Developing a therapy for Marfan Syndrome using CRISPR-based mRNA translation enhancement system

Marfan Syndrome (MFS) is an autosomal dominant disorder characterized by cardiovascular phenotypes and caused by mutations in Fibrillin-1 (FBN1) gene. Approximately 40% of MFS cases are haploinsufficient. The aim of this project is to increase mRNA translation of fibrillin-1 in cell lines derived from patients with MFS. For these studies, I propose to develop a CRISPR-based RNA-targeting translation enhancement system to rescue the molecular and cellular phenotype produced by FBN1 loss of function (LOF) mutation. The specific aims are: 1) To activate FBN1 mRNA translation in human cells with CRISPR/Cas13 2) To rescue FBN1 haploinsufficiency in Marfan Syndrome patient-derived iPSCs and vascular smooth muscle cells (VSMCs) 3) To use allele-specific mRNA targeting to restore fibrillin-1 function in Marfan Syndrome cells carrying dominant negative mutations This project will involve the use of both experimental and computational techniques. The research will require flow cytometry, fluorescence microscopy, recombinant DNA cloning, mammalian cell culture, including iPSC generation and VSMC differentiation from patient derived cells, RNA-seq, ribosome profiling, qPCR, Western blotting, gRNA library preparation, and other techniques. Reporter assays will be completed in HEK293 cells. Computationally, the project will involve developing machine-learning models for predicting gRNA specificity. The objective of this project is to achieve robust upregulation of mRNA translation of FBN1 without affecting translation or abundance of other mRNAs. I will use a fluorescence reporter for high-throughput screening during optimization. Once optimized, the goal is to use patient derived cell lines with mutations in FBN1, and attempt rescue of molecular phenotype. Finally, the project will aim for targeting specific alleles in missense mutation background of FBN1 through development of deep learning models of gRNA mismatch tolerance. (AHA Program: Postdoctoral Fellowship)