Steric-blocking AntiSense Oligonucleotide (ASO) Discovery to Selectively Correct NF1 Haploinsufficiency

项目摘要。神经纤维瘤病（NF 1）是一种常染色体显性遗传病， 3000名新生儿，包括全球超过200万人，症状包括“良性”肿瘤，骨发育不良， 认知/社交缺陷，以及对癌症和心血管疾病的高度易感性。电流 治疗方法靶向NF 1信号传导的特定下游组分，例如MEK抑制 在肿瘤中，未能解决与NF 1相关的广泛信号传导和症状。由于NF 1 特征在于单倍不足（例如，缺乏功能性NF 1蛋白），而且已经 显示调节NF 1蛋白水平（Paschou 2012; Zhu 2020），我们在此提出开发特异性RNA- 靶向反义寡核苷酸（ASO），以促进增加NF 1蛋白水平，目的是 归一化下游信号（Ras等）从而减轻疾病根源的NF 1症状。 我们提出这是一种新的NF 1药物发现途径，对广泛的NF 1患者有潜在的影响。 和症状，以预防的方式，和一个适用于广泛的独特的NF 1 基因突变已经被发现。 研究背景。通过转染增加NF 1蛋白表达已经显示逆转 NF 1蛋白丢失导致的Ras异常激活（沃利斯，2018; Mellert，2018）。此外，蛋白质增加 在其他遗传性疾病如Willams-Beuren综合征和主动脉瓣上狭窄中的表达， 补偿单倍不足（Giordano，2012）。最后，克服其他常染色体单倍不足， 显性条件（Sim 1; Pax 6基因）已经显示出在体内纠正症状的能力（Matharu，et. 2019年; Rabiee，2020）。NF 1的表达受多种miRNAs的调控，这为识别和靶向NF 1提供了机会。 在关键细胞类型中调节NF 1蛋白表达的miRNA。RNA靶向阿索的发展可能导致 选择性地阻断这种抑制并纠正NF 1单倍不足。 具体目标。1)使用计算机分析（例如，TargetScan应用程序和出版物评论），我们确定了34个 候选miRNAs，并在SA 1靶点中确定每种miRNAs在三种不同的靶点中调节NF 1表达的能力。 与NF 1相关的关键细胞类型。2)基于最佳miRNA候选物，设计并验证空间阻断ASO 其选择性阻断NF 1疾病相关细胞类型中NF 1表达的miRNA介导的抑制。验证 在三种NF 1相关细胞类型中增加NF 1蛋白表达。3)一旦NF 1表达增加 蛋白质的建立，验证了NF 1空间阻断ASO进一步正常化NF 1依赖性信号转导， 永生化的雪旺氏细胞、原代成纤维细胞和iPSC衍生的NF 1 +/-细胞，评估以下： Ras/mTOR信号传导（pERK、ELK-1、AKT等），和B）对细胞增殖的影响。计划目标是优先考虑 2-3 SBIR II期IND前评价的候选ASO。

PROJECT ABSTRACT. Neurofibromatosis (NF1) is an autosomal dominant genetic disorder impacting 1 in 3000 births, including over 2M people worldwide, with symptoms including ‘benign’ tumors, bone dysplasia, cognitive/social deficits, and a very high pre-disposition to both cancers and cardiovascular disease. Current therapeutic approaches target specific downstream components of NF1 signaling, for example MEK inhibition in tumors, failing to address the broad range of signaling and symptoms associated with NF1. Given that NF1 is characterized by haploinsufficiency (e.g., lack of functional NF1 protein), and also that miRNA's have been shown to regulate NF1 protein levels (Paschou 2012; Zhu 2020), we propose here to develop specific RNA- targeted anti-sense oligonucleotides (ASOs) to promote increased NF1 protein levels, with the goal of normalizing downstream signaling (Ras, etc.) and thus alleviating NF1 symptoms at the root cause of disease. We propose this as a novel path of NF1 drug discovery, with potential impact on a broad range of NF1 patients and symptoms, in a preventative manner, and one that is applicable to the wide spectrum of unique NF1 genetic mutations already identified. Research Background. Increasing NF1 protein expression via transfection has been shown to reverse abnormal Ras activation resulting from NF1 protein loss (Wallis, 2018; Mellert, 2018). Also, increased protein expression in other genetic conditions such as Willams-Beuren Syndrome, and Supravalvular Aortic Stenosis, compensates for haploinsufficiency (Giordano, 2012). Lastly, overcoming haploinsufficiency in other autosomal dominant conditions (Sim1; Pax6 genes) has shown an ability in vivo to correct symptoms (Matharu, et. al. 2019; Rabiee, 2020). NF1 expression is regulated by multiple miRNAs, providing the opportunity to identify and target miRNAs that regulate NF1 protein expression in key cell types. Development of RNA-targeted ASO’s could lead to selectively blocking this repression and correcting NF1 haploinsufficiency. Specific Aims. 1) Using in silico analysis (e.g., TargetScan application and publication reviews) we identify 34 candidate miRNAs, and in SA1 target determining the ability of each to regulate NF1 expression across three key cell types relevant to NF1. 2) Based on top miRNA candidates, design and validate steric-blocking ASOs that selectively block miRNA-mediated inhibition of NF1 expression in NF1 disease-relevant cell types. Validate increased NF1 protein expression across three NF1 relevant cell types. 3) Once increased expression of NF1 protein is established, validate that NF1 steric-blocking ASOs further normalize NF1-dependent signaling utilizing immortalized Schwann, primary fibroblast and iPSC-derived NF1+/- cells, evaluating the following: a) impact on Ras/mTOR signaling (pERK, ELK-1, AKT, etc.), and b) impact on cell proliferation. Program goal is to prioritize 2-3 candidate ASOs for an SBIR Phase 2 pre-IND evaluation.