Increasing NF1 Expression as a Treatment for NF1 Haploinsufficiency

项目摘要 单倍体功能不全在神经纤维瘤病(NF1)中起着关键作用，NF1是一种常染色体显性遗传病 影响到美国超过12万人。目前的治疗方法主要集中在特定的 NF1信号的组成部分，例如抑制肿瘤中的MEK信号通路，因此无法解决 与NF1相关的广泛信号和症状。鉴于NF1的特点是既有 常染色体显性和单倍体不足(缺乏正常蛋白质)，上调蛋白表达 剩余的野生型nf1等位基因有能力补偿突变等位基因造成的功能损失，因此 缓解广泛的NF1症状和总体疾病进展。Infix提议建立一个 荧光素酶报告试验评估NF1在包括50+化合物在内的已知药物中的表达 Infix已经确定了六种与NF1表达增加相关的药物类别。通过确认 这些候选药物激活NF1转录，增加NF1蛋白表达，并使RAS正常化 NF1+/-雪旺细胞中的通路信号，并通过进一步筛选已知药物库中增加的 NF1的表达，我们提出了一条新的NF1药物发现途径，将影响广泛的NF1患者 和症状，以预防的方式，而不考虑NF1基因突变的广泛范围。 研究背景。通过转基因提高NF1的表达逆转RAS的异常激活 由NF1的损失造成(Wallis等人2018年)。在其他遗传条件下的转录激活，如 Willams-Beuren综合征和瓣膜上动脉狭窄补偿单倍体功能不全(Giordano， 等人的研究。2012年)。最后，在另一常染色体显性条件下克服单倍性不足(Sim1诱导 肥胖；小鼠模型)最近使用Crispr/dCas9转录激活子(Ahituv等人)显示。2019年)。 明确的目标。1)构建内源NF1基因工程荧光素酶报告基因检测 表征的，公共可用的，永生化的NF1+/-Schwann细胞系(Wallace等人2016年；发布数据 在Synapse.org)。使用Crispr/dCas9验证使用Infix开发的病毒转录因子进行检测 上调NF1。2)将该NF1荧光素酶报告试验用于筛选Infix显示的50多种已知药物，以 与不同类型细胞/组织中NF1表达的增加有关。接下来，使用这个经过验证的荧光素酶 记者将筛选斯克里普斯研究公司提供的13,000多种已知药物的化合物再用途库 研究所(称为重框)。3)来自Aim 2屏幕的最热门的点击量将被评估，使用两个不朽的 和IPSC来源的NF1+/-Schwann细胞，用于以下方面：a)诱导NF1 mRNA和蛋白的能力 B)对RAS信号的影响(PERK、ELK-1、AKT等)利用一种 靶向定量质谱学蛋白质组学分析，c)它们对雪旺基因表达的更广泛影响 使用RNAseq的细胞，d)对细胞增殖的影响，以及e)基于来自 之前的试验。目标是对不超过3-5名候选人进行优先排序，以进入第二阶段评估。

Project Abstract Haploinsufficiency plays a crucial role in Neurofibromatosis (NF1), an autosomal dominant genetic disorder impacting over 120,000 individuals in the United States. Current therapeutic approaches focus on specific components of NF1 signaling, for example inhibiting MEK signaling pathways in tumors, thus failing to address the broad range of signaling and symptoms associated with NF1. Given that NF1 is characterized by both autosomal dominance and haploinsufficiency (lack of normal protein), upregulating protein expression of the remaining wild-type NF1 allele has the ability to compensate for loss of function from the mutant allele, thus alleviating a broad range of NF1 symptoms and overall disease progression. Infixion proposes to build a luciferase reporting assay to evaluate NF1 expression against known drugs, including 50+ compounds, across six drug classes, already identified by Infixion to correlate with increased NF1 expression. By confirming that these candidate drugs activate NF1 transcription, increase NF1 protein expression, and normalize Ras pathway signaling in NF1 +/- Schwann cells, and by further screening libraries of known drugs for increased NF1 expression, we propose a novel path of NF1 drug discovery that will impact a broad range of NF1 patients and symptoms, in a preventative manner, and without regard to the wide spectrum of NF1 genetic mutations. Research Background. Increasing NF1 expression via transfection reverses abnormal Ras activation resulting from NF1 loss (Wallis, et al. 2018). Transcriptional activation in other genetic conditions such as Willams-Beuren Syndrome, and Supravalvular Aortic Stenosis compensates for haploinsufficiency (Giordano, et al. 2012). Lastly, overcoming haploinsufficiency in another autosomal dominant condition (Sim1 induced obesity; mouse model) was recently shown using a Crispr/dCas9 transcriptional activator (Ahituv, et al. 2019). Specific Aims. 1) Construct a luciferase reporter assay engineered into the endogenous NF1 gene of a well characterized, publically available, immortalized NF1 +/- Schwann cell line (Wallace et al. 2016; data published at Synapse.org). Validate assay using a viral transcription factor developed by Infixion using Crispr/dCas9 to upregulate NF1. 2) Deploy this NF1 luciferase reporter assay to screen 50+ known drugs shown by Infixion to correlate with increased NF1 expression across various cell/tissue types. Next, use this validated luciferase reporter to screen a 13,000+ compound repurposing library of known drugs available from Scripps Research Institute (known as ReFrame). 3) The top hits from Aim 2 screens will be evaluated, utilizing both immortalized and iPSC derived NF1+/- Schwann cells, for the following: a) ability to induce NF1 mRNA and protein expression, using qPCR and Westerns, b) the impact on Ras signaling (pERK, ELK-1, AKT, etc.) utilizing a targeted quantitative mass spec proteomics assay, c) their broader impact on gene expression in Schwann cells utilizing RNAseq, d) impact on cell proliferation, and e) their safety profile based on published data from previous trials. The goal is to prioritize not more than 3-5 candidates to take forward into a Phase 2 evaluation.