Exploitation of enhanced prime editing for blocking aberrant angiogenesis.

INTRODUCTION: Aberrant angiogenesis plays an important part in the development of a variety of human diseases including proliferative diabetic retinopathy, with which there are still numerous patients remaining a therapeutically challenging condition. Prime editing (PE) is a versatile gene editing approach, which offers a novel opportunity to genetically correct challenging disorders. OBJECTIVES: The goal of this study was to create a dominant-negative (DN) vascular endothelial growth factor receptor (VEGFR) 2 by editing genomic DNA with an advanced PE system to block aberrant retinal angiogenesis in a mouse model of oxygen-induced retinopathy. METHODS: An advanced PE system (referred to as PE6x) was established within two lentiviral vectors, with one carrying an enhanced PE guide RNA and a canonical Cas9 nickase fused with an optimized reversal transcriptase, and the other conveying a nicking guide RNA and a DN-MLH1 to improve PE efficiency. Dual non-integrating lentiviruses (NILVs) produced with the two lentiviral PE6x vectors were then employed to create a mutation of VEGFR2 T17967A by editing the Mus musculus VEGFR2 locus in vitro and in vivo, leading to generation of a premature stop codon (TAG, K796stop) to produce DN-VEGFR2, to interfere with the wild type VEGFR2 which is essential for angiogenesis. RESULTS: NILVs targeting VEGFR2 delivered into cultured murine vascular endothelial cells led to 51.06 % VEGFR2 T17967A in the genome analyzed by next generation sequencing and the production of DN-VEGFR2, which was found to hamper VEGF-induced VEGFR2 phosphorylation, as demonstrated by Western blot analysis. Intravitreally injection of the dual NILVs into postnatal day 12 mice in a model of oxygen-induced retinopathy, led to production of retinal DN-VEGFR2 in postnatal day 17 mice which blocked retinal VEGFR2 expression and activation as well as abnormal retinal angiogenesis without interfering with retinal structure and function, as assessed by electroretinography, optical coherence tomography, fundus fluorescein angiography and histology. CONCLUSION: DN-VEGFR2 resulted from editing genomic VEGFR2 using the PE6x system can be harnessed to treat intraocular pathological angiogenesis.

Genome Editing VEGFA Prevents Corneal Neovascularization In Vivo.

Corneal neovascularization (CNV) is a common clinical finding seen in a range of eye diseases. Current therapeutic approaches to treat corneal angiogenesis, in which vascular endothelial growth factor (VEGF) A plays a central role, can cause a variety of adverse side effects. The technology of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 can edit VEGFA gene to suppress its expression. CRISPR offers a novel opportunity to treat CNV. This study shows that depletion of VEGFA with a novel CRISPR/Cas9 system inhibits proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Importantly, subconjunctival injection of this dual AAV-SpCas9/sgRNA-VEGFA system is demonstrated which blocks suture-induced expression of VEGFA, CD31, and α-smooth muscle actin as well as corneal neovascularization in mice. This study has established a strong foundation for the treatment of corneal neovascularization via a gene editing approach for the first time.

An increase in a long noncoding RNA ANRIL in peripheral plasma is an indicator of stable angina.

INTRODUCTION: Stable angina develops during physical activity or stress, and it is typically an aspect of Coronary Heart Disease (CHD) that can lead to arrhythmia, heart failure and even sudden death. ANRIL, an Antisense Noncoding RNA gene in the INK4 Locus, is associated with multiple disorders including CHD; however, expressional levels of ANRIL in between patients with stable angina and myocardial infarction, one of the acute coronary syndrome, have not been clarified yet. METHODS: The authors enrolled 62 patients with myocardial infarction and 59 with stable angina before primary percutaneous coronary intervention, as well as 48 healthy volunteers. Their peripheral blood was collected for analysis of ANRIL and cardiac troponin I, a traditional diagnostic index of CHD by real-time PCR. RESULTS: The data showed that ANRIL is a better diagnostic indicator than cardiac troponin I in patients with stable angina and that the levels of ANRIL are higher in patients with stable angina than those with the myocardial infarction. DISCUSSION: The levels of ANRIL in peripheral plasma could be used as a good biomarker for stable angina.

