Mutation-Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease.

CRISPR-Cas9 provides a tool to treat autosomal dominant disease by non-homologous end joining (NHEJ) gene disruption of the mutant allele. In order to discriminate between wild-type and mutant alleles, Streptococcus pyogenes Cas9 (SpCas9) must be able to detect a single nucleotide change. Allele-specific editing can be achieved by using either a guide-specific approach, in which the missense mutation is found within the guide sequence, or a protospacer-adjacent motif (PAM)-specific approach, in which the missense mutation generates a novel PAM. While both approaches have been shown to offer allele specificity in certain contexts, in cases where numerous missense mutations are associated with a particular disease, such as TGFBI (transforming growth factor ß-induced) corneal dystrophies, it is neither possible nor realistic to target each mutation individually. In this study, we demonstrate allele-specific CRISPR gene editing independent of the disease-causing mutation that is capable of achieving complete allele discrimination, and we propose it as a targeting approach for autosomal dominant disease. Our approach utilizes natural variants in the target region that contain a PAM on one allele that lies in cis with the causative mutation, removing the constraints of a mutation-dependent approach. Our innovative patient-specific guide design approach takes into account the patient's individual genetic make-up, allowing on- and off-target activity to be assessed in a personalized manner.

A novel role for CRIM1 in the corneal response to UV and pterygium development.

Pterygium is a pathological proliferative condition of the ocular surface, characterised by formation of a highly vascularised, fibrous tissue arising from the limbus that invades the central cornea leading to visual disturbance and, if untreated, blindness. Whilst chronic ultraviolet (UV) light exposure plays a major role in its pathogenesis, higher susceptibility to pterygium is observed in some families, suggesting a genetic component. In this study, a Northern Irish family affected by pterygium but reporting little direct exposure to UV was identified carrying a missense variant in CRIM1 NM_016441.2: c.1235 A > C (H412P) through whole-exome sequencing and subsequent analysis. CRIM1 is expressed in the developing eye, adult cornea and conjunctiva, having a role in cell differentiation and migration but also in angiogenesis, all processes involved in pterygium formation. We demonstrate elevated CRIM1 expression in pterygium tissue from additional individual Northern Irish patients compared to unaffected conjunctival controls. UV irradiation of HCE-S cells resulted in an increase in ERK phosphorylation and CRIM1 expression, the latter further elevated by the addition of the MEK1/2 inhibitor, U0126. Conversely, siRNA knockdown of CRIM1 led to decreased UV-induced ERK phosphorylation and increased BCL2 expression. Transient expression of the mutant H412P CRIM1 in corneal epithelial HCE-S cells showed that, unlike wild-type CRIM1, it was unable to reduce the cell proliferation, increased ERK phosphorylation and apoptosis induced through a decrease of BCL2 expression levels. We propose here a series of intracellular events where CRIM1 regulation of the ERK pathway prevents UV-induced cell proliferation and may play an important role in the in the pathogenesis of pterygium.

Keratin 12 missense mutation induces the unfolded protein response and apoptosis in Meesmann epithelial corneal dystrophy.

Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations.

Keratin disorders: from gene to therapy.

The term 'keratin' is generally accepted to refer to the epithelial keratins of soft and hard epithelial tissues such as: skin, cornea, hair and nail. Since their initial characterization, the total number of mammalian keratins has increased to 54, including 28 type I and 26 type II keratins. Inherited defects that weaken the keratin load-bearing cytoskeleton produce phenotypes characterized by fragility of specific subsets of epithelial tissues. The vast majority of mutations are either missense or small in-frame in-del mutations and disease severity often relates to the position of the mutation in relation to the rod domain. The most complex epithelial structure in humans, the hair follicle, contains trichocyte ('hard') keratin filaments and approximately half of the 54 functional human keratin genes are trichocyte keratins. So far, only four of these have been linked to human genetic disorders: monilethrix, hair-nail ectodermal dysplasia, pseudofolliculitis barbae and woolly hair, while the majority of the hair keratins remain unlinked to human phenotypes. Keratin disorders are a classical group of dominant-negative genetic disorders, representing a large healthcare burden, especially within dermatology. Recent advances in RNA interference therapeutics, particularly in the form of small-interfering RNAs, represent a potential therapy route for keratin disorders through selectively silencing the mutant allele. To date, mutant-specific siRNAs for epidermolysis bullosa simplex, pachyonychia congenita and Messmann epithelial corneal dystrophy-causing missense mutations have been developed and proven to have unprecedented specificity and potency. This could herald the dawn of a new era in translational medical research applied to genetics.

