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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 (Kir7.1) in the RPE. SNCs carrying adenine base editor 8e (ABE8e) mRNA and sgRNA precisely and efficiently corrected the [KCNJ13.sup.W53X/W53X] mutation. Editing in both patient fibroblasts (47%) and human induced pluripotent stem cell-derived RPE (LCA16-iPSC-RPE) (17%) showed minimal off-target editing. We detected functional Kir7.1 channels in the edited LCA16-iPSC-RPE. In the LCA16 mouse model ([Kcnj13.sup.W53X/+[DELTA]R]), RPE cells targeted SNC delivery of ABE8e mRNA preserved normal vision, measured by full-field electroretinogram (ERG). Moreover, multifocal ERG confirmed the topographic measure of electrical activity primarily originating from the edited retinal area at the injection site. Preserved retina structure after treatment was established by optical coherence tomography (OCT). This preclinical validation of targeted ion channel functional rescue, a challenge for pharmacological and genomic interventions, reinforced the effectiveness of nonviral genome-editing therapy for rare inherited disorders., Introduction Leber congenital amaurosis type 16 (LCA16; OMIM #614186) is a severe autosomal recessive inherited retinal dystrophy (IRD) leading to blindness in early life. LCA16 is caused by the loss [...]

The development of fetal organs can be impacted by systemic changes in maternal circulation, with the placenta playing a pivotal role in maintaining pregnancy homeostasis and nutrient exchange. In clinical obstetrics, oxytocin (OXT) is commonly used to induce labor. To explore the potential role of OXT in the placental homeostasis of OXT, we compared OXT levels in neonatal cord blood among neonates (23–42 weeks gestation) whose mothers either received prenatal OXT or experienced spontaneous labor. Our previous research revealed that the oxytocin receptor (OXTR), essential in forming the blood–retina barrier, is expressed in the retinal pigment epithelium (RPE). We hypothesized that perinatal OXT administration might influence the development of the neural retina and its vasculature, offering therapeutic potential for retinal diseases such as retinopathy of prematurity (ROP). Plasma OXT levels were measured using a commercial OXT ELISA kit. Human fetal RPE (hfRPE) cells treated with OXT (10 µM) were assessed for gene expression via RNA sequencing, revealing 14 downregulated and 32 upregulated genes. To validate these differentially expressed genes (DEGs), hfRPE cells were exposed to OXT (0.01, 0.1, 1, or 10 µM) for 12 h, followed by RNA analysis via real-time PCR. Functional, enrichment, and network analyses (Gene Ontology term, FunRich, Cytoscape) were performed to predict the affected pathways. This translational study suggests that OXT likely crosses the placenta, altering fetal OXT concentrations. RNA sequencing identified 46 DEGs involved in vital metabolic and signaling pathways and critical cellular components. Our results indicate that the perinatal administration of OXT may affect neural retina and retinal vessel development, making OXT a potential therapeutic option for developmental eye diseases, including ROP. [ABSTRACT FROM AUTHOR]

PACS1 syndrome, also referred to as Schuurs-Hoeijmakers syndrome, is a multisystemic developmental disorder caused by a specific pathogenic variant in the PACS1 (phosphofurin acidic cluster sorting protein 1) gene. Ocular findings in PACS1 syndrome are known to include iris, retina, optic nerve coloboma, myopia, nystagmus, and strabismus. Here, we present the cases of two patients referred to the University of Wisconsin-Madison Department of Ophthalmology and Visual Sciences for ocular evaluation. The first patient is a 14-month-old female who, at 3 months of age, was found to have a depressed rod and cone response on electroretinogram (ERG), consistent with possible retinal dystrophy (RD). This feature has not been previously described in PACS1 syndrome and joins a growing list of calls for expanding the PACS1 phenotype. The second case illustrates a 5-year-old male referred for ocular screening after diagnosing PACS1 syndrome and underwent ERG without abnormal findings. These cases demonstrate the significant variability in the ophthalmic presentation of PACS1 syndrome and the need for early screening. These novel findings may have implications in understanding the mechanism of the PACS1 protein and its role in retinal ciliary phototransduction in photoreceptors. [ABSTRACT FROM AUTHOR]

The inwardly rectifying [K.sup.+] (Kir) current in mammalian retinal pigment epithelial (RPE) cells, which is largely mediated by Kir7.1 channels, is stable in cells dialyzed with MgATP but runs down when intracellular ATP is depleted. A potential mechanism for this rundown is a decrease in phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]) regeneration by ATP-dependent lipid kinases. Here, we used the whole cell voltage-clamp technique to investigate the membrane PIP2 dependence of Kir channels in isolated bovine RPE cells. When RPE cells were dialyzed with ATP-ffee solution containing [PIP.sub.2] (25-50 [micro]M), rundown persisted hut was markedly reduced. Removal of [Mg.sup.2+] from the pipette solution also slowed rundown, indicating that elevated intracelhilar [Mg.sup.2+] concentration contributes to rundown. Cell dialysis with the [PIP.sub.2] scavenger neomycin in MgATP solution diminished Kir current in a voltage-dependent manner, suggesting that it acted at least in part by blocking the Kir channel. Kir current in MgATP-loaded cells was partially inhibited by bath application of quercetin (100 [micro]M), phenylarsine oxide (100 [micro]M), or wortmannin (50 [micro]M), inhibitors of phosphatidylinositol (PI) kinases, and was completely inhibited by cell dialysis with 2 mM adenosine, a PI4 kinase inhibitor. Both LY-294002 (100 [micro]M), an inhibitor of PI3 kinases, and its inactive analog LY-303511 (100 [micro]M) rapidly and reversibly inhibited Kir current, suggesting that these compounds act as direct channel blockers. We conclude that the activity of Kir channels in the RPE is critically dependent on the regeneration of membrane [PIP.sub.2] by PI4 kinases and that this may explain the dependence of these channels on hydrolyzable ATP. retinal pigment epithelium; Kir7.1; phosphatidylinositol 4-kinases; phosphatidylinositol 4,5-bisphosphate doi: 10.1152/ajpcell.00250.2009

Inward-rectifier potassium channel 7.1 (Kir7.1) is present in the polarized epithelium, including the retinal pigmented epithelium. A single amino acid change at position 153 in the KCNJ13 gene, a substitution of threonine to isoleucine in the Kir7.1 protein, causes blindness. We hypothesized that the disease caused by this single amino acid substitution within the transmembrane protein domain could alter the translation, localization, or ion transport properties. We assessed the effects of amino acid sidechain length, arrangement, and polarity on channel structure and function. We showed that the T153I mutation yielded a full length protein localized to the cell membrane. Whole cell patch-clamp recordings and chord conductance analyses revealed that the T153I mutant channel had negligible K+ conductance and failed to hyperpolarize the membrane potential. However, the mutant channel exhibited enhanced inward current when rubidium was used as a charge carrier, suggesting that an inner pore had formed and the channel was dysfunctional. Substituting with a polar, nonpolar, or short side-chain amino acid did not affect the localization of the protein. Still, it had an altered channel function due to differences in pore radius. Polar side chains (cysteine and serine) with inner pore radii comparable to wildtype exhibited normal inward K+ conductance. Short side chains (glycine and alanine) produced a channel with wider than expected inner pore size and lacked the biophysical characteristics of the wildtype channel. Leucine substitution produced results similar to the T153I mutant channel. This study provides direct electrophysiological evidence for the structure and function of the Kir7.1 channel’s narrow inner pore in regulating conductance. [ABSTRACT FROM AUTHOR]