New Frontiers of Corneal Gene Therapy

Abstract

Corneal diseases are among the most prevalent causes of blindness worldwide. The transparency and clarity of the cornea are guaranteed by a delicate physiological, anatomic, and functional balance. For this reason, all the disorders, including those of genetic origin, that compromise this state of harmony can lead to opacity and eventually vision loss. Many corneal disorders have a genetic etiology, and some are associated with rather rare and complex syndromes. Conventional treatments, such as corneal transplantation, are often ineffective, and to date, many of these disorders are still incurable. Gene therapy carries the promise of being a potential cure for many of these diseases, with solutions and strategies that did not seem possible until a few years ago. With its potential to treat genetic disease by means of deletion, replacement, or editing of a defective gene, the challenge can also be extended to corneal disorders in order to achieve long-term, if not definitive, relief. The aim of this paper is to review the state of the art of the different gene therapy approaches as potential treatments for corneal diseases and the future perspectives for the development of personalized gene-based medicine.

Introduction

In the past two decades, considerable progress has been made on understanding the altered genetic mechanisms underlying the pathogenesis of acquired or hereditary diseases of the eye. This knowledge has led to the identification of novel targets for therapeutic development. Among the various therapeutic approaches, ocular gene therapy seems to be the most promising one.

The majority of eye diseases leading to blindness are caused by the degeneration of the retina. For this reason, retinal disorders have received much attention, and so far hundreds of experimental procedures have been performed for the identification of innovative gene-based interventions.¹ The progress achieved in this field culminated in successful clinical trials started in 2008 for Leber's Congenital Amaurosis (ClinicalTrials.gov number NCT00643747) and on December 19, 2017, the first U.S. Food and Drug Administration–approved gene therapy (Luxturna) was approved for the treatment of retinal diseases caused by mutations in the RPE65 gene.^2,3 However, gene therapy is also suitable for treating other forms of blindness and eye diseases.

According to the World Health Organization, blindness of the cornea is the fourth leading cause of blindness globally and is one of the major causes of visual deficiency after cataract, glaucoma, and age-related macular degeneration. Since the cornea can be cultured for a few weeks ex vivo, it becomes an appropriate model for ex vivo gene therapy as compared with other organs or tissues.⁴ Tolerance to inflammation and the high penetration capacity of each layer, as well as their association with immunological cells and soluble mediators, provide the cornea with strong immune function.^5,6 The corneal epithelial cells and keratocytes secrete cytokines, creating self-immune protection. Therefore, gene therapy represents an ideal strategy for the treatment of diseases related to the cornea, with some of them being very rare and having no current curative treatments available.

In addition to the retina, considerable steps forward have also been made in the field of corneal gene therapy, though not yet enough to reach clinical application. Gene delivery of several therapeutic genes (see Appendix A1 for a description of corneal gene therapy approaches) by means of viral, nonviral, and hybrid vectors (see Appendix A2 for a description of nucleic acid delivery systems for corneal gene therapy) as well as DNA vaccination, enzyme modulation, and various other innovative approaches have been proved to be useful in preclinical settings (see Table 1 for a summary). Lastly, gene editing is increasing its potential applicability and efficacy. In this paper, we critically review the gene administration and gene correction interventions attempted so far and their effects or the challenges posed for treating various corneal disorders.

Table 1.

Gene therapy approaches for corneal disorders

Disease	Incidence	Cause	Clinical manifestation	Therapy	Possible gene therapy approaches
Herpes simplex keratitis	500,000 cases/year (United States)	Herpes simplex virus-1 direct viral invasion or reactivation from latency	Symptoms: redness, discharge, lacrimation, irritation, itching, pain, and photophobia Clinical findings: dendritic lesions, corneal opacity and whitening, keratic precipitates	Topical administration of antivirals and corticosteroids; oral antivirals	Antisense oligonucleotides,¹⁸ morpholinos,¹⁹ siRNA,^22,23 ribozymes,^20,21 aptamers,²⁴ replication-defective virus,²⁵ meganucleases,^26,27 CRISPR/Cas9 editing²
Sjogren syndrome (#24355915)	About 7/100,000 adults	Etiology unknown (potential genetic and environmental factors involved)	Dry eye: ocular and oral dryness due to dysfunction of the nasolacrimal unit (nasolacrimal glands, corneal surface, and eye lids)	Artificial tears, anti-inflammatory agents (lifitegrast, cyclosprine A, rituximanb), and tear-secretion stimulators	Administration of cytokine using viral vectors,^38,40,41 tumor necrosis factor α inhibition by adenoviral transfer³⁹
Mucopolysaccharidosis types IH (Hurler), IH/S (Hurler-Scheie), and IS (Scheie) (#607014, #607015, #607016)	1/100,000 births	Mutations affecting the alpha-L-iduronidase activity on chromosome 4p16.3 (autosomic recessive)	Corneal clouding, glaucoma, retinopathy, and optic nerve abnormalities	Haematopoietic stem cell therapy; enzyme replacement therapy (Laronidase, also known by the brand name of Aldurazyme)	Adeno-associated virus–mediated alpha-L-iduronidase activity administration to human corneas via intrastromal injection⁸²
Mucopolysaccharidosis type VII (also known as Sly syndrome) (#253220)	1/250,000 births	Mutations affecting the beta-glucuronidase activity on chromosome 7q21.11 (autosomic recessive)	Corneal clouding and optic nerve abnormalities	Haematopoietic stem cell therapy	Adenovirus expressing human beta-glucuronidase into the anterior chamber or within the intrastromal region⁸⁴
Increasing corneal graft survival	N/A	Immunologic rejection of the donor cornea	Graft rejection	Anti-inflammatory and immunosuppressive drugs, donor cornea–human leukocyte antigens matching, cornea regrafting	Anti-angiogenesis approaches: (inhibition or regression of blood vessels)^44 –46 Immune response modulation: (adeno-associated virus–mediated gene transfer of CTLA4-Ig, interleukin-10, or interleukin-12)^47 –52 Anti-apoptosis strategies: (lentiviral vectormediated gene delivery of baculoviral p35 or mammalian Bcl-xL or programmed death ligand-1)^54,55
Corneal neovascularization	About 1.4 million cases per year adults	Congenital diseases, contact lens–related hypoxia, inflammatory disorders, chemical burns, limbal stem cell deficiency, allergy, trauma, infectious keratitis, autoimmune diseases, corneal graft rejection	Corneal scarring, stromal edema, lipidic deposition, keratitis, visual impairment, or blindness	Amniotic membrane transplantation, topical nonsteroidal anti-inflammatory and corticosteroid medications, argon and yellow dye laser photocoagulation, photodynamic therapy, cautery, diathermy	Intrastromal corneal injection of naked plasmid DNA encoding vascular endothelial growth factor⁶¹ subconjunctival injection of the recombinant endostatin–adeno-associated virus,¹²⁵ polyplex micelles,⁶⁹ intrastromal injection of a plasmid containing a small hairpin RNA cassette (pSEC.shRNA) against vascular endothelial growth factor-A⁷⁰
Meesmann epithelial corneal dystrophy (#122100)	N/A	Mutations affecting expression of KRT3 and KRT12 genes, on chromosome 12q13.13 and 17q21.2, respectively.	Symptoms: photophobia, blepharospasm, increased tear production, intolerance to contact lenses, transient blurred vision, foreign body sensation, and irregular astigmatism Clinical findings: formation of multiple intraepithelial microcysts in the outermost layer of the cornea	Stem cell transplantation and keratoplasty	Small interfering RNA,^89,97,98 genome editing¹⁰⁰
Ectrodactyly ectodermal dysplasia clefting (#604292)	About 1/900,000 births	Mutations affecting expression of the p63 gene on chromosome 3q27 (autosomic dominant)	Limbal stem cell deficiency characterized by conjunctival epithelial ingrowth, neovascularization, chronic inflammation, opacification, recurrent corneal erosion, persistent ulcers, destruction of basement membrane components, fibrous tissue ingrowth, progressive corneal clouding, and eventually blindness	Scleral lenses, eye drops; haematopoietic stem cell therapy; enzyme replacement therapy (Laronidase, also known by the brand name of Aldurazyme)	Allele-specific gene silencing through small interfering RNA^109,111
Aniridia (#24355915)	About 1/55,000 births	Mutations affecting expression of the PAX6 gene on chromosome 11p13	Ocular abnormalities: foveal and iris hypoplasia, nystagmus, glaucoma, cataracts Corneal abnormalities: aniridic keratopathy, corneal opacification	Lenses, surgical interventions	Premature termination codons read through,^118,126 recombinant pax6,¹²² augmentation therapy using viral vectors,¹²³ genome editing
Fuchs endothelial corneal dystrophy (#613267)	About 1:2000 adults	Trinucleotide repeat expansion in the transcription factor 4 gene	Progression of focal excrescences in the Descemet membrane (guttae) to endothelial cell degeneration and stromal edema, corneal erosions and gradual opacification that results in a visual veiling, cataract	5% salt solution, penetrating keratoplasty, and Descemet's stripping with endothelial keratoplasty.	Antisense oligonucleotide approach¹²⁷ CRISPR-Cas9 treatment.¹²⁴

