FYVE2, a phosphatidylinositol 3-phosphate effector, interacts with the COPII machinery to control autophagosome formation in Arabidopsis.

Autophagy is an intracellular trafficking mechanism by which cytosolic macromolecules and organelles are sequestered into autophagosomes for degradation inside the vacuole. In various eukaryotes including yeast, metazoans, and plants, the precursor of the autophagosome, termed the phagophore, nucleates in the vicinity of the endoplasmic reticulum (ER) with the participation of phosphatidylinositol 3-phosphate (PI3P) and the coat protein complex II (COPII). Here we show that Arabidopsis thaliana FYVE2, a plant-specific PI3P-binding protein, provides a functional link between the COPII machinery and autophagy. FYVE2 interacts with the small GTPase Secretion-associated Ras-related GTPase 1 (SAR1), which is essential for the budding of COPII vesicles. FYVE2 also interacts with ATG18A, another PI3P effector on the phagophore membrane. Fluorescently tagged FYVE2 localized to autophagic membranes near the ER and was delivered to vacuoles. SAR1 fusion proteins were also targeted to the vacuole via FYVE2-dependent autophagy. Either mutations in FYVE2 or the expression of dominant-negative mutant SAR1B proteins resulted in reduced autophagic flux and the accumulation of autophagic organelles. We propose that FYVE2 regulates autophagosome biogenesis through its interaction with ATG18A and the COPII machinery, acting downstream of ATG2.

IL-10-induced modulation of macrophage polarization suppresses outer-blood-retinal barrier disruption in the streptozotocin-induced early diabetic retinopathy mouse model.

Diabetic retinopathy (DR) is associated with ocular inflammation leading to retinal barrier breakdown, vascular leakage, macular edema, and vision loss. DR is not only a microvascular disease but also involves retinal neurodegeneration, demonstrating that pathological changes associated with neuroinflammation precede microvascular injury in early DR. Macrophage activation plays a central role in neuroinflammation. During DR, the inflammatory response depends on the polarization of retinal macrophages, triggering pro-inflammatory (M1) or anti-inflammatory (M2) activity. This study aimed to determine the role of macrophages in vascular leakage through the tight junction complexes of retinal pigment epithelium, which is the outer blood-retinal barrier (BRB). Furthermore, we aimed to assess whether interleukin-10 (IL-10), a representative M2-inducer, can decrease inflammatory macrophages and alleviate outer-BRB disruption. We found that modulation of macrophage polarization affects the structural and functional integrity of ARPE-19 cells in a co-culture system under high-glucose conditions. Furthermore, we demonstrated that intravitreal IL-10 injection induces an increase in the ratio of anti-inflammatory macrophages and effectively suppresses outer-BRB disruption and vascular leakage in a mouse model of early-stage streptozotocin-induced diabetes. Our results suggest that modulation of macrophage polarization by IL-10 administration during early-stage DR has a promising protective effect against outer-BRB disruption and vascular leakage. This finding provides valuable insights for early intervention in DR.

Cone cell dysfunction attenuates retinal neovascularization in oxygen-induced retinopathy mouse model.

Aberrant neovascularization is the most common feature in retinopathy of prematurity (ROP), which leads to the retinal detachment and visual defects in neonates with a low gestational age eventually. Understanding the regulation of inappropriate angiogenic signaling benefits individuals at-risk. Recently, neural activity originating from the specific neural activity has been considered to contribute to retinal angiogenesis. Here, we explored the impact of cone cell dysfunction on oxygen-induced retinopathy (OIR), a mouse model commonly employed to understand retinal diseases associated with abnormal blood vessel growth, using the Gnat2cpfl3 (cone photoreceptor function loss-3) strain of mice (regardless of the sex), which is known for its inherent cone cell dysfunction. We found that the retinal avascular area, hypoxic area, and neovascular area were significantly attenuated in Gnat2cpfl3 OIR mice compared to those in C57BL/6 OIR mice. Moreover, the HIF-1α/VEGF axis was also reduced in Gnat2cpfl3 OIR mice. Collectively, our results indicated that cone cell dysfunction, as observed in Gnat2cpfl3 OIR mice, leads to attenuated retinal neovascularization. This finding suggests that retinal neural activity may precede and potentially influence the onset of pathological neovascularization.

A comprehensive genotype-phenotype study in 203 individuals with retinoblastoma.