Phosphoinositide 3-kinase as a therapeutic target in angiogenic disease.

Phosphoinositide 3-kinases (PI3Ks) generate lipids that control multitudinous intracellular cell signaling events which participate in cell survival and proliferation. In addition, PI3K signaling also contributes to metabolism, immunity, angiogenesis and cardiovascular homeostasis, and many diseases. The diverse actions of PI3K stem from the existence of their various isoforms and a variety of protein effectors. Hence, PI3K isoform-specific inhibitors have already achieved a wonderful effect on treating cancer. Herein, we summarize the molecular mechanism of PI3K inhibitors in preventing the permeability of vessels and neovascularization. Additionally, we briefly illustrate how PI3K signaling modulates blood vessel growth and discuss the different roles that PI3K isoforms play in angiogenesis.

Leverage of nuclease-deficient CasX for preventing pathological angiogenesis.

Gene editing with a CRISPR/Cas system is a novel potential strategy for treating human diseases. Pharmacological inhibition of phosphoinositide 3-kinase (PI3K) δ suppresses retinal angiogenesis in a mouse model of oxygen-induced retinopathy. Here we show that an innovative system of adeno-associated virus (AAV)-mediated CRISPR/nuclease-deficient (d)CasX fused with the Krueppel-associated box (KRAB) domain is leveraged to block (81.2% ± 6.5%) in vitro expression of p110δ, the catalytic subunit of PI3Kδ, encoded by Pik3cd. This CRISPR/dCasX-KRAB (4, 269 bp) system is small enough to be fit into a single AAV vector. We then document that recombinant AAV serotype (rAAV)1 efficiently transduces vascular endothelial cells from pathologic retinal vessels, which show high expression of p110δ; furthermore, we demonstrate that blockade of retinal p110δ expression by intravitreally injected rAAV1-CRISPR/dCasX-KRAB targeting the Pik3cd promoter prevents (32.1% ± 5.3%) retinal p110δ expression as well as pathological retinal angiogenesis in a mouse model of oxygen-induced retinopathy. These data establish a strong foundation for treating pathological angiogenesis by AAV-mediated CRISPR interference with p110δ expression.

Ligand-independent activation of platelet-derived growth factor receptor ß promotes vitreous-induced contraction of retinal pigment epithelial cells.

BACKGROUND: Epiretinal membranes in patients with proliferative vitreoretinopathy (PVR) consist of extracellular matrix and a number of cell types including retinal pigment epithelial (RPE) cells and fibroblasts, whose contraction causes retinal detachment. In RPE cells depletion of platelet-derived growth factor (PDGF) receptor (PDGFR)ß suppresses vitreous-induced Akt activation, whereas in fibroblasts Akt activation through indirect activation of PDGFRα by growth factors outside the PDGF family (non-PDGFs) plays an essential role in experimental PVR. Whether non-PDGFs in the vitreous, however, were also able to activate PDGFRß in RPE cells remained elusive. METHODS: The CRISPR/Cas9 technology was utilized to edit a genomic PDGFRB locus in RPE cells derived from an epiretinal membrane (RPEM) from a patient with PVR, and a retroviral vector was used to express a truncated PDGFRß short of a PDGF-binding domain in the RPEM cells lacking PDGFRß. Western blot was employed to analyze expression of PDGFRß and α-smooth muscle actin, and signaling events (p-PDGFRß and p-Akt). Cellular assays (proliferation, migration and contraction) were also applied in this study. RESULTS: Expression of a truncated PDGFRß lacking a PDGF-binding domain in the RPEM cells whose PDGFRB gene has been silent using the CRISPR/Cas9 technology restores vitreous-induced Akt activation as well as cell proliferation, epithelial-mesenchymal transition, migration and contraction. In addition, we show that scavenging reactive oxygen species (ROS) with N-acetyl-cysteine and inhibiting Src family kinases (SFKs) with their specific inhibitor SU6656 blunt the vitreous-induced activation of the truncated PDGFRß and Akt as well as the cellular events related to the PVR pathogenesis. These discoveries suggest that in RPE cells PDGFRß can be activated indirectly by non-PDGFs in the vitreous via an intracellular pathway of ROS/SFKs to facilitate the development of PVR, thereby providing novel opportunities for PVR therapeutics. CONCLUSION: The data shown here will improve our understanding of the mechanism by which PDGFRß can be activated by non-PDGFs in the vitreous via an intracellular route of ROS/SFKs and provide a conceptual foundation for preventing PVR by inhibiting PDGFRß transactivation (ligand-independent activation).

