Identification of highly potent and selective HTRA1 inhibitors.

High temperature requirement A serine peptidase 1 (HTRA1) is a serine protease involved in an array of signaling pathways. It is also responsible for the regulation of protein aggregates via refolding, translocation, and degradation. It has subsequently been found that runaway proteolytic HTRA1 activity plays a role in a variety of diseases, including Age-Related Macular Degeneration (AMD), osteoarthritis, and Rheumatoid Arthritis. Selective inhibition of serine protease HTRA1 therefore offers a promising new strategy for the treatment of these diseases. Herein we disclose structure-activity-relationship (SAR) studies which identify key interactions responsible for binding affinity of small molecule inhibitors to HTRA1. The study results in highly potent molecules with IC50's less than 15 nM and excellent selectivity following a screen of 35 proteases.

Exploring the Role of HtrA Family Genes in Cancer: A Systematic Review.

PURPOSE: HtrA1, HtrA2, HtrA3 and HtrA4 appear to be involved in the development of pathologies such as cancer. This systematic review reports the results of a literature search performed to compare the expression of HtrA family genes and proteins in cancer versus non-cancer tissues and cell lines, assess relationships between HtrA expression and cancer clinical features in cancer, and analyse the molecular mechanism, by which HtrA family affects cancer. METHODS: The literature search was conducted according to the PRISMA statement among four databases (PubMed, Web of Science, Embase and Scopus). RESULTS: A total of 38 articles met the inclusion criteria and involved the expression of HtrA family members and concerned the effect of HtrA expression on cancer and metastasis development or on the factor that influences it. Additionally, 31 reports were retrieved manually. Most articles highlighted that HtrA1 and HtrA3 exhibited tumour suppressor activity, while HtrA2 was associated with tumour growth and metastasis. There were too few studies to clearly define the role of the HtrA4 protease in tumours. CONCLUSION: Although the expression of serine proteases of the HtrA family was dependent on tumour type, stage and the presence of metastases, most articles indicated that HtrA1 and HtrA3 expression in tumours was downregulated compared with healthy tissue or cell lines. The expression of HtrA2 was completely study dependent. The limited number of studies on HtrA4 expression made it impossible to draw conclusions about differences in expression between healthy and tumour tissue. The conclusions drawn from the study suggest that HtrA1 and HtrA3 act as tumour suppressors.

Pathophysiology of cerebral small vessel disease: a journey through recent discoveries.

Cerebral small vessel disease (cSVD) encompasses a heterogeneous group of age-related small vessel pathologies that affect multiple regions. Disease manifestations range from lesions incidentally detected on neuroimaging (white matter hyperintensities, small deep infarcts, microbleeds, or enlarged perivascular spaces) to severe disability and cognitive impairment. cSVD accounts for approximately 25% of ischemic strokes and the vast majority of spontaneous intracerebral hemorrhage and is also the most important vascular contributor to dementia. Despite its high prevalence and potentially long therapeutic window, there are still no mechanism-based treatments. Here, we provide an overview of the recent advances in this field. We summarize recent data highlighting the remarkable continuum between monogenic and multifactorial cSVDs involving NOTCH3, HTRA1, and COL4A1/A2 genes. Taking a vessel-centric view, we discuss possible cause-and-effect relationships between risk factors, structural and functional vessel changes, and disease manifestations, underscoring some major knowledge gaps. Although endothelial dysfunction is rightly considered a central feature of cSVD, the contributions of smooth muscle cells, pericytes, and other perivascular cells warrant continued investigation.

Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region.

Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1. Crystal structures, coupled with biochemical studies, reveal that the CKPs do not interact in a substrate-like manner but bind to a cryptic pocket at the S1' site region of HTRA1 and abolish catalysis by stabilizing a non-competent active site conformation. The opening and closing of this cryptic pocket is controlled by the gatekeeper residue V221, and its movement is facilitated by the absence of a constraining disulfide bond that is typically present in trypsin fold serine proteases, thereby explaining the remarkable selectivity of the CKPs. Our findings reveal an intriguing mechanism for modulating the activity of HTRA1, and highlight the utility of CKP-based phage display platforms in uncovering potent and selective inhibitors against challenging therapeutic targets.

Circ_0007445 inhibits trophoblast cell proliferation, migration and invasion by mediating the miR-4432/HTRA1 axis in preeclampsia.

