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Patients with Zellweger spectrum disorder (ZSD) commonly present with vision loss due to mutations in PEX genes required for peroxisome assembly and function. Here, we evaluate PEX1 retinal gene augmentation therapy in a mouse model of mild ZSD bearing the murine equivalent (PEX1-p[Gly844Asp]) of the most common human mutation. Experimental adeno-associated virus 8.cytomegalovirus.human PEX1.hemagglutinin (AAV8.CMV.HsPEX1.HA) and control AAV8.CMV.EGFP vectors were administered by subretinal injection in contralateral eyes of early (5-week-old)- or later (9-week-old)-stage retinopathy cohorts. HsPEX1.HA protein was expressed in the retina with no gross histologic side effects. Peroxisomal metabolic functions, assessed by retinal C26:0 lysophosphatidylcholine (lyso-PC) levels, were partially normalized after therapeutic vector treatment. Full-field flash electroretinogram (ffERG) analyses at 8 weeks post-injection showed a 2-fold improved retinal response in the therapeutic relative to control vector-injected eyes. ffERG improved by 1.6- to 2.5-fold in the therapeutic vector-injected eyes when each cohort reached 25 weeks of age. At 32 weeks of age, the average ffERG response was double in the therapeutic relative to control vector-injected eyes in both cohorts. Optomotor reflex analyses trended toward improvement. These proof-of-concept studies represent the first application of gene augmentation therapy to treat peroxisome biogenesis disorders and support the potential for retinal gene delivery to improve vision in these patients.

Background Conventional diagnosis of fragile X syndrome (FXS) is based on a combination of fragment analysis (FA) and Southern blotting (SB); however, this diagnostic approach is time- and labor-intensive and has pitfalls such as the possibility of missing large number alleles. Triplet repeat primed PCR (TP-PCR) is a current alternative used to overcome these limitations. We evaluated the diagnostic usefulness of TP-PCR compared with the conventional diagnostic protocol consisting of FA and/or SB in terms of allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers. Methods From November 2013 to March 2018, 458 patients (326 males, 132 females) were simultaneously examined using FA and/or SB and TP-PCR by detecting CGG repeat numbers in FMR1 gene and diagnosed as per American College of Medical Genetics guidelines. Results The TP-PCR results showed high concordance with the FA and/or SB results for all three aspects (allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers). TP-PCR detected CGG expansions ≥200 in all full mutation (FM) allele cases in male patients, as well as both the normal allele (NL) and FM allele in female carriers. In premutation (PM) allele carriers, the TP-PCR results were consistent with the FA and/or SB results. In terms of zygosity concordance in female genetic carriers, 12 NL cases detected by TP-PCR showed a merged peak consisting of two close heterozygous peaks; however, this issue was resolved using a 10-fold dilution. Conclusions TP-PCR may serve as a reliable alternative method for FXS diagnosis.

Despite the wide application of next-generation sequencing, Sanger sequencing still plays a necessary role in clinical laboratories. However, recent developments in the field of bioinformatics have focused mostly on next-generation sequencing, while tools for Sanger sequencing have shown little progress. In this study, SnackVar (https://github.com/Young-gonKim/SnackVar, last accessed June 22, 2020), a novel graphical user interface-based software for Sanger sequencing, was developed. All types of variants, including heterozygous insertion/deletion variants, can be identified by SnackVar with minimal user effort. The featured reference sequences of all of the genes are prestored in SnackVar, allowing for detected variants to be precisely described based on coding DNA references according to the nomenclature of the Human Genome Variation Society. Among 88 previously reported variants from four insertion/deletion-rich genes (BRCA1, APC, CALR, and CEBPA), the result of SnackVar agreed with reported results in 87 variants [98.9% (93.0%; 99.9%)]. The cause of one incorrect variant calling was proven to be erroneous base callings from poor-quality trace files. Compared with commercial software, SnackVar required less than one-half of the time taken for the analysis of a selected set of test cases. We expect SnackVar to be a cost-effective option for clinical laboratories performing Sanger sequencing.