Anatomy of the cornea

The adult human cornea is made up of five layers: (i) the epithelium, composed of 4–6 cell layers of nonkeratinized, nonsecretory, stratified squamous epithelium (40–50 μm); (ii) the Bowman's layer, an acellular structure of randomly arranged collagen fibrils (8–12 μm); (iii) the stroma, which makes up the bulk of the cornea and is essentially a collagenous matrix, with interspersed keratocytes; (iv) the Descemet membrane, the basement membrane of the corneal endothelial cells; and (v) the endothelium, a single layer of polygonal cells made up of simple squamous epithelium.

From an embryological point of view, the corneal epithelium is derived from the surface ectoderm at approximately 5–6 weeks of gestation. Corneal epithelial cells undergo systematic evolution, apoptosis, and desquamation. Their physiological homeostasis occurs about every 5–7 days after terminal differentiation of epithelial stem cells (undifferentiated unipotent) located at the corneo-scleral limbus.⁷ Alterations in epithelial cells cause at least three different diseases: Meesmann corneal dystrophy, aniridia, and ectrodactyly–ectodermal dysplasia–clefting (EEC) syndrome.

The major constituents of the stroma are the collagen fibers, which are disposed in regular lamellar sheets populated by keratocytes and mesenchymal cells and guarantee the high transparency of the cornea. An extremely rare cause of bilateral corneal opacity is the congenital stromal corneal dystrophy, an autosomal dominant form of corneal dystrophy, characterized by progressively numerous opaque flaky or feathery areas of clouding in the stroma.⁸

The epithelium and the stroma are usually considered as the most favored locations for gene delivery.

The endothelium has the vital function to keep the cornea transparent. In a normal cornea, the liquid equilibrium is guaranteed by the endothelial exchange/pumping excess of liquid and Na⁺/K⁺. Endothelium dysfunction results in excess accumulation of water in the corneal stroma, leading to the stromal swelling or edema resulting therefore in loss of transparency. The corneal endothelial dystrophies, comprising of (i) Fuchs endothelial corneal dystrophy, (ii) posterior polymorphous corneal dystrophy, and (iii) congenital hereditary endothelial dystrophy, generally represent the defects of neural crest terminal differentiation. Fuchs endothelial corneal dystrophy is a degenerative, bilateral, often asymmetric, and slowly progressive autosomal dominant disorder. It is characterized by a pleomorphic, attenuated, dysfunctional and degenerated corneal endothelium with the progressive formation of corneal guttae. Recent reports suggest that missense mutations in zinc finger E-box-binding homeobox 1 (ZEB1) may be responsible for at least some cases of late-onset Fuchs.⁹

Gene therapy approaches to corneal disorders

Examples of corneal disorders for which gene therapy has been considered as a potential treatment are described below.

Noninherited disorders

Herpes simplex keratitis

Herpes simplex virus (HSV), after primary infection of mucous membranes, is able to establish lifelong infections by remaining latent in the peripheral nerve ganglia. During latency, the virus is maintained in an episomal form, and most of the viral genes are not expressed, with the exception of those belonging to the latency associated transcript (LAT) region. HSV can periodically reactivate, and new virions are produced that are transported back to the site of primary infection.

HSVs commonly cause oral and genital lesions, but HSV type 1 (HSV-1) may also be responsible for a variety of ocular diseases. Herpes simplex keratitis (HSK) is the most common infectious cause of blindness in the developed world, and it is also a major determinant of corneal graft rejection after transplantation. In the United States, the incidence of new cases of HSV eye infections is approximately 500,000 cases/year,¹⁰ even if clinical manifestations appear only in 20–30% of cases.¹¹

The virus initially infects the corneal epithelium, then enters in sensory nerve endings and travels along axons to reach the trigeminal ganglion: the disease is most often due to reactivation of a latent infection of trigeminal sensory neurons. Differently from other infectious keratitis (e.g., fungal or bacterial keratitis), HSK may become chronic or recurrent,¹² and the main source of recurrent disease is viral reactivation due to triggers such as ultraviolet exposure, stress, ocular surgery, hormonal factors.^13,14

There are three main subtypes of HSK: epithelial, stromal, and endothelial.¹⁵ Epithelial HSK is the most common type, and it occurs after direct viral invasion. This form presents with dendritic lesions with swollen borders and intraepithelial cell infiltration. The stromal subtype is the consequence of an immune response against the virus and is characterized by corneal opacity and whitening. Lastly, endothelial keratitis may arise either from secondary inflammation caused by HSV-1 or from direct infection of endothelial cells and manifests with keratic precipitates and iritis.

HSK symptoms include redness, discharge, lacrimation, irritation, itching, pain, and photophobia.¹⁵ The occurrence is usually unilateral, but a small fraction of affected people may also experience bilateral disease, especially younger and immunocompromised patients.¹⁶

The treatment for HSK is mainly topical, and involves administration of antivirals such as acyclovir, ganciclovir and trifluridine, with acyclovir as the first-line treatment. Moreover, topical corticosteroids as adjuvant therapy may be employed.¹² Oral antivirals are also currently in use, and include acyclovir, valacyclovir and famciclovir.¹⁷ Nevertheless, current antiviral treatments target HSV DNA polymerase, thus they are active in the replicative viral stage but unable to clear latent HSV from the host. Moreover, any decrease in drug bioavailability and in virus sensitivity to the treatment may cause therapy failure. The study of molecular strategies that could be durably effective in the cells and avoid sensitivity issues led to the development of different gene therapy strategies targeting HSV.

