Probes for INS

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.

Background Retinoblastoma, the most common pediatric intraocular malignancy, can develop during embryogenesis, with most children being diagnosed at 3-4 years of age. Multimodal therapies are typically associated with high levels of cytotoxicity and side effects. Therefore, the development of novel treatments with minimal side effects is crucial. Magnolol has a significant anti-tumor effect on various cancers. However, its antitumor effect on retinoblastoma remains unclear. Purpose The study aimed to determine the effects of magnolol on the regulation of EMT, migration, invasion, and cancer progression in retinoblastoma and the modulation of miR-200c-3p expression and the Wnt/ zinc finger E-box binding homeobox 1 (ZEB1)/E-cadherin axis in vivo and in vitro. Methods The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay was used to evaluate magnolol-induced cell toxicity in the Y79 retinoblastoma cell line. Flow cytometry and immunostaining assays were performed to investigate the magnolol-regulated mitochondrial membrane potential and the intracellular and mitochondrial reactive oxygen species levels in Y79 retinoblastoma cells. Orthotopic and subcutaneous xenograft experiments were performed in eight-week-old male null mice to study retinoblastoma progression and metastasis. In situ hybridization and quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays were performed to evaluate the level of the anti-cancer miRNA miR-200c-3p. The mRNA and protein levels of E-cadherin, β-catenin, α-smooth muscle actin (α-SMA), fibronectin-1, and ZEB1 were analyzed using RT-qPCR, immunoblot, immunocytochemistry, and immunohistochemistry assays in vitro and in vivo. Results Magnolol increased E-cadherin levels and reduced the activation of the EMT signaling pathway, EMT, tumor growth, metastasis, and cancer progression in the Y79 retinoblastoma cell line as well as in the orthotopic and subcutaneous xenograft animal models. Furthermore, magnolol increased the expression of miR-200c-3p. Our results demonstrate that miRNA-200c-3p inhibits EMT progression through the Wnt16/β-catenin/ZEB1/E-cadherin axis, and the ZEB1 silencing response shows that miR-200c-3p regulates ZEB1-mediated EMT in retinoblastoma. Conclusion Magnolol has an antitumor effect by increasing E-cadherin and miRNA-200c-3p expression to regulate ZEB1-mediated EMT and cancer progression in retinoblastoma. The anti-tumor effect of magnolol by increasing E-cadherin and miRNA-200c-3p expression to regulate ZEB1-mediated EMT and cancer progression in retinoblastoma has been elucidated for the first time.