Efficient Gene Editing at Major CFTR Mutation Loci

Abstract

Cystic fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Nuclease-mediated precise gene editing (PGE) represents a promising therapy for CF, for which an efficient strategy that is free of viral vector, drug selection, and reporter enrichment (VDR free) is desirable. Here we compared different transfection methods (lipofectamine versus electroporation) and formats (plasmid DNA versus ribonucleoprotein) in delivering the CRISPR/Cas9 elements along with single-stranded oligodeoxynucleotides (ssODNs) to clinically relevant cells targeting major CFTR mutation loci. We demonstrate that, among different combinations, electroporation of CRISPR/Cas9 and guide RNA (gRNA) ribonucleoprotein (Cas9 RNP) is the most effective one. By using this VDR-free method, 4.8% to 27.2% efficiencies were achieved in creating dF508, G542X, and G551D mutations in a wild-type induced pluripotent stem cell (iPSC) line. When it is applied to a patient-derived iPSC line carrying the dF508 mutation, a greater than 20% precise correction rate was achieved. As expected, genetic correction leads to the restoration of CFTR function in iPSC-derived proximal lung organoids, as well as in a patient-derived adenocarcinoma cell line CFPAC-1. The present work demonstrates the feasibility of gene editing-based therapeutics toward monogenic diseases such as CF.

Application of Genetically Engineered Pigs in Biomedical Research

“…Currently, the research focuses on improving the CF gene therapy with the use of the CRISPR/Cas9 system. These efforts are focused on increasing the delivery efficiency of CRISPR/Cas9 elements to target locus and obtaining sustained expression of the CFTR transgene [19,20]. It was demonstrated that precise integration of the human CFTR gene at a porcine safe harbor locus through CRISPR/Cas9-induced HDR-mediated knock-in allowed the achievement of persistent in vitro expression of the transgene in transduced cells.…”

Gene editing and CRISPR in the clinic: current and future perspectives

“…Genome editors present a new possibility for therapeutic treatment of CF, although the low efficacy with gene therapy delivery agents studied to date may also apply to the delivery of genome editors. Repair of CF mutations has been demonstrated with ZFNs and Cas9 in vitro as well as in patient induced pluripotent stem cells [133][134][135][136][137][138][139][140], although testing in animal models has not yet been performed. CF is an interesting and challenging disease as the CF transmembrane conductance regulator (CTFR) gene is very large and there are numerous mutations associated with the disease.…”

“…CF is an interesting and challenging disease as the CF transmembrane conductance regulator (CTFR) gene is very large and there are numerous mutations associated with the disease. Several gene editing strategies have been used to treat CF including Cas9 targeted removal of mutations that lead to nonfunctional protein splicing [135], ZFNs and Cas9 used to repair the F508 mutation [134,[137][138][139][140], ZFNs used for the targeted addition of exons 11-27 of the CTFR gene into exon 11 as a functional gene correction [136], and Cas9 utilized to add a functional copy of the CTFR gene into an AAVS1 safe site [133].…”

Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors

“…To list a few examples of preclinical advances in CRISPR therapy, multiple groups have been successful in treating hereditary tyrosinemia I, using Cas9, in disease models by correcting disease-causing FAH mutations [ 47 , 48 ] or knocking-out HPD [ 49 ]. Similarly, researchers applied Cas9 to effectively correct a disease causing mutation in CFTR to treat monogenic cystic fibrosis in patient-derived induced pluripotent stem cells [ 50 , 51 ]. Cas13 was developed as an antiviral to target the genomes of RNA viruses such as Influenza and SARS-CoV-2 in human lung epithelial cells to combat airway diseases [ 52 ].…”