BACKGROUND: : Circular RNAs (circRNAs) have been shown to be extensively involved in preeclampsia progression. At present, the role of circ_0007445 in preeclampsia progression is not clear. METHODS: A total of 30 preeclampsia patients and 30 normal pregnant women were recruited in our study. The function of trophoblast cells was explored to clarify the role and mechanism of circ_0007445 on the preeclampsia progression. The expression of circ_0007445, microRNA (miR)-4432 and high temperature requirement A1 (HTRA1) was analyzed by quantitative real-time PCR. The proliferation, migration and invasion of trophoblast cells were determined by cell counting kit 8 assay, EdU assay, colony formation assay, flow cytometry, and transwell assay. Protein expression was examined by western blot analysis. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay were used to assess RNA interaction relationships. RESULTS: Our data suggested that circ_0007445 had increased expression in preeclampsia patients. Knockdown of circ_0007445 enhanced trophoblast cell proliferation, migration and invasion. MiR-4432 was lowly expressed in preeclampsia patients, and it could be sponged by circ_0007445. MiR-4432 inhibitor overturned the promotion effects of circ_0007445 knockdown on trophoblast cell functions. HTRA1 was highly expressed in preeclampsia patients, and it could be targeted by miR-4432. HTRA1 overexpression could also reverse the proliferation, migration and invasion of trophoblast cells promoted by miR-4432 mimic. In addition, circ_0007445 positively regulated HTRA1 through targeting miR-4432. CONCLUSION: :Our results suggested that circ_0007445 facilitated the development of preeclampsia by suppressing trophoblast cell function through miR-4432/HTRA1 axis.

Inhibition of the serine protease HtrA1 by SerpinE2 suggests an extracellular proteolytic pathway in the control of neural crest migration.

We previously showed that SerpinE2 and the serine protease HtrA1 modulate fibroblast growth factor (FGF) signaling in germ layer specification and head-to-tail development of Xenopus embryos. Here, we present an extracellular proteolytic mechanism involving this serpin-protease system in the developing neural crest (NC). Knockdown of SerpinE2 by injected antisense morpholino oligonucleotides did not affect the specification of NC progenitors but instead inhibited the migration of NC cells, causing defects in dorsal fin, melanocyte, and craniofacial cartilage formation. Similarly, overexpression of the HtrA1 protease impaired NC cell migration and the formation of NC-derived structures. The phenotype of SerpinE2 knockdown was overcome by concomitant downregulation of HtrA1, indicating that SerpinE2 stimulates NC migration by inhibiting endogenous HtrA1 activity. SerpinE2 binds to HtrA1, and the HtrA1 protease triggers degradation of the cell surface proteoglycan Syndecan-4 (Sdc4). Microinjection of Sdc4 mRNA partially rescued NC migration defects induced by both HtrA1 upregulation and SerpinE2 downregulation. These epistatic experiments suggest a proteolytic pathway by a double inhibition mechanism.SerpinE2 ┤HtrA1 protease ┤Syndecan-4 → NC cell migration.

Levels of the HtrA1 Protein in Serum and Vitreous Humor Are Independent of Genetic Risk for Age-Related Macular Degeneration at the 10q26 Locus.

Purpose: The purpose of this study was to determine if levels of the HtrA1 protein in serum or vitreous humor are influenced by genetic risk for age-related macular degeneration (AMD) at the 10q26 locus, age, sex, AMD status, and/or AMD disease severity, and, therefore, to determine the contribution of systemic and ocular HtrA1 to the AMD disease process. Methods: A custom-made sandwich ELISA assay (SCTM ELISA) for detection of the HtrA1 protein was designed and compared with three commercial assays (R&D Systems, MyBiosource 1 and MyBiosource 2) using 65 serum samples. Concentrations of HtrA1 were thereafter determined in serum and vitreous samples collected from 248 individuals and 145 human donor eyes, respectively. Results: The SCTM ELISA demonstrated high specificity, good recovery, and parallelism within its linear detection range and performed comparably to the R&D Systems assay. In contrast, we were unable to demonstrate the specificity of the two assays from MyBioSource using either recombinant or native HtrA1. Analyses of concentrations obtained using the validated SCTM assay revealed that genetic risk at the 10q26 locus, age, sex, or AMD status are not significantly associated with altered levels of the HtrA1 protein in serum or in vitreous humor (P > 0.05). Conclusions: HtrA1 levels in serum and vitreous do not reflect the risk for AMD associated with the 10q26 locus or disease status. Localized alteration in HTRA1 expression in the retinal pigment epithelium, rather than systemic changes in HtrA1, is the most likely driver of elevated risk for developing AMD among individuals with risk variants at the 10q26 locus.

