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

项目摘要。神经纤维瘤病(NF1)是一种常染色体显性遗传疾病，影响1 3000名新生儿，包括全球200多万人，症状包括良性肿瘤，骨发育不良， 认知/社交缺陷，以及癌症和心血管疾病的极高易感性。当前 治疗方法针对NF1信号的特定下游成分，例如MEK抑制 在肿瘤方面，未能解决与NF1相关的广泛信号和症状。鉴于NF1 以单倍体不足为特征(例如，缺乏功能性NF1蛋白)，而且miRNA已经 显示调节NF1蛋白水平(Paschou 2012；朱2020)，我们在这里建议开发特定的RNA- 靶向反义寡核苷酸(ASO)以促进NF1蛋白水平的增加，目标是 标准化下行信令(RAS等)从而从根本上缓解NF1症状。 我们建议这是一条发现NF1药物的新途径，对广泛的NF1患者具有潜在的影响 和症状，以预防的方式，并且适用于独特的NF1的广泛范围 已经确定的基因突变。 研究背景。通过转基因增加NF1蛋白的表达已被证明是逆转的 NF1蛋白丢失导致的RAS异常激活(Wallis，2018；Mellert，2018)。此外，蛋白质含量增加 在其他遗传性疾病中的表达，如Willams-Beuren综合征和瓣膜上动脉狭窄， 弥补单倍体不足(佐丹诺，2012)。最后，克服其他常染色体单倍体不足 显性条件(Sim1；Pax6基因)在体内显示出纠正症状的能力(Matharu，et.艾尔2019年； Rabiee，2020)。NF1的表达受多个miRNAs调控，为识别和靶向提供了机会 在关键细胞类型中调节NF1蛋白表达的miRNAs。以RNA为靶点的ASO的开发可能会引领 选择性地阻断这种抑制并纠正NF1单倍体不足。 明确的目标。1)在计算机分析(例如，TargetScan应用程序和出版评论)中，我们确定了34 候选miRNAs，并在SA1靶标中确定每个miRNAs调控NF1表达的能力 与NF1相关的关键细胞类型。2)基于热门的miRNA候选序列，设计并验证空间阻断ASO 选择性地阻断miRNA介导的对NF1疾病相关细胞类型中NF1表达的抑制。验证 NF1蛋白在三种NF1相关细胞类型中的表达增加。3)一旦NF1表达增加 蛋白质被建立，验证NF1空间阻断ASO进一步正常化NF1依赖的信号利用 永生化雪旺细胞、原代成纤维细胞和IPSC来源的NF1/-细胞，评估以下方面：a)对 RAS/mTOR信号(PERK、ELK-1、AKT等)，以及b)对细胞增殖的影响。计划目标是确定优先顺序 2-3个候选ASO进行SBIR第二阶段IND前评估。

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.