where targeted DNA breaks are introduced by programmable nucleases such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) nucleases.⁶⁶ These DNA breaks are repaired by homology-directed repair or the more error-prone non-homologous end joining, resulting in either nucleotide deletion, correction, or addition. Despite tremendous promise of these strategies, their use has been limited, as the commonly used Streptococcus pyogenes Cas9 (SpCas9) is ~4.2 kb, which limits the addition of regulatory elements considering the 4.8 kb packaging constraint of the AAV vector. The BRILLIANCE trial for LCA10 involved one of the first in vivo uses of CRISPR/CAS-9 editing, although the trial was halted due to a small target population.^67,68 Despite limited current use, future advances in gene-editing technology hold tremendous promise for new therapeutics.

Most gene therapy trials favor gene replacement or gene augmentation. Here, a normal copy of the gene is inserted to replace a defective gene or is introduced to increase expression of a specific protein, similar to delivering a protein in the form of a therapy. The gene replacement strategy is most suitable for treating recessive loss-of-function and haploinsufficiency disease-causing gene mutations. A notable example includes Luxturna, which uses an AAV2 vector to deliver an RPE65 gene cassette to treat LCA.⁶⁹

Gene Therapy in Clinical Trials

Previous clinical trials examined potential gene silencing strategies to treat glaucoma and NAION, using Bamosiran and QPI-1007, respectively.^65,70 However, the most commonly utilized strategy is gene addition. A review of ClinicalTrials.gov reveals that most currently ongoing gene therapy clinical trials for optic neuropathies target LHON, possibly due to the simplicity of using gene editing to target the single nucleotide mutation underpinning this disease (Table 1).

Table 1.

A review of ClinicalTrials.Gov for gene therapy clinical trials targeting optic neuropathies

Disease	Intervention	Mechanism	Phase	Clinicaltrials.gov ID	Status
Glaucoma	Bamosiran	siRNA against β2-adrenergic receptors	I/II	NCT01227291	Completed
	Bamosiran	siRNA against β2-adrenergic receptors	II	NCT02250612	Completed
	Bamosiran	siRNA against β2-adrenergic receptors	II	NCT01739244	Completed
LHON	scAAV2-P1ND4v2	rAAV2/2-ND4	I	NCT02161380	Active, not recruiting
	rAAV2-ND4	rAAV2/2-ND4	II/III	NCT03153293	Active, not recruiting
	GS010	rAAV2/2-ND4	III	NCT03293524	Active, not recruiting
	GS010	rAAV2/2-ND4	NA	NCT03672968	Available
	NR082	rAAV2/2-ND4	I/II/III	NCT04912843	Recruiting
	rAAV2-ND4	rAAV2/2-ND4	III	NCT03428178	Unknown
	rAAV2-ND4	rAAV2/2-ND4	I/II	NCT01267422	Completed
	GS010	rAAV2/2-ND4	I/II	NCT02064569	Completed
	GS010	rAAV2/2-ND4	III	NCT02652767	Completed
	GS010	rAAV2/2-ND4	III	NCT02652780	Completed
	GS010	rAAV2/2-ND4	III	NCT03406104	Completed
NAION	QPI-1007	siRNA against caspase 2	I	NCT01064505	Completed
	QPI-1007	siRNA against caspase 2	II/III	NCT02341560	Completed

LHON, Leber Hereditary Optic Neuropathy; NAION, Nonarteritic anterior ischemic optic neuropathy; siRNA, small interfering RNA; rAAV2, recombinant adeno-associated virus serotype 2.

LHON is a primary mitochondrial disorder with prevalence ranging from 1 in 30,000 to 1 in 50,000 in Northern Europe.⁷¹ LHON is characterized by a painless subacute to rapidly progressive vision loss, typically in one eye followed by sequential involvement of the other eye.⁷² Three missense point mutations (m.11778G>A [MT-ND4], m.3460G>A [MT-ND1], m.14484T>C [MT-ND6]) account for up to 90% of LHON cases in Caucasian populations.⁷³ These mitochondrial DNA mutations affect complex I subunits in the mitochondrial respiratory chain resulting in impaired ATP synthesis, excessive reactive oxygen species (ROS) generation, and subsequent apoptosis of RGCs that can be recapitulated in animal models.⁷⁴ Due to their small-caliber, axons of the papillomacular bundle are preferentially affected by energy depletion.^75,76

