Therapeutic blood-brain barrier modulation and stroke treatment by a bioengineered FZD4-selective WNT surrogate in mice.

Derangements of the blood-brain barrier (BBB) or blood-retinal barrier (BRB) occur in disorders ranging from stroke, cancer, diabetic retinopathy, and Alzheimer's disease. The Norrin/FZD4/TSPAN12 pathway activates WNT/ß-catenin signaling, which is essential for BBB and BRB function. However, systemic pharmacologic FZD4 stimulation is hindered by obligate palmitoylation and insolubility of native WNTs and suboptimal properties of the FZD4-selective ligand Norrin. Here, we develop L6-F4-2, a non-lipidated, FZD4-specific surrogate which significantly improves subpicomolar affinity versus native Norrin. In Norrin knockout (NdpKO) mice, L6-F4-2 not only potently reverses neonatal retinal angiogenesis deficits, but also restores BRB and BBB function. In adult C57Bl/6J mice, post-stroke systemic delivery of L6-F4-2 strongly reduces BBB permeability, infarction, and edema, while improving neurologic score and capillary pericyte coverage. Our findings reveal systemic efficacy of a bioengineered FZD4-selective WNT surrogate during ischemic BBB dysfunction, with potential applicability to adult CNS disorders characterized by an aberrant blood-brain barrier.

Cilia-associated wound repair mediated by IFT88 in retinal pigment epithelium.

Primary cilia are conserved organelles that integrate extracellular cues into intracellular signals and are critical for diverse processes, including cellular development and repair responses. Deficits in ciliary function cause multisystemic human diseases known as ciliopathies. In the eye, atrophy of the retinal pigment epithelium (RPE) is a common feature of many ciliopathies. However, the roles of RPE cilia in vivo remain poorly understood. In this study, we first found that mouse RPE cells only transiently form primary cilia. We then examined the RPE in the mouse model of Bardet-Biedl Syndrome 4 (BBS4), a ciliopathy associated with retinal degeneration in humans, and found that ciliation in BBS4 mutant RPE cells is disrupted early during development. Next, using a laser-induced injury model in vivo, we found that primary cilia in RPE reassemble in response to laser injury during RPE wound healing and then rapidly disassemble after the repair is completed. Finally, we demonstrated that RPE-specific depletion of primary cilia in a conditional mouse model of cilia loss promoted wound healing and enhanced cell proliferation. In summary, our data suggest that RPE cilia contribute to both retinal development and repair and provide insights into potential therapeutic targets for more common RPE degenerative diseases.

An efficient inducible RPE-Selective cre transgenic mouse line.

Cre-mediated modulation of gene function in the murine retinal pigment epithelium (RPE) has been widely used, but current postnatal RPE-selective Cre driver lines suffer from limited recombination efficiency and/or ectopic or mosaic expression. We sought to generate a transgenic mouse line with consistently efficient RPE-selective Cre activity that could be temporally regulated. We used ϕC31 integrase to insert a DNA construct encoding a human BEST1 promoter fragment driving a Cre recombinase estrogen receptor fusion (BEST1-CreERT2) at the Rosa26 locus of C57BL/6J mice. Rosa26BEST1-CreERT2 mice were bred with a tdTomato reporter line and to mice with a Cre-conditional allele of Tfam. 4-hydroxytamoxifen or vehicle was delivered by four consecutive daily intraperitoneal injections. TdTomato was robustly expressed in the RPE of mice of both sexes for inductions beginning at P14 (males 90.7 ± 4.5%, females 84.7 ± 3.2%) and at 7 weeks (males 84.3 ± 7.0%, females 82 ± 3.6%). <0.6% of Muller glia also expressed tdTomato, but no tdTomato fluorescence was observed in other ocular cells or in multiple non-ocular tissues, with the exception of sparse foci in the testis. No evidence of retinal toxicity was observed in mice homozygous for the transgene induced beginning at P14 and assessed at 7-10 months. RPE-selective ablation of Tfam beginning at P14 led to reduced retinal thickness at 8 months of age and diminished retinal electrical responses at 12 months, as expected. These findings demonstrate that we have generated a mouse line with consistently efficient, tamoxifen-mediated postnatal induction of Cre recombination in the RPE and a small fraction of Muller glia. This line should be useful for temporally regulated modulation of gene function in the murine RPE.

