To investigate the impact of trabecular bypass surgery targeted to angiographically determined high- vs. low-aqueous humor outflow areas on outflow facility (C) and intraocular pressure (IOP). Ex vivo comparative study. Postmortem ex vivo porcine and human eyes. Porcine (n = 14) and human (n = 13) whole globes were acquired. In both species, anterior segments were dissected, mounted onto a perfusion chamber, and perfused using Dulbecco's phosphate buffered solution containing glucose in a constant flow paradigm to achieve a stable baseline. Fluorescein was perfused into the anterior chamber and used to identify baseline segmental high- and low-flow regions of the conventional outflow pathways. The anterior segments were divided into 2 groups, and a 5 mm needle goniotomy was performed in either a high- or low-flow area. Subsequently, C and IOP were quantitatively reassessed and compared between surgery in baseline "high-flow" and "low-flow" region eyes followed by indocyanine green angiography. Outflow facility. In all eyes, high- and low-flow segments could be identified. Performing a 5-mm goniotomy increased outflow facility to a variable extent depending on baseline flow status. In the porcine high-flow group, C increased from 0.31 ± 0.09 to 0.39 ± 0.09 μL/mmHg/min (P = 0.12). In the porcine low-flow group, C increased from 0.29 ± 0.03 to 0.56 ± 0.10 μL/mmHg/min (P < 0.001). In the human high-flow group, C increased from 0.38 ± 0.20 to 0.41 ± 0.20 μL/mmHg/min (P = 0.02). In the human low-flow group, C increased from 0.25 ± 0.11 to 0.32 ± 0.11 μL/mmHg/min (<0.001). There was statistically significant greater increase in C for eyes where surgery was targeted to baseline low-flow regions in both porcine (0.07 ± 0.09 vs. 0.27 ± 0.13, P = 0.007 μL/mmHg/min, high vs low flow) and human eyes (0.03 ± 0.03 vs. 0.07 ± 0.02, P = 0.03 μL/mmHg/min, high vs. low flow). Targeting surgery to low-flow areas of the trabecular meshwork yields higher overall facility increase and IOP reduction compared to surgery in high-flow areas. Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factorNEUROG2was combined with RGC-expressed TFs (ATOH7,ISL1,andPOU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection.

Retinogenesis involves the transformation of the anterior developing brain into organized retinal lamellae coordinated by intricate gene signalling networks. This complex process has been investigated in several model organisms such as birds, fish, mammals and amphibians, yet many facets of retinal development are different in humans and remain unexplored. In this regard, human pluripotent stem cell (hPSC)-derived 3D retinal organoids and Next Generation Sequencing (NGS) have emerged as key technologies that have facilitated the discovery of previously unknown details about cell fate specification and gene regulation in the retina. Here we utilized hPSCs integrated with fluorescent reporter genes (SIX6-p2A-eGFP/CRX-p2A-h2b-mRuby3) to generate retinal organoids and carry out bulk RNA sequencing of samples encompassing the majority of retinogenesis (D0-D280). This data set will serve as a valuable reference for the vision research community to characterize differentially expressed genes in the developing human eye.

Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds.

Retinogenesis involves the specification of retinal cell types during early vertebrate development. While model organisms have been critical for determining the role of dynamic chromatin and cell-type specific transcriptional networks during this process, an enhanced understanding of the developing human retina has been more elusive due to the requirement for human fetal tissue. Pluripotent stem cell (PSC) derived retinal organoids offer an experimentally accessible solution for investigating the developing human retina. To investigate cellular and molecular changes in developing early retinal organoids, we developed SIX6-GFP and VSX2-tdTomato (or VSX2-h2b-mRuby3) dual fluorescent reporters. When differentiated as 3D organoids these expressed GFP at day 15 and tdTomato (or mRuby3) at day 25, respectively. This enabled us to explore transcriptional and chromatin related changes using RNA-seq and ATAC-seq from pluripotency through early retina specification. Pathway analysis of developing organoids revealed a stepwise loss of pluripotency, while optic vesicle and retina pathways became progressively more prevalent. Correlating gene transcription with chromatin accessibility in early eye field development showed that retinal cells underwent a clear change in chromatin landscape, as well as gene expression profiles. While each dataset alone provided valuable information, considering both in parallel provided an informative glimpse into the molecular nature eye development.

Small extracellular vesicles (EVs) deliver nucleic acid and protein therapeutics that promote tissue repair, however, there are few approaches to direct the tropism of EVs to specific tissues. Here, we address this challenge by engineering EVs to display a peptide library on the surface of EVs that is linked to barcoded guide RNA payloads (gRNAs). We show how these EVs can be administered systemically into mouse models and the cellular uptake of these gRNA-bearing EVs assessed by recovery of the small RNAs followed by PCR amplification and barcode sequencing. Since the design of the peptide library was linked to specific barcodes encoded on the same plasmid, subsequent sequencing of barcode sequences recovered from tissues revealed profiles of EV uptake associated with the display of specific peptide sequences on the surface of EVs. Therefore, candidate peptide motifs were cloned for validation using in vivo and in vitro readouts. In addition to the barcode-based tissue profiling, gRNA-laded EVs mediated functional gene editing in the Cre-loxP R26 LSL-tdTomato reporter mouse model in vivo. These studies demonstrated how EV display linked with barcoded gRNA payloads can address barriers to EV-based delivery of gene editing therapies.