Investigating Adverse Genomic and Regulatory Changes Caused by Replacement of the Full-length CFTR cDNA Using Cas9 and AAV

Abstract

A "Universal strategy" replacing the full-length CFTR cDNA may treat >99% of people with cystic fibrosis (pwCF) regardless of their specific mutations. Cas9-based gene editing was used to insert the CFTR cDNA and a truncated CD19 (tCD19) enrichment tag at the CFTR locus in airway basal stem cells. This strategy restores CFTR function to non-CF levels. Here, we investigate the safety of this approach by assessing genomic and regulatory changes after CFTR cDNA insertion. Safety was first assessed by quantifying genetic rearrangements using CAST-seq. After validating restored CFTR function in edited and enriched airway cells, the CFTR locus open chromatin profile was characterized using ATAC-seq. The regenerative potential and differential gene expression in edited cells was assessed using scRNA-seq. CAST-seq revealed a translocation in ∼0.01% of alleles primarily occurring at a non-oncogenic off-target site and large INDELs in 1% of alleles. The open chromatin profile of differentiated airway epithelial cells showed no appreciable changes except in the region corresponding to the CFTR cDNA and tCD19 cassette indicating no detectable changes in gene regulation. Edited stem cells produced the same types of airway cells as controls with minimal alternations in gene expression. Overall, the Universal strategy showed minor undesirable genomic changes.