Several approaches have been attempted focusing on the downregulation of viral or cellular gene expression, in order to affect viral replication and to prevent the development of herpetic lesions and diseases. In particular HSV-1 specific transcripts have been targeted by a plethora of approaches, including antisense phosphorotiates, peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), and ribozymes.^18

–21

The first successful inhibition of HSV-1 replication by specific targeting of viral mRNA has been achieved by using antisense phosphorothioate oligonucleotides designed to inhibit the translation of HSV-1 transcripts. A screening of 100 phosphorothioate 20-mer oligonucleotides directed against different HSV-1 target genes led to the identification of 6 oligos significantly inhibiting viral replication, the most active being directed against the translation initiation site of the immediate early protein IE 110, involved in transcriptional activation of later viral genes.¹⁸

Viral translation has subsequently been targeted also by PPMO, single-stranded nucleic acid analog reducing gene expression through steric blockage of complementary RNA. Targeting the translation-start sites of HSV-1 ICP10 and ICP27 mRNA has been reported to decrease viral replication in vitro and to reduce ocular disease in HSV-1 infected mice¹⁹

Another possible approach is the degradation of HSV-1 transcripts by ribozymes, RNA enzymes cleaving and splicing RNA molecules. The HSV-1 late gene UL20 proved to be a valuable target for hammerhead ribozymes expressed by adenoviral vectors in both cell cultures and mice, suggesting that cleaving mRNA of essential late genes may represent an effective therapeutic strategy against HSV infection.²⁰ More recently, an adeno-associated virus (AAV) vector has been employed in eyes of rabbits with latent HSV-1 infection for the delivery of LAT-targeting ribozymes: this treatment appeared to block viral reactivation in more than 60% of infected eyes, thus showing the relevance of LAT for HSV reactivation and providing evidences of the potential of the approach for treatment of HSV infections.²¹

As mentioned above, gene silencing has been employed not only for targeting the expression of viral genes, but also against pathways induced by HSV-1 infection. As demonstrated by Kim and colleagues, small interfering RNA (siRNA) targeting vascular endothelial growth factor (VEGF) pathway genes may significantly inhibit excessive ocular angiogenesis, a phenomenon frequently resulting in pathological neovascularization in HSV-related eye disease.²² Moreover, antisense oligonucleotides have been tested against interferon gamma (IFN-γ) mRNA: In a murine model of HSV-1 eye infection, a topical treatment with IFNγ-antisense oligonucleotides induced an improvement in incidence and progress of HSK, by reducing the number of inflammatory cells in the site of infection without affecting antiviral defenses.²³

Aptamers are functionally active nucleic acids that can bind to a wide range of molecules, including complex targets, and are often employed against viral proteins. As demonstrated by in vitro antiviral assays, the gD protein of HSV-1, that mediates virus entry into host cells, may be bound with high affinity by an RNA aptamer, with consequent inhibition of viral entrance and replication. This finding suggests that the identified aptamer could be further investigated for the design of topical products reducing the risk of HSV-1 infection through physical contact.²⁴

Replication-defective and attenuated viruses have been frequently studied as potential vaccines in animal models. In 2004, Augustinova and colleagues generated a replication-defective, dominant- negative HSV-1 strain that acted as a replication inhibitor for itself and for wild-type HSV-1 in a mouse ocular model, showing a vaccine potential due to its capability of eliciting both innate and cell-mediated immunity.²⁵

Latent HSV-1 may be targeted by rare-cutting endonucleases such as meganucleases: these enzymes are able to recognize large DNA sequences (>12 bp) and can be engineered to obtain tailored specificity. The use of meganucleases to cleave regions of HSV genome has been reported by Grosse and colleagues, demonstrating that meganucleases engineered to target HSV-1 could prevent the infection of cultured cells by both recombinant and wild-type viral strains. Moreover, the cleavage could determine the consequent degradation and clearance of cleaved molecules, thus leading also to the disappearance of nonreplicative episomal sequences in HSV-1 latency stage. The authors suggest that their strategy could be particularly useful for the prevention of recurrent infections during corneal transplantation, since introduction of meganucleases into the graft before transplantation could provide additional protection.²⁶ This concept has been furtherly highlighted in another study employing a recombinant adeno-associated virus to deliver anti-HSV-1 meganucleases to human corneas ex vivo before transplantation into herpes keratitis patients to avoid reinfection and graft rejection.²⁷

The CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats [CRISPR] and CRISPR-associated protein 9) system has been also tested as a strategy to target pathogenic viruses. Herpesviruses genomes may be altered in specific regions in order to impair viral replication, as demonstrated in a recent study showing a marked drop in production of HSV-1 infectious particles in cells carrying CRISPR-Cas9 systems directed against multiple essential HSV-1 protein-coding genes simultaneously. The system might also abrogate the replication of HSV-1 reactivated from quiescence, although it was not able to directly engineer the quiescent genome, due to the highly methylated state of latent HSV-1 DNA, that makes the genome unreachable by the CRISPR-Cas9 machinery.²⁸

The interest in developing many different gene therapy strategies against HSK throughout the years, spacing from the first gene silencing approaches up to CRISPR-Cas9 editing, highlights the clinical relevance of herpetic disease in the eye. Further studies focusing on the degradation of latent viral genomes or on the inhibition of HSV reactivation from latency are needed in order to eradicate chronic and recurrent forms of this pathology.

Dry eyes: Sjögren's syndrome dry eyes

Described for the first time in 1933, the Sjogren syndrome (SS) is a chronic systemic autoimmune disease, affecting the exocrine glands associated or not with autoimmune rheumatic disorders. The typical ocular and oral sickness is due to lymphocytic infiltrations of lacrimal and salivary glands.^29
–31 Dry eye, or keratoconjunctivitis sicca, is associated to the dysfunction of the nasolacrimal unit composed by nasolacrimal glands, corneal surface, and eye lids. Principal ocular symptoms are discomfort, visual disturbance, and dryness followed by photophobia, itching, and tearing.³² The prevalence of SS is estimated as 7 per 100,000 people, more commonly in females and Caucasians.^32,33 The pathogenic mechanism is based on an autoimmune response: the induced lacrimal gland dysfunction decreases the production of tear film, with a consequent corneal epithelial erosion. The epithelial erosion is unlighted by the reduction in Goblet cells and mucin production in conjunctiva, where artificial tear administration could provide comfort for patients and avoid corneal damage.^29,34 Although the etiology is still unknown, different potential mechanisms have been revealed, involving genetic and environmental factors virus infection in susceptible subjects.^32,35 Initially, T cells were considered as the principal actors in the autoimmune process, but now there are evidence of an involvement of B cells in the pathophysiology of primary SS.³⁶ Antigens from various sources, like autoantigens, mimicry and apoptosis, presented to T cells, generate inflammation and immune response. It has been also demonstrated the dysfunction in B and Treg cells and the upregulation of human leukocyte antigen (HLA) genes. Therapies, abrogating or modulating the inflammatory response, could play a key role in decreasing the inflammatory symptoms of the keratoconjunctivitis sicca.