AMP-independent activator of AMPK for treatment of mitochondrial disorders.

Mitochondrial diseases are a clinically heterogenous group of disorders caused by respiratory chain dysfunction and associated with progressive, multi-systemic phenotype. There is no effective treatment or cure, and no FDA-approved drug for treating mitochondrial disease. To identify and characterize potential therapeutic compounds, we developed an in vitro screening assay and identified a group of direct AMP-activated protein kinase (AMPK) activators originally developed for the treatment of diabetes and metabolic syndrome. Unlike previously investigated AMPK agonists such as AICAR, these compounds allosterically activate AMPK in an AMP-independent manner, thereby increasing specificity and decreasing pleiotropic effects. The direct AMPK activator PT1 significantly improved mitochondrial function in assays of cellular respiration, energy status, and cellular redox. PT1 also protected against retinal degeneration in a mouse model of photoreceptor degeneration associated with mitochondrial dysfunction and oxidative stress, further supporting the therapeutic potential of AMP-independent AMPK agonists in the treatment of mitochondrial disease.

Depletion of Mitochondrial DNA in Differentiated Retinal Pigment Epithelial Cells.

We investigated the effects of treating differentiated retinal pigment epithelial (RPE) cells with didanosine (ddI), which is associated with retinopathy in individuals with HIV/AIDS. We hypothesized that such treatment would cause depletion of mitochondrial DNA and provide insight into the consequences of degradation of RPE mitochondrial function in aging and disease. Treatment of differentiated ARPE-19 or human primary RPE cells with 200 µM ddI for 6-24 days was not cytotoxic but caused up to 60% depletion of mitochondrial DNA, and a similar reduction in mitochondrial membrane potential and NDUFA9 protein abundance. Mitochondrial DNA-depleted RPE cells demonstrated enhanced aerobic glycolysis by extracellular flux analysis, increased AMP kinase activation, reduced mTOR activity, and increased resistance to cell death in response to treatment with the oxidant, sodium iodate. We conclude that ddI-mediated mitochondrial DNA depletion promotes a glycolytic shift in differentiated RPE cells and enhances resistance to oxidative damage. Our use of ddI treatment to induce progressive depletion of mitochondrial DNA in differentiated human RPE cells should be widely applicable for other studies aimed at understanding RPE mitochondrial dysfunction in aging and disease.

Association of a Primary Open-Angle Glaucoma Genetic Risk Score With Earlier Age at Diagnosis.

IMPORTANCE: Genetic variants associated with primary open-angle glaucoma (POAG) are known to influence disease risk. However, the clinical effect of associated variants individually or in aggregate is not known. Genetic risk scores (GRS) examine the cumulative genetic load by combining individual genetic variants into a single measure, which is assumed to have a larger effect and increased power to detect relevant disease-related associations. OBJECTIVE: To investigate if a GRS that comprised 12 POAG genetic risk variants is associated with age at disease diagnosis. DESIGN, SETTING, AND PARTICIPANTS: A cross-sectional study included individuals with POAG and controls from the Glaucoma Genes and Environment (GLAUGEN) study and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) study. A GRS was formulated using 12 variants known to be associated with POAG, and the alleles associated with increasing risk of POAG were aligned in the case-control sets. In case-only analyses, the association of the GRS with age at diagnosis was analyzed as an estimate of disease onset. Results from cohort-specific analyses were combined with meta-analysis. Data collection started in August 2012 for the NEIGHBOR cohort and in July 2008 for the GLAUGEN cohort and were analyzed starting in March 2018. MAIN OUTCOMES AND MEASURES: Association of a 12 single-nucleotide polymorphism POAG GRS with age at diagnosis in individuals with POAG using linear regression. RESULTS: The GLAUGEN study included 976 individuals with POAG and 1140 controls. The NEIGHBOR study included 2132 individuals with POAG and 2290 controls. For individuals with POAG, the mean (SD) age at diagnosis was 63.6 (9.8) years in the GLAUGEN cohort and 66.0 (13.7) years in the NEIGHBOR cohort. For controls, the mean (SD) age at enrollment was 65.5 (9.2) years in the GLAUGEN cohort and 68.9 (11.4) years in the NEIGHBOR cohort. All study participants were European white. The GRS was strongly associated with POAG risk in case-control analysis (odds ratio per 1-point increase in score = 1.24; 95% CI, 1.21-1.27; P = 3.4 × 10-66). In case-only analyses, each higher GRS unit was associated with a 0.36-year earlier age at diagnosis (ß = -0.36; 95% CI, -0.56 to -0.16; P = 4.0 × 10-4). Individuals in the top 5% of the GRS had a mean (SD) age at diagnosis of 5.2 (12.8) years earlier than those in the bottom 5% GRS (61.4 [12.7] vs 66.6 [12.9] years; P = 5.0 × 10-4). CONCLUSIONS AND RELEVANCE: A higher dose of POAG risk alleles was associated with an earlier age at glaucoma diagnosis. On average, individuals with POAG with the highest GRS had 5.2-year earlier age at diagnosis of disease. These results suggest that a GRS that comprised genetic variants associated with POAG could help identify patients with risk of earlier disease onset impacting screening and therapeutic strategies.

