Skip to content
Ussing Chamber Systems for Epithelial Barrier Research  |        USA Customer Service: 858-451-8845   |        Email: office@piusa.net
Close

Exon-skipping antisense oligonucleotides for cystic fibrosis therapy

by John Thompson 18 Aug 2022
Exon-skipping antisense oligonucleotides for cystic fibrosis therapy

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity but is expressed at a very low level due to nonsense-mediated messenger RNA (mRNA) decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable, and read-through drugs are inefficient partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site–targeting ASOs induced the expression of CFTR mRNA without the premature-termination-codon–containing exon 23 (CFTR-Δex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-Δex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.

 

"

Ussing Chamber Assay.

The 16HBEge cells were grown as an electrically tight monolayer on Snapwell filter supports (Corning, 3801), as described (38), and both serosal and mucosal membranes were exposed to the ASOs for 4 d and to CFTR correctors VX-809, VX-661, or VX-445 for 24 h before the assays (Selleckchem, S1565, S7059, and S8851, respectively). The Snapwell inserts were transferred to an Ussing chamber (P2302, Physiologic Instruments, Inc.). The serosal side only was superfused with 5 mL HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]–buffered physiological saline buffer; on the mucosal side, 5 mL CF-HEPES–buffered physiological saline was used (137 mM Na-gluconate, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose; pH adjusted to 7.4 with N-methyl-d-glucamine) to create a transepithelial chloride-ion gradient. After clamping the transepithelial voltage to 0 mV, the short-circuit current (ISC) was measured with a Physiologic Instruments VCC MC6 epithelial voltage clamp while maintaining the buffer temperature at 37 °C. Baseline activity was recorded for 20 min before agonists (final concentrations: 10 µM forskolin [Sigma, F6886] and 10 µM VX-770 [Selleckchem, S1144]) and inhibitor (final concentration: 20 µM CFTRinh-172 [Sigma, C2992]) were applied sequentially at 10-min intervals to both serosal and mucosal surfaces. Agonists/inhibitors were added from 200× to 1,000× stock solutions. Data acquisition was performed with ACQUIRE & ANALYZE Revision II (Physiologic Instruments)."
SEE THE WHOLE ARTICLE HERE:  https://www.pnas.org/doi/10.1073/pnas.2114858118

Recently Viewed

Recently Viewed Products
Back To Top
Close
Edit Option
Close
Notify Me
Close
is added to your shopping cart.
Close
Close
Login
Close
Order (0)