Patients with Zellweger spectrum disorder (ZSD) commonly present with vision loss due to mutations in PEX genes required for peroxisome assembly and function. Here, we evaluate PEX1 retinal gene augmentation therapy in a mouse model of mild ZSD bearing the murine equivalent (PEX1-p[Gly844Asp]) of the most common human mutation. Experimental adeno-associated virus 8.cytomegalovirus.human PEX1.hemagglutinin (AAV8.CMV.HsPEX1.HA) and control AAV8.CMV.EGFP vectors were administered by subretinal injection in contralateral eyes of early (5-week-old)- or later (9-week-old)-stage retinopathy cohorts. HsPEX1.HA protein was expressed in the retina with no gross histologic side effects. Peroxisomal metabolic functions, assessed by retinal C26:0 lysophosphatidylcholine (lyso-PC) levels, were partially normalized after therapeutic vector treatment. Full-field flash electroretinogram (ffERG) analyses at 8 weeks post-injection showed a 2-fold improved retinal response in the therapeutic relative to control vector-injected eyes. ffERG improved by 1.6- to 2.5-fold in the therapeutic vector-injected eyes when each cohort reached 25 weeks of age. At 32 weeks of age, the average ffERG response was double in the therapeutic relative to control vector-injected eyes in both cohorts. Optomotor reflex analyses trended toward improvement. These proof-of-concept studies represent the first application of gene augmentation therapy to treat peroxisome biogenesis disorders and support the potential for retinal gene delivery to improve vision in these patients., Graphical abstract, Using our PEX1-G844D mouse model for Zellweger spectrum disorder, we evaluated AAV8.CMV.HsPEX1 retinal gene therapy, which improved peroxisomal functions and retinal response after subretinal administration. These proof-of-concept studies represent the first application of gene therapy to treat peroxisome biogenesis disorders and support the potential to improve vision in these patients.

Despite the wide application of next-generation sequencing, Sanger sequencing still plays a necessary role in clinical laboratories. However, recent developments in the field of bioinformatics have focused mostly on next-generation sequencing, while tools for Sanger sequencing have shown little progress. In this study, SnackVar (https://github.com/Young-gonKim/SnackVar, last accessed June 22, 2020), a novel graphical user interface-based software for Sanger sequencing, was developed. All types of variants, including heterozygous insertion/deletion variants, can be identified by SnackVar with minimal user effort. The featured reference sequences of all of the genes are prestored in SnackVar, allowing for detected variants to be precisely described based on coding DNA references according to the nomenclature of the Human Genome Variation Society. Among 88 previously reported variants from four insertion/deletion-rich genes (BRCA1, APC, CALR, and CEBPA), the result of SnackVar agreed with reported results in 87 variants [98.9% (93.0%; 99.9%)]. The cause of one incorrect variant calling was proven to be erroneous base callings from poor-quality trace files. Compared with commercial software, SnackVar required less than one-half of the time taken for the analysis of a selected set of test cases. We expect SnackVar to be a cost-effective option for clinical laboratories performing Sanger sequencing.

Item does not contain fulltext Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an approximately 20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.

Crumbs polarity complex proteins are essential for cellular and tissue polarity, and for adhesion of epithelial cells. In epithelial tissues deletion of any of three core proteins disrupts localization of the other proteins, indicating structural and functional interdependence among core components. Despite previous studies of function and co-localization that illustrated the properties that these proteins share, it is not known whether an individual component of the complex plays a distinct role in a unique cellular and developmental context. In order to investigate this question, we primarily used confocal imaging to determine the expression and subcellular localization of the core Crumbs polarity complex proteins during ocular development. Here we show that in developing ocular tissues core Crumbs polarity complex proteins, Crb, Pals1 and Patj, generally appear in an overlapping pattern with some exceptions. All three core complex proteins localize to the apical junction of the retinal and lens epithelia. Pals1 is also localized in the Golgi of the retinal cells and Patj localizes to the nuclei of the apically located subset of progenitor cells. These findings suggest that core Crumbs polarity complex proteins exert common and independent functions depending on cellular context.

Within the next decade, we will see many gene therapy clinical trials for eye diseases, which may lead to treatments for thousands of visually impaired people around the world. To target retinal diseases that affect specific cell types, several recombinant adeno-associated virus (AAV) serotypes have been generated and used successfully in preclinical mouse studies. Because there are numerous anatomic and physiologic differences between the eyes of mice and "men" and because surgical delivery approaches and immunologic responses also differ between these species, this study evaluated the transduction characteristics of two promising new serotypes, AAV7m8 and AAV8BP2, in the retinas of animals that are most similar to those of humans: non-human primates (NHPs). We report that while AAV7m8 efficiently targets a variety of cell types by subretinal injection in NHPs, transduction after intravitreal delivery was mostly restricted to the inner retina at lower doses that did not induce an immune response. AAV8BP2 targets the cone photoreceptors efficiently but bipolar cells inefficiently by subretinal injection. Additionally, transduction by both serotypes in the anterior chamber of the eye and the optic pathway of the brain was observed post-intravitreal delivery. Finally, we assessed immunogenicity, keeping in mind that these AAV capsids may be used in future clinical trials. We found that AAV8BP2 had a better safety profile compared with AAV7m8, even at the highest doses administered. These studies underscore the differences in AAV transduction between mice and primates, highlighting the importance of careful evaluation of therapeutic vectors in NHPs prior to moving to clinical trials.

