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Exon-Skipping Antisense Oligonucleotides for H3.3K27M-Altered Diffuse Midline Glioma Therapy
Abstract:
Diffuse midline gliomas (DMGs) are a deadly class of pediatric high-grade brain cancers. Approximately 80% of pontine DMGs feature a dominant, somatic, heterozygous point mutation in the non-canonical histone H3.3-coding gene H3-3A . This dominant-negative mutation replaces lysine 27 with methionine (K27M) and prevents global K27 di- and tri-methylation of all wild-type histone H3 proteins. We aimed to target the H3.3K27M onco-histone pre-mRNA with splice-switching antisense oligonucleotides (ASOs) designed to promote skipping of H3-3A exon 2, as this constitutive exon comprises both the K27M mutation and the natural in-frame start codon of the gene. The lead ASO identified in a systematic screen specifically induced H3-3A exon 2 skipping, did not affect expression or splicing of the paralog gene H3-3B -which also encodes histone H3.3-and restored global H3K27me3 marks in patient-derived DMG cells grown as neurospheres. In a patient-derived orthotopic xenograft tumor mouse model, the lead ASO reduced proliferation and extended survival. Our results show the potential of exon-skipping ASOs targeting H3-3A exon 2 as a therapeutic option for H3.3K27M-altered DMG. More generally, they exemplify the strategy of using ASOs to induce skipping of a constitutive exon to effectively achieve gene downregulation.
Insights
This study developed splice-switching antisense oligonucleotides (ASOs) to target the H3.3K27M mutation in diffuse midline gliomas (DMGs). The ASO therapy promoted exon skipping, reduced tumor proliferation, and extended survival in preclinical models.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Diffuse midline gliomas (DMGs) are aggressive pediatric brain tumors.
- Most DMGs harbor a specific H3.3 gene mutation (K27M) that disrupts histone methylation.
- Targeting this mutation is crucial for developing effective therapies.
Purpose of the Study:
- To develop and evaluate splice-switching antisense oligonucleotides (ASOs) targeting the H3.3K27M mutation.
- To investigate the therapeutic potential of inducing exon skipping in the H3-3A gene.
Main Methods:
- Systematic screening to identify lead ASO targeting H3-3A exon 2.
- Assessing ASO efficacy in patient-derived DMG cells and xenograft models.
- Evaluating effects on H3-3A splicing, H3K27me3 marks, tumor proliferation, and survival.
Main Results:
- The lead ASO successfully induced H3-3A exon 2 skipping.
- Global H3K27me3 marks were restored in DMG cells.
- ASO treatment reduced tumor proliferation and extended survival in a mouse model.
Conclusions:
- Exon-skipping ASOs targeting H3-3A exon 2 show therapeutic potential for H3.3K27M-altered DMGs.
- This approach demonstrates a viable strategy for gene downregulation via ASO-induced exon skipping.