Current treatment options for LHON include idebenone (Raxone), a benzoquinone analog that substitutes the defective molecular electron carriers.¹⁶ Additionally, clinical trials using gene therapy strategies are seeking to recover or halt progression of vision loss in patients with the specific m.117788G>A mutation. RESCUE⁵⁵ and REVERSE⁵⁶ were two masked, sham-control Phase III randomized clinical trials that examined the efficacy of gene therapy in LHON patients with less than six months of vision loss, and between 6 months and 1 year from onset of vision loss, respectively. LHON patients received either an intravitreal injection of lenadogene nolparvovec (rAAV2/2-ND4), a replication-defective, recombinant AAV2 containing modified cDNA encoding a human wild-type mitochondrial ND4 protein, or were given a sham treatment. Intravitreal injection allowed for direct delivery of the therapy to macular RGCs. Mitochondrial targeting sequences incorporated in the vector enabled translocation of the ND4 protein into the mitochondrial matrix. At two years follow up, rAAV2/2-ND4 improved visual acuity not only in injected eyes, but also in sham-controlled eyes,⁷⁷ potentially due to transfer from the treated eye to the untreated eye via the optic chiasm as suggested by a non-human primate biodistribution study.⁷⁸ Interim results of the RESTORE trial, which aims to assess the long-term durability of rAAV2/2-ND4, showed progressive and sustained improvement up to 4 years after vision loss.⁷⁹ These studies suggest gene therapy for LHON could be well tolerated and safe.

Promising Pre-clinical Gene Candidates for RGC Neuroprotection

In vitro and pre-clinical studies have focused on genes that influence RGC regeneration and survival with potential therapeutic benefits across various optic neuropathies. While this is not intended as an exhaustive review, several genes have been identified as promising candidates due to their role in inflammation, apoptosis, cell stress responses, and/or cell survival.

Anti-apoptotic genes:

Following the initial insult in many optic neuropathy models, RGC loss occurs due to programmed cell death.^80–82 In the intrinsic pathway, mitochondrial depolarization and permeabilization induces cytochrome c release, resulting in downstream caspase activation. Studies have examined blocking apoptotic pathways through suppression of pro-apoptotic kinases or upregulation of anti-apoptotic factors, with the primary focus on the B-cell lymphoma 2 (Bcl2) gene family. Upregulation of pro-survival Bcl-extra-large (Bcl-x_L) prevents neuronal death,^83–85 with AAV2-mediated overexpression preventing RGC degeneration in vivo⁸⁶ and in a mouse model of glaucoma.⁸⁷ In contrast, pro-apoptotic Bcl-2 Associated X-protein (Bax) knockout mice show Bax plays an essential role in neuronal death but not in axonal degeneration.^88–91

While apoptosis in RGCs occurs primarily through the intrinsic pathway, continued RGC death in Bax-deficient mice treated with excitotoxins points towards the extrinsic apoptotic pathway as an alternative mechanism.⁹¹ Glutamate-induced excitotoxicity, implicated in glaucoma pathogenesis, may be contributed by the extrinsic apoptotic pathway.^92,93 Studies have evaluated the role of Fas ligand in RGC death. One study induced glaucoma through expression of only membrane-bound Fas ligand, which is pro-apoptotic. RGC death was rescued by gene therapy producing soluble Fas ligand,⁹⁴ suggesting that targeting members of the extrinsic pathway could lead to neuroprotection through disruption of apoptosis.

Neurotrophins:

One theory behind RGC apoptosis is a contribution from prolonged deprivation of neurotrophic factors.^95,96 Neurotrophins are a family of closely-related proteins that promote growth, development and survival of neurons. Brain-derived neurotrophic factor (BDNF), for example, exhibits neuroprotective effects on axotomized RGCs.^97–99 In glaucoma, apoptosis may occur secondary to obstruction of retrograde axonal transport of BDNF due to acute IOP elevation.^96,100,101 Exogenous supplementation of BDNF provides only short-lived neuroprotection with rapid loss of rescued cells after the neurotrophin is discontinued, due to downregulation of BDNF receptor expression.^98,102 In contrast, gene therapy provides a potential alternative for long-term overexpression of BDNF or its receptor.^103–105 Downstream signaling induced by BDNF is mediated by mitogen-activated protein kinase (MAPK) family proteins that are activated through phosphorylation, and suppressed by dephosphorylation by enzymes such as dual-specificity phosphatase 14 (Dusp14). Experiments demonstrated increased RGC survival in Dusp14 knockout mice subjected to anterior ischemic optic neuropathy.¹⁰⁶ Other promising neurotrophic factors include ciliary neurotrophic factor (CNTF), which has the additional benefit of stimulating axonal regeneration,¹⁰⁷ glial cell line-derived neurotrophic factor (GDNF), and nerve growth factor (NGF). This is an active area of research, with a number of clinical trials investigating neurotrophins. There is currently 1 completed phase 4, 2 completed phase 3, 11 completed phase 2, and 8 completed phase 1 trials examining treatments for ophthalmologic pathology,⁴¹ with final results pending for most studies.

