Increasing NF1 Expression as a Treatment for NF1 Haploinsufficiency

增加 NF1 表达作为 NF1 单倍体不足的治疗方法

基本信息

批准号：
10304338
负责人：
Clifford Dustin Rubinstein
金额：
$ 5.2万
依托单位：
INFIXION BIOSCIENCE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-21 至 2021-04-17
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10304338
关键词：
Address Alleles Biological Assay Cell Line Cell Proliferation Cells Complex DNA Sequence Alteration Data Disease Progression Dominant Genetic Conditions ELK1 gene Engineering Environment Evaluation FRAP1 gene Gene Expression Genetic Diseases Genetic Transcription Goals Individual Libraries Luciferases MEKs Mutation NF1 gene Neurofibromatoses Neurofibromatosis 1 Obese Mice Other Genetics Pathway interactions Patients Pharmaceutical Preparations Play Proteins Proteomics Proto-Oncogene Proteins c-akt Publishing Ras Signaling Pathway Reporter Reporting Research Research Institute Role Safety Schwann Cells Signal Pathway Signal Transduction Specificity Supravalvular aortic stenosis Symptoms Synapses Therapeutic Tissues Transcription Coactivator Transcriptional Activation Transfection United States Viral Williams Syndrome associated symptom base drug candidate drug discovery drug repurposing induced pluripotent stem cell loss of function mRNA Expression mouse model mutant novel phase 2 testing protein expression screening small molecule small molecule libraries tool transcription factor transcriptome sequencing tumor

项目摘要

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.

项目摘要单倍体不足在神经纤维瘤病 (NF1)（一种常染色体显性遗传疾病）中起着至关重要的作用影响美国超过 120,000 人。目前的治疗方法侧重于特定的 NF1 信号传导的组成部分，例如抑制肿瘤中的 MEK 信号传导途径，因此无法解决与 NF1 相关的广泛信号传导和症状。鉴于 NF1 的特点是常染色体显性和单倍体不足（缺乏正常蛋白质），上调剩余的野生型 NF1 等位基因能够补偿突变等位基因的功能损失，因此缓解广泛的 NF1 症状和总体疾病进展。 Infixion提议建立一个荧光素酶报告测定可评估已知药物（包括 50 多种化合物）的 NF1 表达 Infixion 已确定六类药物与 NF1 表达增加相关。通过确认这些候选药物可激活 NF1 转录、增加 NF1 蛋白表达并使 Ras 正常化 NF1 +/- Schwann 细胞中的通路信号传导，并通过进一步筛选已知药物库以增加 NF1 表达，我们提出了一条 NF1 药物发现的新途径，这将影响广泛的 NF1 患者和症状，以预防的方式，而不考虑广泛的 NF1 基因突变。研究背景。通过转染增加 NF1 表达可逆转 Ras 异常激活 NF1 丢失所致（Wallis 等人，2018）。其他遗传条件下的转录激活，例如 Willams-Beuren 综合征和瓣膜上主动脉瓣狭窄可补偿单倍体不足（Giordano，等人。 2012）。最后，克服另一种常染色体显性遗传条件下的单倍体不足（Sim1 诱导肥胖;最近使用 Crispr/dCas9 转录激活剂（Ahituv 等人，2019）进行了展示。具体目标。 1) 构建一个荧光素酶报告基因检测，将其导入孔的内源性 NF1 基因中特征化、公开可用的永生化 NF1 +/- Schwann 细胞系（Wallace 等人，2016 年；数据已发表在 Synapse.org）。使用 Infixion 开发的病毒转录因子使用 Crispr/dCas9 验证检测上调 NF1。 2) 部署此 NF1 荧光素酶报告基因检测来筛选 Infixion 展示的 50 多种已知药物与各种细胞/组织类型中 NF1 表达增加相关。接下来，使用经过验证的荧光素酶记者筛选 Scripps Research 提供的 13,000 多种已知药物的化合物再利用库研究所（称为 ReFrame）。 3) 将利用不朽的内容来评估 Aim 2 屏幕上的热门歌曲和 iPSC 衍生的 NF1+/- Schwann 细胞，具有以下特点：a) 诱导 NF1 mRNA 和蛋白质的能力表达，使用 qPCR 和 Westerns，b) 利用 a) 对 Ras 信号传导（pERK、ELK-1、AKT 等）的影响靶向定量质谱蛋白质组学测定，c) 它们对雪旺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) 基于已发表数据的安全性概况以前的试验。目标是优先考虑不超过 3-5 名候选人进入第二阶段评估。