CRISPRa-Based Strategies for NF1
Abstract
This project is proposed as a collaboration between the Walker Lab at the Center for Genomic Medicine, Massachusetts General Hospital, and Infixion Bioscience, a drug discovery startup focused on developing drugs to radically improve life for NF1 patients by correcting a known root cause of symptoms, namely the insufficient amount of normal (wild-type) NF1 protein. NF1 Genetic Background: Like other human genes, the NF1 gene has both a paternal and maternal copy. For people who do not have an NF1 mutation, each NF1 gene copy (or allele) produces ~50% of the NF1 protein (neurofibromin) needed to be healthy. However, for people with NF1, one of these two NF1 alleles is abnormal, producing either defective or no protein. The result is much less wild-type neurofibromin protein in every cell of the body. NF1 is considered an autosomal dominant genetic disorder because this abnormality in just one gene allele is enough to cause NF1 symptoms. Biological theory suggests that neurofibromin from this mutant allele either directly causes NF1 symptoms (e.g., is pathogenic) or that the lack of wild-type protein is the root cause. The lack of a normal protein leading to disease is referred to as haploinsufficiency. The idea to keep in mind is that people with NF1 do produce wild-type NF1 protein, but their one normal gene simply doesn’t make enough. Rationale for Approaching NF1 by Targeting Haploinsufficiency: Many scientific publications have provided solid evidence that most NF1 symptoms are either caused directly by NF1 protein haploinsufficiency (i.e., cognitive/social deficits), or that this haploinsufficient condition drives symptom progression (i.e., tumor progression). It’s also well accepted that NF1 haploinsufficiency leads to dysregulation of many biological pathways (i.e., Ras/Mapk, HCN1, CRMP2, etc.), further explaining the wide variety of symptoms experienced by people with NF1. By correcting the underlying NF1 protein deficiency, the possibility exists to improve a wide range of NF1 symptoms. Additional research into other autosomal dominant and haploinsufficient genetic disorders, such as Willams-Beuren Syndrome, Supravalvular Aortic Stenosis and Sim1 (Giordano, et al.; Matharu, et al.), has shown that upregulation of a single normal allele can lead to improving symptoms. More specifically, the ability to correct dysregulation by simply eliminating haploinsufficiency has been demonstrated at the cellular level for NF1 (Mellert, et al., Wallis, et al.). Finally, the mechanisms that regulate gene expression from the NF1 allele have been well characterized, with clear evidence that NF1 gene expression can be regulated by several biological agents and mechanisms, including cytokines, histone acetylation, DNA methylation, and peptide hormones. So the approach proposed here is absolutely feasible. Research Goals: Upregulating expression of neurofibromin in a variety of tissues to correct the underlying root cause of NF1, rather than treating individual symptoms or downstream pathway, is predicted to impact patients possessing any of the over 4,000 unique NF1 mutations already identified. Given this background, this research proposes to develop and study a novel CRISPR-based reagent to increase neurofibromin expression from the wild-type NF1 allele, thus compensating for the lack of normal NF1 protein produced by the mutated allele, and then to test this new reagent in NF1 patient derived cells, and in a mouse model of NF1. Project Research Aims: Using a cutting-edge technique (Gro-Seq), we will first study exactly where production of NF1 protein begins (aka. transcription start site). Using this information will use CRISPRa/dCas9 (gene activating) technology, along with a proprietary reporter cell line from Infixion Bioscience, to identify the ideal locations in the NF1 gene promoter region that can be activated to increase NF1 gene expression and subsequent protein production. Finally, in Ai
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 04, 2024
- Source ID
- HT94252310490
Entities
People
- James Walker
Organizations
- Massachusetts General Hospital
- United States Army