Hippo-Yap signaling has been implicated in organ size determination via its regulation of cell proliferation, growth and apoptosis (Pan, 2007). The vertebrate lens comprises only two major cell types, lens progenitors and differentiated fiber cells, thereby providing a relatively simple system for studying size-controlling mechanisms. In order to investigate the role of Hippo-Yap signaling in lens size regulation, we conditionally ablated Yap in the developing mouse lens. Lens progenitor-specific deletion of Yap led to near obliteration of the lens primarily due to hypocellularity in the lens epithelium (LE) and accompanying lens fiber (LF) defects. A significantly reduced LE progenitor pool resulted mainly from failed self-renewal and increased apoptosis. Additionally, Yap-deficient lens progenitor cells precociously exited the cell cycle and expressed the LF marker, β-Crystallin. The mutant progenitor cells also exhibited multiple cellular and subcellular alterations including cell and nuclear shape change, organellar polarity disruption, and disorganized apical polarity complex and junction proteins such as Crumbs, Pals1, Par3 and ZO-1. Yap-deficient LF cells failed to anchor to the overlying LE layer, impairing their normal elongation and packaging. Furthermore, our localization study results suggest that, in the developing LE, Yap participates in the cell context-dependent transition from the proliferative to differentiation-competent state by integrating cell density information. Taken together, our results shed new light on Yap's indispensable and novel organizing role in mammalian organ size control by coordinating multiple events including cell proliferation, differentiation, and polarity.

Oxidative stress contributes to the loss of neurons in many disease conditions as well as during normal aging; however, small-molecule agents that reduce oxidation have not been successful in preventing neurodegeneration. Moreover, even if an efficacious systemic reduction of reactive oxygen and/or nitrogen species (ROS/NOS) could be achieved, detrimental side effects are likely, as these molecules regulate normal physiological processes. A more effective and targeted approach might be to augment the endogenous antioxidant defense mechanism only in the cells that suffer from oxidation. Here, we created several adeno-associated virus (AAV) vectors to deliver genes that combat oxidation. These vectors encode the transcription factors NRF2 and/or PGC1a, which regulate hundreds of genes that combat oxidation and other forms of stress, or enzymes such as superoxide dismutase 2 (SOD2) and catalase, which directly detoxify ROS. We tested the effectiveness of this approach in 3 models of photoreceptor degeneration and in a nerve crush model. AAV-mediated delivery of NRF2 was more effective than SOD2 and catalase, while expression of PGC1a accelerated photoreceptor death. Since the NRF2-mediated neuroprotective effects extended to photoreceptors and retinal ganglion cells, which are 2 very different types of neurons, these results suggest that this targeted approach may be broadly applicable to many diseases in which cells suffer from oxidative damage.

Purpose Familial exudative vitreoretinopathy (FEVR) is a rare, hereditary visual disorder. The gene TSPAN12 is associated with autosomal dominant inheritance of FEVR. The prevalence and impact of large deletions/duplications of TSPAN12 on FEVR patients is unknown. To glean better insight of TSPAN12 on FEVR pathology, herein, we describe three FEVR patients with TSPAN12 deletions. Methods Thirty-three Korean FEVR patients, who previously screened negative for TSPAN12 mutations, mutations in other FEVR-associated genes such as NDP, FZD4, LRP5, and large deletions and duplications of NDP, FZD4, and LRP5, were selected for TSPAN12 large deletion and duplication analyses. Semiquantitative multiplex PCR for TSPAN12 gene dosage analyses were performed, followed by droplet digital PCR (ddPCR) for validation. Results Among the 33 patients, three patients were confirmed to carry large TSPAN12 deletions. Two of them had whole-gene deletions of TSPAN12, and the other patient possessed a deletion of TSPAN12 in exon 4. FEVR severity detected in these patients was not more severe than in a patient with TSPAN12 point mutation. Conclusions Regarding previously reported proportions of FEVR-associated genes contributing to the disorder's autosomal dominant inheritance pattern in Korea, we determined that patients with TSPAN12 large deletions were more common than patients with single nucleotide variants in TSPAN12. Evaluating TSPAN12 large deletions and duplications should be considered in FEVR screening and diagnosis as well as in routine genetic workups for FEVR patients.