Retinoblastoma is the most common intraocular tumor in children and is caused by biallelic inactivation of the RB1 gene. The identification of RB1 germline variants in patients with retinoblastoma and their families is critical for early diagnosis and prevention. In this study, genetic testing was conducted on the genomic DNA of 203 patients with retinoblastoma using a combined approach of direct sequencing and multiplex ligation-dependent probe amplification (MLPA) assays for genotype-phenotype correlation studies. Sixty-five germline variants were identified in 80 of the 203 patients, with 67 bilateral and 13 unilateral retinoblastoma cases. The variant detection rates in the bilateral and unilateral cases were 88% and 10%, respectively. Eighteen novel variants were identified. Variants were classified according to their presence, mutation pattern, location, molecular consequences, and pathogenicity. Subsequently, the genotypes and phenotypes of the 203 patients were evaluated. Variants were associated with age at diagnosis (p < 0.001), laterality (p < 0.001), and tumor size (p = 0.010). The molecular consequences of the variants were related to laterality (p < 0.001) and tumor size (p = 0.001). The pathogenicity of the variants was associated with age at diagnosis (p = 0.001), laterality (p = 0.0212), treatment response (p = 0.0470), and tumor size (p = 0.002). These results suggest that patient phenotypes are associated with the inherent characteristics of germline RB1 variants. These findings indicate the potential application of genetic testing results in clinical practice.

Ultrasonographic monitoring of fetal eye growth parameters throughout gestation in the common marmoset (Callithrix jacchus).

The common marmoset (Callithrix jacchus) is considered an ideal species for developing genetically modified nonhuman primates (NHP) models of human disease, particularly eye disease. They have been proposed as a suitable bridge between rodents and other NHP models due to their similar ophthalmological features to humans. Prenatal ultrasonography is an accurate and reliable diagnostic tool for monitoring fetal development and congenital malformation. We monitored fetal eye growth and development using noninvasive ultrasonography in 40 heads of clinically normal fetuses during pregnancy to establish the criteria for studying congenital eye anomalies in marmosets. The coronal, sagittal, and transverse planes were useful to identify the facial structures for any associated abnormalities. For orbital measurements, biorbital distance (BOD), ocular diameter (OD), interorbital distance (IOD), and total axial length (TAL) were measured in the transverse plane and carefully identified for intraorbital structures. As a result, high correlations were observed between delivery-based gestational age (GA) and biparietal diameter (BPD), BOD, OD, and TAL. The correlation assessments based on BOD provide more reliable results for monitoring eye growth and development in normal marmosets than any other parameters since BOD has the highest correlation coefficient according to both delivery-based GA and BPD among ocular measurements. In conclusion, orbital measurements by prenatal ultrasonography provide reliable indicators of marmoset eye growth, and it could offer early diagnostic criteria to facilitate the development of eye disease models and novel therapies such as genome editing technologies in marmosets.

Blockade of mTORC1-NOX signaling pathway inhibits TGF-ß1-mediated senescence-like structural alterations of the retinal pigment epithelium.

The retinal pigment epithelium (RPE) undergoes characteristic structural changes and epithelial-mesenchymal transition (EMT) during normal aging, which are exacerbated in age-related macular degeneration (AMD). Although the pathogenic mechanisms of aging and AMD remain unclear, transforming growth factor-ß1 (TGF-ß1) is known to induce oxidative stress, morphometric changes, and EMT as a senescence-promoting factor. In this study, we examined whether intravitreal injection of TGF-ß1 into the mouse eye elicits senescence-like morphological alterations in the RPE and if this can be prevented by suppressing mammalian target of rapamycin complex 1 (mTORC1) or NADPH oxidase (NOX) signaling. We verified that intravitreal TGF-ß1-induced stress fiber formation and EMT in RPE cells, along with age-associated morphometric changes, including increased variation in cell size and reduced cell density. In RPE cells, exogenous TGF-ß1 increased endogenous expression of TGF-ß1 and upregulated Smad3-ERK1/2-mTORC1 signaling, increasing reactive oxygen species (ROS) production and EMT. We demonstrated that inhibition of the mTORC1-NOX4 pathway by pretreatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-dependent protein kinase, or GKT137831, a NOX1/4 inhibitor, decreased ROS generation, prevented stress fiber formation, attenuated EMT, and improved the regularity of the RPE structure in vitro and in vivo. These results suggest that intravitreal TGF-ß1 injection could be used as a screening model to investigate the aging-related structural and functional changes to the RPE. Furthermore, the regulation of TGF-ß-mTORC1-NOX signaling could be a potential therapeutic target for reducing pathogenic alterations in aged RPE and AMD.