Therefore, specific cytokines with anti-inflammatory effects could represent potential genes to be used in gene therapy approaches for SS treatment. In this context, the study of vector-specific reporter gene expression patterns allowed to conclude that vaccinia and adenovirus are efficient vectors for gene transfer into lacrimal gland tissue in primary culture.³⁷ Another study reported a partial, but transient, suppression of SS-like features of the disease in rabbit, after injection of an adenoviral vector carrying the vIL-10 gene.³⁸ Trousdale et al. showed that adenovirus-mediated gene therapy treatment of rabbits with established autoimmune dacryoadenitis, using AdTNFRIp55-Ig, resulted in improvement of clinical features, such as an increase in basal tear production, an increase in tear stability, and a reduction in surface corneal defects.³⁹ Different mouse models, treated with adenoviral administration of erythropoietin, known for its role in retinal protection and for its vasculo-proliferative characteristics (useful to facilitate wound healing in several diseases with neurotrophic or inflammatory keratopathy)⁴⁰ or mucin 5AC, fundamental for the lacrimal fluid stability,⁴¹ showed the tolerability and the success of gene transfer for SS. Zhang et al. demonstrated the topical neutralization of IFN-γ produced by CD4+ T cells, thus preventing Goblet cells loss by modulating apoptosis and maintaining interleukin (IL)-13 levels in a mouse model dry eye model.⁴² These preliminary results show the potential of gene therapy for the treatment of SS.

Gene therapy approaches for corneal graft survival

Even though corneal transplantation is the most successful example of tissue transplant worldwide, immunologic rejection remains a leading cause of graft failure, with a prevalence varying from 5% to 40%. To manage immune-mediated graft rejection, two main gene therapy-based strategies can be envisaged: (i) inhibiting or regressing corneal vascularization and (ii) preventing or reversing immune-mediated graft rejection. In addition, apoptosis has been identified as one of the mechanisms leading to corneal cell death and consequent diminished graft survival.⁴³ Again, approaches based on gene therapy might lead to treatments reducing the chances of graft failure after transplantation.

Anti-angiogenesis strategies

Vascularized beds are a serious hazard to grafting with the likely consequence that corneas are inundated with inflammatory cells, activated antigen presenting cells, and T cells that release cytokines and chemokines, leading to further amplification of the immune response and eventually graft rejection. Inhibition or regression of blood vessels is therefore the main strategy to maintain corneal clarity and survival. Improvements in graft survival have been achieved by knocking down neuropilin-2 through RNA interference,⁴⁴ targeting Flt23k,⁴⁵ or, more generally, reducing the amount of free VEGF-A,⁴⁶ thus leading to the inhibition of heme- and lymph-angiogenesis and eventually a decrease of activated T-cell influx into the corneal graft.

Immune response modulation

Since graft rejection is primarily due to an overwhelming immune response against antigens in the donor cornea, several molecular candidates have been tested in order to modulate the immune response. Adenovirus vector-mediated gene transfer of cytotoxic T-lymphocyte antigen 4 protein has been shown to prevent activation of T cells and effectively prolong graft survival.^47,48 Similarly, adenoviral vectors have been used to deliver IL-10 and IL-12 to ovine corneas leading to higher rates of graft survival.^49

–52 However, the use of these vectors remains an issue, as highly immunogenic. A further strategy to modulate the immune response would be to induce or facilitate tolerance. Recently, lentivirus-mediated transduction of indoleamine 2,3 dioxygenase (thought to lead to T cell arrest in the G1 phase and therefore implicated in ocular immune privilege) to corneal endothelial cells (ECs) ex vivo prior to corneal transplantation was shown to prolong corneal graft survival in mice significantly.⁵³

Anti-apoptosis strategies

A further cause of corneal graft rejection is due to the loss of corneal ECs during storage in eye banks or after transplantation. Corneal ECs have limited proliferative capacity, therefore once lost, their contribution to maintaining corneal transparency is compromised. Cell apoptosis is believed to be a cause of EC loss and successful inhibition of the apoptotic pathways has proven to promote endothelial cell survival, thus suggesting a crucial role of apoptosis in graft survival/rejection.⁵⁴ Lentiviral vector-mediated gene delivery of baculoviral p35 or mammalian Bcl-xL to corneal endothelium during various storage conditions showed increased endothelial cell survival, thus indicating that the ex vivo modification of corneal allografts might increase the availability of tissues for transplantation.⁵⁴ Similarly, ex vivo transduction of rat corneas with lentiviral vectors expressing programmed death ligand-1 significantly increased graft survival and led to a decrease in IL-6 and IFN-γ compared to controls.⁵⁵ As an alternative, a future possibility could envisage transfecting a cDNA encoding cytoprotective molecules for reduction of oxidation.

Considering that human donated corneas are stored in eye banks for some weeks before they are transplanted, ex vivo genetic modification during storage might represent a solution to minimize the chances of immunological rejection and therefore increase in vivo graft survival. This could eventually become a potential new treatment for high risk patients, who had previous and multiple graft rejections and are not responding to corneal HLA-matching either.

Corneal neovascularization

Corneal neovascularization (CNV) is a sight-threatening condition characterized by the ingrowth of new vessels from the limbus caused by the loss of the limbal stem cell barrier and it is becoming increasingly common worldwide with an estimated incidence rate of 1.4 million cases per year.⁵⁶ CNV occurs in a wide variety of corneal pathologies including congenital diseases, contact lens-related hypoxia, inflammatory disorders, chemical burns, limbal stem cell deficiency, allergy, trauma, infectious keratitis, autoimmune diseases, and corneal graft rejection.⁵⁷ CNV can lead to corneal scarring, stromal edema, lipidic deposition, and keratitis, resulting in significant visual impairment or blindness.⁵⁸ Angiogenesis is initiated when the balance between angiogenic and antiangiogenic factors is shifted toward angiogenic factors.^57,59,60 The clinical management of CNV represents a big challenge since current pharmacotherapeutic and surgical options are not always effective. Several approaches including amniotic membrane transplantation, topical nonsteroidal anti-inflammatory and corticosteroid medications, argon and yellow dye laser photocoagulation, photodynamic therapy, cautery, and diathermy have been used to shut new corneal vessels.

