Abstract
Cystic Fibrosis (CF) is caused by a diverse set of mutations distributed across the approximately 250 thousand base pairs of the CFTR gene locus, of which at least 382 are disease-causing (CFTR2.org). Although a variety of editing tools are now available for correction of individual mutations, a strong justification can be made for a more universal gene insertion approach, in principle capable of correcting virtually all CFTR mutations. Provided that such a methodology is capable of efficiently correcting relevant stem cells of the airway epithelium, this could potentially provide life-long correction for the lung. In this Perspective we highlight several requirements for efficient gene insertion into airway epithelial stem cells. In addition, we focus on specific features of the transgene construct and the endogenous CFTR locus that influence whether the inserted gene sequences will give rise to robust and physiologically relevant levels of CFTR function in airway epithelium. Finally, we consider how in vitro gene insertion methodologies may be adapted for direct in vivo editing.
Highlights
Cystic Fibrosis (CF) is an inherited recessive disease that results from mutations in the Cystic fibrosis transmembrane conductance regulator (CFTR) gene (Sharma and Cutting, 2020)
Given this efficient gene insertion ex vivo in airway basal cells, we wished to demonstrate the competence of edited cells to establish a well-differentiated airway epithelium in vivo
Recent developments in various methodologies, including gene editing, ex vivo expansion of airway basal cells, and in vivo delivery of editing reagents to the lung, offer hope that effective, universal, targeted gene insertion-based therapies may eventually be developed for the CF airway
Summary
Cystic Fibrosis (CF) is an inherited recessive disease that results from mutations in the Cystic fibrosis transmembrane conductance regulator (CFTR) gene (Sharma and Cutting, 2020). To correct for all or most CFTR mutations, it likely would be necessary to target integration of the partial CFTR cDNA sequences into the most upstream region of CFTR sequences, for example from exon 1 thru intron 2 If this can be achieved while retaining the native CFTR chromatin structure and regulatory sequences, it has the possibility of restoring appropriate cell-type specific expression. Quantitative RT-PCR demonstrated that the majority (58.0—89.9%) of CFTR transcripts in these homozygous intron 8 TI clones exhibited the desired splicing into the corrective human codon-optimized exon 9 sequences; the remaining transcripts reflected splicing across the corrective exon sequences directly into endogenous exon 9 sequences Given this efficient gene insertion ex vivo in airway basal cells, we wished to demonstrate the competence of edited cells to establish a well-differentiated airway epithelium in vivo. Since the NHEJ repair mechanism, as well as other targeting approaches, are active in non-cycling cells, such methods may be required, e.g. HITI (Suzuki et al, 2016), prime editing (Anzalone et al, 2019), twin prime editing (Anzalone et al, 2021), transposonencoded CRISPR/Cas system (Klompe et al, 2019), and fusion of CRISPR/Cas nickase to both a reverse transcriptase and serine integrase (Ioannidi et al, 2021)
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