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Mutations in the rhodopsin (RHO) gene are the predominant causes of autosomal dominant retinitis pigmentosa (adRP). Given the diverse gain-of-function mutations, therapeutic strategies targeting specific sequences face significant challenges. Here, we provide a universal approach to conquer this problem: we have devised a CRISPR-Cas12i-based, mutation-independent gene knockout and replacement compound therapy carried by a dual AAV2/8 system. In this study, we successfully delayed the progression of retinal degeneration in the classic mouse disease model RhoP23H, and also RhoP347S, a new native mouse mutation model we developed. Our research expands the horizon of potential options for future treatments of RHO-mediated adRP. [ABSTRACT FROM AUTHOR]

Abstract Background Rhodopsin (RHO) is the most well-known genetic cause of autosomal dominant retinitis pigmentosa (adRP). This study aimed to investigate the genetic cause of a large Chinese adRP family and assess the pathogenicity of the detected RHO mutant. Methods Routine ocular examinations were conducted on all participants. Next-generation sequencing with targeted capture was performed to screen mutations in 179 genes associated with hereditary retinal diseases and 10 candidate genes. Variants detected by NGS were validated by Sanger sequencing and evaluated for pathogenicity. Fragments of mutant and wild-type RHO were cloned into the pEGFP-N1 vector and were transfected into different cell lines to observe the cellular localization of the Rhodopsin-GFP fusion protein and evaluate the expression of endoplasmic reticulum (ER) stress markers. RT-PCR analysis was used to detect transfected the splicing of X box-binding protein 1 (XBP1) mRNA, which is a critical factor affecting ER stress. Results Genetic analysis identified a heterozygous missense variant, RHO, c.284 T > C (p.L95P) in this adRP family. Another RHO variant (p.P53R) that we reported previously was also included in further functional assessment. Both misfolded mutant proteins accumulated in the ER in a manner similar to that noted for the classic mutant P23H. Spliced XBP1 was observed in cells transfected with mutants, indicating an increase in ER stress. Conclusions Although the p.L95P variant is not a novel change, it was the first variant to be functionally evaluated and reported in Chinese RP patients. The results in our study provide significant evidence to classify the p.L95P mutation as a class II mutation.

Retinitis pigmentosa (RP) consists of a group of inherited, retinal degenerative disorders and is characterized by progressive loss of rod photoreceptors and eventual degeneration of cones in advanced stages, resulting in vision loss or blindness. Gene therapy has been effective in treating autosomal recessive RP (arRP). However, limited options are available for patients with autosomal dominant RP (adRP). In vivo gene editing may be a therapeutic option to treat adRP. We previously rescued vision in neonatal adRP rats by the selective ablation of the Rhodopsin S334ter transgene following electroporation of a CRISPR/Cas9 vector. However, the translational feasibility and long-term safety and efficacy of ablation therapy is unclear. To this end, we show that AAV delivery of a CRISPR/Cas9 construct disrupted the Rhodopsin P23H transgene in postnatal rats, which rescued long-term vision and retinal morphology. [ABSTRACT FROM AUTHOR]

The manifestation of intermediate uveitis (IU) in patients with retinitis pigmentosa (RP) is uncommon and poses diagnostic and management challenges. In this case, we describe the clinical features and management outcomes in an RP patient with a novel homozygous splice site mutation in PRPF8. A 21-year-old male presented with unilateral decrease of vision in the right eye for 1 week. Retinal dystrophy features were present in the left eye. After 2 weeks of topical steroid therapy, near-total resolution of IU was achieved and vision improved to 20/30. Signs of (RP) were present bilaterally, with the right eye more affected than the left. Genetic testing indicated a novel homozygous c. 3061-6_3061-3del mutation in the PRPF8 gene. IU in young patients with RP can be effectively treated with a short course of topical steroids, sparing the need for systemic immunosuppressives. After the improvement in IU, the right eye showed more advanced RP changes. [ABSTRACT FROM AUTHOR]

