Cystic Fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis trans membrane conductance regulator (CFTR) gene. The disease affects multiple organs, with lung disease producing most CF morbidity and mortality. Major progress has been made over the past decade in CF drug development. Greater than 90% patients now benefit from mono or combination therapy of several CFTR potentiators and correctors. However, these compounds neither permanently cure the disease nor address the unmet need of the 10% null mutation carriers. Gene editing therapy (GETx) represents a promising strategy to permanently cure the disease. In 2019 we reported efficient gene editing on major CFTR loci in human induced pluripotent stem(iPS) cells. In 2020, we reported the development of mi-spCas9 that has extraordinary homology directed repair capacity. Built on these two major lines of work, here we propose to develop and optimize a novel mi-saCas9 variant that is suitable for in vivo GETx of CF. In Aim 1, we will developmi-saCas9-KKH-A that is particular useful for correcting CF-causing CFTR mutations. In Aim 2we will test two GETx strategies for CF: (i) a large size gene knock-in strategy that is suitable for correcting all CFTR mutations; and (ii) a donor free strategy that is suitable for correcting compound heterozygous CFTR mutations. Successful completion of the proposed Phase I work will set a solid foundation for Phase II, in which we plan to evaluate the efficacy and safety of themi-saCas9-KKH-A based GETx strategies in preclinical CF animal models.
Public Health Relevance Statement: Project narrative: Cystic Fibrosis (CF) is a genetic disease caused by mutations in the CFTR gene, affecting multiple organs with lung disease producing most morbidity and mortality. Here we propose experiments to develop novel gene editing therapy strategies to correct different CFTR mutations, potentially with improved efficacy and safety outcome, to cure this devastating disease.
Project Terms: Dependovirus, Adeno-Associated Viruses, Dependoparvovirus, adeno associated virus group, Affect, Alleles, Allelomorphs, Amino Acids, aminoacid, Biological Assay, Assay, Bioassay, Biologic Assays, Complementary DNA, cDNA, Cells, Cell Body, Chromosomes, Combined Modality Therapy, Multimodal Therapy, Multimodal Treatment, combination therapy, combined modality treatment, combined treatment, multi-modal therapy, multi-modal treatment, Cystic Fibrosis, Mucoviscidosis, Disease, Disorder, Elements, Epithelial Cells, Exons, Fibroblasts, Foundations, Future, Genes, Regulator Genes, Transcriptional Regulatory Elements, regulatory gene, trans acting element, Human, Modern Man, Lung, Lung Respiratory System, pulmonary, Lung diseases, Pulmonary Diseases, Pulmonary Disorder, disease of the lung, disorder of the lung, lung disorder, Michigan, Morbidity - disease rate, Morbidity, mortality, Mutation, Genetic Alteration, Genetic Change, Genetic defect, genome mutation, Organoids, Patients, Peptides, Play, Domestic Rabbit, Rabbits, Rabbits Mammals, Oryctolagus cuniculus, Safety, S aureus, S. aureus, Staph aureus, Staphylococcus aureus, Swelling, Technology, Testing, Universities, Vision, Sight, visual function, Work, Cystic Fibrosis Transmembrane Conductance Regulator, CFTR, CFTR Protein, cystic fibrosis transmembrane regulator, Businesses, Mediating, Point Mutation, base, Organ, improved, Site, Solid, repaired, repair, Phase, Variant, Variation, Licensing, RAD51 protein, Rad51 recombinase, Sister, cell type, nuclease, homologous recombination, Animal Models and Related Studies, model of animal, model organism, Animal Model, novel, Reporting, Coding System, Code, drug development, Address, Preclinical Models, Pre-Clinical Model, in vivo, Small Business Technology Transfer Research, STTR, follow-up, Active Follow-up, active followup, follow up, followed up, followup, Development, developmental, pre-clinical, preclinical, insertion/deletion mutation, indel, insertion-deletion, insertion-deletion mutation, insertion/deletion, next generation, efficacy evaluation, efficacy analysis, efficacy assessment, efficacy examination, evaluate efficacy, examine efficacy, cystic fibrosis patients, CF patients, individuals with CF, individuals with cystic fibrosis, patients with CF, patients with cystic fibrosis, airway epithelium, Respiratory Epithelium, Structure of respiratory epithelium, adeno-associated viral vector, AAV vector, adeno-associated virus vector, Delta F508 mutation, F508 deletion, F508 mutation, F508-del, F508del, ÎF508, induced pluripotent stem cell, iPS, iPSC, iPSCs, inducible pluripotent stem cell, mutation carrier, disease-causing mutation, null mutation, Knock-in, knockin, experimental study, experiment, experimental research, recruit, Genetic Diseases, genetic condition, genetic disorder, therapeutic genome editing, gene-editing therapy, genome editing based therapy, genome editing therapy, genome editing treatment, genome editing-based therapeutics, therapeutic editing, rare genetic disorder, rare genetic disease, safety outcomes, efficacy outcomes, delivery vehicle, delivery vector
