Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines.

Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species.

Transcriptomic Analysis of Metastatic Uveal Melanoma and Differences in Male and Female Patients.

Background/aim: Uveal melanoma is an ocular malignancy whose prognosis severely worsens following metastasis. In order to improve the understanding of molecular physiology of metastatic uveal melanoma, we identified genes and pathways implicated in metastatic vs non-metastatic uveal melanoma.

Patients And Methods: A previously published dataset from Gene Expression Omnibus (GEO) was used to identify differentially expressed genes between metastatic and non-metastatic samples as well as to conduct pathway and perturbagen analyses using Gene Set Enrichment Analysis (GSEA), EnrichR, and iLINCS.

Heterogeneity in the progression of retinal pathologies in mice harboring patient mimicking Impg2 mutations.

Biallelic mutations in interphotoreceptor matrix proteoglycan 2 (IMPG2) in humans cause retinitis pigmentosa (RP) with early macular involvement, albeit the disease progression varies widely due to genetic heterogeneity and IMPG2 mutation type. There are currently no treatments for IMPG2-RP. To aid preclinical studies toward eventual treatments, there is a need to better understand the progression of disease pathology in appropriate animal models.

Nonviral base editing of KCNJ13 mutation preserves vision in a model of inherited retinal channelopathy.

Clinical genome editing is emerging for rare disease treatment, but one of the major limitations is the targeting of CRISPR editors' delivery. We delivered base editors to the retinal pigmented epithelium (RPE) in the mouse eye using silica nanocapsules (SNCs) as a treatment for retinal degeneration. Leber congenital amaurosis type 16 (LCA16) is a rare pediatric blindness caused by point mutations in the KCNJ13 gene, a loss of function inwardly rectifying potassium channel (Kir7.

A versatile laser-induced porcine model of outer retinal and choroidal degeneration for preclinical testing.

Over 30 million people worldwide suffer from untreatable vision loss and blindness associated with childhood-onset and age-related eye diseases caused by photoreceptor (PR), retinal pigment epithelium (RPE), and choriocapillaris (CC) degeneration. Recent work suggests that RPE-based cell therapy may slow down vision loss in late stages of age-related macular degeneration (AMD), a polygenic disease induced by RPE atrophy. However, accelerated development of effective cell therapies is hampered by the lack of large-animal models that allow testing safety and efficacy of clinical doses covering the human macula (20 mm2).

Re-formation of synaptic connectivity in dissociated human stem cell-derived retinal organoid cultures.

Human pluripotent stem cell (hPSC)-derived retinal organoids (ROs) can efficiently and reproducibly generate retinal neurons that have potential for use in cell replacement strategies [Capowski ,  146, dev171686 (2019)]. The ability of these lab-grown retinal neurons to form new synaptic connections after dissociation from ROs is key to building confidence in their capacity to restore visual function. However, direct evidence of reestablishment of retinal neuron connectivity via synaptic tracing has not been reported to date.

Human retinal organoids harboring IMPG2 mutations exhibit a photoreceptor outer segment phenotype that models advanced retinitis pigmentosa.

Interphotoreceptor matrix proteoglycan 2 (IMPG2) mutations cause a severe form of early-onset retinitis pigmentosa (RP) with macular involvement. IMPG2 is expressed by photoreceptors and incorporated into the matrix that surrounds the inner and outer segments (OS) of rods and cones, but the mechanism of IMPG2-RP remains unclear. Loss of Impg2 function in mice produces a mild, late-onset photoreceptor phenotype without the characteristic OS loss that occurs in human patients.

Systemic immunosuppression promotes survival and integration of subretinally implanted human ESC-derived photoreceptor precursors in dogs.

Regenerative therapies aimed at replacing photoreceptors are a promising approach for the treatment of otherwise incurable causes of blindness. However, such therapies still face significant hurdles, including the need to improve subretinal delivery and long-term survival rate of transplanted cells, and promote sufficient integration into the host retina. Here, we successfully delivered in vitro-derived human photoreceptor precursor cells (PRPCs; also known as immature photoreceptors) to the subretinal space of seven normal and three rcd1/PDE6B mutant dogs with advanced inherited retinal degeneration.

autofluorescence lifetime assay of a photoreceptor stimulus response in mouse retina and human retinal organoids.

Photoreceptors are the key functional cell types responsible for the initiation of vision in the retina. Phototransduction involves isomerization and conversion of vitamin A compounds, known as retinoids, and their recycling through the visual cycle. We demonstrate a functional readout of the visual cycle in photoreceptors within stem cell-derived retinal organoids and mouse retinal explants based on spectral and lifetime changes in autofluorescence of the visual cycle retinoids after exposure to light or chemical stimuli.

Human photoreceptors switch from autonomous axon extension to cell-mediated process pulling during synaptic marker redistribution.

Photoreceptors (PRs) are the primary visual sensory cells, and their loss leads to blindness that is currently incurable. Although cell replacement therapy holds promise, success is hindered by our limited understanding of PR axon growth during development and regeneration. Here, we generate retinal organoids from human pluripotent stem cells to study the mechanisms of PR process extension.

Cone photoreceptors in human stem cell-derived retinal organoids demonstrate intrinsic light responses that mimic those of primate fovea.

High-definition vision in humans and nonhuman primates is initiated by cone photoreceptors located within a specialized region of the retina called the fovea. Foveal cone death is the ultimate cause of central blindness in numerous retinal dystrophies, including macular degenerative diseases. 3D retinal organoids (ROs) derived from human pluripotent stem cells (hPSCs) hold tremendous promise to model and treat such diseases.

CRISPR Generated SIX6 and POU4F2 Reporters Allow Identification of Brain and Optic Transcriptional Differences in Human PSC-Derived Organoids.