Various angiogenic factors mediate CNV, including VEGF, basic fibroblast growth factor, matrix metalloproteinase, platelet-derived growth factors, and IL-1. Among these, VEGF is reported to be the primary mediator of neovascularization in the eye and is elevated in inflamed vascularized corneas of rats and humans.^61,62 For these reasons, many studies on gene delivery to the cornea have been focused on inhibiting the VEGF signaling pathway in animal models of chemical burn-induced corneal neovascularization. The intrastromal corneal injection of naked plasmid DNA encoding VEGF and the soluble receptor Flt-1 in mouse showed that the plasmid-injected corneas remained clear and free of inflammation, thus demonstrating that the expression of the genes injected into cornea is extraordinarily rapid.⁶³ In addition, a plasmid DNA encoding Kringle 5 of plasminogen and endostatin were effectively transferred to the rat cornea by subconjunctival injection with electroporation, showing the inhibition of alkali-induced corneal neovascularization.⁶⁴ Another study showed that Parstatin, a 41-mer peptide produced by proteolytic cleavage during activation of the PAR1 receptor, is believed to inhibit angiogenesis. Intravitreal or subconjunctival administration of Parstatin peptide reduced CNV in mice and interrupted the progress of CNV in rat corneas with chemical burns. It was demonstrated that combined blockage of VEGF and FGF2 receptor with Parstatin may have higher efficiency towards blocking ERK1/2. It was also reported that its application reduced the inflammatory cell count in rat corneas after chemical cauterization, thus suggesting that it could be a suppressant for inflammation in cornea.⁶⁵ The antiangiogenic effects of the brain-specific angiogenesis inhibitor 1 (BAI1-ECR) gene has been investigated in an in vivo rabbit model of corneal angiogenesis, by means of subconjunctival injection of the BAI1-ECR gene mixed with nonliposomal lipid, showing an effective reduction of corneal neovascularization induced by chemical and mechanical denudation and thus suggesting that the BAI1-ECR protein can be used as an angiogenesis suppressor in the eye.⁶⁶

Alternative approaches towards the treatments of CNV are represented by viral vectors encoding anti-angiogenic genes. The use of a recombinant adeno-associated viral (rAAV) vector carrying endostatin gene as an anti-angiogenic strategy was evaluated in a mouse model, where CNV was induced by silver nitrate cauterization.⁶⁷ The recombinant endostatin-AAV was administrated by subconjunctival injection and it proved to be an efficacious means of delivering the endostatin gene, since the transgene expression was stable for over 8 months with minimal immune reaction and successfully inhibited neovascularization. Another study showed the delivery of the decorin, a small leucine-rich proteoglycan able to modulate angiogenesis in nonocular tissues, to the rabbit stroma with AAV5, after CNV was induced with VEGF using a micro-pocket assay.⁶⁸ A significant inhibition of VEGF-induced corneal angiogenesis was observed, thus demonstrating the potential use of decorin gene therapy for treating corneal angiogenesis in vivo, without any major side effect.

Gene delivery by means of nanoparticles or nanomaterials also represents a promising method to treat CNV, as well as the polyplex micelles, obtained with the complex of plasmid DNA with synthetic cationic polymers.⁶⁹ A block copolymer, poly (ethylene glycol) (PEG)-block-polycation carrying ethylenediamine units in the side chain (PEG-b-P[Asp(DET)]), generated a polyplex micelle via polyion complex formation with plasmid DNA. After injecting PEG-b-P[Asp(DET)] micelles in the subconjunctival space of mice, the reporter gene expression was monitored and was found prolonging gene expression with reduced cytotoxicity. Gene transfer by the same polyplex micelle carrying a sflt-1–containing plasmid also proved considerable inhibition of CNV in mice making it a potential source for treatment of CNV.

Another study demonstrated that intrastromal injection of a plasmid containing a small hairpin RNA cassette against VEGF-A-loaded poly (lactic co-glycolic acid) nanoparticles was an effective, nonviral, and nontoxic form of anti-angiogenic therapeutic strategy in vivo for murine CNV. This approach provides an attractive option for the treatment of human CNV.⁷⁰

Recently, Lu et al. identified potential anti-angiogenic corneal microRNAs (miRNAs) that can be delivered via recombinant adeno-associated viruses (rAAVs) into injured corneas to block angiogenesis.⁷¹ One of these, miR-204, showed strong anti-angiogenic properties and has proved to be effective in reducing vascolarization of injured mouse cornea.

In conclusion, gene therapy approaches by means of subconjunctival, intracorneal or topical delivery of anti-angiogenic genes using plasmids, viral vectors and nanoparticles have proven to be safe and effective in inhibiting experimental CNV and pave the way for the treatment of patients with CNV.

Inherited disorders

Mucopolysaccharidosis

Mucopolysaccharidosis (MPSs) is an inherited group of disorders caused by defects in lysosomal enzymes responsible for the degradation of glycosaminoglycans (GAGs) and is characterized by a high range of clinical manifestations. MPSs are classified on the basis of the enzymes, which are dysregulated and include MPS I (Hurler, Scheie and Hurler/Scheie), MPS II (Hunter), MPS III (Sanfilippo A, B, C, and D), MPS IV (Morquio A and Morquio B), MPS VI (Maroteaux-Lamy), MPS VII (Sly), and MPS IX Natowicz). Symptoms vary and include coarse face central nervous system impairment, hearing loss, respiratory problems, valvular heart disease, hepatosplenomegaly, skeletal dysplasia, gait abnormality, and corneal clouding.

Therapies like hematopoietic stem cell transplantation (HSCT), enzyme replacement therapies (ERT) and various surgical interventions are currently available for patients with MPS. While some advantages have been shown and a slower progression of the disease observed, limitations do exist. ERT requires weekly injections, is costly and has limited penetration to the bone and central nervous system. HSCT may not be applicable to all the patients because of the limited availability of matched donors and complications such as the graft-versus-host disease. Therefore, more effective and feasible therapies for MPS are urgently required.

Gene therapy might represent a promising approach for treating patients with MPS and has been under investigation for the last three decades. Phase 1/2 gene therapy clinical trials for some types of MPS (MPS I, II, IIIA, IIIB, and VI) are ongoing or scheduled in the United States, some European countries and Australia (reviewed by Sawamoto et al.⁷²). Despite all these studies, it remains controversial whether the ocular manifestation of MPS are influenced by HSCT, ERT or gene therapy.^73,74 The proposed gene therapy-based strategies for the treatment of the ocular manifestations of MPS include both systemic and local approaches (i.e., delivered to the cornea or the retina), but findings are so far limited to experimental investigations in animal models (mainly mice, cats and dogs with MPS I and VII as they share many of the clinical, biochemical, and histopathological features of humans).

Systemic gene therapy approaches

Studies have been conducted in newborn and adult mice with MPS I or MPS VII using various vectors, dosages, and outcome measures (reviewed by Ponder and Auricchio⁷⁵). High serum enzyme activity levels resulted in profound effects on the eyes of both newborn and adult mice at 6–8 months after treatment with retroviral vectors.^76,77 Correction of the dark-adapted electroretinogram and GAG storage in the corneal stroma and endothelium were normalized in a dose-dependent fashion.⁷⁶ Similar storage improvements in the cornea and retinal pigmented epithelium were found in MPS VII mice at 6 months after neonatal retroviral vector mediated gene transfer, which correlated with to the enzyme expression levels.⁷⁷ In contrast, intravenous administration of AAV vectors to MPS VII newborn mice resulted in low expression levels upon ageing, thus suggesting that only juvenile or adult mice should be treated in order to avoid the loss of extra-chromosomal AAV vector in the liver as hepatocyte divide during growth.^78
–80

Success of systemic gene transfer in preventing the ocular manifestations of MPS was also demonstrated in studies with larger animal models. Ponder and colleagues showed that, when MPS VII dogs were injected intravenously at 2–3 days of age with a retroviral vector containing the human alpha1-antitrypsin promoter upstream of the canine beta-glucuronidase (GUSB) cDNA, little or no corneal clouding were observed. A likely explanation is that the prevention of corneal clouding is probably due to the uptake of enzyme from the blood via the prelimbic capillaries by the corneal stromal cells.⁸¹ In conclusion, systemic gene therapy with integrating vectors could be an ideal treatment, as it would lead to widespread and persistent enzyme activity in vivo, even if high vector dosages might have to be used. However, safety concerns will have to be addressed before moving into clinical settings.