Highly Differentiated Human Fetal RPE Cultures Are Resistant to the Accumulation and Toxicity of Lipofuscin-Like Material.

Purpose: The accumulation of undigestible autofluorescent material (UAM), termed lipofuscin in vivo, is a hallmark of aged RPE. Lipofuscin derives, in part, from the incomplete degradation of phagocytized photoreceptor outer segments (OS). Whether this accumulated waste is toxic is unclear. We therefore investigated the effects of UAM in highly differentiated human fetal RPE (hfRPE) cultures. Methods: Unmodified and photo-oxidized OS were fed daily to confluent cultures of ARPE-19 RPE or hfRPE. The emission spectrum, composition, and morphology of resulting UAM were measured and compared to in vivo lipofuscin. Effects of UAM on multiple RPE phenotypes were assessed. Results: Compared to ARPE-19, hfRPE were markedly less susceptible to UAM buildup. Accumulated UAM in hfRPE initially resembled the morphology of lipofuscin from AMD eyes, but compacted and shifted spectrum over time to resemble lipofuscin from healthy aged human RPE. UAM accumulation mildly reduced transepithelial electrical resistance, ketogenesis, certain RPE differentiation markers, and phagocytosis efficiency, while inducing senescence and rare, focal pockets of epithelial-mesenchymal transition. However, it had no effects on mitochondrial oxygen consumption rate, certain other RPE differentiation markers, secretion of drusen components or polarity markers, nor cell death. Conclusions: hfRPE demonstrates a remarkable resistance to UAM accumulation, suggesting mechanisms for efficient OS processing that may be lost in other RPE culture models. Furthermore, while UAM alters hfRPE phenotype, the effects are modest, consistent with conflicting reports in the literature on the toxicity of lipofuscin. Our results suggest that healthy RPE may adequately adapt to and tolerate lipofuscin accumulation.

Genetic analyses of human fetal retinal pigment epithelium gene expression suggest ocular disease mechanisms.

The retinal pigment epithelium (RPE) serves vital roles in ocular development and retinal homeostasis but has limited representation in large-scale functional genomics datasets. Understanding how common human genetic variants affect RPE gene expression could elucidate the sources of phenotypic variability in selected monogenic ocular diseases and pinpoint causal genes at genome-wide association study (GWAS) loci. We interrogated the genetics of gene expression of cultured human fetal RPE (fRPE) cells under two metabolic conditions and discovered hundreds of shared or condition-specific expression or splice quantitative trait loci (e/sQTLs). Co-localizations of fRPE e/sQTLs with age-related macular degeneration (AMD) and myopia GWAS data suggest new candidate genes, and mechanisms by which a common RDH5 allele contributes to both increased AMD risk and decreased myopia risk. Our study highlights the unique transcriptomic characteristics of fRPE and provides a resource to connect e/sQTLs in a critical ocular cell type to monogenic and complex eye disorders.

Abnormal mTORC1 signaling leads to retinal pigment epithelium degeneration.