Enhancement of the anti-Salmonella immune response in CD154-deficient mice by an attenuated, IFN-γ-expressing, strain of Salmonella enterica serovar Typhimurium.

Previously, we demonstrated that cell-cell communications via the CD40-CD154 pathway play a critical role in the induction of type 1 cytokine responses, including IL-12 and IFN-γ, which in turn greatly influence the response to Salmonella infections. Mice genetically deficient in the expression of CD154 exhibited markedly increased susceptibility to infection by an attenuated, double auxotrophic (aroA-aroD-) strain, designated BRD509, of Salmonella enterica Serovar Typhimurium. In the present study, we used a strain of Salmonella engineered to express murine IFN-γ, designated GIDIFN, in order to assess its potential to enhance the host's immune response in CD154-deficient animals. We demonstrate that infection of animals with GIDIFN results in markedly enhanced anti-bacterial response, as evidenced by the significant reduction in bacterial loads in target organs and decreased animal mortality. This was associated with a more robust proinflammatory cytokine response, including IL-6, IL-12, TNF-α and IFN-γ. In protection studies, GIDIFN strain was demonstrably superior than the BRD509 strain in affording protection against virulent Salmonella challenge in naïve CD154-/- mice. Interestingly, however, infection with GIDIFN failed to correct the isotype switching defect in CD154-/- mice, suggesting that the enhanced immunity triggered by GIDIFN strain occurs independently of humoral immune responses. These findings demonstrate that GIDIFN has immunopotentiating effects on the host's immune response and provide direct evidence for the utility of IFN-γ-expressing attenuated Salmonella in enhancement of immune responsiveness in immunodeficient hosts.

Delivery of siRNA to the Eye: Protocol for a Feasibility Study to Assess Novel Delivery System for Topical Delivery of siRNA Therapeutics to the Ocular Surface.

Drug delivery to the eye remains a real challenge due to the presence of ocular anatomical barriers and physiological protective mechanisms. The lack of effective siRNA delivery mechanism has hampered the real potential of RNAi therapy, but recent literature suggests that nanocarrier systems show great promise in enhancing siRNA bioavailability and reducing the need for repeated intraocular injections. A diverse range of materials are under exploration worldwide, including natural and synthetic polymers, liposomes, peptides, and dendrimeric nanomaterials. This chapter describes a simple workflow for feasibility assessment of a proposed ocular surface siRNA delivery system. Gel retardation assay is used for investigation of optimal siRNA to carrier loading ratio. Fluorescent siRNA allows for initial in vitro testing of cellular uptake to corneal epithelial cells and investigation of in vivo siRNA delivery into mouse cornea by live animal imaging and fluorescence microscopy.

Excimer laser surface ablation - a review.

Corneal surface laser ablation procedures for the correction of refractive error have enjoyed a resurgence of interest, especially in patients with a possible increased risk of complications after lamellar surgery. Improvements in the understanding of corneal biomechanical changes, the modulation of wound healing, laser technology including ablation profiles and different methods for epithelial removal have widened the scope for surface ablation. This article discusses photorefractive keratectomy, trans-epithelial photorefractive keratectomy, laser-assisted sub-epithelial keratomileusis and epithelial-laser-assisted in situ keratomileusis.

Topical siRNA delivery to the cornea and anterior eye by hybrid silicon-lipid nanoparticles.

The major unmet need and crucial challenge hampering the exciting potential of RNAi therapeutics in ophthalmology is to find an effective, safe and non-invasive means of delivering siRNA to the cornea. Although all tissues of the eye are accessible by injection, topical application is preferable for the frequent treatment regimen that would be necessary for siRNA-induced gene silencing. However, the ocular surface is one of the more complex biological barriers for drug delivery due to the combined effect of short contact time, tear dilution and poor corneal cell penetration. Using nanotechnology to overcome the challenges, we developed a unique silicon-based delivery platform for ocular delivery of siRNA. This biocompatible hybrid of porous silicon nanoparticles and lipids has demonstrated an ability to bind nucleic acid and deliver functional siRNA to corneal cells both in vitro and in vivo. Potent transfection of human corneal epithelial cells with siRNA-ProSilic® formulation was followed by a successful downregulation of reporter protein expression. Moreover, siRNA complexed with this silicon-based hybrid and applied in vivo topically to mice eyes penetrated across all cornea layers and resulted in a significant reduction of the targeted protein expression in corneal epithelium. In terms of siRNA loading capacity, system versatility, and potency of action, ProSilic provides unique attributes as a biodegradable delivery platform for therapeutic oligonucleotides.

