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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.

Purpose: To study the performance of a completely synthetic organ culture (OC) preservation system containing recombinant human serum albumin (rHSA) for preservation of human donor corneas., Methods: Twenty-four paired donor corneas were randomly collected, and one cornea from each donor was preserved in synthetic (experimental) and serum-based media (control). The tissues were assessed at day 0; after 6 days of preservation at room temperature (RT) in Cornea Trans ® and Cornea Prep II ® ; after 28 days at 31°C in Cornea Syn ® [with rHSA] and Cornea Max ® [with foetal calf serum (FCS)] and; 4-day post deswelling in Cornea Trans ® and Cornea Jet ® . Thickness was determined with optical coherence tomography (OCT) and transparency with a validated, custom device. Morphology, endothelial cell density (ECD) and mortality were observed after treating the tissues with Trypan blue and sucrose. Glucose uptake by the cells was analysed. Data were compared using non-parametric paired Wilcoxon tests with p < 0.05 deemed significant. Histology using periodic acid-schiff (PAS), expressions of p63, CK12, αSMA and ZO-1 were analysed, and cell apoptosis postpreservation was studied., Results: Corneas stored in synthetic media showed a higher and statistically significant value as compared to serum-based media in terms of viable endothelial cell density (VECD), mortality, morphology and glucose uptake postpreservation. Histology showed presence of all the layers, all the markers were expressed, and no apoptosis was observed in either media., Conclusion: The new synthetic preservation system containing rHSA (and other confidential constituents) showed better preservation effects than traditional media containing serum., (© 2017 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly. The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision(4,5,6). The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina(5,6). The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve(5,6). The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

Macular corneal dystrophy (MCD; OMIM 217800) is a rare autosomal recessive inherited disorder caused by mutations in the carbohydrate sulfotransferase 6 (CHST6) and characterised by the presence of unsulfated keratan sulfate proteoglycans (KSPGs) forming abnormal deposits that eventually lead to visual impairment. The aim of this study is to understand in which corneal cells CHST6 and KSPGs are expressed and exert their activity. Expression and localization of CHST6, keratan sulfate (KS) and proteins of the KSPGs, such as mimecan and lumican, were assessed both in human cornea sections and in cultured primary keratinocytes (n = 3) and keratocytes (n = 4). Immunohistochemistry, semiquantitative RT-PCR, in situ RNA hybridization and HPLC analysis of glycosaminoglycans were used as read-outs. In human corneas KS was predominantly found in the stroma, but absent, or barely detectable, in the corneal epithelium. A similar pattern of distribution was found in the epidermis, with KS mainly localised in the derma. As expected, in the cornea CHST6 (the gene encoding the enzyme which transfers sulfate residues onto KSPGs) was found expressed in the suprabasal, but not basal, layers of the epithelium, in the stroma and in the endothelium. Analyses of KS by means of HPLC showed that in vitro cultured stromal keratocytes express and secrete more KS than keratinocytes, thus mirroring results observed in vivo. Similarly expression of the CHST6 gene and of KS proteoglycans such as mimecan, lumican is limited to stromal keratocytes. Unlike keratocytes, corneal keratinocytes do not synthesize mimecan or lumican, and express very little, if none, CHST6. Any drug/gene therapy or surgical intervention aimed at curing this rare genetic disorder must therefore involve and target stromal keratocytes. If coupled to the accuracy of HPLC-based assay that we developed to determine the amount of KS in serum, our findings could lead to more targeted therapeutic treatments of the ocular features in MCD patients., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Purpose: To reconstruct a human hemicornea in vitro by means of limbal stem cells cultured onto human keratoplasty lenticules (HKLs) and to obtain a natural corneal graft for clinical applications., Methods: Limbal stem cells were seeded onto HKLs with or without the presence of feeder layers of lethally irradiated 3T3-J2 cells and compared with the current "gold standard" scaffold, i.e., the fibrin glue. The effects of the scaffold on the preservation of stemness and/or induction of differentiation pathways were investigated through analysis of a variety of markers, including p63 and DeltaNp63alpha for stemness, 14-3-3sigma for early differentiation, keratins 3, 14, 12, and 19 to determine cell phenotype, and alpha6, beta1, and beta4 integrins to evaluate interactions with the stroma. Integrity of the stroma was assessed through analysis of keratan sulfate, CD-34 and aldehyde dehydrogenase 3A1 (ALDH3A1) (for keratocytes), visual system homeobox 1 (VSX1), and alpha-smooth muscle actin (alpha-SMA) (for fibroblasts and myofibroblasts). The structural properties of the reconstructed "hemicornea" were investigated through scanning electron microscopy. To evaluate the preservation of the stemness potential, cells were trypsinized from each scaffold and clonogenic/proliferative characteristics analyzed., Results: Limbal stem cells expanded onto HKLs gave rise to a stratified squamous keratinized epithelium morphologically similar to that of normal corneas. The resulting corneal epithelium was characterized by basal expression of p63 and DeltaNp63alpha, while expression of 14-3-3sigma, keratin 3, and keratin 12 was found in the upper cell layers. The basal cuboidal epithelial cells were anchored to the basement membrane and expressed keratin 14 and alpha6, beta1, and beta4 integrins. In the stroma of HKLs, keratocytes maintained the biosynthetic and phenotypic appearances typical of resting/quiescent cells and expressed keratan sulfate, CD-34, and ALDH3A1. Fibroblastic transformation was observed with the appearance of VSX1 and alpha-SMA. Scanning electron microscopy analysis showed that HKLs maintained their native conformation with collagen fibrils interconnected to the network and parallel to the corneal surface. HKLs did not alter the clonogenic/proliferative capacity of limbal stem cells. No differences were seen when HKL was compared to fibrin glue, one of the scaffolds currently used for limbal stem cell transplantation., Conclusions: Our findings demonstrate that HKL could be a suitable scaffold for corneal epithelial stem cells as they were shown to proliferate, express differentiation markers, and bind to the underlying stroma with no alterations in clonogenic potential. HKLs have some advantages over currently used scaffolds, such as the possibility to allow cell growth with no feeder layers, to be freeze dried, and to preserve the integrity and viability of stromal keratocytes. The development of a tissue-engineered "hemicornea" might offer new therapeutic perspectives to patients affected by total limbal stem cell deficiency with stromal scarring.

