Investigating the Implications of CFTR Exon Skipping Using a Cftr Exon 9 Deleted Mouse Model

Abstract

Introduction: Severity and disease progression in people with Cystic Fibrosis (CF) is typically dependent on their genotype. One potential therapeutic strategy for people with specific mutations is exon skipping with antisense oligonucleotides (AO). CFTR exon 9 is an in-frame exon and hence the exclusion of this exon would excise only 31 amino acids but not alter the reading frame of the remaining mRNA. Splice mutations 1209 + 1 G > C and 1209 + 2 T > G were documented to cause CFTR exon 9 skipping and these variants were reported to manifest as a milder CF disease, therefore exon 9 skipping could be beneficial for people with class I mutations that affect exon 9 such as p.Trp401X. While the impact of exon 9 skipping on gene expression and cellular pathways can be studied in cells in vitro, trace amount of full-length normal or mutated material could confound the evaluation. To overcome this limitation, the impact of CFTR exon 9 skipping on disease phenotype and severity is more effectively evaluated in a small animal model. It was hypothesised that antisense oligonucleotide-mediated skipping this particular exon could result in a "mild mouse CF phenotype". Methods: Cftr exon 9 deleted mice were generated using homologous recombination. Survival of homozygous (Cftr ^Δ9/Δ9 ) and heterozygous (Cftr ^Δ9/+ ) mice was compared to that of other CF mouse models, and lung and intestinal organ histology examined for any pathologies. Primary airway epithelial cells (pAECs) were harvested from Cftr ^Δ9/Δ9 mice and cultured at the Air Liquid Interface for CFTR functional assessment using Ussing Chamber analysis. Results: A Cftr ^Δ9/Δ9 mouse model presented with intestinal obstructions, and at time of weaning (21 days). Cftr ^Δ9/Δ9 mice had a survival rate of 83% that dropped to 38% by day 50. Histological sections of the small intestine from Cftr ^Δ9/Δ9 mice showed more goblet cells and mucus accumulation than samples from the Cftr ^Δ9/+ littermates. Airway epithelial cell cultures established from Cftr ^Δ9/Δ9 mice were not responsive to forskolin stimulation. Summary: The effect of Cftr exon 9 deletion on Cftr function was assessed and it was determined that the encoded Cftr isoform did not result in a milder "mouse CF disease phenotype," suggesting that Cftr exon 9 is not dispensable, although further investigation in human CF pAECs would be required to confirm this observation.

Keywords: cystic fibrosis transmembrane conductance regulator; exon deletion; exon skipping therapy; mouse model; transgenic mouse.

FIGURE 1

Survival plot of Cftr ^Δ9/Δ9 mice. At time of weaning (21 days) Cftr ^Δ9/Δ9 mice had a survival of 83% which dropped to 38% by day 50, with most mice succumbing to intestinal obstruction or malnutrition (solid line). Heterozygous mice did not show decreased survival with 89% survival at day 50 until day 150 when monitoring ended (dashed line). n = 24.

FIGURE 2

Hematoxylin and Eosin and Alcian Blue staining of representative lung and intestinal sections from Cftr ^Δ9/Δ9 and Cftr ^Δ9/+ (control) mice. (A–F) Hematoxylin and Eosin stained (A). Lung section for Cftr ^Δ9/Δ9 mouse. (B). Lung section for Cftr ^Δ9/+ mouse. (C). Small intestinal section from Cftr ^Δ9/Δ9 mouse. (D). Intestinal section from Cftr ^Δ9/+ mouse. (E). Crypts of Lieberkühn section from Cftr ^Δ9/Δ9 mouse. (F). Lieberkühn section from Cftr ^Δ9/+ mouse. (G–J) Alcain blue stained (G). a Cftr ^Δ9/Δ9 mouse and (H). Cftr ^Δ9/+ mouse showing Crypts of Lieberkühn. Sections of small intestine from (I). Cftr ^Δ9/Δ9 mouse and (J). Cftr ^Δ9/+ mouse, showing goblet cells. n = 4. Images (A,B), (E–H) were taken at ×40 (scale bar 20 micrometres) and (C,D), (I,J) were taken at ×20 (scale bar 50 micrometres).

FIGURE 3

CftrΔ9/Δ9 deleted airway epithelial cell morphology. (A). Airway epithelial cells grown in monolayer had standard cobble stone appearance. Scale bar: 30 micrometres (B). Airway epithelial cells grown at the air-liquid interface with the dense packed growth morphology. Scale bar 30 micrometres. n = 4.

FIGURE 4

Cftr ^Δ9/Δ9 epithelial cell CFTR function. (A,B). Ussing chamber tracing showing change in short circuit current (Isc) in response to drug stimulation on Cftr ^Δ9/Δ9 ALI cultures pre-treated for 24 h with DMSO. (C,D). Ussing chamber tracing showing change in Isc in response to drug stimulation on homozygote Cftr exon 9 deleted ALI cultures pre-treated for 24 h with Lumacaftor. (E). Summary of changes in Isc in response to amiloride, forskolin and CFTR inhibitor-172 for Cftr ^Δ9/Δ9 ALI cultures pre-treated with DMSO (Black bars) or Lumacaftor (Grey bars) (Isc, n = 2). Pre-treatment with DMSO (black bar) pre-treatment with Lumacaftor (blue bar), Amiloride addition (green bar), Forskolin stimulation (yellow bar), Ivacaftor stimulation (orange bar), CFTR inhibitor-172 (CI-red bar).