Personalised genome editing - The future for corneal dystrophies.

The potential of personalised genome editing reaching the clinic has come to light due to advancements in the field of gene editing, namely the development of CRISPR/Cas9. The different mechanisms of repair used to resolve the double strand breaks (DSBs) mediated by Cas9 allow targeting of a wide range of disease causing mutations. Collectively, the corneal dystrophies offer an ideal platform for personalised genome editing; the majority of corneal dystrophies are monogenic, highly penetrant diseases with a known pattern of inheritance. This genetic background coupled with the accessibility, ease of visualisation and immune privilege status of the cornea make a gene editing strategy for the treatment of corneal dystrophies an attractive option. Off-target cleavage is a major concern for the therapeutic use of CRISPR/Cas9, thus current efforts in the gene editing field are focused on improving the genome-wide specificity of Cas9 to minimise the risk of off-target events. In addition, the delivery of CRISPR/Cas9 to different tissues is a key focus; various viral and non-viral platforms are being explored to develop a vehicle that is highly efficient, specific and non-toxic. The rapid pace and enthusiasm with which CRISPR/Cas9 has taken over biomedical research has ensured the personalised medicine revolution has been realised. CRISPR/Cas9 has recently been utilised in the first wave of clinical trials, and the potential for a genome editing therapy to treat corneal dystrophies looks promising. This review will discuss the current status of therapeutic gene editing in relation to the corneal dystrophies.

Towards personalised allele-specific CRISPR gene editing to treat autosomal dominant disorders.

CRISPR/Cas9 holds immense potential to treat a range of genetic disorders. Allele-specific gene disruption induced by non-homologous end-joining (NHEJ) DNA repair offers a potential treatment option for autosomal dominant disease. Here, we successfully delivered a plasmid encoding S. pyogenes Cas9 and sgRNA to the corneal epithelium by intrastromal injection and acheived long-term knockdown of a corneal epithelial reporter gene, demonstrating gene disruption via NHEJ in vivo. In addition, we used TGFBI corneal dystrophies as a model of autosomal dominant disease to assess the use of CRISPR/Cas9 in two allele-specific systems, comparing cleavage using a SNP-derived PAM to a guide specific approach. In vitro, cleavage via a SNP-derived PAM was found to confer stringent allele-specific cleavage, while a guide-specific approach lacked the ability to distinguish between the wild-type and mutant alleles. The failings of the guide-specific approach highlights the necessity for meticulous guide design and assessment, as various degrees of allele-specificity are achieved depending on the guide sequence employed. A major concern for the use of CRISPR/Cas9 is its tendency to cleave DNA non-specifically at "off-target" sites. Confirmation that S. pyogenes Cas9 lacks the specificity to discriminate between alleles differing by a single base-pair regardless of the position in the guide is demonstrated.

Potential Effects of Corneal Cross-Linking upon the Limbus.

Corneal cross-linking is nowadays the most used strategy for the treatment of keratoconus and recently it has been exploited for an increasing number of different corneal pathologies, from other ectatic disorders to keratitis. The safety of this technique has been widely assessed, but clinical complications still occur. The potential effects of cross-linking treatment upon the limbus are incompletely understood; it is important therefore to investigate the effect of UV exposure upon the limbal niche, particularly as UV is known to be mutagenic to cellular DNA and the limbus is where ocular surface tumors can develop. The risk of early induction of ocular surface cancer is undoubtedly rare and has to date not been published other than in one case after cross-linking. Nevertheless it is important to further assess, understand, and reduce where possible any potential risk. The aim of this review is to summarize all the reported cases of a pathological consequence for the limbal cells, possibly induced by cross-linking UV exposure, the studies done in vitro or ex vivo, the theoretical bases for the risks due to UV exposure, and which aspects of the clinical treatment may produce higher risk, along with what possible mechanisms could be utilized to protect the limbus and the delicate stem cells present within it.

Can the accuracy of multifocal intraocular lens power calculation be improved to make patients spectacle free?