Autophagy inducers lead to transient accumulation of autophagosomes in Arabidopsis roots.

KEY MESSAGE: This study reveals that plant roots show a rapid termination of autophagy induction, offering a plant model for studying how excessive autophagy is deterred. In eukaryotes, autophagy is an intracellular mechanism that is important for recycling nutrients by degrading various macromolecules and organelles in vacuoles and lysosomes. Autophagy is induced when the nutrient supply to plant cells is limited. The protein kinase target of rapamycin (TOR) complex negatively regulates autophagy when nutrients are present in adequate amounts. The TOR inhibitor AZD8055 is an autophagy inducer that is useful for studying starvation-induced autophagy in plant cells. The mechanism by which AZD8055 increases the autophagic flux in plant cells has not been studied in detail. Here, we show that AZD8055-induced autophagy requires phosphatidylinositol 3-kinase activity and canonical AUTOPHAGY-RELATED (ATG) genes in Arabidopsis thaliana. Autophagic flux rapidly increased in seedlings treated with AZD8055. Unexpectedly, autophagy induction was transient in root cells and terminated earlier than in cotyledon cells. Transient induction is partly caused by a temporary effect of AZD8055 on phagophore initiation. These findings indicate a TOR-independent mechanism for terminating autophagy induction, thereby paving the way for elucidating how excess autophagy is prevented in plant roots.

Vapor-Mediated Infiltration of Nanocatalysts for Low-Temperature Solid Oxide Fuel Cells Using Electrosprayed Dendrites.

Electrode architecturing for fast electrochemical reaction is essential for achieving high-performance of low-temperature solid oxide fuel cells (LT-SOFCs). However, the conventional droplet infiltration technique still has limitations in terms of the applicability and scalability of nanocatalyst implementation. Here, we develop a novel two-step precursor infiltration process and fabricate high-performance LT-SOFCs with homogeneous and robust nanocatalysts. This novel infiltration process is designed based on the principle of a reversible sol-gel transition where the gelated precursor dendrites are uniformly deposited onto the electrode via controlled nanoscale electrospraying process then resolubilized and infiltrated into the porous electrode structure through subsequent humidity control. Our infiltration technique reduces the cathodic polarization resistance by 18% compared to conventional processes, thereby achieving an enhanced peak power density of 0.976 W cm-2 at 650 °C. These results, which provide various degrees of freedom for forming nanocatalysts, exhibit an advancement in LT-SOFC technology.

Antitumor Activity of Novel Signal Transducer and Activator of Transcription 3 Inhibitors on Retinoblastoma.

Signal transducer and activator of transcription 3 (STAT3) is a plausible therapeutic target in the treatment of retinoblastoma, the most common intraocular malignant tumor in children. STAT3, a transcription factor of several genes related to tumorigenesis, is activated in retinoblastoma tumors as well as other cancers. In this study, we investigated the structure-activity relationship of a library of STAT3 inhibitors, including a novel series of derivatives of the previously reported compound with a Michael acceptor (compound 1). We chose two novel STAT3 inhibitors, compounds 11 and 15, from the library based on their inhibitory effects on the phosphorylation and transcription activity of STAT3. These STAT3 inhibitors effectively suppressed the phosphorylation of STAT3 and inhibited the expression of STAT3-related genes CCND1, CDKN1A, BCL2, BCL2L1, BIRC5, MYC, MMP1, MMP9, and VEGFA Intraocularly administered STAT3 inhibitors decreased the degree of tumor formation in the vitreous cavity of BALB/c nude mice of an orthotopic transplantation model. It is noteworthy that compounds 11 and 15 did not induce in vitro and in vivo toxicity on retinal constituent cells and retinal tissues, respectively, despite their potent antitumor effects. We suggest that these novel STAT3 inhibitors be used in the treatment of retinoblastoma. SIGNIFICANCE STATEMENT: The current study suggests the novel STAT3 inhibitors with Michael acceptors possess antitumor activity on retinoblastoma, the most common intraocular cancer in children. Based on detailed structure-activity relationship studies, we found a 4-fluoro and 3-trifluoro analog (compound 11) and a monochloro analog (compound 15) of the parental compound (compound 1) inhibited STAT3 phosphorylation, leading to suppressed retinoblastoma in vitro and in vivo.