Background: Rhodopsin (RHO) is the most well-known genetic cause of autosomal dominant retinitis pigmentosa (adRP). This study aimed to investigate the genetic cause of a large Chinese adRP family and assess the pathogenicity of the detected RHO mutant.Methods: Routine ocular examinations were conducted on all participants. Next-generation sequencing with targeted capture was performed to screen mutations in 179 genes associated with hereditary retinal diseases and 10 candidate genes. Variants detected by NGS were validated by Sanger sequencing and evaluated for pathogenicity. Fragments of mutant and wild-type RHO were cloned into the pEGFP-N1 vector and were transfected into different cell lines to observe the cellular localization of the Rhodopsin-GFP fusion protein and evaluate the expression of endoplasmic reticulum (ER) stress markers. RT-PCR analysis was used to detect transfected the splicing of X box-binding protein 1 (XBP1) mRNA, which is a critical factor affecting ER stress.Results: Genetic analysis identified a heterozygous missense variant, RHO, c.284 T > C (p.L95P) in this adRP family. Another RHO variant (p.P53R) that we reported previously was also included in further functional assessment. Both misfolded mutant proteins accumulated in the ER in a manner similar to that noted for the classic mutant P23H. Spliced XBP1 was observed in cells transfected with mutants, indicating an increase in ER stress.Conclusions: Although the p.L95P variant is not a novel change, it was the first variant to be functionally evaluated and reported in Chinese RP patients. The results in our study provide significant evidence to classify the p.L95P mutation as a class II mutation. [ABSTRACT FROM AUTHOR]

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy, and 15–25% of RP is transmitted as an autosomal dominant (ad) trait. The objectives of this study were to establish the variant profile in a large cohort of adRP families and to elucidate the variant spectrum of each adRP gene in Chinese patients. A total of 138 probands clinically diagnosed with RP as a presumed autosomal dominant trait were recruited. All probands underwent ophthalmic examinations by specialists. A combination of molecular screening methods, including targeted next-generation sequencing, Sanger DNA sequencing, and multiplex ligation probe amplification assay, was used to detect variants. We identified heterozygous variants of 11 adRP genes in 73 probands, hemizygous, or heterozygous variants of X-linked RP genes in six patients, compound heterozygous variants of autosomal recessive RP genes in three pseudodominant families, and one heterozygous variant of one ad cone and rod dystrophy gene in one proband. One proband was found carrying both variants in RPGR and FAM161A. The overall detection rate was 59.4% (82/138). We detected 72 distinct disease-causing variants involving 16 RP genes and one cone-rod dystrophy gene; 33 of these variants have not been reported previously. Disease-causing variants were identified in the adRP genes in 52.9% of the families, followed by 4.3% in the X-linked RP genes, and 2.2% in the autosomal recessive genes. The most frequent mutant genes were RHO, PRPF31, RP1, SNRNP200, and PRPF8, which explained up to 78.0% of the genetically diagnosed families. Most of the variants identified in adRP genes were missense, and copy number variations were common (7/20) in the PRPF31 gene. We established the profile of the mutated genes and the variant spectrum of adRP genes in a large cohort of Chinese patients, providing essential information for genetic counseling and future development of therapeutics for retinal dystrophy inherited as a dominant trait.

Purpose: To identify structural and functional outcome measures among patients with Rho-positive autosomal dominant Retinitis Pigmentosa (adRP) to aid neuroprotection trial design. Methods: This was a retrospective cohort study of 52 patients with Rho-positive adRP. We measured Goldmann Visual Fields (GVF) constriction in four sectors (nasal, temporal, inferior, superior), and sectoral Ellipsoid Zone (EZ) width degeneration using Spectral Domain Optical Coherence Tomography (OCT) scans. Disease progression trajectories were projected using mixed effects modeling. Results: Superior GVF was most constricted at presentation and had the shallowest trajectory (less steep negative slope); Inferior GVF was less constricted (corrected p <.001) and had a steeper negative slope (corrected p =.019) than superior GVF. Temporal EZ was most stable on OCT with a relatively shallow negative trajectory (corrected p =.011). Conclusions: Patients' superior visual fields presented with more constriction and subsequently had a shallow negative slope suggesting the corresponding inferior retina may be "burned out" at presentation. Targeted therapies for adRP will likely show a greater efficacy signal if delivered to the superior and nasal retina, which may demonstrate more change on OCT and GVF over the course of a neuroprotection trial. Translational Relevance: Mixed effects analysis of sectoral visual field constriction and EZ degeneration in Rho-positive adRP can prove useful in monitoring therapeutic efficacy and identifying targets for local therapies. [ABSTRACT FROM AUTHOR]