Genetics and Age-Related Macular Degeneration: A Practical Review for Clinicians.

Age-related macular degeneration (AMD) is a multifactorial genetic disease, with at least 52 identifiable associated gene variants at 34 loci, including variants in complement factor H (CFH) and age-related maculopathy susceptibility 2/high-temperature requirement A serine peptidase-1 (ARMS2/HTRA1). Genetic factors account for up to 70% of disease variability. However, population-based genetic risk scores are generally more helpful for clinical trial design and stratification of risk groups than for individual patient counseling. There is some evidence of pharmacogenetic influences on various treatment modalities used in AMD patients, including Age-Related Eye Disease Study (AREDS) supplements, photodynamic therapy (PDT), and anti-vascular endothelial growth factor (anti-VEGF) agents. However, there is currently no convincing evidence that genetic information plays a role in routine clinical care.

HTRA1 disaggregates α-synuclein amyloid fibrils and converts them into non-toxic and seeding incompetent species.

Parkinson's disease (PD) is closely linked to α-synuclein (α-syn) misfolding and accumulation in Lewy bodies. The PDZ serine protease HTRA1 degrades fibrillar tau, which is associated with Alzheimer's disease, and inactivating mutations to mitochondrial HTRA2 are implicated in PD. Here, we report that HTRA1 inhibits aggregation of α-syn as well as FUS and TDP-43, which are implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The protease domain of HTRA1 is necessary and sufficient for inhibiting aggregation, yet this activity is proteolytically-independent. Further, HTRA1 disaggregates preformed α-syn fibrils, rendering them incapable of seeding aggregation of endogenous α-syn, while reducing HTRA1 expression promotes α-syn seeding. HTRA1 remodels α-syn fibrils by targeting the NAC domain, the key domain catalyzing α-syn amyloidogenesis. Finally, HTRA1 detoxifies α-syn fibrils and prevents formation of hyperphosphorylated α-syn accumulations in primary neurons. Our findings suggest that HTRA1 may be a therapeutic target for a range of neurodegenerative disorders.

Identification of Genetic Variants for Risk Prediction and Early Diagnosis of Age-Related Macular Degeneration in the Taiwanese Population.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly worldwide. The prevalence and phenotypes of AMD differ among populations, including between people in Taiwan and other regions. We performed a genome-wide association study to identify genetic variants and to develop genetic models to predict the risk of AMD development and progression in the Taiwanese population. In total, 4039 patients with AMD and 16,488 non-AMD controls (aged ≥ 65 years) were included. We identified 31 AMD-associated variants (p < 5 × 10-8) on chromosome 10q26, surrounding PLEKHA1-ARMS2-HTRA1. Two genetic models were constructed using the clump and threshold method. Model 1 included the single nucleotide polymorphism rs11200630 and showed a 1.31-fold increase in the risk of AMD per risk allele (95% confidence interval (CI) = 1.20-1.43, p < 0.001). In model 2, 1412 single-nucleotide polymorphisms were selected to construct a polygenic risk score (PRS). Individuals with the top 5% PRS had a 1.40-fold higher AMD risk compared with that of individuals with a PRS in the bottom quartile (95% CI = 1.04-1.89, p = 0.025). Moreover, the PRS in the upper quartile was related to a decreased age at AMD diagnosis by 0.62 years (95% CI = -1.15, -0.09, p = 0.023). Both genetic models provide useful predictive power for populations at high risk of AMD, affording a basis for identifying patients requiring close follow-up and early intervention.

Secretome from Magnetically Stimulated Muscle Exhibits Anticancer Potency: Novel Preconditioning Methodology Highlighting HTRA1 Action.