Antioxidants:

Increased oxidative stress marked by overproduction of ROS is an important risk factor in multiple optic neuropathies.^108–111 ROS are generated through the electron transport chain as a byproduct of aerobic metabolic processes, and are neutralized by antioxidant defense systems. Excessive ROS can compromise cellular integrity and inhibit enzymes involved in mitochondrial energy production. Promising prospects include SIRT1, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), catalase, and yeast single-subunit NADH-quinone oxidoreductase (NDI1). SIRT1 is a ubiquitously expressed NAD+-dependent deacetylase that reduces oxidative stress, increases mitochondrial biogenesis, and protects against apoptosis.^25,112–115 Neuroprotective effects have been documented in mouse models of optic neuritis using SIRT1 activating compounds.^116,117 Further studies delivered SIRT1 as a gene therapy and demonstrated its efficacy in reducing RGC death in traumatic optic neuropathy, optic neuritis, and glaucoma.^{26,29,118,119} Other work with experimental models of glaucoma and traumatic optic neuropathy highlighted the significance of declining redox factors, linked to decreased expression of NAD+-synthetic enzyme, NMNAT2. Notably, levels of NAD+ were decreased, and supplementation with NAD+ and its precursors increased RGC survival. In vivo studies of NMNAT2 gene therapy show preserved visual function in glaucoma and traumatic optic neuropathy.¹²⁰ Catalase is an antioxidant enzyme responsible for detoxification of hydrogen peroxide to water and oxygen. Studies using an optic neuritis disease model and viral-mediated gene transfer of catalase have shown decreases in demyelination and optic nerve head swelling.¹²¹ Similar optic neuritis disease modeling has also shown success of NDI1 in reducing loss of RGCs, optic nerve axons, and retinal electrical activity using in vivo delivery of NDI1 packaged in a self-complementary AAV through intravitreal injections in mice, after onset of visual defects.¹²²

Challenges

While gene therapy provides a promising avenue of treatment, several key challenges and issues remain. There are inherent safety risks associated with gene therapy, as it remains difficult to halt once treatment has been delivered. Toxicity and adverse effects remain a persistent concern as the use of AAV can induce a significant inflammatory response, albeit less severe than that caused by use of adenoviruses.^32,123 Although AAVs are intrinsically less immunogenic, toxicity may arise from the capsid and promoter types, although the level is also dependent on the nature of the transgene.^124–126 In one clinical trial, one of six patients treated with an AAV expressing Rab escort-protein 1 for choroideremia developed a marked decline in visual function after a presumed intra-retinal immune response.¹²⁷

Besides inflammation, integrating viruses come with their own additional risk of insertional mutagenesis. Leukemogenesis has been reported in clinical trials where retroviral vectors were used to treat X-linked SCID³⁴ and Wiskott-Aldrich syndrome.¹²⁸ Due to their predominantly episomal expression, AAVs greatly reduce chances of insertional mutagenesis, but have integrated into genomic DNA at low frequency in animal models, leading to increased incidence of hepatic carcinoma.^129–131

Another major barrier for gene therapy is the comparatively high price due to the cost of manufacturing.¹³² While Alipogene tiparvovec (Glybera) was successful in being the first approved gene therapy in Europe, it was discontinued due to its cost, approximately $1 million per treatment.^36,133 Despite the high upfront cost of $425,000 per eye, a cost-effectiveness analysis for voretigene neparvovec (Luxturna) suggested that gene therapy may be cost-effective compared to standard of care in the scenario of 0% reduction in treatment effect over a lifetime, although cost-effectiveness remained highly sensitive to duration of treatment effect.^134,135

Future Directions

Over the past few decades, the number of clinical trials has steadily increased. Registries of gene therapy clinical trials include ClinicalTrials.gov, the World Health Organization’s International Clinical Trials Registry Platform, and American Society of Gene & Cell Therapy (ASGCT) Clinical Trials. Based on estimates of current growth, the FDA estimates approving 10 to 20 cell and gene therapy products a year by 2025.¹³⁶ However, this increase in numbers of clinical trials comes with its own concerns, and a critical examination of methodology and results must be undertaken when interpreting conclusions from these trials. The RESCUE, REVERSE, and current ongoing trials represent the efficacy as well as the all but assured growth of gene therapy in treating optic neuropathies. With the large number of preclinical studies continuing to grow and advance towards clinical applications, the hope for true neurorestoration and neuroprotection in optic neuropathies as the new standard will continue to take form. As gene therapy develops as a field, studies may lead to a future where gene therapy becomes a first-line treatment. Understanding basic principles behind gene therapy strategies may help clinicians make informed decisions about referring potential patients for clinical trials, and ultimately for treatment with new therapies once they are approved.