RPE-Directed Gene Therapy Improves Mitochondrial Function in Murine Dry AMD Models.

Age-related macular degeneration (AMD) is the most common cause of blindness in the aged population. However, to date there is no effective treatment for the dry form of the disease, representing 85-90% of cases. AMD is an immensely complex disease which affects, amongst others, both retinal pigment epithelium (RPE) and photoreceptor cells and leads to the progressive loss of central vision. Mitochondrial dysfunction in both RPE and photoreceptor cells is emerging as a key player in the disease. There are indications that during disease progression, the RPE is first impaired and RPE dysfunction in turn leads to subsequent photoreceptor cell degeneration; however, the exact sequence of events has not as yet been fully determined. We recently showed that AAV delivery of an optimised NADH-ubiquinone oxidoreductase (NDI1) gene, a nuclear-encoded complex 1 equivalent from S. cerevisiae, expressed from a general promoter, provided robust benefit in a variety of murine and cellular models of dry AMD; this was the first study employing a gene therapy to directly boost mitochondrial function, providing functional benefit in vivo. However, use of a restricted RPE-specific promoter to drive expression of the gene therapy enables exploration of the optimal target retinal cell type for dry AMD therapies. Furthermore, such restricted transgene expression could reduce potential off-target effects, possibly improving the safety profile of the therapy. Therefore, in the current study, we interrogate whether expression of the gene therapy from the RPE-specific promoter, Vitelliform macular dystrophy 2 (VMD2), might be sufficient to rescue dry AMD models.

Next-Generation Sequencing Applications for Inherited Retinal Diseases.

Inherited retinal diseases (IRDs) represent a collection of phenotypically and genetically diverse conditions. IRDs phenotype(s) can be isolated to the eye or can involve multiple tissues. These conditions are associated with diverse forms of inheritance, and variants within the same gene often can be associated with multiple distinct phenotypes. Such aspects of the IRDs highlight the difficulty met when establishing a genetic diagnosis in patients. Here we provide an overview of cutting-edge next-generation sequencing techniques and strategies currently in use to maximise the effectivity of IRD gene screening. These techniques have helped researchers globally to find elusive causes of IRDs, including copy number variants, structural variants, new IRD genes and deep intronic variants, among others. Resolving a genetic diagnosis with thorough testing enables a more accurate diagnosis and more informed prognosis and should also provide information on inheritance patterns which may be of particular interest to patients of a child-bearing age. Given that IRDs are heritable conditions, genetic counselling may be offered to help inform family planning, carrier testing and prenatal screening. Additionally, a verified genetic diagnosis may enable access to appropriate clinical trials or approved medications that may be available for the condition.

Fibrotic Changes to Schlemm's Canal Endothelial Cells in Glaucoma.

Previous studies have shown that glaucomatous Schlemm's canal endothelial cells (gSCECs) are stiffer and associated with reduced porosity and increased extracellular matrix (ECM) material compared to SCECs from healthy individuals. We hypothesised that Schlemm's canal (SC) cell stiffening was a function of fibrotic changes occurring at the inner wall of SC in glaucoma. This study was performed in primary cell cultures isolated from the SC lumen of human donor eyes. RNA and protein quantification of both fibrotic and endothelial cell markers was carried out on both healthy and gSCECs. Functional assays to assess cell density, size, migration, proliferation, and mitochondrial function of these cells were also carried out. Indeed, we found that gSCECs deviate from typical endothelial cell characteristics and exhibit a more fibrotic phenotype. For example, gSCECs expressed significantly higher protein levels of the fibrotic markers α-SMA, collagen I-α1, and fibronectin, as well as significantly increased protein expression of TGFß-2, the main driver of fibrosis, compared to healthy SCECs. Interestingly, we observed a significant increase in protein expression of endothelial marker VE-cadherin in gSCECs, compared to healthy SCECs. gSCECs also appeared to be significantly larger, and surprisingly proliferate and migrate at a significantly higher rate, as well as showing significantly reduced mitochondrial activity, compared to healthy SCECs.