PURPOSE: To optimise intraocular lens (IOL) power calculation techniques for a segmental multifocal IOL, LENTIS™ MPlus(®) (Oculentis GmbH, Berlin, Germany) and assess outcomes. METHODS: A retrospective consecutive non-randomised case series of patients receiving the MPlus(®) IOL following cataract surgery or clear lens extraction was performed at a privately owned ophthalmic hospital, Midland Eye, Solihull, UK. Analysis was undertaken of 116 eyes, with uncomplicated lens replacement surgery using the LENTIS™ MPlus(®) lenses. Pre-operative biometry data were stratified into short (<22.00 mm) and long axial lengths (ALs) (≥22.00 mm). IOL power predictions were calculated with SRK/T, Holladay I, Hoffer Q, Holladay II and Haigis formulae and compared to the final manifest refraction. These were compared with the OKULIX ray tracing method and the stratification technique suggested by the Royal College of Ophthalmologists (RCOphth). RESULTS: Using SRK/T for long eyes and Hoffer Q for short eyes, 64% achieved postoperative subjective refractions of ≤±0.25 D, 83%≤±0.50 D and 93%≤±0.75 D, with a maximum predictive error of 1.25D. No specific calculation method performed best across all ALs; however for ALs under 22 mm Hoffer Q and Holliday I methods performed best. CONCLUSIONS: Excellent but equivalent overall refractive results were found between all biometry methods used in this multifocal IOL study. For eyes with ALs under 22 mm Hoffer Q and Holliday I performed best. Current techniques mean that patients are still likely to need top up glasses for certain situations.

Protein Composition of TGFBI-R124C- and TGFBI-R555W-Associated Aggregates Suggests Multiple Mechanisms Leading to Lattice and Granular Corneal Dystrophy.

PURPOSE: Transforming growth factor beta-induced (TGFBI)-related dystrophies constitute the most common heritable forms of corneal dystrophy worldwide. However, other than the underlying genotypes of these conditions, a limited knowledge exists of the exact pathomechanisms of these disorders. This study expands on our previous research investigating dystrophic stromal aggregates, with the aim of better elucidating the pathomechanism of two conditions arising from the most common TGFBI mutations: granular corneal dystrophy type 1 (GCD1; R555W) and lattice corneal dystrophy type 1 (LCD1; R124C). METHODS: Patient corneas with GCD1 and LCD1 were stained with hematoxylin and eosin and Congo red to visualize stromal nonamyloid and amyloid deposits, respectively. Laser capture microdissection was used to isolate aggregates and extracted protein was analyzed by mass spectrometry. Proteins were identified and their approximate abundances were determined. Spectra of TGFBIp peptides were also recorded and quantified. RESULTS: In total, three proteins were found within GCD1 aggregates that were absent in the healthy control corneal tissue. In comparison, an additional 18 and 24 proteins within stromal LCD1 and Bowman's LCD1 deposits, respectively, were identified. Variances surrounding the endogenous cleavage sites of TGFBIp were also noted. An increase in the number of residues experiencing cleavage was observed in both GCD1 aggregates and LCD1 deposits. CONCLUSIONS: The study reveals previously unknown differences between the protein composition of GCD1 and LCD1 aggregates, and confirms the presence of the HtrA1 protease in LCD1-amyloid aggregates. In addition, we find mutation-specific differences in the processing of mutant TGFBIp species, which may contribute to the variable phenotypes noted in TGFBI-related dystrophies.

Protection of corneal epithelial stem cells prevents ultraviolet A damage during corneal collagen cross-linking treatment for keratoconus.

BACKGROUND/AIMS: Cross-linking of the cornea is usually carried out at a young age as a treatment to manage ectasia. The corneal limbal region contains delicate long-lived stem cells, which could potentially be deleteriously affected by Ultraviolet A (UV-A) radiation. Damage to these stem cells may not demonstrate as a clinical problem for many years subsequent to cross-linking treatment. UV-A radiation is known to have potential mutagenic effects upon mammalian DNA and can result in cancer. METHODS: Cultured corneal epithelial cells and ex vivo corneal tissue were treated with the standard clinical cross-linking protocol for UV-A irradiation. 8-hydroxydeoxyguansoine (8-OHdG) and cyclin-dependent kinase inhibitor genes (CDKN1A and CDKN2A) were assayed as markers of DNA damage using immunohistochemistry, ELISA and quantitative real time PCR. RESULTS: Staining of treated limbal tissue demonstrated the presence of 8-OHdG within p63 positive basal limbal cells. Levels of 8-OHdG and CDKN1A mRNA were found to be significantly increased in cultured corneal epithelial cells and limbal epithelial cells but no increase was demonstrated with the use of a polymethyl methylacrylate protective cover. CONCLUSIONS: This study provides evidence that oxidative nuclear DNA damage can occur through cross-linking in layers of corneal epithelial cells at the limbus and that this can be easily prevented by covering the limbus.

siRNA silencing of the mutant keratin 12 allele in corneal limbal epithelial cells grown from patients with Meesmann's epithelial corneal dystrophy.