Giant Y79 retinoblastoma cells contain functionally active T-type calcium channels.

Retinoblastoma is the most common malignant intraocular tumor in children. Y79 human retinoblastoma cells are in vitro models of retinal tumors used for drug screening. Undifferentiated Y79 cells originate from a primitive multi-potential neuroectodermal cell and express neuronal and glial properties. However, the nature of cellular heterogeneity in Y79 cells is unclear because functional methods to characterize neurons or glial cells have not been employed to Y79 cells. Here, we perform patch-clamp recordings to characterize electrophysiological properties in retinoblastoma cells. We identified a population of large-sized Y79 cells (i.e., giant cells, ~ 40-µm diameter), hyperpolarized resting membrane potential (-54 mV), and low input resistance (~ 600 MΩ), indicating electrically mature cells. We also found that giant Y79 cells contain increased density of T-type calcium channels. Finally, we found that T-type calcium channels are active only in giant cells suggesting that cancer treatments aimed to prevent calcium influx in retinoblastomas should be tested in giant cells.

ZnFe2 O4 Dendrite/SnO2 Helix 3D Hetero-Structure Photoanodes for Enhanced Photoelectrochemical Water Splitting: Triple Functions of SnO2 Nanohelix.

An array of SnO2 nanohelix structures is employed to fabricate a SnO2 helix@ZnFe2 O4 dendrite core-shell 3D heterostructure photoanode for photoelectrochemical (PEC) water splitting. The SnO2 helix provides triple critical functions to enhance the PEC performance of the photoanode. First, it scatters the incident light to achieve a higher light harvesting efficiency. Second, it provides a facile electron pathway as an electron transfer layer (ETL) while blocking hole transport to mitigate charge recombination in the bulk of ZnFe2 O4 . Finally, it becomes a template for the formation of ZnFe2 O4 dendrite nanostructure shell. The ZnFe2 O4 dendrite/SnO2 helix photoanode exhibits a remarkable increase in incident photon-to-electron conversion efficiency compared to unmodified ZnFe2 O4 with no ETL and modified one with "flat" SnO2 ETL. The surface of the ZnFe2 O4 /SnO2 helix photoanode is further modified with TiO2 passivation layer and NiFeOx oxygen evolution co-catalyst to achieve one of the best PEC performances among reported ZnFe2 O4 -based photoanodes.

Importance of extracutaneous organ involvement in determining the clinical severity and prognosis of incontinentia pigmenti caused by mutations in the IKBKG gene.

Incontinentia pigmenti (IP) is a rare X-linked skin disease caused by mutations in the IKBKG gene, which is required for activation of the nuclear factor-kappa B signalling pathway. Multiple systems can be affected with highly variable phenotypic expressivity. We aimed to clarify the clinical characteristics observed in molecularly confirmed Korean IP patients. The medical records of 25 females confirmed as IP by molecular genetic analysis were retrospectively reviewed. The phenotypic score of extracutaneous manifestations was calculated to assess the disease severity. The IKBKG gene partial deletion or intragenic mutations were investigated using long-range PCR, multiplex ligation-dependent probe amplification and direct sequencing methods. Among the 25 individuals, 18 (72%) were sporadic cases. All patients showed typical skin manifestations at birth or during the neonatal period. Extracutaneous findings were noted in 17 (68%) patients; ocular manifestations (28%), neurological abnormalities (28%), hair abnormalities (20%), dental anomalies (12%), nail dystrophy (8%). The common exon 4-10 IKBKG deletion was observed in 20 (80%) patients. In addition, five intragenic sequence variants were identified, including three novel variants. The phenotype scores were highly variable, ranging from abnormal skin pigmentation only to one or more extracutaneous features, although no significant difference was observed for each clinical characteristic between the group with sequence variants and that with common large deletion. Our cohort with IP showed heterogeneity of extracutaneous manifestations and high incidence of sporadic cases. Long-term monitoring with multidisciplinary management is essential for evaluating the clinical status, providing adequate genetic counselling and understanding the genotype-phenotype correlation in IP.

Effect of Viscosity on the Formation of Porous Polydimethylsiloxane for Wearable Device Applications.

