A BAC Transgene Expressing Human CFTR under Control of Its Regulatory Elements Rescues Cftr Knockout Mice

Abstract

Small-molecule modulators of cystic fibrosis transmembrane conductance regulator (CFTR) biology show promise in the treatment of cystic fibrosis (CF). A Cftr knockout (Cftr KO) mouse expressing mutants of human CFTR would advance in vivo testing of new modulators. A bacterial artificial chromosome (BAC) carrying the complete hCFTR gene including regulatory elements within 40.1 kb of DNA 5′ and 25 kb of DNA 3′ to the gene was used to generate founder mice expressing hCFTR. Whole genome sequencing indicated a single integration site on mouse chromosome 8 (8qB2) with ~6 gene copies. hCFTR+ offspring were bred to murine Cftr KO mice, producing hCFTR+/mCftr− (H+/m−) mice, which had normal survival, growth and goblet cell function as compared to wild-type (WT) mice. Expression studies showed hCFTR protein and transcripts in tissues typically expressing mCftr. Functionally, nasal potential difference and large intestinal short-circuit (I sc ) responses to cAMP stimulation were similar in magnitude to WT mice, whereas small intestinal cAMP ΔI sc responses were reduced. A BAC transgenic mouse with functional hCFTR under control of its regulatory elements has been developed to enable the generation of mouse models of hCFTR mutations by gene editing for in vivo testing of new CF therapies.

Animal and Cell Culture Models for Cystic Fibrosis

“…These mice can be broadly classified into three categories: i) Cftr null or knockout (KO) models, 7,26 ii) strains with human CFcausing mutations introduced into the mouse Cftr gene (eg, Phe508del, Gly551Asp, and G542X), 38 and iii) transgenic models [eg, gut-corrected models, mice expressing human CFTR, and b-epithelial sodium channel (b-ENaC) mice]. 9,27,39 Cftr Knockout and Mutant Models Phenotype severity varies among CF mouse strains as a result of the method used to disrupt the Cftr gene, the type of mutation introduced, and the levels of Cftr mRNA produced. 8 Despite these variations, all CF mouse models generally recapitulate similar disease characteristics, including nasal electrophysiological defects, intestinal disease, reduced survival, impaired growth, and malformations of the male reproductive tract.…”

“…These mice demonstrate robust hCFTR protein function in tissues that typically express mouse Cftr, such as the intestinal mucosa and airways. 9 By use of gene editing, the hCFTR mouse model is now being used to generate a range of mouse models that harbor human CF-causing mutations. 47 Although these models are still undergoing phenotype characterization (http://www.cfmice.org/cfmouse-strains.html, last accessed September 16, 2020), they have potential to provide great value for the CF research field, particularly for testing therapeutics that target CFTR.…”

“…Considering the high priority to identify and develop effective treatments for patients with nonsense mutations, it is expected that animal models with these types of CFTR variants are likely to become more common. The generation of humanized CF models is also an area that is gaining momentum with the successful development of hCFTR-expressing mice 9 and recent generation of a rat model with the hG551D mutation. 13 Because this approach provides benefits for testing humanspecific mechanisms of gene function, regulation, and response to therapeutics in an in vivo system, 47 humanization of other CF models is predicted to occur in the near future.…”

On the Corner of Models and Cure: Gene Editing in Cystic Fibrosis

“…Two other transgenic models have been generated using yeast artificial chromosomes (YAC) ( Manson et al (1997) ) and recently also bacterial artificial chromosomes (BAC) ( Gawenis et al, 2019 ). They have as most prominent advantage the possibility to incorporate the entire h CFTR gene, including all necessary regulatory elements that control its expression, which is impossible using plasmid or viral vector technology.…”

“…They have as most prominent advantage the possibility to incorporate the entire h CFTR gene, including all necessary regulatory elements that control its expression, which is impossible using plasmid or viral vector technology. The BAC transgenic mouse with functional hCFTR was generated with the specific purpose to enable the subsequent generation of mouse models with hCFTR mutations, to support future in vivo testing of new CF therapies, pharmacologic or gene therapeutic ( Gawenis et al, 2019 ). Phenotypically, all CF-specific organ pathologies were rescued to WT levels, with the exception of the abnormal dentition.…”