Toward an elucidation of the molecular genetics of inherited retinal degenerations.

While individually classed as rare diseases, hereditary retinal degenerations (IRDs) are the major cause of registered visual handicap in the developed world. Given their hereditary nature, some degree of intergenic heterogeneity was expected, with genes segregating in autosomal dominant, recessive, X-linked recessive, and more rarely in digenic or mitochondrial modes. Today, it is recognized that IRDs, as a group, represent one of the most genetically diverse of hereditary conditions - at least 260 genes having been implicated, with 70 genes identified in the most common IRD, retinitis pigmentosa (RP). However, targeted sequencing studies of exons from known IRD genes have resulted in the identification of candidate mutations in only approximately 60% of IRD cases. Given recent advances in the development of gene-based medicines, characterization of IRD patient cohorts for known IRD genes and elucidation of the molecular pathologies of disease in those remaining unresolved cases has become an endeavor of the highest priority. Here, we provide an outline of progress in this area.

Prevention of autosomal dominant retinitis pigmentosa by systemic drug therapy targeting heat shock protein 90 (Hsp90).

Retinitis pigmentosa (RP) is the most prevalent cause of registered visual handicap among working aged populations of developed countries. Up to 40% of autosomal dominant cases of disease are caused by mutations within the rhodopsin, RDS-peripherin and inosine 5'-monophosphate dehydrogenase type 1 (IMPDH1) genes, at least 30 mutations within which give rise to proteins that cause disease pathology by misfolding and aggregation. Given the genetic complexity of this disease, therapies that simultaneously target multiple mutations are of substantial logistic and economic significance. We show here, in a murine model of autosomal dominant RP (RP10) involving expression of an Arg224Pro mutation within the IMPDH1 gene, that treatment with the low-molecular-weight drug, 17-allylamino-17-demethoxygeldanamycin (17-AAG), an ansamycin antibiotic that binds to heat shock protein Hsp90, activating a heat shock response in mammalian cells, protects photoreceptors against degeneration induced by aggregating mutant IMPDH1 protein, systemic delivery of this low-molecular-weight drug to the retina being facilitated by RNA interference-mediated modulation of the inner-blood retina barrier. 17-AAG has an orphan drug status and is in current clinical use for the treatment of non-ocular diseases. These data show that a single low-molecular-weight drug has the potential to suppress a wide range of mutant proteins causing RP.

AAV-PHP.eB transduces both the inner and outer retina with high efficacy in mice.

Recombinant adeno-associated virus (AAV) vectors are one of the main gene delivery vehicles used in retinal gene therapy approaches; however, there is a need to further improve the efficacy, tropism, and safety of these vectors. In this study, using a CMV-EGFP expression cassette, we characterize the retinal utility of AAV-PHP.eB, a serotype recently developed by in vivo directed evolution, which can cross the blood-brain barrier and target neurons with high efficacy in mice. Systemic and intravitreal delivery of AAV-PHP.eB resulted in the high transduction efficacy of retinal ganglion and horizontal cells, with systemic delivery providing pan-retinal coverage of the mouse retina. Subretinal delivery transduced photoreceptors and retinal pigment epithelium cells robustly. EGFP expression (number of transduced cells and mRNA levels) were similar when the retinas were transduced systemically or intravitreally with AAV-PHP.eB or intravitreally with AAV2/2. Notably, in photoreceptors, EGFP fluorescence intensities and mRNA levels were 50-70 times higher, when subretinal injections with AAV-PHP.eB were compared to AAV2/8. Our results demonstrate the pan-retinal transduction of ganglion cells and extremely efficient transduction of photoreceptor and retinal pigment epithelium cells as the most valuable features of AAV-PHP.eB in the mouse retina.

siRNA targeting Schlemm's canal endothelial tight junctions enhances outflow facility and reduces IOP in a steroid-induced OHT rodent model.