Human pluripotent stem cells (PSCs) represent a powerful tool to investigate human eye development and disease. When grown in 3D, they can self-assemble into laminar organized retinas; however, variation in the size, shape and composition of individual organoids exists. Neither the microenvironment nor the timing of critical growth factors driving retinogenesis are fully understood.

Outer Retinal Cell Replacement: Putting the Pieces Together.

Retinal degenerative diseases (RDDs) affecting photoreceptors (PRs) are one of the most prevalent sources of incurable blindness worldwide. Due to a lack of endogenous repair mechanisms, functional cell replacement of PRs and/or retinal pigmented epithelium (RPE) cells are among the most anticipated approaches for restoring vision in advanced RDD. Human pluripotent stem cell (hPSC) technologies have accelerated development of outer retinal cell therapies as they provide a theoretically unlimited source of donor cells.

Ultrathin micromolded 3D scaffolds for high-density photoreceptor layer reconstruction.

Polymeric scaffolds are revolutionizing therapeutics for blinding disorders affecting the outer retina, a region anatomically and functionally defined by light-sensitive photoreceptors. Recent engineering advances have produced planar scaffolds optimized for retinal pigment epithelium monolayer delivery, which are being tested in early-stage clinical trials. We previously described a three-dimensional scaffold supporting a polarized photoreceptor monolayer, but photoreceptor somata typically occupy multiple densely packed strata to maximize light detection.

Necrotizing myositis in a rectus muscle arising in the setting of long-standing Langerhans cell histiocystosis and recent dabrafenib treatment.

Purpose: to describe an unusual case of necrotizing myositis in a rectus muscle, possibly related to BRAF inhibitor therapy.

Observations: An 18-year old man with neurodegenerative Langerhans cell histiocytosis (LCH), recently started on the BRAF inhibitor dabrafenib, presented with right eye pain. Magnetic resonance imaging (MRI) orbits revealed a rectus muscle mass concerning for LCH recurrence or malignancy.

Human iPSC Modeling Reveals Mutation-Specific Responses to Gene Therapy in a Genotypically Diverse Dominant Maculopathy.

Dominantly inherited disorders are not typically considered to be therapeutic candidates for gene augmentation. Here, we utilized induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. Gene augmentation in iPSC-RPE fully restored BEST1 calcium-activated chloride channel activity and improved rhodopsin degradation in an iPSC-RPE model of recessive bestrophinopathy as well as in two models of dominant Best disease caused by different mutations in regions encoding ion-binding domains.

Imaging Transplanted Photoreceptors in Living Nonhuman Primates with Single-Cell Resolution.

Stem cell-based transplantation therapies offer hope for currently untreatable retinal degenerations; however, preclinical progress has been largely confined to rodent models. Here, we describe an experimental platform for accelerating photoreceptor replacement therapy in the nonhuman primate, which has a visual system much more similar to the human. We deployed fluorescence adaptive optics scanning light ophthalmoscopy (FAOSLO) to noninvasively track transplanted photoreceptor precursors over time at cellular resolution in the living macaque.

PAX6D instructs neural retinal specification from human embryonic stem cell-derived neuroectoderm.

PAX6 is essential for neural retina (NR) and forebrain development but how PAX6 instructs NR versus forebrain specification remains unknown. We found that the paired-less PAX6, PAX6D, is expressed in NR cells during human eye development and along human embryonic stem cell (hESC) specification to retinal cells. hESCs deficient for PAX6D failed to enter NR specification.

Lentiviral mediated RPE65 gene transfer in healthy hiPSCs-derived retinal pigment epithelial cells markedly increased RPE65 mRNA, but modestly protein level.

The retinal pigment epithelium (RPE) is a monolayer of cobblestone-like epithelial cells that accomplishes critical functions for the retina. Several protocols have been published to differentiate pluripotent stem cells into RPE cells suitable for disease modelling and therapy development. In our study, the RPE identity of human induced pluripotent stem cell (hiPSC)-derived RPE (iRPE) was extensively characterized, and then used to test a lentiviral-mediated RPE65 gene augmentation therapy.

Investigating cone photoreceptor development using patient-derived NRL null retinal organoids.

Photoreceptor loss is a leading cause of blindness, but mechanisms underlying photoreceptor degeneration are not well understood. Treatment strategies would benefit from improved understanding of gene-expression patterns directing photoreceptor development, as many genes are implicated in both development and degeneration. Neural retina leucine zipper (NRL) is critical for rod photoreceptor genesis and degeneration, with NRL mutations known to cause enhanced S-cone syndrome and retinitis pigmentosa.

A combined RNA-seq and whole genome sequencing approach for identification of non-coding pathogenic variants in single families.

Inherited retinal degenerations (IRDs) are at the focus of current genetic therapeutic advancements. For a genetic treatment such as gene therapy to be successful, an accurate genetic diagnostic is required. Genetic diagnostics relies on the assessment of the probability that a given DNA variant is pathogenic.

The Role of FGF9 in the Production of Neural Retina and RPE in a Pluripotent Stem Cell Model of Early Human Retinal Development.

Purpose: To investigate the role of fibroblast growth factors (FGFs) in the production of neural retina (NR) and retinal pigmented epithelium (RPE) in a human pluripotent stem cell model of early retinal development.

Methods: Human induced pluripotent stem cell (hiPSC) lines from an individual with microphthalmia caused by a functional null mutation (R200Q) in visual system homeobox 2 (VSX2), a transcription factor involved in early NR progenitor cell (NRPC) production, and a normal sibling were differentiated along the retinal and forebrain lineages using an established protocol. Quantitative and global gene expression analyses (microarray and RNAseq) were used to investigate endogenous FGF expression profiles in these cultures over time.