Briefly (10 min) exposing C2C12 myotubes to low amplitude (1.5 mT) pulsed electromagnetic fields (PEMFs) generated a conditioned media (pCM) that was capable of mitigating breast cancer cell growth, migration, and invasiveness in vitro, whereas the conditioned media harvested from unexposed myotubes, representing constitutively released secretome (cCM), was less effective. Administering pCM to breast cancer microtumors engrafted onto the chorioallantoic membrane of chicken eggs reduced tumor volume and vascularity. Blood serum collected from PEMF-exposed or exercised mice allayed breast cancer cell growth, migration, and invasiveness. A secretome preconditioning methodology is presented that accentuates the graded anticancer potencies of both the cCM and pCM harvested from myotubes, demonstrating an adaptive response to pCM administered during early myogenesis that emulated secretome-based exercise adaptations observed in vivo. HTRA1 was shown to be upregulated in pCM and was demonstrated to be necessary and sufficient for the anticancer potency of the pCM; recombinant HTRA1 added to basal media recapitulated the anticancer effects of pCM and antibody-based absorption of HTRA1 from pCM precluded its anticancer effects. Brief and non-invasive PEMF stimulation may represent a method to commandeer the secretome response of muscle, both in vitro and in vivo, for clinical exploitation in breast and other cancers.

Microbleeds in Heterozygous HTRA1-Related Cerebral Small Vessel Disease.

This case report describes the evaluation of a 44-year-old man with a history of headache, dizziness, and imbalance and imaging that showed lacunar infarctions and bilateral white matter hyperintensities.

Possible involvement of HtrA1 serine protease in the onset of osteoporotic bone extracellular matrix changes.

High-temperature requirement A1 (HtrA1), a multidomain serine protease acting on Extracellular matrix (ECM) rearrangement, is also secreted by osteoblasts and osteoclasts. Recent and conflicting literature highlights HtrA1's role as a controller of bone remodeling, proposing it as a possible target for pathologies with unbalanced bone resorption, like Osteoporosis (OP). To add knowledge on this molecule function in bone physiopathology, here we compared HtrA1 distribution in the ECM of healthy (H) and OP bone tissue, also examining its localization in the sites of new bone formation. HtrA1 was homogeneously expressed in the mature bone ECM of H tissue showing a 55.6 ± 16.4% of the stained area, with a significant (p=0.0001) decrease in OP percentage stained area (21.1 ± 13.1). Moreover, HtrA1 was present in the endosteum and cells involved in osteogenesis, mainly in those "entrapped" in woven bone, whereas osteocytes in mature lamellar bone were negative. Based on our previous observation in OP tissue of a significantly increased expression of Decorin and Osteocalcin, both involved in bone mineralization and remodeling and equally substrates for HtrA1, we speculate that HtrA1 by controlling the proper amount of Decorin and Osteocalcin favors normal bone maturation and mineralization. Besides, we suggest that late-osteoblasts and pre-osteocytes secrete HtrA1 in the adjacent matrix whilst proceeding with their maturation and that HtrA1 expression is further modified during the remodeling from woven to the lamellar bone. Overall, our data suggest HtrA1 as a positive regulator of bone matrix formation and maturation: its reduced expression in mature OP bone, affecting protein content and distribution, could hamper correct bone remodeling and mineralization.

Identification of osteoarthritis-characteristic genes and immunological micro-environment features through bioinformatics and machine learning-based approaches.

BACKGROUND: Osteoarthritis (OA) is a multifaceted chronic joint disease characterized by complex mechanisms. It has a detrimental impact on the quality of life for individuals in the middle-aged and elderly population while also imposing a significant socioeconomic burden. At present, there remains a lack of comprehensive understanding regarding the pathophysiology of OA. The objective of this study was to examine the genes, functional pathways, and immune infiltration characteristics associated with the development and advancement of OA. METHODS: The Gene Expression Omnibus (GEO) database was utilized to acquire gene expression profiles. The R software was employed to conduct the screening of differentially expressed genes (DEGs) and perform enrichment analysis on these genes. The OA-characteristic genes were identified using the Weighted Gene Co-expression Network Analysis (WGCNA) and the Lasso algorithm. In addition, the infiltration levels of immune cells in cartilage were assessed using single-sample gene set enrichment analysis (ssGSEA). Subsequently, a correlation analysis was conducted to examine the relationship between immune cells and the OA-characteristic genes. RESULTS: A total of 80 DEGs were identified. As determined by functional enrichment, these DEGs were associated with chondrocyte metabolism, apoptosis, and inflammation. Three OA-characteristic genes were identified using WGCNA and the lasso algorithm, and their expression levels were then validated using the verification set. Finally, the analysis of immune cell infiltration revealed that T cells and B cells were primarily associated with OA. In addition, Tspan2, HtrA1 demonstrated a correlation with some of the infiltrating immune cells. CONCLUSIONS: The findings of an extensive bioinformatics analysis revealed that OA is correlated with a variety of distinct genes, functional pathways, and processes involving immune cell infiltration. The present study has successfully identified characteristic genes and functional pathways that hold potential as biomarkers for guiding drug treatment and facilitating molecular-level research on OA.

