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Corneal epithelial dystrophies are typically characterized by symptoms such as pain, light sensitivity, and corneal opacification leading to impaired vision. The development of gene therapy for such conditions has been hindered by an inability to achieve sustained and extensive gene transfer, as the epithelium is highly replicative and has evolved to exclude foreign material. We undertook a comprehensive study in mice aiming to overcome these impediments. Direct injection of lentiviral vector within the stem cell niche resulted in centripetal streaks of epithelial transgene expression sustained for1 year, indicating limbal epithelial stem cell transduction in situ. The extent of transgene expression varied markedly but at maximum covered 26% of the corneal surface. After intrastromal injection, adeno-associated viral (AAV) vectors were found to penetrate Bowman's membrane and mediate widespread, but transient (12-16 days), epithelial transgene expression. This was sufficient, when applied within a Cre/lox system, to result in recombined epithelium covering up to approximately 80% of the corneal surface. Lastly, systemic delivery of AAV2/9 in neonatal mice resulted in extensive corneal transduction, despite the relative avascularity of the tissue. These findings provide the foundations of a gene therapy toolkit for the corneal epithelium, which might be applied to correction of inherited epithelial dystrophies.

Purpose Endothelial cell density (ECD) is the principal factor determining the success of corneal transplants. Here we explored a strategy to increase corneal ECD in human explants via modulation of the ZO-1/ZONAB pathway. In multiple cell types, ZO-1 maintains G1 cell cycle arrest via cytoplasmic sequestration of the mitosis-inducing transcription factor ZONAB. In this study, we assessed the effects of lentiviral vector-mediated downregulation of ZO-1 or overexpression of ZONAB upon ECD and the integrity of the endothelial monolayer. Methods HIV-based lentiviral vectors were used to deliver either constitutively expressed ZONAB (LNT-ZONAB), or a small hairpin RNA targeting ZO-1 (LNT-shZO1). Human corneal specimens were bisected and each half was exposed to either treatment or control vector. After 1 week in ex vivo culture, effects were assessed by quantitative RT-PCR, immunohistochemistry, and ECD assessment. Results LNT-shZO1 achieved an ∼45% knockdown of ZO-1 mRNA in corneal endothelial cells cultured ex vivo, reduced ZO-1 staining, and did not affect morphologic endothelial monolayer integrity. The proliferative effect of LNT-shZO1 correlated with control ECD but not with donor age. Within a low-ECD cohort an ∼30% increase in ECD was observed. LNT-ZONAB achieved a >200-fold overexpression of ZONAB mRNA, which led to an ∼25% increase in ECD. Conclusions ZO-1 downregulation or ZONAB upregulation increases corneal ECD via interference with contact inhibition and cell cycle control. With further development, such approaches might provide a means for improving ECD in donor corneas before transplantation.

Corneal transplantation has been performed successfully for over 100 years. Normally, HLA typing and systemic immunosuppressive drugs are not utilized, yet 90% of corneal allografts survive. In rodents, corneal allografts representing maximal histoincompatibility enjoy >50% survival even without immunosuppressive drugs. By contrast, other categories of transplants are invariably rejected in such donor/host combinations. The acceptance of corneal allografts compared to other categories of allografts is called immune privilege. The cornea expresses factors that contribute to immune privilege by preventing the induction and expression of immune responses to histocompatibility antigens on the corneal allograft. Among these are soluble and cell membrane molecules that block immune effector elements and also apoptosis of T lymphocytes. However, some conditions rob the corneal allograft of its immune privilege and promote rejection, which remains the leading cause of corneal allograft failure. Recent studies have examined new strategies for restoring immune privilege to such high-risk hosts.

