Bao, Gang2022-09-232022-09-232022-052022-06-01May 2022Chokshi, Mithil. "Genome Editing Strategies to Cure Cystic Fibrosis." (2022) Diss., Rice University. <a href="https://hdl.handle.net/1911/113325">https://hdl.handle.net/1911/113325</a>.https://hdl.handle.net/1911/113325Cystic Fibrosis (CF) is a recessive genetic disorder with fatal consequences caused by over 1,700 mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene. We studied gene editing in patient-derived induced pluripotent stem cells (iPSCs) by correcting the most prevalent disease-causing mutation, F508del, a 3-bp deletion that prevents CFTR protein to fold properly. The challenges in editing primary and stem cells have caused current approaches to focus on using drug-based selection and fluorescence-based enrichment of the edited cells. We systematically optimized different parameters to obtain highest reported repair rate of 20% allelic HDR at the F508del locus in the iPSCs and conducted an extensive search for off-target activity of the tested gRNA. The cells were then differentiated to epithelia to show recovery of CFTR expression and function. We further optimized the CRISPR / Cas9 machinery to increase the repair rates to above 50%, which is comparable or higher than virus-based editing approaches. To translate our approaches in vivo we are further engineering AAVs to target lung cell types of interest for delivery of the optimized gene editing machinery for an in vivo therapy. These approaches were tested in novel gene-edited human bronchial epithelia-based cell models containing a halide-sensitive fluorescence reporter to conduct high-throughput assays for the CFTR channel activity. We have made such cell models for multiple classes of CF to enable faster discovery of new therapies to cure CF. Finally, CF is a multifaceted disease with fatal lung inflammation and infection cycles; we use gene edited iPSCs developed above to test the hypothesis that CF lung macrophages are hyperinflammatory and poorly bactericidal to perpetuate the inflammation and infection.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.Cystic FibrosisGene EditingYFPMacrophagesiPSCsCFTRCRISPR / Cas9Thesis2022-09-23