Probes for INS

RESULTS : Cell clustering revealed that Myrf deficiency altered cell type distributions with reductions in RPE cells at all timepoints. Cell cycle dynamics were stable, consistent with increased cell death in mutants. There was also a compensatory increase in retinal progenitor (RPC) population at P0, without alteration in overall cell cycle dynamics. Differential gene expression analysis and PANTHER gene ontology-term analysis revealed down regulation of key pathways in mutant RPE cells, including melanosome biogenesis, cytoskeleton, and extracellular matrix. EM analysis and immunofluorescence staining of RPE flatmounts confirmed structural defects in RPE and disorganization of photoreceptor outer segments, loss of melanosomes, and alterations in novel structural proteins in the apical RPE. Compensatory upgregulation of _Prss56_, another gene implicated in nanophthalmos, was found in the RPC population.

RESULTS : In rodent eye (both rat and mouse), CFH mRNA is strongly expressed in the retinal pigment epithelium with some expression also found in inner nuclear (INL) and retinal ganglion cell (RGC) layers of the retina. C3 mRNA is expressed mainly in RGC, INL of retina, ciliary body, corneal epithelium with some expression is also found in rodent retinal pigment epithelium layer. However, in human eye, CFH and C3 mRNA are strongly expressed in the choroid. Some expression is also found in RGC, INL layer of retina, ONH, sclera, cornea endothelial and stroma; and ciliary body. There is no C3 or CFH signal detected in RPE cells.

Purpose : ADOA is the most common inherited optic neuropathy, starting in the first decade of life and resulting in severe and progressive visual decline due to loss of RGCs. Most patients harbor loss-of-function mutations in the OPA1 gene that lead to haploinsufficiency. Reduced OPA1 protein levels result in impaired mitochondrial function in RGCs leading to cell death. Currently, there is no treatment for patients with ADOA. Targeted Augmentation of Nuclear Gene Output (TANGO) ASOs, such as STK-002, reduce the inclusion of a non-productive, alternatively spliced exon in OPA1, and leverage the wild-type allele to increase productive OPA1 mRNA and protein. We previously demonstrated that TANGO ASOs can increase OPA1 protein levels in human cell lines, rabbit retina, and ADOA patient fibroblasts. In this study, we evaluated ASO localization and OPA1 protein levels in the retina following intravitreal administration of STK-002 to NHPs. Methods : Cynomolgus monkeys (N=22) received bilateral intravitreal injections of vehicle or STK-002. Eyes were collected at 4 or 8 weeks after injection. Retinas were isolated for molecular analyses and whole globes were prepared for histology. Retinal OPA1 mRNA and protein were measured using qPCR (Taqman) and enzyme-linked immunosorbent assay (ELISA), respectively. A hybridization ELISA (HELISA) method was used to quantitate STK-002 levels in retina. Whole globes were sent for custom assay development and detection of STK-002 by miRNAscope™ in situ hybridization (ISH), and detection of OPA1 protein by immunofluorescence (IF). Results : Retinal exposure of STK-002 increased in a dose-dependent manner and remained high at the last timepoint evaluated (Week 8). STK-002 also dose-dependently increased protein levels at Week 4, ranging from 31 to 47% compared to vehicle, and levels were maintained at Week 8. ISH and IF analysis demonstrated that both STK-002 and OPA1 protein levels increased in RGCs, the target cells for ADOA. Conclusions : STK-002 produced a dose-dependent and persistent increase in OPA1 protein expression in the retinas of NHPs. ASO-induced increase in OPA1 protein levels in RGCs represents a potentially disease-modifying therapy for patients with ADOA.

RESULTS : Uniform Manifold Approximation and Projection clustering identified distinct expression signatures from the ganglion cell layer(GCL), inner nuclear layer(INL), retinal pigment epithelium (RPE)/choroid/sclera, optic nerve, and ciliary body (Fig, 1) but not the outer nuclear layer(ONL) which was contaminated with expression from other layers. Our findings highlight Clu, C4b, Apoe, and C1qa genes (z-score 3.0, 2.4, 2.3, and 2.2) as potential markers of disease in the RPE. Gene Set Enrichment analysis between rd6 and WT eyes showed upregulation of glycolysis and carbon metabolism pathways in the GCL and Rap1, Hippo and lysosome pathways in the RPE/Choroid/sclera. The ribosomal pathway was downregulated in these layers. No significant pathways were found in the INL, ciliary body or optic nerve.