Medical devices, which enhance the quality of life, have experienced a gradual increase in demand. Various research groups have attempted to incorporate soft materials such as skin into wearable devices. We developed a stretchable substrate with high elasticity by forming a porous structure on polydimethylsiloxane (PDMS). To optimize the porous structure, we propose a manufacturing process that utilizes a high-pressure steam with different viscosities (400, 800, 2100, and 3000 cP) of an uncured PDMS solution. The proposed method simplifies the manufacturing of porous structures and is cost-effective compared to other technologies. Porous structures of various viscosities were formed, and their electrical and mechanical properties evaluated. Porous PDMS (3000 cP) was formed in a sponge-like three-dimensional porous structure, compared to PDMS formed by other viscosities. The elongation of porous PDMS (3000 cP) was increased by up to 30%, and the relative resistance changed to less than 1000 times with the maximum strain test. The relative resistance increased the initial resistance (R0) by approximately 10 times during the 1500-times repeated cycling tests with 30% strain. As a result, patch-type wearable devices based on soft materials can provide an innovative platform that can connect with the human skin for robotics applications and for continuous health monitoring.

Activation of ERK1/2-mTORC1-NOX4 mediates TGF-ß1-induced epithelial-mesenchymal transition and fibrosis in retinal pigment epithelial cells.

Transforming growth factor-ß (TGF-ß) plays a crucial role in the development of epithelial to mesenchymal transition (EMT) and fibrosis, particularly in an ocular disorder such as proliferative vitreoretinopathy (PVR). However, the key molecular mechanism underlying its pathogenesis remains unknown. In the present study, using cultured ARPE-19 cells, we determined that TGF-ß initiates a signaling pathway through extracellular signal-regulated kinase (ERK)-mammalian target of rapamycin complex 1 (mTORC1) that stimulates trans-differentiation and fibrosis of retinal pigment epithelium. Blocking this pathway by a TGF-ßRI, ERK or mTORC1 inhibitor protected cells from EMT and fibrotic protein expression. TGF-ß1 treatment increased reactive oxygen species (ROS) via NOX4 upregulation, which acts downstream of ERK and mTORC1, as the ROS scavenger N-acetylcysteine and a pan-NADPH oxidase (NOX) inhibitor DPI dissipated excess ROS generation. TGF-ß1-induced oxidative stress resulted in EMT and fibrotic changes, as NAC and DPI prevented α-SMA, Col4α3 expression and cell migration. All these inhibitors blocked the downstream pathway activation in addition to clearly preventing the activation of its upstream molecules, indicating the presence of a feedback loop system that may boost the upstream events. Furthermore, the FDA-approved drug trametinib (10 nM) blunted TGF-ß1-induced mTORC1 activation and downstream pathogenic alterations through ERK1/2 inhibition, which opens a therapeutic avenue for the treatment of PVR in the future.

Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration.

RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene-specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody- and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension.

Arabidopsis cargo receptor NBR1 mediates selective autophagy of defective proteins.

Aggrephagy, a type of selective autophagy that sequesters protein aggregates for degradation in the vacuole, is an important protein quality control mechanism, particularly during cell stress. In mammalian cells, aggrephagy and several other forms of selective autophagy are mediated by dedicated cargo receptors such as NEIGHBOR OF BRCA1 (NBR1). Although plant NBR1 homologs have been linked to selective autophagy during biotic stress, it remains unclear how they impact selective autophagy under non-stressed and abiotic stress conditions. Through microscopic and biochemical analysis of nbr1 mutants expressing autophagy markers and an aggregation-prone reporter, we tested the connection between NBR1 and aggrephagy in Arabidopsis. Although NBR1 is not essential for general autophagy, or for the selective clearance of peroxisomes, mitochondria, or the ER, we found that NBR1 is required for the heat-induced formation of autophagic vesicles. Moreover, cytoplasmic puncta containing aggregation-prone proteins, which were rarely observed in wild-type plants, were found to accumulate in nbr1 mutants under both control and heat stress conditions. Given that NBR1 co-localizes with these cytoplasmic puncta, we propose that Arabidopsis NBR1 is a plant aggrephagy receptor essential for maintaining proteostasis under both heat stress and non-stress conditions.

Astrocytic cystine/glutamate antiporter is a key regulator of erythropoietin expression in the ischemic retina.

