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

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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.