Genome Editing Inhibits Retinal Angiogenesis in a Mouse Model of Oxygen-Induced Retinopathy.

This protocol describes a novel approach harnessing the technology of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9-based gene editing for treating retinal angiogenesis. In this system, adeno-associated virus (AAV)-mediated CRISPR/Cas9 was employed to edit the genome of vascular endothelial growth factor receptor (VEGFR)2 in retinal vascular endothelial cells in a mouse model of oxygen-induced retinopathy. The results showed that genome editing of VEGFR2 suppressed pathological retinal angiogenesis. This mouse model mimics a critical aspect of abnormal retinal angiogenesis in patients with neovascular diabetic retinopathy and retinopathy of prematurity, indicating genome editing has high potential for treating angiogenesis-associated retinopathies.

NFκB-Mediated Expression of Phosphoinositide 3-Kinase δ Is Critical for Mesenchymal Transition in Retinal Pigment Epithelial Cells.

Epithelial mesenchymal transition (EMT) plays a vital role in a variety of human diseases including proliferative vitreoretinopathy (PVR), in which retinal pigment epithelial (RPE) cells play a key part. Transcriptomic analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was up-regulated in human RPE cells upon treatment with transforming growth factor (TGF)-ß2, a multifunctional cytokine associated with clinical PVR. Stimulation of human RPE cells with TGF-ß2 induced expression of p110δ (the catalytic subunit of PI3Kδ) and activation of NFκB/p65. CRISPR-Cas9-mediated depletion of p110δ or NFκB/p65 suppressed TGF-ß2-induced fibronectin expression and activation of Akt as well as migration of these cells. Intriguingly, abrogating expression of NFκB/p65 also blocked TGF-ß2-induced expression of p110δ, and luciferase reporter assay indicated that TGF-ß2 induced NFκB/p65 binding to the promoter of the PIK3CD that encodes p110δ. These data reveal that NFκB/p65-mediated expression of PI3Kδ is essential in human RPE cells for TGF-ß2-induced EMT, uncovering hindrance of TGF-ß2-induced expression of p110δ as a novel approach to inhibit PVR.

Genome Editing of Pik3cd Impedes Abnormal Retinal Angiogenesis.

Abnormal angiogenesis is associated with myriad human diseases, including proliferative diabetic retinopathy (PDR). Signaling transduction through phosphoinositide 3-kinases (PI3Ks) plays a critical role in angiogenesis. Herein, we showed that p110δ, the catalytic subunit of PI3Kδ, was highly expressed in pathological retinal vascular endothelial cells (ECs) in a mouse model of oxygen-induced retinopathy (OIR) and in fibrovascular membranes from patients with PDR. To explore novel intervention with PI3Kδ expression, we developed a recombinant dual adeno-associated viral (rAAV) system for delivering CRISPR/Cas9 in which Streptococcus pyogenes (Sp) Cas9 expression was driven by an endothelial specific promoter of the intercellular adhesion molecule 2 (pICAM2) to edit genomic Pik3cd, the gene encoding p110δ. We then demonstrated that infection of cultured mouse vascular ECs with the dual rAAV1s of rAAV1-pICAM2-SpCas9 and rAAV1-SpGuide targeting genomic Pik3cd resulted in 80% DNA insertion/deletion in the locus of genomic Pik3cd and 70% depletion of p110δ expression. Furthermore, we showed that in the mouse model of OIR editing retinal Pik3cd with the dual rAAV1s resulted in not only a significant decrease in p110δ expression, and Akt activation, but also a dramatic reduction in pathological retinal angiogenesis. These findings reveal that Pik3cd editing is a novel approach to treating abnormal retinal angiogenesis.

An increase in a long noncoding RNA ANRIL in peripheral plasma is an indicator of stable angina

Abstract Introduction: Stable angina develops during physical activity or stress, and it is typically an aspect of Coronary Heart Disease (CHD) that can lead to arrhythmia, heart failure and even sudden death. ANRIL, an Antisense Non-coding RNA gene in the INK4 Locus, is associated with multiple disorders including CHD; however, expressional levels of ANRIL in between patients with stable angina and myocardial infarction, one of the acute coronary syndrome, have not been clarified yet. Methods: The authors enrolled 62 patients with myocardial infarction and 59 with stable angina before primary percutaneous coronary intervention, as well as 48 healthy volunteers. Their peripheral blood was collected for analysis of ANRIL and cardiac troponin I, a traditional diagnostic index of CHD by real-time PCR. Results: The data showed that ANRIL is a better diagnostic indicator than cardiac troponin I in patients with stable angina and that the levels of ANRIL are higher in patients with stable angina than those with the myocardial infarction. Discussion: The levels of ANRIL in peripheral plasma could be used as a good biomarker for stable angina.

GAGE is a method for identification of plant species based on whole genome analysis and genome editing.

Whole genomes of plants should be ideal databases for their species identification, but unfortunately there was no such method before this exploration. Here we report a plant species identification method based on the whole Genome Analysis and Genome Editing (GAGE). GAGE searches for target sequences from the whole genome of the subject plant and specifically detects them by employing a CRISPR/Cas12a system. Similar to how Mendel chose Pisum sativum (pea), we selected Crocus sativus (saffron) to establish GAGE, in which we constructed a library containing all candidate target sequences. Taking a target sequence in the ITS2 region as an example, we confirmed the feasibility, specificity, and sensitivity of GAGE. Consequently, we succeeded in not only using GAGE to identify Cr. sativus and its adulterants, but also executing GAGE in the plants from different classes including angiosperms, gymnosperms, ferns, and lycophytes. This sensitive and rapid method is the first plant species identification method based on the whole genome and provides new insights into the application of the whole genome in species identification.

The Key Network of mRNAs and miRNAs Regulated by HIF1A in Hypoxic Hepatocellular Carcinoma Cells.

Purpose: Hypoxia plays an essential role in the progression of hepatocellular carcinoma (HCC), whereas hypoxia inducible factor-1 (HIF-1) is the key transcription factor allowing HCC to survive hypoxia. The aim of this study was to define the essential mRNAs and miRNAs regulated by HIF1A and dissect their functions, interactions, and tumor-infiltrating immune cells in HCC. Methods: A human HCC cell line HepG2 was used as a cell model of HCC. The CRISPR/Cas9 system was used to knock out HIF1A in HepG2 cells, and RNA sequencing was utilized to characterize differentially expressed mRNAs and miRNAs in the HIF1A-knockout HepG2 cells; the identified candidates were then analyzed by GO annotation and KEGG pathway enrichment to study their function and establish a PPI network. Quantitative (q) PCR was used to verify if there were significant differences in the expression of mRNAs, and the association of the selected mRNAs expression with immune cell infiltration levels was further analyzed using The Cancer Genome Atlas (TCGA) pan-cancer data. Results: Using RNA-sequencing, we discovered that there were 1535 mRNAs differentially expressed (adjusted p < 0.05, |fold change|>1.5) in the HIF1A-knockout HepG2 cells, among which there were 644 mRNAs upregulated and 891 mRNAs downregulated. GO annotation and KEGG pathway enrichment showed that these mRNAs were involved in glycolysis/gluconeogenesis, PI3K-Akt signaling pathways, and HIF-1 signaling pathways. In addition, we found that there were 309 miRNAs differentially expressed (adjusted p < 0.05, |fold change|>1.5) in the HIF1A-knockout HepG2 cells, of which there were 213 miRNAs upregulated and 96 miRNAs downregulated. Our further analyses uncovered that these miRNA putative targets were involved in the hippo signaling pathway, axon guidance, and tight junction. Moreover, the construction and analysis of the PPI network showed that OASL, IL6, and TAF1 were recognized as hub genes with the highest connectivity degrees. Importantly, in the HIF1A-knockout HepG2 cells, our qRT-PCR data confirmed the selected mRNA changes revealed by RNA-sequencing, and with TCGA pan-cancer data, we revealed that the expressional levels of these three genes, LUM, SCOC, and CCL2, were associated with immune cell infiltration levels. Conclusion: The identified potential key network of mRNAs and miRNAs regulated by HIF1A in the HCC cells suggests a key role of HIF1A in the tumorigenesis of HCC.

Idelalisib inhibits experimental proliferative vitroretinopathy.

Proliferative vitreoretinopathy (PVR) is a fibrotic eye disease that develops after rhegmatogenous retinal detachment surgery and open-globe traumatic injury. Idelalisib is a specific inhibitor of phosphoinositide 3-kinase (PI3K) δ. While PI3Kδ is primarily expressed in leukocytes, its expression is also considerably high in retinal pigment epithelial (RPE) cells, which play a crucial part in the PVR pathogenesis. Herein we show that GeoMx Digital Spatial Profiling uncovered strong expression of fibronectin in RPE cells within epiretinal membranes from patients with PVR, and that idelalisib (10 µM) inhibited Akt activation, fibronectin expression and collagen gel contraction induced by transforming growth factor (TGF)-ß2 in human RPE cells. Furthermore, we discovered that idelalisib at a vitreal concentration of 10 µM, a non-toxic dose to the retina, prevented experimental PVR induced by intravitreally injected RPE cells in rabbits assessed by experienced ophthalmologists using an indirect ophthalmoscope plus a + 30 D fundus lens, electroretinography, optical coherence tomography and histological analysis. These data suggested idelalisib could be harnessed for preventing patients from PVR.

A Novel Role of IL13Rα2 in the Pathogenesis of Proliferative Vitreoretinopathy.

Proliferative vitreoretinopathy (PVR), an inflammatory and fibrotic blinding disease, is still a therapeutic challenge. Retinal pigment epithelial (RPE) cells dislodged in the vitreous play a central role in the PVR pathogenesis. To identify potential novel contributors to the pathogenesis of PVR, we investigated a profile of vitreous-induced changes in ARPE-19 cells by RNA sequencing. Bioinformatics analysis of the sequencing data showed that there were 258 genes up-regulated and 835 genes down-regulated in the ARPE-19 cells treated with human vitreous. Among these genes, there were three genes related to eye disease with more than threefold changes. In particular, quantitative PCR and western blot results showed that interleukin 13 receptor (IL13R)α2 that is over-expressed in a variety of cancers was up-regulated more than three times in the vitreous-treated ARPE-19 cells. Immunofluorescence analysis indicated that interleukin-13 receptor subunit α2 (IL13Rα2) was highly expressed in ARPE-19 cells within epiretinal membranes from patients with PVR. Importantly, blocking IL13Rα2 with its neutralizing antibody significantly inhibited vitreous-induced contraction of ARPE-19 cells, suggesting a novel role of IL13Rα2 in the PVR pathogenesis. These findings will improve our understanding of the molecular mechanisms by which PVR develops and provides potential targets for PVR therapeutics.

The MDM2 Single-Nucleotide Polymorphism T309G Is Associated With the Development of Epimacular Membranes.

Purpose: To investigate the role of the mouse double minute 2 (MDM2) gene single-nucleotide polymorphism (SNP) T309G in the development of epimacular membranes (EMMs) by analyzing the genotype distribution and consistency of the polymorphism in paired membrane-blood samples. Methods: This was a cross-sectional genetic association study of patients with proliferative vitreoretinopathy (PVR) or EMMs. PVR membranes (PVRMs), internal limiting membranes (ILMs) (PVR-ILMs) and blood samples (PVR-blood) from patients with PVR, and EMMs, EMM-ILMs and EMM-blood from patients with EMMs were collected. The genotype of all samples was determined by Sanger sequencing. Sex composition, mean age, the genotype distribution of MDM2 T309G, the allelic frequency of the MDM2 SNP309 G allele (% G) and the somatic mutation rate at the MDM2 T309G locus (% M) were analyzed and compared. The PVR and healthy Chinese donor groups were used as controls for different comparisons. Results: The EMM group of 62 patients was older than the PVR group of 61 patients by an average of 8.87 years (p < 0.0001), but the two groups were statistically similar in the sex composition (p = 0.1754). Importantly, G allele carriers were at a higher risk of developing EMMs than non-G allele carriers (p = 0.0479; OR = 2.047). Moreover, EMM-blood exhibited a significantly higher % G than blood samples from healthy Chinese donors (EMM-blood: 56.78%, donors: 45.61%; p = 0.0256; OR = 1.567). Regarding membrane-blood consistency, % M was significantly different between PVRMs and EMMs (PVRMs: 2.63%, EMMs: 21.57%; p = 0.0097; OR = 10.18) but not between different types of ILMs (PVR-ILMs: 18.18%, EMM-ILMs: 29.17%; p = 0.6855). Furthermore, EMMs (p = 0.0053; OR = 8.250) and EMM-ILMs (p = 0.0233; OR = 14.40) from patients with preoperative macular holes were more predisposed toward somatic mutations at the MDM2 T309G locus than those from patients without preoperative macular holes. Conclusions: MDM2 T309G is associated with the development of EMMs. Herein, the MDM2 SNP309 G allele is first reported as an associated factor of EMMs in a Chinese population. In addition, EMMs and ILMs are genetically unstable at the MDM2 T309G locus, especially when complicated with preoperative macular holes.

The roles of mouse double minute 2 (MDM2) oncoprotein in ocular diseases: A review.

Mouse double minute 2 (MDM2), an E3 ubiquitin ligase and the primary negative regulator of the tumor suppressor p53, cooperates with its structural homolog MDM4/MDMX to control intracellular p53 level. In turn, overexpression of p53 upregulates and forms an autoregulatory feedback loop with MDM2. The MDM2-p53 axis plays a pivotal role in modulating cell cycle control and apoptosis. MDM2 itself is regulated by the PI3K-AKT and RB-E2F-ARF pathways. While amplification of the MDM2 gene or overexpression of MDM2 (due to MDM2 SNP T309G, for instance) is associated with various malignancies, numerous studies have shown that MDM2/p53 alterations may also play a part in the pathogenetic process of certain ocular disorders. These include cancers (retinoblastoma, uveal melanoma), fibrocellular proliferative diseases (proliferative vitreoretinopathy, pterygium), neovascular diseases, degenerative diseases (cataract, primary open-angle glaucoma, age-related macular degeneration) and infectious/inflammatory diseases (trachoma, uveitis). In addition, MDM2 is implicated in retinogenesis and regeneration after optic nerve injury. Anti-MDM2 therapy has shown potential as a novel approach to treating these diseases. Despite major safety concerns, there are high expectations for the clinical value of reformative MDM2 inhibitors. This review summarizes important findings about the role of MDM2 in ocular pathologies and provides an overview of recent advances in treating these diseases with anti-MDM2 therapies.

Chalcomoracin prevents vitreous-induced activation of AKT and migration of retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells are the major cell type in the epi- or sub-retinal membranes in the pathogenesis of proliferative vitreoretinopathy (PVR), which is a blinding fibrotic eye disease and still short of effective medicine. The purpose of this study is to demonstrate whether Chalocomoracin (CMR), a novel purified compound from fungus-infected mulberry leaves, is able to inhibit vitreous-induced signalling events and cellular responses intrinsic to PVR. Our studies have revealed that the CMR IC50 for ARPE-19 cells is 35.5 µmol/L at 72 hours, and that 5 µmol/L CMR inhibits vitreous-induced Akt activation and p53 suppression; in addition, we have discovered that this chemical effectively blocks vitreous-stimulated proliferation, migration and contraction of ARPE-19 cells, suggesting that CMR is a promising PVR prophylactic.