Local gene therapy approaches

Some of the concern of systemic gene therapy approaches could be overcome with local delivery of gene transfer agents to the eye. Examples of potential administration routes include the subretinal and intravitreal injections, or injections into the anterior chamber or periocularly. A few examples of such applications are reviewed below.

Vance and colleagues showed that AAV-mediated cytomegalovirus-driven alpha-L-iduronidase (IDUA) administration to normal human donor cornea explants via intrastromal injection led to widespread transduction with >10-fold increase in IDUA activity. Given the ability of secreted IDUA to cross-correct neighboring cells, these data suggest that the strategy adopted should result in normal levels of IDUA when MPS I patient corneas will be treated.⁸² Since corneal transplants are met with high rejection rates in MPS I children,⁸³ this strategy could prevent/reverse MPS I-associated cornea blindness, which is mainly caused by accumulation of chondroitin and dermatan sulphate GAGs, known to alter the uniform distribution, organization and size of collagen fibrils.

In animal models of MPS, all studies of local gene therapy have reported a reduction of histopathological aberrations and storage of GAGs in the cornea, ciliary body, and retina. This was accomplished by benefits on corneal clouding and retinal function improvements, thus suggesting the efficacy of strategies based on local gene transfer, although longer-term evaluations are needed.⁷⁵ In mice with MPS type VII, administration of an adenovirus expressing human GUSB under the control of the CAG promoter into the anterior chamber or within the intrastromal region of the cornea showed to be successful for the treatment of corneal clouding. Widespread transgene expression was observed when the adenoviral vector was administered inside the cornea after lamellar keratotomy and rapid elimination of the lysosomal storage in the corneal keratocytes was seen occurring. Furthermore, intrastromal vector administration did not generate significant levels of anti-adenovirus neutralizing antibodies, and secondary vector administration was effective, thus suggesting that a direct intraocular administration of adenoviral vectors could be beneficial for treatment of MPS VII.⁸⁴ In a more recent report, Serratrice and colleagues showed that the pathology in the canine MPS VII cornea improved after the injection in the cornea of a helper-dependent canine adenovirus type 2 vector carrying the human GUSB cDNA under the control of a Rous sarcoma virus promoter.⁸⁵

In conclusion, since the clinical trials with HST and/or ERT have not yet demonstrated whether there is any beneficial effect on the ocular manifestations of the disease, local delivery of gene therapeutics might become a supplemental therapy to reverse and/or prevent MPS-associated corneal blindness.

Meesmann epithelial corneal dystrophy

Meesmann epithelial corneal dystrophy (MECD, OMIM 122100) is a rare autosomal dominant inherited disease caused by heterozygous mutations in keratin 3 (KRT3) (12q13.13) or KRT12 (17q21.2) genes, encoding respectively, corneal specific keratins 3 and 12 (k3 and k12⁸⁶). K3 and k12 are the unit of the intermediate filament cytoskeleton of corneal epithelial cells, providing structure and stability⁸⁷ and consequently, their malfunction causes mechanical fragility of the anterior corneal epithelium. MECD, whose frequency is unknown, is characterized by the formation of myriads of intraepithelial microcysts of variable distribution and density in the outermost layer of the cornea that can appear early in life and increase in number with age.⁸⁸ In most cases, MECD remains asymptomatic while in some cases, microcysts can break, causing the typical symptoms, including photophobia, blepharospasm, increased tear production, intolerance to contact lenses, transient blurred vision, foreign body sensation, and irregular astigmatism. All mutations are found in the highly conserved helix initiation or termination motifs, located at the N- and C-termini of the keratin protein, and implicated in heterodimerization, protein coiling, and keratin fiber assembly.^89,90 At least 24 mutations have been described for MECD (21 mutations in KRT12 gene and 3 in KRT3 gene), the majority of which are missense point mutations.^{86,89,91
–93} Nevertheless, the mechanism underlying the formation of corneal microcysts and the genotype phenotype correlation remain poorly understood. Stem cell transplantation and keratoplasty are the only options available when rare severe phenotypes with corneal scarring cause loss of visual acuity.^94,95 Unfortunately, after penetrating keratoplasty, pathology reappearance can occur, due to resident mutant limbal stem cells that with time foster corneal surface regrowth.⁹⁴ To date, there is no therapy available to address MECD pathology. As a dominant-negative disease, gene therapy strategies for MECD should rely on mutant allele silencing or knock out. In fact, only expression of one K12 wild type allele, is required for normal corneal epithelial function, as demonstrated by studies on heterozygous mice (+/−) for Krtl.12 gene, which show a phenotype without corneal clinical manifestations.⁹⁶ Therefore, allele-specific siRNA therapy, designed to ablate the mutant allele, might represent a possible treatment. Importantly, allele-specific siRNAs have been successfully developed against the severe heterozygous missense mutation Leu132Pro⁸⁹ and the milder and most common European mutation Arg135Thr^97,98 in KRT12 gene. Both siRNAs specifically silenced the mutant alleles expression, without neither affecting the wild-type allele or other keratins. Cytoskeleton dysfunction was reversed both in a cell culture model system as well as in established cultures of MECD corneal epithelial limbal stem cells.^97
–99 These inhibitors will have to be further tested before translating therapy into patients but might represent a viable treatment option for MECD. Recently, a mouse model of MECD with the severe Leu132Pro mutation has been generated by homologous recombination, providing an in vivo model to test potential therapies.⁹⁰ The phenotype of this model closely mirrors that of MECD patients: a corneal epithelium with cell fragility, destratification of the basal layer, and the formation of microcysts that might rupture at the corneal surface. In this model, an alternative but very innovative and efficacious approach for mutant allele ablation has been recently applied using the CRISPR-Cas9 technology.¹⁰⁰ In this pioneering work, the authors aimed at developing an allele-specific genome-editing strategy for the Leu132Pro mutation by exploiting the occurrence of a novel protospacer adjacent motif site in the mutant allele, caused by the single base change T>C, and absent in the wild type allele, making this strategy very safe. This Cas9-based gene-editing system was found to be allele specific when injected intrastromally in vivo. The efficiency of nonhomologous end joining was of 38.5%, with deletions of up to 53 nucleotides, most of which were predicted cause frameshifts, thus demonstrating the potential of this approach for combined gene and cell therapy.¹⁰⁰

