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性疾病，影响 1 % 3000 名新生儿，包括全球超过 200 万人，患有“良性”肿瘤、骨发育不良、 认知/社交缺陷，以及非常容易患癌症和心血管疾病。当前 治疗方法针对 NF1 信号传导的特定下游成分，例如 MEK 抑制 在肿瘤中，未能解决与 NF1 相关的广泛信号传导和症状。鉴于 NF1 其特点是单倍体不足（例如，缺乏功能性 NF1 蛋白），并且 miRNA 已被 显示可以调节 NF1 蛋白水平（Paschou 2012；Zhu 2020），我们在此建议开发特定的 RNA- 靶向反义寡核苷酸（ASO）促进 NF1 蛋白水平增加，目的是 使下游信号（Ras 等）正常化，从而从根本上缓解 NF1 症状。 我们建议将此作为 NF1 药物发现的新途径，对广泛的 NF1 患者具有潜在影响 和症状，以预防的方式，并且适用于广泛的独特 NF1 基因突变已经确定。 研究背景。通过转染增加 NF1 蛋白表达已被证明可以逆转 NF1 蛋白丢失导致 Ras 异常激活（Wallis，2018；Mellert，2018）。另外，增加蛋白质 在其他遗传疾病中的表达，例如 Willams-Beuren 综合征和瓣上主动脉瓣狭窄， 补偿单倍体不足（Giordano，2012）。最后，克服其他常染色体的单倍体不足 显性条件（Sim1；Pax6 基因）已显示出体内纠正症状的能力（Matharu 等人，2019 年； 拉比，2020）。 NF1 表达受多种 miRNA 调节，提供了识别和靶向的机会 调节关键细胞类型中 NF1 蛋白表达的 miRNA。 RNA 靶向 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。 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 候选 miRNA，并在 SA1 目标中确定每个 miRNA 调节三个中 NF1 表达的能力 与 NF1 相关的关键细胞类型。 2) 基于顶级 miRNA 候选者，设计并验证空间阻断 ASO 在 NF1 疾病相关细胞类型中选择性阻断 miRNA 介导的 NF1 表达抑制。验证 三种 NF1 相关细胞类型的 NF1 蛋白表达增加。 3) NF1表达一旦增加 蛋白建立后，验证 NF1 空间阻断 ASO 进一步使 NF1 依赖性信号传导正常化 永生化雪旺细胞、原代成纤维细胞和 iPSC 衍生的 NF1/- 细胞，评估以下内容：a) 对 Ras/mTOR 信号传导（pERK、ELK-1、AKT 等），b) 对细胞增殖的影响。计划目标是优先考虑 2-3 个候选 ASO 用于 SBIR 第 2 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.