Development of a versatile paired prime editing toolkit for CFTR gene correction - PROJECT SUMMARY Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene are the primary cause of cystic fibrosis (CF) and are associated with many other genetic diseases. Thus, CFTR is a compelling therapeutic target for treating various CFTR-related disorders. Genome editing techniques can correct the defective genes at their native locus and permanently restore the gene function, potentially offering a one-time curative treatment for CF. However, given that over 1,700 CF-causing mutations have been identified in the CFTR gene, mutation- specific genome editing methods, such as base editing, may not be suitable for correcting the wide range of CFTR mutations. To address these limitations, we propose to develop a more generalizable genome editing strategy for CFTR correction. Because most CFTR mutations occur within the exons or at the exon-intron boundary, we propose to replace the frequently mutated exons with their wild-type counterpart. Therefore, a single set of gene editing agents can correct all the mutations within the same exon. Prime editing (PE) employs an extended guide RNA (pegRNA) as a template for a conjugated reverse transcriptase to incorporate desired edits into the targeted genomic site. Recently, we devised a paired prime editing strategy to program the replacement of target genomic sequences without requiring a DNA donor in vitro and in vivo. Building upon this work, the goal of this proposal is to develop paired prime editing-based sequence replacement strategies to correct a broad spectrum of CFTR mutations, regardless of the mutation types or locations. We hypothesize that replacing the defective CFTR exons with corrected ones will effectively restore CFTR protein expression and function in vitro and in vivo. In the proposed research, we will utilize a donor-free sequence replacement strategy to replace individual CFTR exons without creating double-stranded DNA breaks or requiring a DNA donor (Aim 1). To further expand the editing scope and efficiency of the paired prime editing approaches in inserting or replacing large DNA sequences, we will develop a paired PE-based homology-independent insertion strategy and apply it to correct CFTR mutations caused by large deletions or occurred in the large exons (Aim 2). Finally, we will explore the therapeutic applications of the newly-established paired prime editing-mediated gene correction strategy by delivering the genome editing agents to the lungs of a CF mouse model (Aim 3). Successful completion of this project will result in a gene therapy strategy tailored for CF and provide a universal gene correction framework for treating genetic diseases of high allelic heterogeneity.
