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

Lara R Gawenis,Calvin U Cotton,Ann Harris,Jürgen Bosch,Jinghua Liu,Daniel R Mchugh,Dana M Valerio,Mitchell L Drumm,Ashlee M Strubberg,Austin E Gillen,George Kotzamanis,Craig A Hodges,Michael J Mutolo,Alexander Miron,Lane L Clarke,Nancy M Walker

doi:10.1038/s41598-019-48105-4

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 (Isc) responses to cAMP stimulation were similar in magnitude to WT mice, whereas small intestinal cAMP ΔIsc 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.

Highlights

cystic fibrosis (CF) patients that benefit the most have type III mutations of at least one allele where CFTR protein is made in sufficient amounts and traffics to the plasma membrane but has defective channel gating, e.g., G551D
Offspring from the human CFTR (hCFTR) founder were bred into murine Cftr (mCftr) KO mice to generate hCFTR+/mCftr− (H+/m−) mice
One apparent exception is the enamel organ because the phenotype of white teeth was not corrected by the hCFTR transgene

Summary

Introduction

CF patients that benefit the most have type III mutations of at least one allele where CFTR protein is made in sufficient amounts and traffics to the plasma membrane but has defective channel gating, e.g., G551D. Based on the earlier studies and interest in the development of a gene therapy construct, a 250.3 kb bacterial artificial chromosome (BAC) transgene was engineered containing the entire coding region of hCFTR, 40.1 kb of DNA 5′ and 25 kb of DNA 3′ to the gene[9] Though this BAC lacks the topologically associating domain (TAD) boundaries for CFTR (at −80.1 kb and +48.9 kb10–12) it contains the majority of key tissue-specific control elements for the locus. This construct was used to develop a BAC transgenic mouse model expressing hCFTR by its endogenous regulatory elements with the purpose of introducing different CF mutations by gene editing and subsequent in vivo testing of CFTR modulators and other therapeutic modalities for the treatment of cystic fibrosis

Objectives

Methods

Results

Conclusion