Related Experiment Video
Updated: Mar 25, 2026
Characterizing Exon Skipping Efficiency in DMD Patient Samples in Clinical Trials of Antisense Oligonucleotides
Published on: May 7, 2020
Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept
Julie W Rutten1, Hans G Dauwerse1, Dorien J M Peters2
1Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands.
Insights
This study introduces a novel exon skipping therapy to correct NOTCH3 mutations causing CADASIL, a hereditary stroke disorder. The approach successfully eliminated mutant domains, offering a potential treatment for most CADASIL cases.
Area of Science:
- Genetics and Molecular Biology
- Neurology
- Biotechnology
Background:
- Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetic small vessel disease.
- It results from NOTCH3 gene mutations causing toxic protein accumulation in blood vessels.
- Current treatments for CADASIL are limited, necessitating novel therapeutic strategies.
Purpose of the Study:
- To develop a gene therapy approach to correct NOTCH3 mutations in CADASIL.
- To investigate the feasibility of excluding mutant epidermal growth factor-like repeat (EGFr) domains from NOTCH3 protein.
- To establish a potential therapeutic strategy for CADASIL by preventing toxic protein aggregation.
Main Methods:
- Utilized in silico studies to identify key NOTCH3 exons for skipping.
- Employed antisense-mediated pre-mRNA exon skipping to eliminate mutant EGFr domains.
- Designed and tested antisense oligonucleotides targeting specific NOTCH3 exons.
- Validated the approach in patient-derived cells, assessing protein processing, ligand binding, and signaling.
Main Results:
- Exon skipping successfully removed mutant EGFr domains from NOTCH3.
- Skipped NOTCH3 proteins maintained normal processing, ligand binding, and activation.
- Antisense oligonucleotides effectively induced exon skipping in patient-derived cells.
- The strategy showed proof of concept for a novel therapeutic approach for CADASIL.
Conclusions:
- Exon skipping is a viable strategy to correct NOTCH3 mutations in CADASIL.
- This approach offers a potential therapeutic avenue for up to 94% of CADASIL-causing mutations.
- Further development could lead to a rational treatment for this debilitating hereditary condition.
Abstract:
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, is a hereditary cerebral small vessel disease caused by characteristic cysteine altering missense mutations in the NOTCH3 gene. NOTCH3 mutations in CADASIL result in an uneven number of cysteine residues in one of the 34 epidermal growth factor like-repeat (EGFr) domains of the NOTCH3 protein. The consequence of an unpaired cysteine residue in an EGFr domain is an increased multimerization tendency of mutant NOTCH3, leading to toxic accumulation of the protein in the (cerebro)vasculature, and ultimately reduced cerebral blood flow, recurrent stroke and vascular dementia. There is no therapy to delay or alleviate symptoms in CADASIL. We hypothesized that exclusion of the mutant EGFr domain from NOTCH3 would abolish the detrimental effect of the unpaired cysteine and thus prevent toxic NOTCH3 accumulation and the negative cascade of events leading to CADASIL. To accomplish this NOTCH3 cysteine correction by EGFr domain exclusion, we used pre-mRNA antisense-mediated skipping of specific NOTCH3 exons. Selection of these exons was achieved using in silico studies and based on the criterion that skipping of a particular exon or exon pair would modulate the protein in such a way that the mutant EGFr domain is eliminated, without otherwise corrupting NOTCH3 structure and function. Remarkably, we found that this strategy closely mimics evolutionary events, where the elimination and fusion of NOTCH EGFr domains led to the generation of four functional NOTCH homologues. We modelled a selection of exon skip strategies using cDNA constructs and show that the skip proteins retain normal protein processing, can bind ligand and be activated by ligand. We then determined the technical feasibility of targeted NOTCH3 exon skipping, by designing antisense oligonucleotides targeting exons 2-3, 4-5 and 6, which together harbour the majority of distinct CADASIL-causing mutations. Transfection of these antisense oligonucleotides into CADASIL patient-derived cerebral vascular smooth muscle cells resulted in successful exon skipping, without abrogating NOTCH3 signalling. Combined, these data provide proof of concept for this novel application of exon skipping, and are a first step towards the development of a rational therapeutic approach applicable to up to 94% of CADASIL-causing mutations.
