Cynthia A. Berlinicke

Inducible CreERT-mediated gene editing is a powerful tool for targeted gene manipulation, but systemic tamoxifen (Tam) administration can cause off-target effects and toxicity. Here we demonstrate that topical administration of 4-hydroxytamoxifen (4-OHT) eye drops in Plp1-CreERT/tdTomato^f/f mice effectively induces CreERT-mediated recombination specifically in oligodendrocytes of the optic nerve, optic chiasm, and adjacent CNS regions including the optic tract, hippocampus, and corpus callosum. This localized approach achieves robust tdTomato expression in over 80% of oligodendrocytes without detectable retinal structural or functional abnormalities, as confirmed by electroretinography and fundus imaging. These findings demonstrate a safe, non-invasive method for spatially restricted gene editing in the visual pathway and certain brain areas, providing a valuable platform for investigating oligodendrocyte biology and demyelinating diseases while minimizing systemic side effects associated with Tam treatment.

Epithelial-mesenchymal transition (EMT), which is well known for its role in embryonic development, malignant transformation, and tumor progression, has also been implicated in a variety of retinal diseases, including proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), and diabetic retinopathy. EMT of the retinal pigment epithelium (RPE), although important in the pathogenesis of these retinal conditions, is not well understood at the molecular level. We and others have shown that a variety of molecules, including the co-treatment of human stem cell-derived RPE monolayer cultures with transforming growth factor beta (TGF–β) and the inflammatory cytokine tumor necrosis factor alpha (TNF–α), can induce RPE–EMT; however, small molecule inhibitors of RPE–EMT have been less well studied. Here, we demonstrate that BAY651942, a small molecule inhibitor of nuclear factor kapa-B kinase subunit beta (IKKβ) that selectively targets NF-κB signaling, can modulate TGF–β/TNF–α-induced RPE–EMT. Next, we performed RNA-seq studies on BAY651942 treated hRPE monolayers to dissect altered biological pathways and signaling events. Further, we validated the effect of IKKβ inhibition on RPE–EMT-associated factors using a second IKKβ inhibitor, BMS345541, with RPE monolayers derived from an independent stem cell line. Our data highlights the fact that pharmacological inhibition of RPE–EMT restores RPE identity and may provide a promising approach for treating retinal diseases that involve RPE dedifferentiation and EMT.

Glaucoma is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is one of the major risk factors for glaucoma onset and progression, and available pharmaceutical interventions are exclusively targeted at IOP lowering. However, degeneration of retinal ganglion cells (RGCs) may continue to progress despite extensive lowering of IOP. A complementary strategy to IOP reduction is the use of neuroprotective agents that interrupt the process of cell death by mechanisms independent of IOP. Here, we describe an ion complexation approach for formulating microcrystals containing ~50% loading of a protein kinase inhibitor, sunitinib, to enhance survival of RGCs with subconjunctival injection. A single subconjunctival injection of sunitinib-pamoate complex (SPC) microcrystals provided 20 weeks of sustained retina drug levels, leading to neuroprotection in a rat model of optic nerve injury. Furthermore, subconjunctival injection of SPC microcrystals also led to therapeutic effects in a rat model of corneal neovascularization. Importantly, therapeutically relevant retina drug concentrations were achieved with subconjunctival injection of SPC microcrystals in pigs. For a chronic disease such as glaucoma, a formulation that provides sustained therapeutic effects to complement IOP lowering therapies could provide improved disease management and promote patient quality of life.

Injury and loss of oligodendrocytes can cause demyelinating diseases such as multiple sclerosis. To improve our understanding of oligodendrocyte development, which could facilitate development of remyelination-based treatment strategies, we performed single-cell-transcriptomic-analysis of developing human oligodendrocyte-precursor-cells (hOPCs). We engineered knock-in hESC-reporter lines in which an Identification-and-Purification tag is expressed under control of the endogenous, OPC-specific, PDGFRα promoter, and performed time-course single-cell-RNA-sequencing of purified hOPCs. Our analysis uncovered marked transcriptional heterogeneity of PDGFR α + hOPCs and identified regulatory genes and networks that control their differentiation and myelination competence. Pseudotime trajectory analysis revealed two distinct trajectories for the development of oligodendrocytes vs astrocytes from hOPCs. We also identified novel transcription factors and other genes that developing hOPCs potentially use to choose between oligodendrocyte vs astrocyte lineages. In addition, pathway enrichment analysis followed by pharmacological intervention of those pathways confirm that mTOR and cholesterol biosynthesis signaling pathways are involved in maturation of oligodendrocytes from hOPCs.

Recent advances in stem cell engineering have led to an explosion in the use of organoids as model systems for studies in multiple biological disciplines. Together with breakthroughs in genome engineering and the various omics, organoid technology is making possible studies of human biology that were not previously feasible. For vision science, retinal organoids derived from human stem cells allow differentiating and mature human retinal cells to be studied in unprecedented detail. In this review, we examine the technologies employed to generate retinal organoids and how organoids are revolutionizing the fields of developmental and cellular biology as they pertain to the retina. Furthermore, we explore retinal organoids from a clinical standpoint, offering a new platform with which to study retinal diseases and degeneration, test prospective drugs and therapeutic strategies, and promote personalized medicine. Finally, we discuss the range of possibilities that organoids may bring to future retinal research and consider their ethical implications.

Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in zebrafish and mouse RP models, reasoning drugs effective across species may translate better clinically. We first performed a large-scale phenotypic drug screen using a larval zebrafish model of inducible RP. 2,934 compounds, mostly human-approved drugs, were tested across six concentrations. Statistically, 113 compounds achieved “hit” status. Secondary tests of 42 high-priority hits confirmed eleven lead compounds. Nine leads were then evaluated in mouse RP models, with six exhibiting neuroprotective effects. An analysis of potential mechanisms of action suggested complementary activities. Paired lead compound assays in zebrafish showed additive neuroprotective effects for the majority. These results highlight the value of cross-species phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for patients with RP and IRDs.

High content analysis (HCA) has become a leading methodology in phenotypic drug discovery efforts. Typical HCA workflows include imaging cells using an automated microscope and analyzing the data using algorithms designed to quantify one or more specific phenotypes of interest. Due to the richness of high content data, unappreciated phenotypic changes may be discovered in existing image sets using interactive machine-learning based software systems. Primary postnatal day four retinal cells from the photoreceptor (PR) labeled QRX-EGFP reporter mice were isolated, seeded, treated with a set of 234 profiled kinase inhibitors and then cultured for 1 week. The cells were imaged with an Acumen plate-based laser cytometer to determine the number and intensity of GFP-expressing, i.e. PR, cells. Wells displaying intensities and counts above threshold values of interest were re-imaged at a higher resolution with an INCell2000 automated microscope. The images were analyzed with an open source HCA analysis tool, PhenoRipper (Rajaram et al., Nat Methods 9:635–637, 2012), to identify the high GFP-inducing treatments that additionally resulted in diverse phenotypes compared to the vehicle control samples. The pyrimidinopyrimidone kinase inhibitor CHEMBL-1766490, a pan kinase inhibitor whose major known targets are p38α and the Src family member lck, was identified as an inducer of photoreceptor neuritogenesis by using the open-source HCA program PhenoRipper. This finding was corroborated using a cell-based method of image analysis that measures quantitative differences in the mean neurite length in GFP expressing cells. Interacting with data using machine learning algorithms may complement traditional HCA approaches by leading to the discovery of small molecule-induced cellular phenotypes in addition to those upon which the investigator is initially focusing.