Related Experiment Video

Updated: Mar 30, 2026

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts
07:02

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

Published on: May 11, 2018

14.5K

Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides

Susana Igreja1, Luka A Clarke1, Hugo M Botelho1

  • 1University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Lisboa, Portugal.

Human Mutation
|November 11, 2015
PubMed

Insights

Antisense oligonucleotides (AONs) show promise for treating cystic fibrosis (CF) by correcting faulty CF transmembrane conductance regulator (CFTR) gene splicing. This RNA-based therapy restored normal exon 16 inclusion, improving CFTR protein function.

Area of Science:

  • Genetics
  • Molecular Biology
  • RNA Therapeutics

Background:

  • Cystic Fibrosis (CF) is a life-threatening genetic disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene.
  • Approximately 13% of CFTR mutations impact pre-mRNA splicing, a therapeutic area that has been relatively neglected.
  • A specific CFTR splicing mutation, c.2657+5G>A in IVS16, leads to aberrant splicing, producing transcripts lacking exon 16 alongside wild-type transcripts.

Purpose of the Study:

  • To investigate the efficacy of an RNA-based antisense oligonucleotide (AON) strategy for correcting the aberrant pre-mRNA splicing caused by the CFTR c.2657+5G>A mutation.
  • To assess the impact of AON treatment on RNA splicing, CFTR protein levels, localization, and function.

Main Methods:

  • Design and synthesis of two AONs (AON1/2) targeting the mutant pre-mRNA IVS16 region.
  • Utilizing a stable HEK293 Flp-In cell line expressing a single copy of the c.2657+5G>A mutant CFTR minigene.
  • Assessing splicing correction at the RNA level, CFTR protein expression, plasma membrane localization, and protein function.
  • Development of a novel two-color CFTR splicing reporter minigene for quantitative monitoring via automated microscopy.

Main Results:

  • Treatment with AON1 significantly restored exon 16 inclusion in the CFTR pre-mRNA to 95% in mutant cells.
  • This splicing correction led to increased levels of correctly localized CFTR protein at the plasma membrane.
  • Enhanced CFTR protein function was observed in AON1-treated cells.
  • The novel reporter minigene enabled quantitative assessment of splicing efficiency.

Conclusions:

  • The tested antisense oligonucleotide (AON) strategy is a promising therapeutic approach for correcting alternative splicing defects in the CFTR gene.
  • This RNA-based therapy effectively restores normal splicing, leading to improved CFTR protein function and localization.
  • The developed splicing reporter system provides a valuable tool for quantitative assessment of splicing-modulating therapies.
Keywords:
CFTRantisense oligonucleotidecystic fibrosisexon skippingsplicing mutation

More Related Videos

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders10:28

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Published on: April 3, 2021

7.3K
Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells10:06

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Published on: April 26, 2017

9.5K

Related Concept Videos

Cystic Fibrosis: Pathogenesis01:23

Cystic Fibrosis: Pathogenesis

Cystic fibrosis (CF), an autosomal recessive disorder, significantly affects the function of exocrine glands. This genetically inherited disease is characterized by the production of thick and sticky mucus, which can severely affect various organs and systems in the body.
CF is primarily caused by a genetic mutation in a chromosome 7 gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common gene mutation leading to CF is the ΔF508 mutation,...
1.1K
Alternative RNA Splicing02:18

Alternative RNA Splicing

Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...
26.3K
Cystic Fibrosis: Management01:24

Cystic Fibrosis: Management

Cystic fibrosis (CF) is an autosomal recessive disorder that predominantly affects individuals of Northern European descent, occurring at a rate of 1 in 3500. It is caused by a genetic mutation in a gene on chromosome 7, most commonly the ΔF508 mutation, that codes for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. This results in thicker mucus secretions and obstruction pathologies in multiple organs, including the lungs and sinuses.
Sinus disease and chronic...
620
RNA Splicing01:32

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
61.4K
RNA Splicing01:32

RNA Splicing

20.2K
CRISPR01:59

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
59.1K