Retinal pigment epithelial (RPE) degeneration is potentially involved in the pathogenesis of several retinal degenerative diseases. mTORC1 signaling is shown as a crucial regulator of many biological processes and disease progression. In this study, we aimed at investigating the role of mTORC1 signaling in RPE degeneration. Methods: Western blots were conducted to detect mTORC1 expression pattern during RPE degeneration. Cre-loxP system was used to generate RPE-specific mTORC1 activation mice. Fundus, immunofluorescence staining, transmission electron microscopy, and targeted metabolomic analysis were conducted to determine the effects of mTORC1 activation on RPE degeneration in vivo. Electroretinography, spectral-domain optical coherence tomography, and histological experiments were conducted to determine the effects of mTORC1 activation on choroidal and retinal function in vivo. Results: RPE-specific activation of mTORC1 led to RPE degeneration as shown by the loss of RPE-specific marker, compromised cell junction integrity, and intracellular accumulation of lipid droplets. RPE degeneration further led to abnormal choroidal and retinal function. The inhibition of mTORC1 signaling with rapamycin could partially reverse RPE degeneration. Targeted metabolomics analysis further revealed that mTORC1 activation affected the metabolism of purine, carboxylic acid, and niacin in RPE. Conclusion: This study revealed that abnormal activation of mTORC1 signaling leads to RPE degeneration, which could provide a promising target for the treatment of RPE dysfunction-related diseases.

Method for measuring extracellular flux from intact polarized epithelial monolayers.

Purpose: The Seahorse XFp platform is widely used for metabolic assessment of cultured cells. Current methods require replating of cells into specialized plates. This is problematic for certain cell types, such as primary human fetal RPE (hfRPE) cells, which must be cultured for months to become properly differentiated. Our goal was to overcome this limitation by devising a method for assaying intact cell monolayers with the Seahorse XFp, without the need for replating. Methods: Primary hfRPE cells were differentiated by prolonged culture on filter inserts. Triangular sections of filters with differentiated cells attached were excised, transferred to XFp cell culture miniplate wells, immobilized at the bottoms, and subjected to mitochondrial stress tests. Replated cells were measured for comparison. Differentiated hfRPE cells were challenged or not with bovine photoreceptor outer segments (POS), and mitochondrial stress tests were performed 3.5 h later, after filter excision and transfer to assay plates. Results: Differentiated hfRPE cells assayed following filter excision demonstrated increased maximal respiration, increased spare respiration capacity, and increased extracellular acidification rate (ECAR) relative to replated controls. hfRPE cells challenged with POS exhibited increased maximal respiration and spare capacity, with no apparent change in the ECAR, relative to untreated controls. Conclusions: We have developed a method to reproducibly assay intact, polarized monolayers of hfRPE cells with the Seahorse XFp platform and have shown that the method yields more robust metabolic measurements compared to standard methods and is suitable for assessing the consequences of prolonged perturbations of differentiated cells. We expect our approach to be useful for a variety of studies involving metabolic assessment of adherent cells cultured on filters.

Testosterone Pathway Genetic Polymorphisms in Relation to Primary Open-Angle Glaucoma: An Analysis in Two Large Datasets.

Purpose: Sex hormones may be associated with primary open-angle glaucoma (POAG), although the mechanisms are unclear. We previously observed that gene variants involved with estrogen metabolism were collectively associated with POAG in women but not men; here we assessed gene variants related to testosterone metabolism collectively and POAG risk. Methods: We used two datasets: one from the United States (3853 cases and 33,480 controls) and another from Australia (1155 cases and 1992 controls). Both datasets contained densely called genotypes imputed to the 1000 Genomes reference panel. We used pathway- and gene-based approaches with Pathway Analysis by Randomization Incorporating Structure (PARIS) software to assess the overall association between a panel of single nucleotide polymorphisms (SNPs) in testosterone metabolism genes and POAG. In sex-stratified analyses, we evaluated POAG overall and POAG subtypes defined by maximum IOP (high-tension [HTG] or normal tension glaucoma [NTG]). Results: In the US dataset, the SNP panel was not associated with POAG (permuted P = 0.77), although there was an association in the Australian sample (permuted P = 0.018). In both datasets, the SNP panel was associated with POAG in men (permuted P ≤ 0.033) and not women (permuted P ≥ 0.42), but in gene-based analyses, there was no consistency on the main genes responsible for these findings. In both datasets, the testosterone pathway association with HTG was significant (permuted P ≤ 0.011), but again, gene-based analyses showed no consistent driver gene associations. Conclusions: Collectively, testosterone metabolism pathway SNPs were consistently associated with the high-tension subtype of POAG in two datasets.