PURPOSE: The aim of this study is to further assess our previously reported keratin 12 (K12)-Leu132Pro specific siRNA in silencing the mutant allele in Meesmann's Epithelial Corneal Dystrophy (MECD) in experimental systems more akin to the in vivo situation through simultaneous expression of both wild-type and mutant alleles. METHODS: Using KRT12 exogenous expression constructs transfected into cells, mutant allele specific knockdown was quantified using pyrosequencing and infrared Western blot analysis, while the silencing mechanism was assessed by a modified rapid amplification of cDNA ends (5'RACE) method. Corneal limbal biopsies taken from patients suffering from MECD were used to establish cultures of MECD corneal limbal epithelial stem cells and the ability of the siRNA to silence the endogenous mutant KRT12 allele was assessed by a combination of pyrosequencing, qPCR, ELISA, and quantitative-fluorescent immunohistochemistry (Q-FIHC). RESULTS: The siRNA displayed a potent and specific knockdown of K12-Leu132Pro at both the mRNA and protein levels with exogenous expression constructs. Analysis by the 5'RACE method confirmed siRNA-mediated cleavage. In the MECD cells, an allele-specific knockdown of 63% of the endogenous mutant allele was observed without effect on wild-type allele expression. CONCLUSIONS: Combined with an effective delivery vehicle this siRNA approach represents a viable treatment option for prevention of the MECD pathology observed in K12-Leu132Pro heterozygous individuals.

Development of allele-specific gene-silencing siRNAs for TGFBI Arg124Cys in lattice corneal dystrophy type I.

PURPOSE: This study aimed to investigate the potency and specificity of short-interfering RNA (siRNA) treatment for TGFBI-Arg124Cys lattice corneal dystrophy type I (LCDI) using exogenous expression constructs in model systems and endogenous gene targeting in an ex vivo model using corneal epithelial cell cultures. METHODS: A panel of 19 TGFBI-Arg124Cys-specific siRNAs were assessed by a dual-luciferase reporter assay. Further assessment using pyrosequencing and qPCR was used to identify the lead siRNA; suppression of mutant TGFBIp expression was confirmed by Western blot and Congo red aggregation assays. An ex vivo model of LCDI was established using limbal biopsies from corneal dystrophy patients harboring the Arg124Cys mutation. Treatment efficiency of the siRNA was assessed for the inhibition of the mutant allele in the primary patient's corneal epithelial cells using pyrosequencing, quantitative PCR (qPCR), and an ELISA. RESULTS: A lead siRNA was identified, and demonstrated to be potent and specific in inhibiting the TGFBI-Arg124Cys mutant allele at the mRNA and protein levels. Besides high allele specificity, siRNA treatment achieved a 44% reduction of the endogenous Arg124Cys allele in an ex vivo model of LCDI. CONCLUSIONS: We have identified a lead siRNA specific to the TGFBI-Arg124Cys mutant allele associated with LCDI. Silencing of exogenous TGFBI was observed at mRNA and protein levels, and in an ex vivo model of LCDI with an efficient suppression of the endogenous mutant allele. This result indicates the potential of siRNA treatment as a personalized medicine approach for the management of heritable TGFBI-associated corneal dystrophies.

Allele-specific siRNA silencing for the common keratin 12 founder mutation in Meesmann epithelial corneal dystrophy.

PURPOSE: To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment. METHODS: siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5' rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting. RESULTS: The lead siRNA, with an IC(50) of thirty picomolar, showed no keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein. CONCLUSIONS: A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms.