Retinitis pigmentosa (RP) consists of a group of inherited, retinal degenerative disorders and is characterized by progressive loss of rod photoreceptors and eventual degeneration of cones in advanced stages, resulting in vision loss or blindness. Gene therapy has been effective in treating autosomal recessive RP (arRP). However, limited options are available for patients with autosomal dominant RP (adRP). In vivo gene editing may be a therapeutic option to treat adRP. We previously rescued vision in neonatal adRP rats by the selective ablation of the Rhodopsin S334ter transgene following electroporation of a CRISPR/Cas9 vector. However, the translational feasibility and long-term safety and efficacy of ablation therapy is unclear. To this end, we show that AAV delivery of a CRISPR/Cas9 construct disrupted the Rhodopsin P23H transgene in postnatal rats, which rescued long-term vision and retinal morphology.

The organisms have the capacity to sense and adapt to their surroundings for their life in a dynamic environment. In response to amino acid starvation, cells activate a rectifying physiological program, termed the integrated stress response (ISR), to restore cellular homeostasis. General controlled non-repressed (GCN2) kinase is a master regulator of the ISR and modulates protein synthesis in response to amino acid starvation. We previously established the GCN2/ATF4/4E-BP pathway in development and aging. Here, we investigated the tissue-specific roles of GCN2 upon dietary restriction of amino acid in a Drosophila model. The knockdown of GCN2 in the gut and fat body, an energy sensing organ in Drosophila , abolished the beneficial effect of GCN2 in lifespan extension upon dietary restriction of amino acids. Proteome analysis in an autosomal dominant retinitis pigmentosa (ADRP) model showed that dietary restriction of amino acids regulates the synthesis of proteins in several pathways, including mitochondrial translation, mitochondrial gene expression, and regulation of biological quality, and that gcn2 -mutant flies have reduced levels of these mitochondria-associated proteins, which may contribute to retinal degeneration in ADRP. These results indicate that the tissue-specific regulation of GCN2 contributes to normal physiology and ADRP progression. • Tissue-specific knockdown of gcn2 abolishes the beneficial effect of dietary restriction on lifespan. • Dietary restriction in Drosophila model for ADRP regulates the proteins expression in mitochondria and regulation of biological quality. • The expression of proteins associated with rhodopsin trafficking and phototransduction were differentially regulated by dietary restriction. [ABSTRACT FROM AUTHOR]

Background: Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations that is estimated to affect more than 1.5 million people worldwide. RP is characterized by retinal pigment deposits visible on fundus examination, abnormal electroretinogram and progressive retinal dysfunction. Aim: The present work aimed to identify the possible mutations in the rhodopsin gene (RHO) among Egyptian RP patients as well as identifying the different inheritance patterns of those patients. Subjects and methods: Thirty diagnosed retinitis pigmentosa patients were enrolled in the study. Inheritance forms of RP were identified by recording full family history, the coding regions of the rhodopsin gene were sequenced using blood-derived genomic DNA samples donated by patients and fifteen healthy controls. Results: A high percentage of autosomal recessive cases was reported. Also a high parental consanguinity rates were evident. Sequencing of rhodopsin gene revealed no mutations among the study population. Conclusion: Rhodopsin mutations are scarcely associated with the autosomal recessive RP, suggesting that wide scale studies are needed to determine the genetic variations involved in RP and particularly in the autosomal recessive inheritance.

Aged and damaged mitochondria can be selectively degraded by specific autophagic elimination, termed mitophagy. Defects in mitophagy have been increasingly linked to several diseases including neurodegenerative diseases, metabolic diseases and other aging-related diseases. However, the molecular mechanisms of mitophagy are not fully understood. Here, we identify PRPF8 (pre-mRNA processing factor 8), a core component of the spliceosome, as an essential mediator in hypoxia-induced mitophagy from an RNAi screen based on a fluorescent mitophagy reporter, mt-Keima. Knockdown of PRPF8 significantly impairs mitophagosome formation and subsequent mitochondrial clearance through the aberrant mRNA splicing of ULK1, which mediates macroautophagy/autophagy initiation. Importantly, autosomal dominant retinitis pigmentosa (adRP)-associated PRPF8 mutant R2310K is defective in regulating mitophagy. Moreover, knockdown of other adRP-associated splicing factors, including PRPF6, PRPF31 and SNRNP200, also lead to ULK1 mRNA mis-splicing and mitophagy defects. Thus, these findings demonstrate that PRPF8 is essential for mitophagy and suggest that dysregulation of spliceosome-mediated mitophagy may contribute to pathogenesis of retinitis pigmentosa. [ABSTRACT FROM AUTHOR]