Genetic modification of dendritic-cell (DC) function is an attractive approach to treat disease, either using mature DCs (mDCs) to immunize patients, or immature DCs (iDCs) to induce tolerance. Viral vectors are efficient at transducing DCs, and we have investigated the effect of transduction with a variety of viral vectors on the phenotype and function of DCs. Adenovirus (Ad), human immunodeficiency virus (HIV), equine anemia virus (EIAV), and Moloney murine leukemia virus (MMLV) all up-regulate costimulatory molecules and major histocompatibility complex (MHC) class II expression on DCs, as well as, in the case of Ad and lentiviral vectors, inducing production of Th1 and proinflammatory cytokines. Following transduction there is activation of double-stranded (ds) RNA-triggered pathways resulting in interferon (IFN) α/β production. In addition, the function of virally infected DCs is altered; iDCs have an increased, and mDCs a decreased, ability to stimulate a mixed lymphocyte reaction (MLR). Viral transduction of mDCs results in up-regulation of the indoleamine 2,3-dioxygenase (IDO) enzyme, which down-regulates T-cell responsiveness. Inhibition of IDO restores the ability of mDCs to stimulate an MLR, indicating that IDO is responsible for the modulation of mDC function. These data have important implications for the use of viral vectors in the transduction of DCs.

Purpose To assess the effect of standardized, paired arcuate keratotomy (AK) on the change in astigmatism in postkeratoplasty eyes. Setting Moorfields Eye Hospital, London, United Kingdom. Methods A retrospective review was conducted of 20 eyes of 19 patients having the same AK procedure regardless of the magnitude of the preoperative astigmatism. Each eye had a pair of 60-degree arc length incisions placed in the corneal stroma. The incisions were 600 μm deep and 6.0 mm apart. The preoperative and postoperative refractions and complications were analyzed. Astigmatic change was analyzed without regard to axis, as surgically induced refractive change, and using a modified polar plot of change in astigmatism. Results The mean cylinder was reduced from −10.99 diopters (D) ± 4.26 (SD) to −3.33 ± 2.18 D. There was no significant change in the mean spherical equivalent. There was a strong correlation between the magnitude of the preoperative cylinder and the magnitude of the change in astigmatism ( R 2 = 0.76). In 3 eyes, the surgically induced axis of astigmatism was more than 15 degrees from that expected. Conclusions In postkeratoplasty eyes, the change in the magnitude of astigmatism induced by standardized AK was proportional to the preoperative magnitude of astigmatism. Arcuate nomograms for congenital astigmatism have no role in the management of astigmatism in postkeratoplasty eyes.

Objective: This study describes the clinical features, management, and outcome of 19 patients who had severe Acanthamoeba sclerokeratitis (ASK) unresponsive to conventional management, requiring systemic immunosuppression to control disease. Design: Retrospective, noncomparative, interventional case series. Participants: Records of all patients with Acanthamoeba keratitis treated at Moorfields Eye Hospital between 1989 and 2000 were reviewed. From more than 200 patients, 19 who developed ASK treated with systemic immunosuppression were identified. Main Outcome Measures: Visual acuity, level of pain, and degree of inflammation were recorded after immunosuppressive treatment. Results: ASK requiring immunosuppression occurred in 20 eyes of 19 patients (11 males and 8 females). The mean age (mean ± standard deviation) at onset was 38.6 ± 13.2 years. On presentation, best-corrected visual acuity was counting fingers or worse in 11 eyes (55%), 6/18 to 6/60 in 5 eyes (25%), and 6/12 or better in 4 eyes (20%). The mean time between onset of initial symptoms of Acanthamoeba keratitis and commencement of systemic immunosuppression was 4.8 ± 3.5 months. The mean duration of immunosuppression required to control inflammation was 7.2 ± 3.9 months. Severe scleritic pain remained uncontrolled in two patients and resulted in enucleation. Best-corrected visual acuity at final follow-up was counting fingers or worse in eight eyes (40%), 6/18 to 6/60 in six eyes (30%), and 20/40 or better in six eyes (30%). The mean follow-up period after resolution of inflammation was 24.3 ± 20.9 months (range, 0.2-59.7 months). Conclusions: ASK is an uncommon complication of Acanthamoeba keratitis. The scleritis associated with this infection seems to be an immune-mediated response. After topical amebicidal treatment, systemic immunosuppression may be required to control the pain and tissue destruction associated with ASK.