Assessment of Murine Retinal Function by Electroretinography.

The electroretinogram (ERG) is a sensitive and noninvasive method for testing retinal function. In this protocol, we describe a method for performing ERGs in mice. Contact lenses on the mouse cornea measure the electrical response to a light stimulus of photoreceptors and downstream retinal cells, and the collected data are analyzed to evaluate retinal function.

Genetic correlations between intraocular pressure, blood pressure and primary open-angle glaucoma: a multi-cohort analysis.

Primary open-angle glaucoma (POAG) is the most common chronic optic neuropathy worldwide. Epidemiological studies show a robust positive relation between intraocular pressure (IOP) and POAG and modest positive association between IOP and blood pressure (BP), while the relation between BP and POAG is controversial. The International Glaucoma Genetics Consortium (n=27 558), the International Consortium on Blood Pressure (n=69 395), and the National Eye Institute Glaucoma Human Genetics Collaboration Heritable Overall Operational Database (n=37 333), represent genome-wide data sets for IOP, BP traits and POAG, respectively. We formed genome-wide significant variant panels for IOP and diastolic BP and found a strong relation with POAG (odds ratio and 95% confidence interval: 1.18 (1.14-1.21), P=1.8 × 10-27) for the former trait but no association for the latter (P=0.93). Next, we used linkage disequilibrium (LD) score regression, to provide genome-wide estimates of correlation between traits without the need for additional phenotyping. We also compared our genome-wide estimate of heritability between IOP and BP to an estimate based solely on direct measures of these traits in the Erasmus Rucphen Family (ERF; n=2519) study using Sequential Oligogenic Linkage Analysis Routines (SOLAR). LD score regression revealed high genetic correlation between IOP and POAG (48.5%, P=2.1 × 10-5); however, genetic correlation between IOP and diastolic BP (P=0.86) and between diastolic BP and POAG (P=0.42) were negligible. Using SOLAR in the ERF study, we confirmed the minimal heritability between IOP and diastolic BP (P=0.63). Overall, IOP shares genetic basis with POAG, whereas BP has limited shared genetic correlation with IOP or POAG.

Age at natural menopause genetic risk score in relation to age at natural menopause and primary open-angle glaucoma in a US-based sample.

OBJECTIVE: Several attributes of female reproductive history, including age at natural menopause (ANM), have been related to primary open-angle glaucoma (POAG). We assembled 18 previously reported common genetic variants that predict ANM to determine their association with ANM or POAG. METHODS: Using data from the Nurses' Health Study (7,143 women), we validated the ANM weighted genetic risk score in relation to self-reported ANM. Subsequently, to assess the relation with POAG, we used data from 2,160 female POAG cases and 29,110 controls in the National Eye Institute Glaucoma Human Genetics Collaboration Heritable Overall Operational Database (NEIGHBORHOOD), which consists of 8 datasets with imputed genotypes to 5.6+ million markers. Associations with POAG were assessed in each dataset, and site-specific results were meta-analyzed using the inverse weighted variance method. RESULTS: The genetic risk score was associated with self-reported ANM (P = 2.2 × 10) and predicted 4.8% of the variance in ANM. The ANM genetic risk score was not associated with POAG (Odds Ratio (OR) = 1.002; 95% Confidence Interval (CI): 0.998, 1.007; P = 0.28). No single genetic variant in the panel achieved nominal association with POAG (P ≥0.20). Compared to the middle 80 percent, there was also no association with the lowest 10 percentile or highest 90 percentile of genetic risk score with POAG (OR = 0.75; 95% CI: 0.47, 1.21; P = 0.23 and OR = 1.10; 95% CI: 0.72, 1.69; P = 0.65, respectively). CONCLUSIONS: A genetic risk score predicting 4.8% of ANM variation was not related to POAG; thus, genetic determinants of ANM are unlikely to explain the previously reported association between the two phenotypes.