Ectrodactyly–ectodermal dysplasia–clefting syndrome

Ectrodactyly–ectodermal dysplasia–clefting (EEC) syndrome is a rare autosomal dominant inherited disease characterized by ectrodactyly (split-hand-foot malformation), ectodermal dysplasia, and cleft lip and palate, affecting skin, nails, hair, teeth, sweat glands, and the ocular ectodermal derivatives.^101,102 The incidence in the population is about 1:900.000, according to the Italian Ministry of Health. While in childhood, clefting and hand deformities are the main clinical features; during early adulthood, ocular problems become the predominant clinical aspect of EEC syndrome.^103,104 Patients often show ocular surface alterations such as recurrent blepharitis and conjunctivitis, superficial microlesions of the cornea, spontaneous corneal perforation and ulceration, defective regeneration, and poor re-epithelialization following trauma or penetrating keratoplasty (PK). They develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. The EEC syndrome is caused by mutations in the p63 gene, an important transcription factor for the normal development of orofacial ectoderma and limbs and for stem cell differentiation in stratified epithelia.¹⁰¹ The p63 gene generates six isoforms and DNp63a is the predominant isoform in the human corneal epithelium¹⁰⁵ The EEC syndrome is mainly caused by point mutations in the DNA binding domain of the p63 gene.¹⁰⁶ Five p63 DNA–binding mutations account for almost 90% of EEC patient cases. The five missense mutations affect arginine residues (R204, R227, R279, R280, and R304) and represent hotspot mutations for EEC.¹⁰⁷ In particular, mutations R379H and R304Q result as the most severe mutations affecting the cornea.¹⁰⁶

Despite the clinical and molecular knowledge accumulated, no definitive cures are currently available to treat the ocular alterations in EEC patients, and the biological knowledge of the disease has only recently been clarified.^104,108,109 Progressive limbal stem cell failure results in visual morbidity in EEC syndrome, ultimately leading to blindness.¹⁰⁴ Common therapeutic strategies, such as PK, are not a solution since in cases of limbal stem cell deficiency as soon as the donor epithelium is exhausted, conjunctivalisation occurs again.¹¹⁰ As EEC syndrome results from heterozygous dominant-negative mutations in the p63 gene, therapeutic strategies based on allele-specific gene silencing through siRNA could specifically inhibit the expression of disease-associated alleles without suppressing the expression of the corresponding wild-type alleles to phenotypically correct the stem cell population. Recently, a systematic screening of locked nucleic acid siRNAs against R279H-p63 allele allowed researchers to identify a number of potent siRNA inhibitors for the mutant allele, able to specifically downregulate the R279H-DNp63a mRNA by approximately 90% without affecting the wild-type p63. Long-term treatment resulted in a longer acquired lifespan, thus counteracting premature stem cell aging in vitro, and was shown to be effective in correcting the epithelial hypoplasia, giving rise to a full-thickness stratified and differentiated epithelium.¹⁰⁸

Similarly, specific siRNAs able to repress the EEC-causing p63 mutant R304W were identified, by using a systemic screening based on a dual-luciferase reported gene assay.¹¹¹ Upon siRNA treatment, the transcriptional function of DNp63-WT allele was restored in induced pluripotent stem cells derived from EEC patient biopsy.

These studies demonstrate the phenotypic correction of mutant stem cells in EEC syndrome by means of siRNA mediated allele specific silencing with restoration of function. Since EEC-related corneal pathology follows a clear clinical course with limbal stem cell deficiency usually manifesting in the second to third decade leading to severe corneal failure in the fourth to fifth decade, this might provide a therapeutic window to correct the EEC defect using genetic preventive strategies. In early corneal disease, with limbal stem cells still present in the limbus, the use of eye drops containing mutant specific siRNAs may be a practical therapeutic option which could prevent blindness.

Aniridia

Aniridia is an infrequent, progressive panocular genetic disorder (incidence about 1:55000). It is characterized by a bilateral alteration in the development of the eye and it is associated with a noticeable foveal and iris hypoplasia, the latter also being the main diagnostic feature of the disease and usually leading to nystagmus. Patients usually develop a variable range of symptoms affecting a number of ocular structures, including cornea, iris, lens, fovea, and optic nerve. Therefore, a number of additional complications are common, including a few sights threatening ones such as glaucoma, cataracts and corneal opacification. Aniridia is a well-documented genetic anomaly that may appear sporadically (approximatively 33% of cases) or within families (approximatively 66% of cases), exhibiting a dominant autosomic inheritance pattern with variable expression amongst the members of a family. The disease is caused by an extremely heterogeneous number of different mutations leading to haploinsufficiency of PAX6 gene, which is located in the short arm of chromosome 11^p13 and is broadly expressed in the development of various eye structures, including the cornea.¹¹²

Indeed, one of the causes of progressive loss of vision and morbidity in aniridia patients is aniridic keratopathy (AAK), which is caused by dysfunctioning limbal stem cells. PAX6 (+/−) small eye mice, despite the fact that they do not fully recapitulate the complexity of the phenotype observed in humans, have been used as an animal model for aniridia. In such disease model, altered expression of cytokeratin 12, the presence of goblet cells on the corneal surface, and modified migratory properties of corneal epithelium could be observed together with a depletion in limbal epithelial stem cells which caused a reduced production of progeny.¹¹³ These experimental findings support the notion that limbal stem cell deficiency plays a causative role in ocular surface failure from aniridia.^114,115 Hence, AAK is an example of primary, intrinsic limbal stem cell deficiency caused by mutations in PAX6 gene.

Due to the progressive nature of the disease, management depends on the clinical manifestations, and it is therefore very variable, mainly including the use of lenses as well as surgical procedures such as goniotomy, cataract extraction, and keratoplasty, and it is generally associated with severe complications and unsatisfactory success rates.¹¹⁶ No pharmacological treatment is available to date, and AAK remains a major issue. However, given the fact that the nonsystemic forms of aniridia depend on defects of a single gene such as PAX6 combined stem cell-gene therapy approaches aimed at correcting PAX6 function could represent an attractive treatment to be pursued.¹¹⁶

A massive bulk of studies investigating the molecular mechanisms underlying the development of aniridia heterogeneous manifestations highlighted a prominent role for the alteration of PAX6 expression levels in different eye districts. Importantly, around 50% of the more than 500 PAX6 mutations causing aniridia are represented by in frame, nonsense mutations inserting premature termination codons (PTCs) within PAX6 sequence.¹¹⁷ This evidence, together with the autosomic dominant nature of aniridia, is consistent with alteration of PAX6 expression levels being responsible for the clinical manifestation, which implies the possibility to reverse the phenotype (i.e., cure the disease) by restoring protein expression in ocular tissues.

This goal could be reached using different strategies, depending on the mutations causing the disease. Indeed, the most promising approach for patients whereby PAX6 expression is altered by the presence of PTCs, is represented by the administration of aminoglycosides, or of small molecules capable of promoting ribosome read thorough at PTCs, and restoring wild-type levels of protein synthesis.

