Increasing NF1 Expression as a Treatment for NF1 Haploinsufficiency

工程摘要 单倍不足在神经纤维瘤病（NF 1）中起着至关重要的作用，NF 1是一种常染色体显性遗传病 影响了美国超过12万人目前的治疗方法集中在特定的 NF 1信号传导的组分，例如抑制肿瘤中的MEK信号传导途径，因此未能解决 与NF 1相关的广泛的信号传导和症状。假设NF 1的特征在于 常染色体显性和单倍不足（缺乏正常蛋白质），上调蛋白质的表达， 剩余的野生型NF 1等位基因具有补偿突变等位基因功能丧失的能力，因此 缓解广泛的NF 1症状和整体疾病进展。Infixion建议建立一个 荧光素酶报告试验，以评估针对已知药物的NF 1表达，包括50+化合物， 六种药物类别，已经被Infixion鉴定为与增加的NF 1表达相关。通过确认 这些候选药物激活NF 1转录，增加NF 1蛋白表达，并使Ras正常化， NF 1 +/-雪旺细胞中的NF 1通路信号传导，并通过进一步筛选已知药物库， NF 1表达，我们提出了一种新的NF 1药物发现途径，将影响广泛的NF 1患者 和症状，以预防的方式，而不考虑广泛的NF 1基因突变。 研究背景。通过转染增加NF 1表达逆转异常Ras活化 由NF 1损失引起（沃利斯等人，2018）。在其他遗传条件下的转录激活，如 Willams-Beuren综合征和瓣上主动脉狭窄补偿了单倍功能不全（Giordano， 等，2012年）。最后，克服另一种常染色体显性遗传条件下的单倍不足（Sim 1诱导）， 肥胖症;小鼠模型）最近显示使用Crispr/dCas 9转录激活剂（Ahituv等人，2019）。 具体目标。1)构建工程化到孔的内源性NF 1基因中的荧光素酶报告基因测定 特征性的、可获得的永生化NF 1 +/-雪旺细胞系（Wallace et al. 2016;数据发表于 在Synapse.org）。使用由Infixion开发的病毒转录因子，使用Crispr/dCas 9进行PCR检测， 上调NF 1。2)部署这种NF 1荧光素酶报告基因检测来筛选Infixion显示的50多种已知药物， 与各种细胞/组织类型中增加的NF 1表达相关。接下来，使用这种经过验证的荧光素酶 报告人筛选了Scripps Research提供的13，000多个已知药物的化合物再利用库 研究所（称为ReFrame）。3)将评估Aim 2屏幕中的热门歌曲， 和iPSC衍生的NF 1 +/-雪旺细胞，用于以下方面： 表达，B）对Ras信号传导（pERK、ELK-1、AKT等）的影响。利用 靶向定量质谱蛋白质组学测定，c）它们对雪旺氏细胞中基因表达的更广泛影响， 利用RNAseq的细胞，d）对细胞增殖的影响，以及e）基于来自以下的已发表数据的它们的安全性特征： 以前的审判。目标是优先考虑不超过3-5名候选人进入第2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.