Systemic or localized application of glucocorticoids (GCs) can lead to iatrogenic ocular hypertension, which is a leading cause of secondary open-angle glaucoma and visual impairment. Previous work has shown that dexamethasone increases zonula occludens-1 (ZO-1) protein expression in trabecular meshwork (TM) cells, and that an antisense oligonucleotide inhibitor of ZO-1 can abolish the dexamethasone-induced increase in trans-endothelial flow resistance in cultured Schlemm's canal (SC) endothelial and TM cells. We have previously shown that intracameral inoculation of small interfering RNA (siRNA) targeting SC endothelial cell tight junction components, ZO-1 and tricellulin, increases aqueous humor outflow facility ex vivo in normotensive mice by reversibly opening SC endothelial paracellular pores. In this study, we show that targeted siRNA downregulation of these SC endothelial tight junctions reduces intraocular pressure (IOP) in vivo, with a concomitant increase in conventional outflow facility in a well-characterized chronic steroid-induced mouse model of ocular hypertension, thus representing a potential focused clinical application for this therapy in a sight-threatening scenario.

Therapeutic benefit derived from RNAi-mediated ablation of IMPDH1 transcripts in a murine model of autosomal dominant retinitis pigmentosa (RP10).

Mutations within the inosine 5'-monophosphate dehydrogenase 1 (IMPDH1) gene cause the RP10 form of autosomal dominant retinitis pigmentosa (adRP), an early-onset retinopathy resulting in extensive visual handicap owing to progressive death of photoreceptors. Apart from the prevalence of RP10, estimated to account for 5-10% of cases of adRP in United States and Europe, two observations render this form of RP an attractive target for gene therapy. First, we show that while recombinant adeno-associated viral (AAV)-mediated expression of mutant human IMPDH1 protein in the mouse retina results in an aggressive retinopathy modelling the human counterpart, expression of a normal human IMPDH1 gene under similar conditions has no observable pathological effect on retinal function, indicating that over-expression of a therapeutic replacement gene may be relatively well tolerated. Secondly, complete absence of IMPDH1 protein in mice with a targeted disruption of the gene results in relatively mild retinal dysfunction, suggesting that significant therapeutic benefit may be derived even from the suppression-only component of an RNAi-based gene therapy. We show that AAV-mediated co-expression in the murine retina of a mutant human IMPDH1 gene together with short hairpin RNAs (shRNA) validated in vitro and in vivo, targeting both human and mouse IMPDH1, substantially suppresses the negative pathological effects of mutant IMPDH1, at a point where, in the absence of shRNA, expression of mutant protein in the RP10 model essentially ablates all photoreceptors in transfected areas of the retina. These data strongly suggest that an RNAi-mediated approach to therapy for RP10 holds considerable promise for human subjects.

Protection of photoreceptors in a mouse model of RP10.

Recombinant adeno-associated viral (rAAV) vectors have recently been widely used for the delivery of therapeutic transgenes in preclinical and clinical studies for inherited retinal degenerative diseases. Interchanging capsid genes between different AAV serotypes has enabled selective delivery of transgene into specific cell type(s) of the retina. The RP10 form of autosomal dominant retinitis pigmentosa (adRP) is caused by missense mutations within the gene encoding inosine 5'-monophosphate dehydrogenase type 1. Here, we report that the use of rAAV2/5 vectors expressing shRNA targeting mutant IMPDH1 prevents photoreceptor degeneration, and preserves synaptic connectivity in a mouse model of RP10.

Non-photoreceptor Expression of Tulp1 May Contribute to Extensive Retinal Degeneration in Tulp1-/- Mice.