Assessing the Association of Mitochondrial Genetic Variation With Primary Open-Angle Glaucoma Using Gene-Set Analyses.

PURPOSE: Recent studies indicate that mitochondrial proteins may contribute to the pathogenesis of primary open-angle glaucoma (POAG). In this study, we examined the association between POAG and common variations in gene-encoding mitochondrial proteins. METHODS: We examined genetic data from 3430 POAG cases and 3108 controls derived from the combination of the GLAUGEN and NEIGHBOR studies. We constructed biological-system coherent mitochondrial nuclear-encoded protein gene-sets by intersecting the MitoCarta database with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We examined the mitochondrial gene-sets for association with POAG and with normal-tension glaucoma (NTG) and high-tension glaucoma (HTG) subsets using Pathway Analysis by Randomization Incorporating Structure. RESULTS: We identified 22 KEGG pathways with significant mitochondrial protein-encoding gene enrichment, belonging to six general biological classes. Among the pathway classes, mitochondrial lipid metabolism was associated with POAG overall (P = 0.013) and with NTG (P = 0.0006), and mitochondrial carbohydrate metabolism was associated with NTG (P = 0.030). Examining the individual KEGG pathway mitochondrial gene-sets, fatty acid elongation and synthesis and degradation of ketone bodies, both lipid metabolism pathways, were significantly associated with POAG (P = 0.005 and P = 0.002, respectively) and NTG (P = 0.0004 and P < 0.0001, respectively). Butanoate metabolism, a carbohydrate metabolism pathway, was significantly associated with POAG (P = 0.004), NTG (P = 0.001), and HTG (P = 0.010). CONCLUSIONS: We present an effective approach for assessing the contributions of mitochondrial genetic variation to open-angle glaucoma. Our findings support a role for mitochondria in POAG pathogenesis and specifically point to lipid and carbohydrate metabolism pathways as being important.

A Common Variant in MIR182 Is Associated With Primary Open-Angle Glaucoma in the NEIGHBORHOOD Consortium.

PURPOSE: Noncoding microRNAs (miRNAs) have been implicated in the pathogenesis of glaucoma. We aimed to identify common variants in miRNA coding genes (MIR) associated with primary open-angle glaucoma (POAG). METHODS: Using the NEIGHBORHOOD data set (3853 cases/33,480 controls with European ancestry), we first assessed the relation between 85 variants in 76 MIR genes and overall POAG. Subtype-specific analyses were performed in high-tension glaucoma (HTG) and normal-tension glaucoma subsets. Second, we examined the expression of miR-182, which was associated with POAG, in postmortem human ocular tissues (ciliary body, cornea, retina, and trabecular meshwork [TM]), using miRNA sequencing (miRNA-Seq) and droplet digital PCR (ddPCR). Third, miR-182 expression was also examined in human aqueous humor (AH) by using miRNA-Seq. Fourth, exosomes secreted from primary human TM cells were examined for miR-182 expression by using miRNA-Seq. Fifth, using ddPCR we compared miR-182 expression in AH between five HTG cases and five controls. RESULTS: Only rs76481776 in MIR182 gene was associated with POAG after adjustment for multiple comparisons (odds ratio [OR] = 1.23, 95% confidence interval [CI]: 1.11-1.42, P = 0.0002). Subtype analysis indicated that the association was primarily in the HTG subset (OR = 1.26, 95% CI: 1.08-1.47, P = 0.004). The risk allele T has been associated with elevated miR-182 expression in vitro. Data from ddPCR and miRNA-Seq confirmed miR-182 expression in all examined ocular tissues and TM-derived exosomes. Interestingly, miR-182 expression in AH was 2-fold higher in HTG patients than nonglaucoma controls (P = 0.03) without controlling for medication treatment. CONCLUSIONS: Our integrative study is the first to associate rs76481776 with POAG via elevated miR-182 expression.

Treatment of retinitis pigmentosa due to MERTK mutations by ocular subretinal injection of adeno-associated virus gene vector: results of a phase I trial.