HtrA3: a promising prognostic biomarker and therapeutic target for head and neck squamous cell carcinoma.

Objective: The dysregulation of the human high-temperature requirement A (HtrA) family of serine proteases is associated with many malignancies. However, there are few reports on HtrAs in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to investigate the expression, prognostic value, and biological functions of HtrAs in HNSCC. Methods: The RNA-sequencing data and clinical data of HNSCC were downloaded from The Cancer Genome Atlas (TCGA) database. The GSE30784 and GSE31056 datasets from the Gene Expression Omnibus (GEO) database were used for further verification. This study explored the differential expression of HtrAs and assessed their potential impact on the prognosis of HNSCC patients using a survival module. Correlations between clinical characteristics and HtrA expression levels were then explored using a Wilcoxon rank sum test. A Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed using "clusterProfile" in the R software. A Pearson/Spearman correlation test was applied to analyze the relationship between HtrAs and immune infiltration level/checkpoint genes. Validation of HtrA expression levels were carried out by RT-PCR and western blot in human squamous carcinoma cell lines (Fadu and Cal-27) and human non-tumorigenic bronchial epithelium cells (BEAS-2B). Finally, through cell transfection, CCK-8, Ki-67 immunofluorescence, and flow cytometry assays, the effect of HtrA3 knockdown on the malignant biological behavior of HNSCC cells was explored. Results: The gene expression levels of HtrAs were significantly upregulated and associated with patient age, TNM stage, clinical stage, and TP53 mutation status in the TCGA-HNSCC cohort. High expressions of HtrA1/3 were associated with shorter overall survival, shorter progress-free interval, and lower disease-specific survival in HNSCC. A nomogram for HtrAs was constructed and validated. HtrA-related genes were significantly enriched in the immune response and cell apoptosis pathway. In addition, the expression of HtrAs showed significant correlations with B cells, M cells, DC cell infiltration, and immune infiltration checkpoint (CD276, TNFRSF14). Validation of HtrA expression was carried out by RT-PCR and western blot. Results of in vitro experiments indicated that HtrA3 gene knockdown inhibits the proliferation of FaDu and Cal-27 cells while concurrently promoting apoptosis. Conclusions: HtrA3 shows significant potential as both a prognostic marker and a promising therapeutic target for HNSCC, highlighting its relevance and importance in future research and potential clinical applications.

Current Views on Chr10q26 Contribution to Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the global aging population. Familial aggregation and genome-wide association (GWA) studies have identified gene variants associated with AMD, implying a strong genetic contribution to AMD development. Two loci, on human Chr 1q31 and 10q26, respectively, represent the most influential of all genetic factors. While the role of CFH at Chr 1q31 is well established, uncertainty remains about the genes ARMS2 and HTRA1, at the Chr 10q26 locus. Since both genes are in strong linkage disequilibrium, assigning individual gene effects is difficult. In this chapter, we review current literature about ARMS2 and HTRA1 and their relevance to AMD risk. Future studies will be necessary to unravel the mechanisms by which they contribute to AMD.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL): A challenging diagnosis and a rare multiple sclerosis mimic.

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is an extremely rare hereditary cerebral small vessel disease caused by homozygous or compound heterozygous mutations in the gene coding for high-temperature requirement A serine peptidase 1 (HtrA1). Given the rare nature of the disease, delays in diagnosis and misdiagnosis are not uncommon. In this article, we reported the first case of CARASIL from Saudi Arabia with a novel homozygous variant c.1156C>T in exon 7 of the HTRA1 gene. The patient was initially misdiagnosed with primary progressive multiple sclerosis and treated with rituximab. CARASIL should be considered in the differential diagnosis of patients with suspected atypical progressive multiple sclerosis who have additional signs such as premature scalp alopecia and low back pain with diffuse white matter lesions in brain MRI. Genetic testing is important to confirm the diagnosis.

Exploring the Therapeutic Potential of Elastase Inhibition in Age-Related Macular Degeneration in Mouse and Human.

