Investigating adverse genomic and regulatory changes caused by replacement of the full-length CFTR cDNA using Cas9 and AAV.

Autor:	Vaidyanathan S; Center for Gene Therapy, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH 43205, USA.; Department of Pediatrics, The Ohio State University, Columbus, OH 43210, USA., Kerschner JL; Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH 44106, USA., Paranjapye A; Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH 44106, USA., Sinha V; Department of Pediatrics, Stanford University, Palo Alto, CA 94305, USA., Lin B; Department of Developmental, Molecular, and Chemical Biology, Tufts University, Boston, MA 02111, USA., Bedrosian TA; Department of Pediatrics, The Ohio State University, Columbus, OH 43210, USA.; Steve and Cindy Rasmussen Institute for Genomic Medicine, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH 43205, USA., Thrasher AJ; Infection, Immunity, and Inflammation Research and Teaching Department, Zayed Centre for Research Into Rare Disease in Children, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK., Turchiano G; Infection, Immunity, and Inflammation Research and Teaching Department, Zayed Centre for Research Into Rare Disease in Children, Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK., Harris A; Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH 44106, USA., Porteus MH; Department of Pediatrics, Stanford University, Palo Alto, CA 94305, USA.
Jazyk:	angličtina
Zdroj:	Molecular therapy. Nucleic acids [Mol Ther Nucleic Acids] 2024 Feb 02; Vol. 35 (1), pp. 102134. Date of Electronic Publication: 2024 Feb 02 (Print Publication: 2024).
DOI:	10.1016/j.omtn.2024.102134
Abstrakt:	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 nononcogenic 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. Competing Interests: M.H.P. serves on the Board of Directors of Graphite Bio and the Scientific Advisory Board of Allogene and serves as an advisor to Versant Ventures. M.H.P. has equity in CRISPR Tx and has equity in and is a founder of Kamau Therapeutics. The companies had no input on the design or execution of the experiments described here and were also not involved in the data analysis or interpretation. (© 2024 The Author(s).)
Databáze:	MEDLINE
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