Mutations in tubby like protein 1 gene (TULP1) are causative of early-onset recessive inherited retinal degenerations (IRDs); similarly, the Tulp1-/- mouse is also characterized by a rapid IRD. Tulp1 mRNA and protein expression was analyzed in wild type mouse retinas and expression data sets (NCBI) during early postnatal development. Comparative histology was undertaken in Tulp1-/-, rhodopsin-/- (Rho-/-) and retinal degeneration slow-/- (Rds-/-) mouse retinas. Bioinformatic analysis of predicted TULP1 interactors and IRD genes was performed. Peak expression of Tulp1 in healthy mouse retinas was detected at p8; of note, TULP1 was detected in both the outer and inner retina. Bioinformatic analysis indicated Tulp1 expression in retinal progenitor, photoreceptor and non-photoreceptor cells. While common features of photoreceptor degeneration were detected in Tulp1-/-, Rho-/-, and Rds-/- retinas, other alterations in bipolar, amacrine and ganglion cells were specific to Tulp1-/- mice. Additionally, predicted TULP1 interactors differed in various retinal cell types and new functions for TULP1 were suggested. A pilot bioinformatic analysis indicated that in a similar fashion to Tulp1, many other IRD genes were expressed in both inner and outer retinal cells at p4-p7. Our data indicate that expression of Tulp1 extends to multiple retinal cell types; lack of TULP1 may lead to primary degeneration not only of photoreceptor but also non-photoreceptor cells. Predicted interactors suggest widespread retinal functions for TULP1. Early and widespread expression of TULP1 and some other IRD genes in both the inner and outer retina highlights potential hurdles in the development of treatments for these IRDs.

AAV-Delivered Tulp1 Supplementation Therapy Targeting Photoreceptors Provides Minimal Benefit in Tulp1-/- Retinas.

With marketing approval of the first ocular gene therapy, and other gene therapies in clinical trial, treatments for inherited retinal degenerations (IRDs) have become a reality. Biallelic mutations in the tubby like protein 1 gene (TULP1) are causative of IRDs in humans; a mouse knock-out model (Tulp1-/-) is characterized by a similar disease phenotype. We developed a Tulp1 supplementation therapy for Tulp1-/- mice. Utilizing subretinal AAV2/5 delivery at postnatal day (p)2-3 and rhodopsin-kinase promoter (GRK1P) we targeted Tulp1 to photoreceptor cells exploring three doses, 2.2E9, 3.7E8, and 1.2E8 vgs. Tulp1 mRNA and TULP1 protein were assessed by RT-qPCR, western blot and immunocytochemistry, and visual function by electroretinography. Our results indicate that TULP1 was expressed in photoreceptors; achieved levels of Tulp1 mRNA and protein were similar to wild type levels at p20. However, the thickness of the outer nuclear layer (ONL) did not improve in treated Tulp1-/- mice. There was a small and transient electroretinography benefit in the treated retinas at 4 weeks of age (not observed by 6 weeks) when using 3.7E8 vg dose. Dark-adapted mixed rod and cone a- and b-wave amplitudes were 24.3 ± 13.5 µV and 52.2 ± 31.7 µV in treated Tulp1-/- mice, which were significantly different (p < 0.001, t-test), from those detected in untreated eyes (7.1 ± 7.0 µV and 9.4 ± 15.1 µV, respectively). Our results indicate that Tulp1 supplementation in photoreceptors may not be sufficient to provide robust benefit in Tulp1-/- mice. As such, further studies are required to fine tune the Tulp1 supplementation therapy, which, in principle, should rescue the Tulp1-/- phenotype.

Properties and Therapeutic Implications of an Enigmatic D477G RPE65 Variant Associated with Autosomal Dominant Retinitis Pigmentosa.

RPE65 isomerase, expressed in the retinal pigmented epithelium (RPE), is an enzymatic component of the retinoid cycle, converting all-trans retinyl ester into 11-cis retinol, and it is essential for vision, because it replenishes the photon capturing 11-cis retinal. To date, almost 200 loss-of-function mutations have been identified within the RPE65 gene causing inherited retinal dystrophies, most notably Leber congenital amaurosis (LCA) and autosomal recessive retinitis pigmentosa (arRP), which are both severe and early onset disease entities. We previously reported a mutation, D477G, co-segregating with the disease in a late-onset form of autosomal dominant RP (adRP) with choroidal involvement; uniquely, it is the only RPE65 variant to be described with a dominant component. Families or individuals with this variant have been encountered in five countries, and a number of subsequent studies have been reported in which the molecular biological and physiological properties of the variant have been studied in further detail, including observations of possible novel functions in addition to reduced RPE65 enzymatic activity. With regard to the latter, a human phase 1b proof-of-concept study has recently been reported in which aspects of remaining vision were improved for up to one year in four of five patients with advanced disease receiving a single one-week oral dose of 9-cis retinaldehyde, which is the first report showing efficacy and safety of an oral therapy for a dominant form of RP. Here, we review data accrued from published studies investigating molecular mechanisms of this unique variant and include hitherto unpublished material on the clinical spectrum of disease encountered in patients with the D477G variant, which, in many cases bears striking similarities to choroideremia.