Purpose: To identify the underlying genetic defect for non-syndromic autosomal dominant retinitis pigmentosa (adRP) with incomplete penetrance in a North Indian family.Methods: Family history and clinical data were collected. Linkage analysis using 72 fluorescently labeled microsatellite markers flanking all the 26 candidate genes known for adRP was performed. Mutation screening in candidate gene at the mapped region was performed by bi-directional DNA sequencing.Results: Positive two-point lod scores > 1.0 (θ = 0.000) suggestive of linkage were obtained with markers D19S572, D19S927 and D19S926 at 19q13.42, in the vicinity of PRPF31 gene. Mutation screening in all the 14 exonic regions and intron-exon boundaries of PRPF31 revealed a novel change, i.e. c.896G>A (p.Cys299Tyr) in exon eight. The observed change segregated in heterozygous form in all the six affected members and in three carriers, consistent with incomplete penetrance. This substitution was not observed in tested 15 unaffected members and in 200 ethnically matched controls.Conclusion: Present study describes mapping of a locus for non-syndromic adRP with incomplete penetrance at 19q13.42 in a North Indian family and identifies a novel missense mutation (p.Cys299Tyr) in PRPF31 localized at the mapped interval. The observed substitution lies in the NOP domain of PRPF31 that exhibit RNA and protein binding surfaces and thus may interfere in the formation of spliceosome complex. Due to p.Cys299Tyr substitution hydrogen bonds are generated, which may result in conformational changes and PRPF31 protein deformity. Present findings further substantiate the role of PRPF31 in adRP with incomplete penetrance and expand the mutation spectrum of PRPF31. [ABSTRACT FROM AUTHOR]

Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin (RHO) gene, translation to the clinic has stalled. Here, we identified a highly efficient shRNA that targets human (and canine) RHO in a mutationindependent manner. In a single adeno-associated viral (AAV) vector we combined this shRNA with a human RHO replacement cDNA made resistant to RNA interference and tested this construct in a naturally occurring canine model of RHO-adRP. Subretinal vector injections led to nearly complete suppression of endogenous canine RHO RNA, while the human RHO replacement cDNA resulted in up to 30% of normal RHO protein levels. Noninvasive retinal imaging showed photoreceptors in treated areas were completely protected from retinal degeneration. Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (>8 mo) follow-up by retinal imaging and electroretinography indicated stable structural and functional preservation. The efficacy of this gene therapy in a clinically relevant large-animal model paves the way for treating patients with RHO-adRP. [ABSTRACT FROM AUTHOR]

Background: Retinitis pigmentosa (RP) is a rare type of inherited retinal dystrophy that can result in progressive vision loss. Molecular diagnosis of RP is challenging due to phenotypic and genotypic heterogeneities. Aims: This study aimed to identify the pathogenic mutations in two Chinese families with autosomal dominant RP (adRP) and in a patient with sporadic RP. Materials and Methods: Peripheral blood DNA samples were obtained from the participants. Targeted next generation sequencing (NGS) was applied to identify mutations in these patients. For pathogenic mutation analyses, stringent NGS data analyses and segregation analyses were applied. Primers were designed to validate the identified mutations by Sanger sequencing analyses. Results: A novel heterozygous insertion frameshift mutation c.1226_1227insA, p.T410Dfs*65, and a novel heterozygous stopgain mutation c.1015C>T, p.Q339* were identified in PRPF31. A known c.527 + 3A>G splicing mutation was identified in one of the adRP-074 families. All mutations were found to co-segregate with the disease, and none of these mutations were detected in 500 control samples. Conclusions: Our data identified two new autosomal dominant mutations in PRPF31 , expanding the mutational spectrum of this gene. [ABSTRACT FROM AUTHOR]

