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

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

Published on: May 11, 2018

Antisense oligonucleotide splicing modulation as a novel Cystic Fibrosis therapeutic approach for the W1282X nonsense

Yifat S Oren¹, Ofra Avizur-Barchad¹, Efrat Ozeri-Galai¹

¹SpliSense Biohouse Labs, Hadassah Ein Kerem, Jerusalem, Israel.

Journal of Cystic Fibrosis : Official Journal of the European Cystic Fibrosis Society

|January 1, 2022

Abstract

Background:

Antisense oligonucleotide- based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to generate skipping over exon 23 of the CFTR transcript, to eliminate the W1282X nonsense mutation and avoid RNA degradation induced by the nonsense mediated mRNA decay mechanism, allowing production of partially active CFTR proteins lacking exon 23.

Methods:

∼80 ASOs were screened in 16HBEge W1282X cells. ASO candidates showing significant exon skipping were assessed for their W1282X allele selectivity and the increase of CFTR protein maturation and function. The effect of a highly potent ASO candidates was further analyzed in well differentiated primary human nasal epithelial cells, derived from a W1282X homozygous patient.

Results:

ASO screening led to identification of several ASOs that significantly decrease the level of CFTR transcripts including exon 23. These ASOs resulted in significant levels of mature CFTR protein and together with modulators restore the channel function following free uptake into these cells. Importantly, a highly potent lead ASOs, efficiently delivered by free uptake, was able to increase the level of transcripts lacking exon 23 and restore the CFTR function in cells from a W1282X homozygote patient.

Conclusion:

The highly efficient exon 23 skipping induced by free uptake of the lead ASO and the resulting levels of mature CFTR protein exhibiting channel function in the presence of modulators, demonstrate the ASO therapeutic potential benefit for CF patients carrying the W1282X mutation with the objective to advance the lead candidate SPL23-2 to proof-of-concept clinical study.

Insights

Antisense oligonucleotides (ASOs) effectively skipped exon 23 in cystic fibrosis cells with the W1282X mutation. This approach restored partially functional CFTR protein, showing therapeutic promise for genetic diseases.

Area of Science:

Genetic medicine
RNA therapeutics
Molecular biology

Background:

Antisense oligonucleotides (ASOs) are emerging therapies for genetic diseases.
The W1282X mutation in the CFTR gene causes cystic fibrosis.
Nonsense-mediated mRNA decay degrades faulty transcripts.

Purpose of the Study:

To develop ASOs for exon skipping of CFTR transcript.
To target the W1282X mutation and bypass nonsense-mediated decay.
To restore partially functional CFTR protein.

Main Methods:

Screened ~80 ASOs in W1282X cells (16HBEge).
Assessed ASOs for exon skipping, W1282X allele selectivity, and CFTR protein function.
Validated lead ASO in patient-derived nasal epithelial cells.

Main Results:

Identified ASOs that significantly reduced exon 23-containing CFTR transcripts.
Achieved significant mature CFTR protein levels and restored channel function with modulators.
Demonstrated lead ASO efficacy in W1282X patient cells via free uptake.

Conclusions:

Lead ASO efficiently induces exon 23 skipping.
Restored CFTR protein exhibits channel function with modulators.
ASO therapy shows potential for W1282X cystic fibrosis patients.

Read full analysis

Keywords:

Antisense oligonucleotides Cystic fibrosis Drug development Exon skipping

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