Advanced late-onset retinitis pigmentosa with dominant-acting D477G RPE65 mutation is responsive to oral synthetic retinoid therapy.

OBJECTIVES: No therapeutic interventions are currently available for autosomal dominant retinitis pigmentosa (adRP). An RPE65 Asp477Gly transition associates with late-onset adRP, reduced RPE65 enzymatic activity being one feature associated with this dominant variant. Our objective: to assess whether in a proof-of-concept study, oral synthetic 9 cis-retinyl acetate therapy improves vision in such advanced disease. METHODS AND ANALYSIS: A phase 1b proof-of-concept clinical trial was conducted involving five patients with advanced disease, aged 41-68 years. Goldmann visual fields (GVF) and visual acuities (VA) were assessed for 6-12 months after 7-day treatment, patients receiving consecutive oral doses (40 mg/m2) of 9-cis-retinyl acetate, a synthetic retinoid replacement. RESULTS: Pathological effects of D477G variant were preliminarily assessed by electroretinography in mice expressing AAV-delivered D477G RPE65, by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme- thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assays on RPE viability and enzyme activity in cultured cells. In addition to a mild dominant effect reflected in reduced electroretinographics in mice, and reduced cellular function in vitro, D477G exhibited reduced enzymatic RPE65 activity in vitro. In patients, significant improvements were observed in GVF from baseline ranging from 70% to 200% in three of five subjects aged 67-68 years, with largest improvements at 7-10 months. Of two GVF non-responders, one had significant visual acuity improvement (5-15 letters) from baseline after 6 months. CONCLUSION: Families with D477G variant have been identified in Ireland, the UK, France, the USA and Canada. Effects of single 7-day oral retinoid supplementation lasted at least 6 months, possibly giving visual benefit throughout remaining life in patients with advanced disease, where gene therapy is unlikely to prove beneficial.

SARM1 deficiency promotes rod and cone photoreceptor cell survival in a model of retinal degeneration.

Retinal degeneration is the leading cause of incurable blindness worldwide and is characterised by progressive loss of light-sensing photoreceptors in the neural retina. SARM1 is known for its role in axonal degeneration, but a role for SARM1 in photoreceptor cell degeneration has not been reported. SARM1 is known to mediate neuronal cell degeneration through depletion of essential metabolite NAD and induction of energy crisis. Here, we demonstrate that SARM1 is expressed in photoreceptors, and using retinal tissue explant, we confirm that activation of SARM1 causes destruction of NAD pools in the photoreceptor layer. Through generation of rho -/- sarm1 -/- double knockout mice, we demonstrate that genetic deletion of SARM1 promotes both rod and cone photoreceptor cell survival in the rhodopsin knockout (rho -/- ) mouse model of photoreceptor degeneration. Finally, we demonstrate that SARM1 deficiency preserves cone visual function in the surviving photoreceptors when assayed by electroretinography. Overall, our data indicate that endogenous SARM1 has the capacity to consume NAD in photoreceptor cells and identifies a previously unappreciated role for SARM1-dependent cell death in photoreceptor cell degeneration.

Dysregulated claudin-5 cycling in the inner retina causes retinal pigment epithelial cell atrophy.