Purpose Conjunctival papillomata are squamous epithelial tumors with a strong association with human papilloma virus (HPV) types 6 and 11. They are benign conjunctival tumors that can be treated by surgical excision. We report a case where topical immunosuppressive therapy modified the local T-cell immunity in the conjunctiva resulting in papilloma development in a patient with keratoconus and a strong atopic history. Methods A case report of a 44-year-old man with a history of severe ocular and generalized atopy is presented. We present the problems encountered in management of his severe ocular allergy and how these impeded the management of his keratoconus. Results Conventional antiallergy topical medication was not producing symptom relief in this patient, and so topical immunosuppression was commenced using ciclosporin ointment 0.2%. This therapy modified the local T-cell immunity in the conjunctiva resulting in the development of papillomata which contributed to the intolerance of contact lens wear for visual rehabilitation of the keratoconus in the patient. These lesions were surgically removed but typically recurred and required further surgical excision. Adjunct cryotherapy was also performed at the time of the surgery to try to stem the recurrence of the papillomas. Conclusions To the best of our knowledge and following a review of the published literature using key databases that include Medline and PubMed, this is the first report confirming the development of conjunctival papillomas secondary to HPV type 6 in a ciclosporin-treated patient.

PURPOSE. To compare outcomes after penetrating keratoplasty (PK) and deep anterior lamellar keratoplasty (DALK) for keratoconus in the United Kingdom.METHODS. Patient outcome data were collected at the time of transplantation and at 1, 2, and 5 years after surgery. Data were analyzed by Kaplan-Meier survival curves, Cox regression, and binary logistic regression to determine the influence of surgical procedure on graft survival and visual outcome.RESULTS. The risk of graft failure for DALK was almost twice that for PK (P = 0.02). Nineteen percent of the DALK failures occurred in the first 30 postoperative days compared with only 2% of PK failures. When these early failures were excluded, there was little difference between the 3-year graft survivals for DALK (92%; 95% confidence interval [CI], 85%-95%) and PK (94%; 95% CI, 92%-95%) (P = 0.8). Although the mean best corrected visual acuity (BCVA) was similar for the two procedures (P = 0.7), 33% of patients who underwent PK achieved a BCVA of 6/6 or better at 2 years compared with only 22% of those who underwent DALK (P < 0.001). Those with DALK were also likely to be more myopic (< -3 D) but there was little difference in scalar cylinder.CONCLUSIONS. DALK had a higher overall failure rate than PK. The difference was largely accounted for by early failures, which appeared to be related to the surgeon's experience. DALK recipients were less likely to achieve BCVA of 6/6 than were PK recipients and were more likely to have -3 D or worse myopia. (Invest Ophthalmol Vis Sci. 2009; 50: 5625-5629) DOI: 10.1167/iovs.09-3994

Background. The expression of chemokines is central to the recruitment of inflammatory cells for graft rejection, and modulation of chemokine action is of potential in preventing graft rejection. We have examined chemokine expression in a murine model of corneal allograft rejection, and also determined the effect of expressing a broad acting chemokine antagonist, viral macrophage inflammatory protein II (vMIP II), on graft survival. Method. The expression of chemokines in a murine model of corneal transplantation was determined by real time RT-PCR and, in the case of regulated on activation normal T-cell expressed and secreted, by ELISA. The plasmid encoding the virally derived chemokine antagonist, vMIP II, was introduced into the corneal endothelial cells using a non-viral vector consisting of liposomes and transferrin. The expression and activity of vMIP II was determined by ELISA and functional assays, and the effect on graft survival noted. Results. After allotransplantation, there was up-regulation of all 11 chemokines examined. After gene delivery, there was expression of active vMIP II for more than 14 days and considerable prolongation of graft survival. This was associated with a decrease in leukocyte infiltration of the stroma of the cells. Conclusion. As expected there was considerable up-regulation of chemokines during allograft rejection. The expression ofvMIP II showed considerable prolongation of graft survival. This is the first time we have observed prolongation of graft survival after a non-viral (as opposed to viral) means of gene delivery and indicates the potential of interfering with chemokine action to prevent corneal graft failure.

Gene transfer to the corneal endothelium has potential for modulating rejection of corneal grafts. It can also serve as a convenient and useful model for gene therapy of other organs. In this article we review the work carried out in our laboratory using both viral and nonviral vectors to obtain gene expression in the cornea.