We report the reconstruction and characterization of a hemicornea (epithelialized stroma), using primary human cells, for use in research and as an alternative to the use of animals in pharmacotoxicology testing. To create a stromal equivalent, keratocytes from human corneas were cultured in collagen-glycosaminoglycan-chitosan foams. Limbal stem cell-derived epithelial cells were seeded on top of these, giving rise to hemi-corneas. The epithelium appeared morphologically similar to its physiological counterpart, as shown by the basal cell expression of p63 isoforms including, in some cases, the stem cell marker p63DeltaNalpha, and the expression of keratin 3 and 14-3-3sigma in the upper cell layers. In addition, the cuboidal basal epithelial cells were anchored to a basement membrane containing collagen IV, laminin 5, and hemidesmosomes. In the stromal part, the keratocytes colonized the porous scaffold, formed a network of interconnecting cells, and synthesized an ultrastructurally organized extracellular matrix (ECM) containing collagen types I, V, and VI. Electron microscopy showed the newly synthesized collagen fibrils to have characteristic periodic striations, with diameters and interfibril spacings similar to those found in natural corneas. Compared to existing models for corneal pharmacotoxicology testing, this new model more closely approaches physiological conditions by including the inducing effects of mesenchyme and cell-matrix interactions on epithelial cell morphogenesis.

The p63 gene generates transactivating and N-terminally truncated transcripts (DeltaNp63) initiated by different promoters. Alternative splicing gives rise to three different C termini, designated alpha, beta, and gamma. In the ocular epithelium, the corneal stem cells, which are segregated in the basal layer of the limbus, contain the alpha isoform but not beta or gamma. Holoclones derived from the limbus are rich in alpha, meroclones contain little, and paraclones contain none. In normal resting corneal epithelium, p63 of all isoforms is absent. Upon corneal wounding, cells originating from the limbus and containing alpha migrate progressively through the epithelium of the peripheral and central cornea. In the absence of an attached limbus, no alpha isoform appears in the corneal epithelium. When migrating cells containing the alpha isoform appear in the wounded corneal epithelium, they are confined to the basal layer, but the suprabasal cells, not only of the cornea but of the limbus as well, contain mRNA encoding beta and gamma. These data support the concept that the alpha isoform of p63 is necessary for the maintenance of the proliferative potential of limbal stem cells and their ability to migrate over the cornea. The beta and gamma isoforms, being suprabasal and virtually absent from the resting limbus, are not stem cell markers but are likely to play a role in epithelial differentiation specifically during the process of corneal regeneration.