Background: Inherited retinal degenerations are a major cause of untreatable blindness in the younger age group. Recent advances in gene therapy using adeno-associated viral (AAV) vectors have raised the possibility of slowing or stopping retinal degenerations with gene replacement in cases of gene deficiency. Materials and methods: In this report, we present a family with autosomal dominant retinitis pigmentosa. A screen for commonADRPgenes was performed with 105 genes targeted. Next generation sequencing was used to identify the mutation which was next confirmed by bidirectional Sanger sequencing. Results: A novel mutation of theTOPORSgene was identified, c.2539C>T p.(Arg847Ter), resulting in a premature termination codon and suggesting haploinsufficiency as the pathological mechanism. Conclusions: Since the cDNA encodingTOPORSis 3,135 nucleotides (within the coding capacity of AAV vectors) and haploinsufficiency is a mechanism relating to inadequate gene expression, gene replacement therapy may be an option for patients with this condition. [ABSTRACT FROM AUTHOR]

Major bottlenecks in development of therapeutic post transcriptional gene silencing ( PTGS ) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme ( hhRz ) or RNA interference ( RNAi ) constructs, using a model retinal degeneration target, human rod opsin ( RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N = G,C,A,U; H = C,A,U) within the target mRNA of secreted alkaline phosphatase ( SEAP ), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening ( HTS ). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a large set of potential VAI-hhRz expression plasmids against diverse NUH↓ cleavage sites uses cultured human HEK293S cells stably expressing a dicistronic Target -IRES- SEAP target fusion mRNA. Broad utility of this rational RNA drug discovery approach is feasible for any ophthalmological disease-relevant mRNA targets and any disease mRNA targets in general. The approach will permit rank ordering of PTGS agents based on potency to identify a lead therapeutic compound for further optimization. [ABSTRACT FROM AUTHOR]

Neuronatin (NNAT) is a small transmembrane proteolipid that is highly expressed in the embryonic developing brain and several other peripheral tissues. This study is the first to provide evidence that NNAT is detected in the adult retina of various adult rod-dominant mammals, including wild-type (WT) rodents, transgenic rodents expressing mutant S334ter, P23H, or T17M rhodopsin, non-human primates, humans, and cone-dominant tree shrews. Immunohistochemical and quantitative real time polymerase chain reaction (qRT-PCR) analyses were applied to detect NNAT. Confocal microscopy analysis revealed that NNAT immunofluorescence is restricted to the outer segments (OSs) of photoreceptors without evidence of staining in other retinal cell types across all mammalian species. Moreover, in tree shrew retinas, we found NNAT to be co-localized with rhodopsin, indicating its predominant expression in rods. The rod-derived expression of NNAT was further confirmed by qRT-PCR in isolated rod photoreceptor cells. We also used these cells to mimic cellular stress in transgenic retinas by treating them with the endoplasmic reticulum stress inducer, tunicamycin. Thus, our data revealed accumulation of NNAT around the nucleus as compared to dispersed localization of NNAT within control cells. This distribution coincided with the partial intracellular mislocalization of NNAT to the outer nuclear layer observed in transgenic retinas. In addition, stressed retinas demonstrated an increase of NNAT mRNA and protein levels. Therefore, our study demonstrated that NNAT is a novel stress-responsive protein with a potential structural and/or functional role in adult mammalian retinas. [ABSTRACT FROM AUTHOR]

Background: Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of inherited retinal degenerations that is estimated to affect more than 1.5 million people worldwide. RP is characterized by retinal pigment deposits visible on fundus examination, abnormal electroretinogram and progressive retinal dysfunction. Aim: The present work aimed to identify the possible mutations in the rhodopsin gene (RHO) among Egyptian RP patients as well as identifying the different inheritance patterns of those patients. Subjects and methods: Thirty diagnosed retinitis pigmentosa patients were enrolled in the study. Inheritance forms of RP were identified by recording full family history, the coding regions of the rhodopsin gene were sequenced using blood-derived genomic DNA samples donated by patients and fifteen healthy controls. Results: A high percentage of autosomal recessive cases was reported. Also a high parental consanguinity rates were evident. Sequencing of rhodopsin gene revealed no mutations among the study population. Conclusion: Rhodopsin mutations are scarcely associated with the autosomal recessive RP, suggesting that wide scale studies are needed to determine the genetic variations involved in RP and particularly in the autosomal recessive inheritance. [ABSTRACT FROM AUTHOR]