Age-related macular degeneration (AMD) is the leading cause of central retinal vision loss worldwide, with an estimated 1 in 10 people over the age of 55 showing early signs of the condition. There are currently no forms of therapy available for the end stage of dry AMD, geographic atrophy (GA). Here, we show that the inner blood-retina barrier (iBRB) is highly dynamic and may play a contributory role in GA development. We have discovered that the gene CLDN5, which encodes claudin-5, a tight junction protein abundantly expressed at the iBRB, is regulated by BMAL1 and the circadian clock. Persistent suppression of claudin-5 expression in mice exposed to a cholesterol-enriched diet induced striking retinal pigment epithelium (RPE) cell atrophy, and persistent targeted suppression of claudin-5 in the macular region of nonhuman primates induced RPE cell atrophy. Moreover, fundus fluorescein angiography in human and nonhuman primate subjects showed increased retinal vascular permeability in the evening compared with the morning. These findings implicate an inner retina-derived component in the early pathophysiological changes observed in AMD, and we suggest that restoring the integrity of the iBRB may represent a novel therapeutic target for the prevention and treatment of GA secondary to dry AMD.

Light in retinitis pigmentosa.

Retinitis pigmentosa (RP) is one of the most genetically heterogeneous inherited disorders. Twelve genes have now been identified in the autosomal dominant form of the disease, including some recently characterized genes that show unprecedented and fascinating traits in both their function and in their expression profiles. These include many widely expressed genes encoding components of the spliceosome and a guanine nucleotide synthesis gene. Intriguingly, the most recently identified dominant gene does not appear to be expressed in the neuronal retina but is expressed in the capillaries of the choroid. In attempting to understand the effects of mutations in these genes, investigators are forced to re-evaluate their thinking on the molecular mechanisms of genetic blindness and to undertake an increasingly inter-disciplinary approach in their analysis of this disease. Recently, this has resulted in significant developments in the elucidation of the molecular pathogenesis of RP.

Reference gene selection for real-time rtPCR in human epidermal keratinocytes.

BACKGROUND: RNA interference represents a powerful tool with which to achieve suppression of specific target mRNA. Real-time rtPCR is a useful technique for assessing levels of mRNA expression. Critically, for real-time rtPCR to yield meaningful results, it is necessary to normalise expression of the gene of interest to stably expressed endogenous control genes. OBJECTIVES: The study involved establishing expression profiles for 11 housekeeping genes in human epidermal keratinocyte cell lines determining their relative stability. Furthermore, the effect of the presence of shRNA on these expression profiles has been established. METHODS: Keratinocytes were transfected using lipid-based transfection or AMAXA nucleofection. Real-time rtPCR was used to establish RNA expression levels. Data analysis was carried out using geNORM. RESULTS: When using HaCaT or adult NHEK cells any combination of 8 of the housekeeping genes would be appropriate for normalisation. In contrast, with juvenile NHEK cells only 4 of the housekeeping genes were found to be sufficiently stable to be deemed appropriate for normalisation of expression data. Furthermore data demonstrated that the expression of housekeeping genes may sometimes be affected by the induction of the RNAi pathway. CONCLUSIONS: The data obtained highlight the importance of characterising housekeeping gene expression profiles in each specific cell type prior to choosing a set of housekeeping genes for expression studies. The results from this study are applicable to researchers working with human epidermal keratinocytes and the experimental approach is more broadly applicable to any researcher carrying out real-time rtPCR.

Manipulating ocular endothelial tight junctions: Applications in treatment of retinal disease pathology and ocular hypertension.

Protein levels of endothelial tight-junctions of the inner retinal microvasculature, together with those of Schlemm's canal, can be readily manipulated by RNA interference (RNAi), resulting in the paracellular clefts between such cells to be reversibly modulated. This facilitates access to the retina of systemically-deliverable low molecular weight, potentially therapeutic compounds, while also allowing potentially toxic material, for example, soluble Amyloid-ß1-40, to be removed from the retina into the peripheral circulation. The technique has also been shown to be highly effective in alleviation of pathological cerebral oedema and we speculate that it may therefore have similar utility in the oedematous retina. Additionally, by manipulating endothelial tight-junctions of Schlemm's canal, inflow of aqueous humour from the trabecular meshwork into the Canal can be radically enhanced, suggesting a novel avenue for control of intraocular pressure. Here, we review the technology underlying this approach together with specific examples of clinical targets that are, or could be, amenable to this novel form of genetic intervention.