Background Mutations of Crb1 gene cause irreversible and incurable visual impairment in humans. This study aims to use an LCA8-like mouse model to identify host-mediated responses that might interfere with survival, retinal integration and differentiation of grafted cells during neonatal cell therapy. Methods Mixed retinal donor cells (1 ~ 2 × 104) isolated from neural retinas of neonatal eGFP transgenic mice were injected into the subretinal space of LCA8-like model neonatal mice. Markers of specific cell types were used to analyze microglial attraction, CSPG induction and retinal cell differentiation. The positions of host retinal cells were traced according to their laminar location during disease progression to look for host cell rearrangements that might inhibit retinal integration of the transplanted cells. Results Transplanted retinal cells showed poor survival and attracted microglial cells, but CSPG was not greatly induced. Retinas of the LCA8 model hosts underwent significant cellular rearrangement, including rosette formation and apical displacement of inner retinal cells. Conclusions Local disease environment, particularly host immune responses to injected cells and formation of a physical barrier caused by apical migration of host retinal cells upon disruption of outer limiting membrane, may impose two major barriers in LCAs cell transplantation therapy.

This paper presents an experimental investigation of the radiation-enhanced diffusion effect for a pure aluminum (Al 1050) surface by using a high-flux ion beam. When a nitrogen-ion beam with a 24 to ∼40 µA/cm2 flux irradiated Al surfaces, the depth of the AlN (aluminum-nitride) layer on the surface, which was formed by N2+-ion-beam irradiation, was identified through XPS (X-ray photoelectron spectroscopy), and the AlN layer related to the radiation-enhanced diffusion effect strongly depended on the flux and fluence of ion-beam. The atomic composition and the atom ratios were identified through AES (Auger electron spectroscopy). Also, the hardness of the high-flux ionbeam-irradiated surface was decreased by 30 ∼ 40% compared with that of the as-received sample due to material softening by grain growth. We measured the contact angle of the surface to identify the relation between radiation-enhanced diffusion and changes in the surface characteristics. The radiation enhanced diffusion due to high-flux ion-beam irradiation was shown to affect the depth of AlN formation, and the formed AlN layer was shown to give a hydrophilic property to the Al surface.

RASSF2 belongs to the Ras-association domain family (RASSF) of proteins, which may be involved in the Hippo signalling pathway. However, the role of RASSF2 in vivo is unknown. Here, we show that Rassf2 knockout mice manifest a multisystemic phenotype including haematopoietic anomalies and defects in bone remodelling. Bone marrow (BM) transplantation showed that Rassf2−/− BM cells had a normal haematopoietic reconstitution activity, indicating no intrinsic haematopoietic defects. Notably, in vitro differentiation studies revealed that ablation of Rassf2 suppressed osteoblastogenesis but promoted osteoclastogenesis. Co-culture experiments showed that an intrinsic defect in osteoblast differentiation from Rassf2−/− osteoblast precursors likely leads to both haematopoiesis and osteoclast defects in Rassf2−/− mice. Moreover, Rassf2 deficiency resulted in hyperactivation of nuclear factor (NF)-κB during both osteoclast and osteoblast differentiation. RASSF2 associated with IκB kinase (IKK) α and β forms, and suppressed IKK activity. Introduction of either RASSF2 or a dominant-negative form of IKK into Rassf2−/− osteoclast or osteoblast precursors inhibited NF-κB hyperactivation and normalized osteoclast and osteoblast differentiation. These observations indicate that RASSF2 regulates osteoblast and osteoclast differentiation by inhibiting NF-κB signalling.

Mutation of the polarity gene Crumbs homolog 1 (CRB1) is responsible for >10% of Leber congenital amaurosis (LCA) cases worldwide; LCA is characterized by early-onset degenerative retinal dystrophy. The role of CRB1 in LCA8 pathogenesis remains elusive since Crb1 mouse mutants, including a null allele, have failed to mimic the early-onset of LCA, most likely due to functional compensation by closely related genes encoding Crb2 and Crb3. Crb proteins form an evolutionarily conserved, apical polarity complex with the scaffolding protein associated with lin-seven 1 (Pals1), also known as MAGUK p55 subfamily member 5 (MPP5). Pals1 and Crbs are functionally inter-dependent in establishing and maintaining epithelial polarity. Pals1 is a single gene in the mouse and human genomes; therefore, we ablated Pals1 to establish a mouse genetic model mimicking human LCA. In our study, the deletion of Pals1 leads to the disruption of the apical localization of Crb proteins in retinal progenitors and the adult retina, validating their mutual interaction. Remarkably, the Pals1 mutant mouse exhibits the critical features of LCA such as early visual impairment as assessed by electroretinogram, disorganization of lamination and apical junctions and retinal degeneration. Our data uncover the indispensible role of Pals1 in retinal development, likely involving the maintenance of retinal polarity and survival of retinal neurons, thus providing the basis for the pathologic mechanisms of LCA8.

In the Li2O-B2O3-SiO2 glasses with Li2O≥30 mol%, substitution of Li2O for B2O3 or SiO2 leads to increase the amount of borate and silicate groups with non-bridging oxygen in glass network; while substitution of B2O3 for SiO2 leads to increase the amount of borate groups with or without non-bridging oxygen and silicate groups with non-bridging oxygen.