RESULTS : PR-specific expression of _PRLΔE1_ was observed in the following canine models of progressive inherited retinal degeneration (IRD): _RPGR_-XLPRA1 and _NPHP5_-LCA. In _RPGR_-XLPRA2 carrier retinas that undergo random X-inactivation, patches of_ PRLΔE1 _expression correlated with patches of PR degeneration. However, we did not observe expression of _PRLΔE1_ 24 hrs and 2 wks after light exposure that triggers acute rod loss in the canine RHO-T4R model of adRP. No _PRLΔE1 _expression was seen either in the _CNGB3_-ACHM3 retina that undergoes extremely slow cone degeneration. In _RPGR-_XLPRA1 and _RPGR-_XLPRA2 dogs subretinally-injected with an AAV-_RPGR_ vector, _PRLΔE1 _was completely absent in treated PRs while robust expression was seen in diseased/untreated areas.

RESULTS : After quality control and data integration, 17,401 nuclei were isolated from 26,471 original droplets, derived from macular samples of 4 patients without retinal disease and 3 patients with POAG. The proportion of retinal ganglion cells in glaucomatous retina was significantly lower than that in healthy retina (p=0.024). An activated subpopulation of Müller glia was identified in both healthy and glaucomatous retina by cell clustering. Cross-species analysis comparing zebrafish and humans identified YAP1 activation as a differentiator between zebrafish and human glial activation. Human retinal explants cultured with N3B1P3C demonstrated significant proliferation of GS+ Muller cells (p=0.044).

RESULTS : All 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7 were preferentially expressed in the basal layer of the cornea and limbus compared to the suprabasal layer (_P_

METHODS : _MgpCre+/-_ mice were bred with _tfap2b+/-_ mice. Male _MgpCre+/-;tfap2b+/- _offspring were then crossed with female _tfap2blox/lox_ mice to obtain the final offspring, the _MgpCre+/-;tfap2b-/lox_ or AP-2β trabecular meshwork region knockout (TMR-KO) mice, as well as littermate controls. A 40 kDA FITC-conjugated dextran tracer was injected into the anterior segment of mutant and control mice. 0.005% LTP eye drops were used for topical treatment of the eye. The mice were euthanized 10 minutes after injection and eyes were enucleated, fixed, and cryosectioned. RNAscope Hiplex Assay was performed to determine changes in key genes in the mutants that are critical for proper functioning of TM and SC.

METHODS : 8-week wild-type mice were used to determine gene (_Htr1b_) expression. RNAscope _in situ_ hybridization (ISH) was performed on retinal cryosections and imaged using confocal microscopy. Whole field flash electroretinograms (ERGs) were used to record scotopic and photopic amplitudes in 22 mice (8 _Htr1b_-/-; 8 _Htr1b_+/-; 6 WT). Positive scotopic threshold response (pSTR), b-wave, and a-wave amplitudes were recorded. Visual behavior was evaluated in _Htr1b_-/- mice and controls by assessing the scotopic and photopic optokinetic response. Gratings of variable spatial frequency or contrast were presented to evaluate spatial frequency threshold and contrast sensitivity threshold, respectively. We performed retinal histology and _in vivo_ SD-OCT imaging on 8-week _Htr1b_-/- animals to quantify retinal layer thickness. Retinal layers (GCL, IPL, INL, OPL, and ONL) were measured as a percentage of total retinal thickness and compared to age and sex-matched wild-type controls.RGC numbers were determined from whole-mount retinas which were prepared and visualized with confocal microscopy. RBPMS-positive cells were counted semi-automatically using an ImageJ extension. Total cell density was normalized to image size.

METHODS : To visualize the BRB _in vivo, _we utilized the transgenic _Tg(l-fabp:DBP-EGFP) _zebrafish model that expresses vitamin D binding protein (a member of the albumin gene family) tagged to GFP. This model displays the integrity of the BRB with GFP-tagged protein localized within the retinal vasculature by 3 days post-fertilization. Breakdown of the BRB is visualized as “leaking” of GFP outside the vasculature. To disrupt RA signaling, zebrafish embryos, larvae, and adults were treated with varying concentrations of DEAB and BMS493, antagonists of retinal dehydrogenase and the RA receptor, respectively. To visualize the Müller cells and endothelial cells, _gfap:GFP_ and _kdrl:GFP_ transgenic fish were used, respectively. RNAScope analysis was used to detect and quantify the expression of _cyp26a1 _in retinas of zebrafish at different developmental stages.