Development of allele-specific therapeutic siRNA for keratin 5 mutations in epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is an incurable, inherited skin-blistering disorder predominantly caused by dominant-negative mutations in the genes encoding keratins K5 or K14. RNA interference, particularly in the form of small interfering RNA (siRNA), offers a potential therapy route for EBS and related keratin disorders by selectively silencing the mutant allele. Here, using a systemic screening system based on a luciferase reporter gene assay, we have developed mutant-specific siRNAs for two independent EBS-causing missense mutations in the K5 gene (p.Ser181Pro and p.Asn193Lys). The specificity of the allele-specific inhibitors identified in the screen was subsequently confirmed at the protein level, where the lead inhibitors were shown to strongly knock down the expression of mutant proteins with negligible effect on wild-type K5 expression. In a cell-based model system, the lead inhibitors were able to significantly reverse the cytoskeletal aggregation phenotype. Overall, this approach shows promise for the treatment of EBS and paves the way for future clinical trials.

Development of allele-specific therapeutic siRNA in Meesmann epithelial corneal dystrophy.

BACKGROUND: Meesmann epithelial corneal dystrophy (MECD) is an inherited eye disorder caused by dominant-negative mutations in either keratins K3 or K12, leading to mechanical fragility of the anterior corneal epithelium, the outermost covering of the eye. Typically, patients suffer from lifelong irritation of the eye and/or photophobia but rarely lose visual acuity; however, some individuals are severely affected, with corneal scarring requiring transplant surgery. At present no treatment exists which addresses the underlying pathology of corneal dystrophy. The aim of this study was to design and assess the efficacy and potency of an allele-specific siRNA approach as a future treatment for MECD. METHODS AND FINDINGS: We studied a family with a consistently severe phenotype where all affected persons were shown to carry heterozygous missense mutation Leu132Pro in the KRT12 gene. Using a cell-culture assay of keratin filament formation, mutation Leu132Pro was shown to be significantly more disruptive than the most common mutation, Arg135Thr, which is associated with typical, mild MECD. A siRNA sequence walk identified a number of potent inhibitors for the mutant allele, which had no appreciable effect on wild-type K12. The most specific and potent inhibitors were shown to completely block mutant K12 protein expression with negligible effect on wild-type K12 or other closely related keratins. Cells transfected with wild-type K12-EGFP construct show a predominantly normal keratin filament formation with only 5% aggregate formation, while transfection with mutant K12-EGFP construct resulted in a significantly higher percentage of keratin aggregates (41.75%; p<0.001 with 95% confidence limits). The lead siRNA inhibitor significantly rescued the ability to form keratin filaments (74.75% of the cells contained normal keratin filaments; p<0.001 with 95% confidence limits). CONCLUSIONS: This study demonstrates that it is feasible to design highly potent siRNA against mutant alleles with single-nucleotide specificity for future treatment of MECD.

Assessment of toric intraocular lens alignment by a refractive power/corneal analyzer system and slitlamp observation.

PURPOSE: To assess the validity of an internal optical path difference map of a refractive power/corneal analyzer system in determining the alignment of toric intraocular lenses (IOLs). SETTINGS: Private practices, Spring Hill, Brisbane, and Chermside, Australia. METHODS: This retrospective study comprised patients with more than 1.5 diopters of preexisting corneal astigmatism who had phacoemulsification and AcrySof toric IOL implantation. Preoperatively, the surgical eye was marked at the slitlamp microscope using a 4-point technique. The desired IOL orientation was marked with a Mendez marker based on the steep corneal axis. The toric IOL axis was measured 3 weeks postoperatively by rotating the slitlamp beam to align with the IOL axis indicator marks and using the Internal OPD Map on the Nidek OPD-Scan system. Uncorrected (UDVA) and corrected (CDVA) distance visual acuities, residual refractive sphere, and residual keratometric and refractive cylinders were also measured at 3 weeks. RESULTS: Postoperatively, the mean UDVA was 0.17 logMAR +/- 0.18 (SD) and the mean CDVA, -0.01 +/- 0.12 logMAR; 88.2% of eyes had a UDVA of 0.3 or better, and no eye lost lines of visual acuity. There was an 82.33% reduction in defocus equivalent and a 64.62% reduction in refractive cylinder. The mean IOL misalignment measured by slitlamp was 2.55 +/- 2.76 degrees and by the internal map, 2.65 +/- 1.98 degrees. The correlation between the 2 methods was highly significant (r = 0.99, P<.001). CONCLUSIONS: Both refractive power/corneal analyzer system and slitlamp observation were reliable and predictable methods of assessing IOL alignment. The 4-point preoperative marking technique yielded clinically acceptable, accurate toric IOL alignment.