A first important proof of principle regarding the feasibility of such an approach came from studies performed using mice whereby one pax6 allele bears a naturally occurring G194X stop codon mutation (UGA). This recapitulates a number of aniridia symptoms observed in humans, including corneal and retinal thickening, as well as underdeveloped lens and impaired vision. Remarkably, topical administration to the mice eyes of the so-called START formulation (sodium chloride, Tween, small molecule ataluren—the compound acting on PTCs and carboxymethylcellulose) postnatally from P14 to P60, restored PAX6 expression levels to wild-type and remarkably reduced the eye abnormalities (leading to a reduction of both retinal and corneal thickness, restoring normal lens size) and improved light stimuli. Very importantly, the treatment did not reverse the phenotype in mice bearing a splice site mutation in pax6, thus demonstrating the specificity of the mode of action of such formulation.

A follow-up study focused on testing the best Ataluren concentration and temporal treatment schedule using the very same small eye mouse model and a combination of histopathological, behavioral and electroretinographic tests.¹¹⁸ The results confirmed the possibility to revert the aniridia phenotype postnatally and highlighted the importance in determining the most appropriate regimen to restore the physiological PAX6 expression levels in different eye districts. Indeed, eye development appears extremely sensitive to alterations in PAX6 expression levels, with just a 2.5-fold protein overexpression causing microphthalmia in mice.¹¹⁹ Clearly, further studies are required for validating such results in humans. In this context, a phase 2, interventional clinical trial aimed at assessing the safety and efficacy of ataluren in the management of aniridia patients, bearing a nonsense mutation in one allele of PAX6 gene started in 2015 (NCT02647359). However, ataluren appears particularly effective in promoting read-through of certain stop codons, also depending on nucleotides surrounding the stop codon itself and the location in the mRNA.¹²⁰ It is therefore hypothesizable that only a small percentage of aniridia patients bearing PTCs would benefit from such approach. For all the other patients, including the few ones bearing mutations in the PAX6 regulatory regions,¹²¹ alternative approaches to restore physiological PAX6 levels would be required. One of them could be the controlled administration of human recombinant pax6 protein. Indeed, a recent report described the ability of bacterially expressed, recombinant human pax protein fused to a C-terminal tag of 11 arginine domain, which allows intracellular protein intake, to restore the phenotypic impairments observed in a cellular aniridia model. Such model is represented by telomerase immortalized limbal stem cells where one PAX6 allele is edited by CRISPR-Cas9 technology to introduce a PTC mutation naturally occurring in aniridia patients, and exhibit defects in cell proliferation and migration.¹²²

Other approaches would involve the irreversible genetic modification of patient's cells to reverse the aniridia phenotype. This could be achieved by augmentation gene therapy, delivering exogenous genetic elements capable of correcting PAX6 expression levels in a spatiotemporal coordinated fashion. The main limitation would be represented by the difficulty to package into a viral vector all the regulatory elements required to ensure the proper PAX6 spatiotemporal expression profile in the patients. Indeed, PAX6 gene expression is controlled by at least 39 cis-regulatory elements, including a rather large number of regulatory regions located even megabases away from the transcriptional start site.¹¹⁶ To overcome this issue, a recent report described the genesis and characterization of PAX6 MiniPromoters. Their reduced size (less than 2 Kps) allowed insertion into rAAV, and regulated expression of a transgene in several mice ocular tissues after transduction.¹²³ Obviously, further optimization of such technology is required to ensure PAX6 expression in human aniridia patients. The last potential approach (i.e. genome editing of PAX6 mutated alleles by CRISPR-Cas9 technology) would have the advantage to potentially cure any aniridia patient regardless of the responsible mutation, but its use would be strongly limited by the necessity to design and optimize hundreds of guides specific to each mutation.¹¹⁷

Figure 1.

Schematic representation of a section of the eye and of the five layers forming the adult human cornea.

Fuchs endothelial corneal dystrophy

Fuchs endothelial corneal dystrophy (FECD) is the most common form of posterior corneal dystrophy; it is characterized by the progression of focal excrescences in the Descemet membrane (also called the guttae) to endothelial cell degeneration and stromal edema. The clinical onset usually occurs during the fifth or sixth decade of life and FECD is slightly more common in women than in men. Patients experience discomfort and painful episodes related to corneal erosions, in association with gradual opacification that results in a visual veiling. Over time, discomfort may decrease, but severe visual acuity may occur, leading to blindness in older patients. The disease is often associated with cataract. Microbial keratitis and corneal neovascularization are very rare complications. Stromal edema causes an anterior bluish-gray opacification of the Descemet membrane, followed by a possible thickening of the cornea stroma, which can take on the ground-glass appearance. The etiology of FECD is not known but appears to be a heterogeneous complex hereditary disease caused by the interaction between genetic and environmental factors. There currently is no cure for FECD. In early stages of the disease, vision may be improved with a 5% salt solution. However, there is no treatment that can actually halt or reverse the course of the disease. Eventually, in some patients with FECD, surgery may be necessary. The two options currently available are: penetrating keratoplasty (PK) and Descemet's stripping with endothelial keratoplasty.

Rare cases with early onset were associated with mutations in the COL8A2, SLC4A11, ZEB1, and LOXHD1 genes.¹²⁴ Approximately 70% of FECD cases are caused by a trinucleotide repeat expansion in the TCF4 gene¹²⁴ that leading to nuclear RNA foci, with the sequestration of splicing factor proteins (MBNL1 and MBNL2) to the foci and altered mRNA processing.

Studies using in vitro and animal models have shown that an antisense oligonucleotide approach leads to a reduction in RNA foci and downstream markers of toxicity. This proof-of-concept study highlights the potential of a targeted antisense oligonucleotide therapy to treat the accessible and tractable corneal tissue affected by this repeat expansion-mediated disease.¹²⁵ Further, with the latest evidence demonstrating pathogenic TCF4 trinucleotide repeat expansions causing defects in endothelial barrier function in a majority of FECD cases, an analogous application of CRISPR-Cas9 treatment for FECD holds significant promise.¹²⁷

Conclusions and future perspectives

Besides corneal transplantation, not many valuable therapies are available nowadays to treat corneal diseases. Thus, gene therapy can be considered as a promising biomedical approach.

Gene therapy is a new branch of regenerative medicine that has matured considerably in the last decade, as demonstrated by the results of several clinical trials for a number of different disorders of genetic origin and by means of a diverse range of gene transfer methods. The lentiviral and AAV vectors are the main gene transfer vectors currently in use, but other approaches, primarily nonvectorial, are also under study because of their lower invasiveness and higher level of safety (see delivery systems Appendix A2 ). The experience and knowledge learnt from the successes, problems and obstacles encountered in recent studies will be indispensable for the innovation of gene therapy approaches of the future. New gene transfer protocols and tools are already under development, which will also contribute to expand the spectrum of diseases that can be treated with gene therapy. Continuous progress towards gene therapy of precision represented by genome editing is the next challenge.

The studies described in this review paper have proved to be valid and effective. Now it would be interesting to move these findings onto clinical settings, to see whether they are clinically valuable as well.

Footnotes

Author Disclosure

The authors declare no competing interests exist.

Appendix A1. Nucleic acids delivered for corneal gene therapy approaches

Appendix B2. Systems used to deliver nucleic acids for corneal gene therapy

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