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

发现立体阻断反义寡核苷酸 (ASO) 以选择性纠正 NF1 单倍体不足

基本信息

批准号：
10789739
负责人：
Michelle Mattson-Hoss
金额：
$ 3.88万
依托单位：
INFIXION BIOSCIENCE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10789739
关键词：
Address Antisense Oligonucleotides Benign Birth Cardiovascular Diseases Cell Proliferation Cells Cognitive Compensation Complex DNA Sequence Alteration Development Developmental Bone Diseases Disease Dominant Genetic Conditions ELK1 gene Evaluation FRAP1 gene Fibroblasts Genes Genetic Diseases Goals In Vitro MAP Kinase Gene MEK inhibition Malignant Neoplasms Mediating MicroRNAs Mutation Neurofibromatoses Neurofibromatosis 1 Other Genetics Patients Persons Phase Predisposition Proteins Proto-Oncogene Proteins c-akt Publications Publishing RNA Repression Research Signal Pathway Signal Transduction Small Business Innovation Research Grant Supravalvular aortic stenosis Symptoms Therapeutic Transfection Williams Syndrome associated symptom autosome cell type design drug development drug discovery improved in silico in vivo induced pluripotent stem cell novel programs protein expression social deficits targeted treatment therapeutic candidate tumor

Address Antisense Oligonucleotides Benign Birth Cardiovascular Diseases Cell Proliferation Cells Cognitive Compensation Complex DNA Sequence Alteration Development Developmental Bone Diseases Disease Dominant Genetic Conditions ELK1 gene Evaluation FRAP1 gene Fibroblasts Genes Genetic Diseases Goals In Vitro MAP Kinase Gene MEK inhibition Malignant Neoplasms Mediating MicroRNAs Mutation Neurofibromatoses Neurofibromatosis 1 Other Genetics Patients Persons Phase Predisposition Proteins Proto-Oncogene Proteins c-akt Publications Publishing RNA Repression Research Signal Pathway Signal Transduction Small Business Innovation Research Grant Supravalvular aortic stenosis Symptoms Therapeutic Transfection Williams Syndrome associated symptom autosome cell type design drug development drug discovery improved in silico in vivo induced pluripotent stem cell novel programs protein expression social deficits targeted treatment therapeutic candidate tumor

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项目摘要

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.

项目摘要。神经纤维瘤病（NF1）是一种影响1的常染色体显性遗传疾病 3000个出生，包括全球超过200万人的症状，包括“良性”肿瘤，骨骼发育不良，认知/社会定义了对癌症和心血管疾病的非常高的前置态。当前的治疗方法的目标是NF1信号的特定特定下游组件，例如MEK抑制在肿瘤中，无法解决与NF1相关的广泛信号传导和符号。鉴于NF1 具有单倍弥补的特征（例如，缺乏功能性NF1蛋白），并且miRNA的特征是证明可以调节NF1蛋白水平（Paschou 2012; Zhu 2020），我们在这里提议开发特定的RNA- 靶向抗义寡核苷酸（ASO）以促进NF1蛋白水平升高，目的是使下游信号传导（RAS等）归一化，从而减轻了疾病根本原因的NF1症状。我们提出这是NF1药物发现的新路径，对广泛的NF1患者有潜在的影响和符号，以预防性的方式，并且适用于广泛的独特NF1 遗传突变已经鉴定出来。研究背景。通过翻译的增加NF1蛋白表达已被证明是反向的 NF1蛋白损失引起的异常RAS激活（Wallis，2018； Mellert，2018）。另外，蛋白质增加在其他遗传条件下的表达，例如Willams-Beuren综合征和上主动脉狭窄，补偿单倍弥补（Giordano，2012年）。最后，在其他常染色体中克服单倍不足主要条件（SIM1； PAX6基因）表现出体内能力纠正症状的能力（Matharu，etal。2019; Rabiee，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。靶向RNA的ASO的开发可能会导致有选择地阻止此表达式并纠正NF1单倍度不足。具体目标。 1）在计算机分析中使用（例如TargetScan应用和出版评论）我们确定34 候选miRNA，在SA1目标中确定每个人在三个中调节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，设计和验证空间阻滞ASOS 在NF1疾病相关的细胞类型中，有选择地阻止miRNA介导的NF1表达抑制。证实在三种NF1相关细胞类型中增加了NF1蛋白的表达。 3）一旦增加了NF1的表达建立蛋白质，验证NF1的阻滞ASOS进一步使使用NF1依赖性信号归一化永生的Schwann，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）和B）对细胞增殖的影响。计划目标是优先 2-3 SBIR 2期预先评估的候选ASO。