Abnormal turnover of the extracellular matrix (ECM) protein elastin has been linked to AMD pathology. Elastin is a critical component of Bruch's membrane (BrM), an ECM layer that separates the retinal pigment epithelium (RPE) from the underlying choriocapillaris. Reduced integrity of BrM's elastin layer corresponds to areas of choroidal neovascularization (CNV) in wet AMD. Serum levels of elastin-derived peptides and anti-elastin antibodies are significantly elevated in AMD patients along with the prevalence of polymorphisms of genes regulating elastin turnover. Despite these results indicating significant associations between abnormal elastin turnover and AMD, very little is known about its exact role in AMD pathogenesis. Here we report on results that suggest that elastase enzymes could play a direct role in the pathogenesis of AMD. We found significantly increased elastase activity in the retinas and RPE cells of AMD mouse models, and AMD patient-iPSC-derived RPE cells. A1AT, a protease inhibitor that inactivates elastase, reduced CNV lesion sizes in mouse models. A1AT completely inhibited elastase-induced VEGFA expression and secretion, and restored RPE monolayer integrity in ARPE-19 monolayers. A1AT also mitigated RPE thickening, an early AMD phenotype, in HTRA1 overexpressing mice, HTRA1 being a serine protease with elastase activity. Finally, in an exploratory study, examining archival records from large patient data sets, we identified an association between A1AT use, age and AMD risk. Our results suggest that repurposing A1AT may have therapeutic potential in modifying the progression to AMD.

Osteoprogenitor-GMP crosstalk underpins solid tumor-induced systemic immunosuppression and persists after tumor removal.

Remote tumors disrupt the bone marrow (BM) ecosystem (BME), eliciting the overproduction of BM-derived immunosuppressive cells. However, the underlying mechanisms remain poorly understood. Herein, we characterized breast and lung cancer-induced BME shifts pre- and post-tumor removal. Remote tumors progressively lead to osteoprogenitor (OP) expansion, hematopoietic stem cell dislocation, and CD41- granulocyte-monocyte progenitor (GMP) aggregation. The tumor-entrained BME is characterized by co-localization between CD41- GMPs and OPs. OP ablation abolishes this effect and diminishes abnormal myeloid overproduction. Mechanistically, HTRA1 carried by tumor-derived small extracellular vesicles upregulates MMP-13 in OPs, which in turn induces the alterations in the hematopoietic program. Importantly, these effects persist post-surgery and continue to impair anti-tumor immunity. Conditional knockout or inhibition of MMP-13 accelerates immune reinstatement and restores the efficacies of immunotherapies. Therefore, tumor-induced systemic effects are initiated by OP-GMP crosstalk that outlasts tumor burden, and additional treatment is required to reverse these effects for optimal therapeutic efficacy.

CRISPR editing demonstrates rs10490924 raised oxidative stress in iPSC-derived retinal cells from patients with ARMS2/HTRA1-related AMD.

Genome-wide association studies (GWAS) have identified genetic risk loci for age-related macular degeneration (AMD) on the chromosome 10q26 (Chr10) locus and are tightly linked: the A69S (G>T) rs10490924 single-nucleotide variant (SNV) and the AATAA-rich insertion-deletion (indel, del443/ins54), which are found in the age-related maculopathy susceptibility 2 (ARMS2) gene, and the G512A (G>A) rs11200638 SNV, which is found in the high-temperature requirement A serine peptidase 1 (HTRA1) promoter. The fourth variant is Y402H complement factor H (CFH), which directs CFH signaling. CRISPR manipulation of retinal pigment epithelium (RPE) cells may allow one to isolate the effects of the individual SNV and thus identify SNV-specific effects on cell phenotype. Clustered regularly interspaced short palindromic repeats (CRISPR) editing demonstrates that rs10490924 raised oxidative stress in induced pluripotent stem cell (iPSC)-derived retinal cells from patients with AMD. Sodium phenylbutyrate preferentially reverses the cell death caused by ARMS2 rs10490924 but not HTRA1 rs11200638. This study serves as a proof of concept for the use of patient-specific iPSCs for functional annotation of tightly linked GWAS to study the etiology of a late-onset disease phenotype. More importantly, we demonstrate that antioxidant administration may be useful for reducing reactive oxidative stress in AMD, a prevalent late-onset neurodegenerative disorder.