Ischemic retinopathies and optic neuropathies are important causes of vision loss. The neuroprotective effect of erythropoietin (EPO) in ischemic neuronal injury and the expression of EPO and its receptor in retinal tissue have been well documented. However, the exact regulatory mechanism of EPO expression in retinal ischemia still remains to be elucidated. In this study, we investigated the role of cystine/glutamate antiporter (system xc-) in the regulation of astrocytic EPO expression by using both in vitro and in vivo models. Under hypoxia, the expression of astrocytic system xc- is up-regulated both in vitro and in vivo. Inhibition of system xc- resulted in depletion of intracellular glutathione (GSH) and decrement of GSH disulfide ratios in human brain astrocytes (HBAs). In HBAs, hypoxia-induced stabilization of hypoxia-inducible factor (Hif)-2α is nearly completely abolished by inhibition of system xc-. Hypoxia-induced up-regulation of astrocytic EPO expression is suppressed by both pharmacological inhibition and siRNA-mediated knockdown of system xc-. In contrast, basal EPO expression under normoxia is not affected by system xc- modulation. In summary, under hypoxia, increased system xc- acts as the major source of intracellular GSH, which helps in stabilizing Hif-2α and subsequent up-regulation of EPO in astrocytes.-Lee, B. J., Jun, H. O., Kim, J. H., Kim, J. H. Astrocytic cystine/glutamate antiporter is a key regulator of erythropoietin expression in the ischemic retina.

Bispecific anti-mPDGFRß x cotinine scFv-Cκ-scFv fusion protein and cotinine-duocarmycin can form antibody-drug conjugate-like complexes that exert cytotoxicity against mPDGFRß expressing cells.

Antibody selection for antibody-drug conjugates (ADCs) has traditionally depended on its internalization into the target cell, although ADC efficacy also relies on recycling of the receptor-ADC complex, endo-lysosomal trafficking, and subsequent linker/antibody proteolysis. In this study, we observed that a bispecific anti-murine platelet-derived growth factor receptor beta (mPDGFRß) x cotinine single-chain variable fragment (scFv)-kappa constant region (Cκ)-scFv fusion protein and cotinine-duocarmycin can form an ADC-like complex to induce cytotoxicity against mPDGFRß expressing cells. Multiple anti-mPDGFRß antibody candidates can be produced in this bispecific scFv-Cκ-scFv fusion protein format and tested for their ability to deliver cotinine-conjugated cytotoxic drugs, thus providing an improved approach for antibody selection in ADC development.

Long-Term Effects of In Vivo Genome Editing in the Mouse Retina Using Campylobacter jejuni Cas9 Expressed via Adeno-Associated Virus.

Genome editing with CRISPR systems provides an unprecedented opportunity to modulate cellular responses in pathological conditions by inactivating undruggable targets, such as transcription factors. Previously, we demonstrated that the smallest Cas9 ortholog characterized to date, from Campylobacter jejuni (CjCas9) targeted to Hif1a and delivered in an adeno-associated virus (AAV) vector, effectively suppressed pathological choroidal neovascularization in the mouse retina. Before implementation of CjCas9 as an in vivo therapeutic modality, it is essential to investigate the long-term effects of target gene disruption via AAV-mediated delivery of CjCas9 in vivo. In this study, histologic and electroretinographic analyses demonstrated that CjCas9 targeted to Hif1a did not induce any definite toxicity in the retina, although the target gene was mutated with a frequency ranging from 45% to 79% in retinal or retinal pigment epithelial cells. Importantly, at 14 months after injection, no indels were detected at potential off-target sites identified using Digenome-seq and Cas-OFFinder, suggesting that long-term expression of CjCas9 does not aggravate off-target effects. Taken together, our results show that intravitreal injection of AAV encoding CjCas9 targeted to Hif1a effectively induced and maintained mutations in retinal tissues for more than 1 year and did not affect retinal histologic integrity or functions.

CRISPR-Pass: Gene Rescue of Nonsense Mutations Using Adenine Base Editors.

A nonsense mutation is a substitutive mutation in a DNA sequence that causes a premature termination during translation and produces stalled proteins, resulting in dysfunction of a gene. Although it usually induces severe genetic disorders, there are no definite methods for inducing read through of premature termination codons (PTCs). Here, we present a targeted tool for bypassing PTCs, named CRISPR-pass, that uses CRISPR-mediated adenine base editors. CRISPR-pass, which should be applicable to 95.5% of clinically significant nonsense mutations in the ClinVar database, rescues protein synthesis in patient-derived fibroblasts, suggesting potential clinical utility.