MERTK is an essential component of the signaling network that controls phagocytosis in retinal pigment epithelium (RPE), the loss of which results in photoreceptor degeneration. Previous proof-of-concept studies have demonstrated the efficacy of gene therapy using human MERTK (hMERTK) packaged into adeno-associated virus (AAV2) in treating RCS rats and mice with MERTK deficiency. The purpose of this study was to assess the safety of gene transfer via subretinal administration of rAAV2-VMD2-hMERTK in subjects with MERTK-associated retinitis pigmentosa (RP). After a preclinical phase confirming the safety of the study vector in monkeys, six patients (aged 14 to 54, mean 33.3 years) with MERTK-related RP and baseline visual acuity (VA) ranging from 20/50 to <20/6400 were entered in a phase I open-label, dose-escalation trial. One eye of each patient (the worse-seeing eye in five subjects) received a submacular injection of the viral vector, first at a dose of 150 µl (5.96 × 10(10)vg; 2 patients) and then 450 µl (17.88 × 10(10)vg; 4 patients). Patients were followed daily for 10 days at 30, 60, 90, 180, 270, 365, 540, and 730 days post-injection. Collected data included (1) full ophthalmologic examination including best-corrected VA, intraocular pressure, color fundus photographs, macular spectral domain optical coherence tomography and full-field stimulus threshold test (FST) in both the study and fellow eyes; (2) systemic safety data including CBC, liver and kidney function tests, coagulation profiles, urine analysis, AAV antibody titers, peripheral blood PCR and ASR measurement; and (3) listing of ophthalmological or systemic adverse effects. All patients completed the 2-year follow-up. Subretinal injection of rAAV2-VMD2-hMERTK was associated with acceptable ocular and systemic safety profiles based on 2-year follow-up. None of the patients developed complications that could be attributed to the gene vector with certainty. Postoperatively, one patient developed filamentary keratitis, and two patients developed progressive cataract. Of these two patients, one also developed transient subfoveal fluid after the injection as well as monocular oscillopsia. Two patients developed a rise in AAV antibodies, but neither patient was positive for rAAV vector genomes via PCR. Three patients also displayed measurable improved visual acuity in the treated eye following surgery, although the improvement was lost by 2 years in two of these patients. Gene therapy for MERTK-related RP using careful subretinal injection of rAAV2-VMD2-hMERTK is not associated with major side effects and may result in clinical improvement in a subset of patients.

Gene Therapy for MERTK-Associated Retinal Degenerations.

MERTK-associated retinal degenerations are thought to have defects in phagocytosis of shed outer segment membranes by the retinal pigment epithelium (RPE), as do the rodent models of these diseases. We have subretinally injected an RPE-specific AAV2 vector, AAV2-VMD2-hMERTK, to determine whether this would provide long-term photoreceptor rescue in the RCS rat, which it did for up to 6.5 months, the longest time point examined. Moreover, we found phagosomes in the RPE in the rescued regions of RCS retinas soon after the onset of light. The same vector also had a major protective effect in Mertk-null mice, with a concomitant increase in ERG response amplitudes in the vector-injected eyes. These findings suggest that planned clinical trials with this vector will have a favorable outcome.

The mTOR Kinase Inhibitor INK128 Blunts Migration of Cultured Retinal Pigment Epithelial Cells.

Retinal pigment epithelium (RPE) cell migration in response to disease has been reported for age-related macular degeneration, proliferative vitreoretinopathy, and proliferative diabetic retinopathy. The complex molecular process of RPE cell migration is regulated in part by growth factors and cytokines, and activation of the PI3/AKT/mTOR signaling pathway. Rapamycin, an allosteric mTOR inhibitor, has been shown to block only one of the primary downstream mTOR effectors, p70 S6 kinase 1, in many cell types. INK128, a selective mTOR ATP binding site competitor, blocks both p70 S6 kinase 1 and a second primary downstream effector, 4E-BP1. We performed scratch assays using differentiated ARPE-19 and primary porcine RPE cells to assess the effect of mTOR inhibition on cell migration. We found that INK128-mediated blocking of both p70 S6 kinase 1 and 4E-BP1 was much more effective at preventing RPE cell migration than rapamycin-mediated inhibition of p70 S6 kinase 1 alone.

Tyro3 Modulates Mertk-Associated Retinal Degeneration.

Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of Mertk-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in Mertk-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. Mertk-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of Mertk. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in Mertk knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of Mertk knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates Mertk-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD.