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.

Prime Editing of Vascular Endothelial Growth Factor Receptor 2 Attenuates Angiogenesis In Vitro.

Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro. Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo.

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.