Single Antisense Oligonucleotides Correct Diverse Splicing Mutations in Hotspot Exons

Bioengineering Basic Medical Sciences Cell Biology Biochemistry Oncology Genetics Life Sciences Medicine
antisense oligonucleotide splicing mutation hotspot exon RNA splicing actionable genes gene mutation therapeutic strategy

Broad-Spectrum Correction of Splicing Mutations in Rare Disease Hotspot Exons by Single Antisense Oligonucleotide: Review of a Recent 2025 PNAS Study

I. Academic Background: The Challenge of Disease-Associated Splicing Mutations and Dilemmas in Antisense Therapy

RNA splicing is a crucial step in the regulation of gene expression in eukaryotes. The vast majority of human genes undergo the removal of introns and the joining of exons during the formation of mature mRNA through the splicing process. This process depends not only on classic cis-elements such as the 5’ and 3’ splice sites, branch points, and polypyrimidine tracts, but also involves numerous splicing enhancers and silencers located within exons and introns. Genetic studies indicate that up to 60% of human pathogenic mutations ultimately cause abnormal protein coding through affecting RNA splicing. Therefore, understanding and correcting abnormal gene splicing is a significant foundational challenge in the diagnosis and treatment of rare genetic diseases, tumors, and other disorders.

However, the reality is complex: First, splicing mutations are not evenly distributed across all exons. Some exons become “hotspot exons,” clearly more prone to aberrant splicing (such as exon skipping) from various mutations, while most exons are relatively “robust.” Second, although antisense oligonucleotides (ASOs) have been approved for precise regulation of RNA splicing in certain diseases (such as DMD, SMA), current ASOs are customized for individual mutations, which is time-consuming, labor-intensive, and costly, making it difficult for rare genotypes or small patient populations to benefit. Third, it remains a significant challenge to systematically and high-throughputly identify all medically relevant splicing mutations and to map the distribution of “splicing-vulnerable” exons.

With this background, the team led by William G. Fairbrother launched this study, aiming to: 1) Determine which medically relevant genes contain splicing-mutation-prone “hotspot exons”; 2) Explore whether a single ASO can broadly correct multiple splicing mutations within the same hotspot exon, thus providing new ideas for precision treatment of rare genetic diseases.

II. Source and Author Team

The paper, entitled “single antisense oligonucleotides correct diverse splicing mutations in hotspot exons,” was co-authored by Chaorui Duan, Stephen Rong, Luke Buerer, Christopher R. Neil, Yu Zhong, Zhuoyang Lyu, Juliann M. Savatt, Natasha T. Strande, William G. Fairbrother, and others. The lead and corresponding authors are from Brown University and institutions such as Geisinger Health. The paper was published on June 16, 2025 in the Proceedings of the National Academy of Sciences of the United States of America (PNAS).

III. Detailed Overview of the Research Process

1. High-Throughput Screening and Dataset Assembly of Splicing Mutations

Researchers first focused on 71 clinically actionable human disease genes (chosen for their high clinical intervention value, giving great practical significance to the study of their splicing abnormalities), incorporating a total of 32,112 exonic single-nucleotide variants (SNVs) from the ClinVar public mutation database and the Geisinger MyCode project. These variants cover different regions within exons and involve various mechanisms of action.

Due to physical constraints of current oligonucleotide synthesis lengths (<230nt), two construction modes were adopted: full-length cloning for short exons (within 120nt), and analysis of a 90nt segment near the 3’ splice site for longer exons (>120nt). This ensures both high-throughput experimentation and cloning reliability.

Additionally, to ensure wide coverage and clinical relevance, included exons were selected based on the definition of “constitutive exons” in the HexEvent database (present in ≥95% of transcripts).

2. Self-Developed High-Throughput Splicing Functional Screening System (MaPSy Platform)

The team developed and refined the Massively Parallel Splicing Assay (MaPSy), with the core process as follows:

  • Employing Agilent high-density oligonucleotide synthesis to generate DNA libraries for all target exons and their mutant versions (wild-type and mutant);
  • Embedding each WT and variant sequence into a standardized three-exon, two-intron minigene reporter framework to form the library;
  • Performing high-throughput Illumina sequencing on cDNA products after transfection into HEK293T cells, with separate counting for each species;
  • Calculating the impact of each mutation on splicing (the log2 ratio of output/input for mutant vs. wild type, yielding the MaPSy splicing score);
  • Using comprehensive statistical testing (such as the mpralm algorithm with FDR control) to categorize and identify “splice-disrupting variants” (SDVs).

This approach quantitatively and at scale assesses the actual splicing impact of tens of thousands of genetic variants, providing a robust foundation for subsequent genetics research and targeted therapy development.

3. Bioinformatics Analysis and Association With Pathogenicity

For the experimentally obtained data, further analyses included:

  • Mapping the mutations against ClinVar annotations and pathogenicity profiles, and comparing experimental results to check the relationship between MaPSy scores and pathogenic/benign phenotypes;
  • Building binary logistic regression models linking MaPSy splicing scores with ClinVar pathogenicity labels, discovering that lower splicing scores correspond to higher pathogenic potential (log-odds coefficient = -0.32, p=1.44e-6), with the association being particularly significant in the “extreme SDV” subset (bottom 2% scores);
  • Incorporating evolutionary biology tools (phyloP, GERP for conservation scoring, CADD for comprehensive pathogenicity scoring), showing that variants with the most severe impact on splicing are rarer and more likely to be purged by natural selection from the population;
  • Analysis of the MyCode cohort revealed a high singleton rate (>40%) among exonic variants severely affecting splicing, further supporting these conclusions.

4. Hotspot Exon Identification and Splicing Vulnerability Assessment

Data mining revealed that splice-disrupting variants are far from evenly distributed and cluster intensely in a small subset of exons (“hotspot exons”). For example, in crucial cancer susceptibility genes like BRCA1 and MLH1, only a handful of exons show a high proportion of SDVs, while most exons are unaffected. Further analysis showed that identical 5mer mutations can have very different effects depending on the exon context, indicating that the local sequence “background” exerts a greater influence on the splicing outcome than the specific mutation type itself.

Innovatively, the team used genome-wide in silico mutagenesis plus the SpliceAI algorithm to systematically scan the entire human coding annotation (CCDS), further confirming that “hotspot exons” are a universal pattern in the distribution of splice-disrupting mutations.

Additionally, by treating cells with the non-specific splicing inhibitor Pladienolide B (Plad B) and performing RNA-seq, they quantitatively measured susceptibility of each exon to drug-induced exon skipping. Results showed that exons most sensitive to Plad B largely overlapped with predicted hotspots, confirming these exons’ intrinsic “splicing vulnerability.”

5. Broad-Spectrum Rescue of Hotspot Exon Splicing Mutations by Single ASO

Having established the existence and high susceptibility of hotspot exons to multiple mutations, the researchers tested a key hypothesis: “Can a single ASO reverse various splicing abnormalities within the same hotspot exon, regardless of mutation type?”

Experimental steps were:

  • Inducing typical hotspot exons in genes such as PTEN, LDLR, VHL, and TSC1 to skip using Plad B, and designing ASOs targeting the upstream 5’ss and/or downstream 3’ss. PCR/qPCR quantification showed that a single ASO (especially targeting downstream 3’ss or upstream 5’ss) could reverse 18%-86% of anomalous skipping;
  • Further, using multiple real SDVs discovered by MaPSy (e.g., three different mutations in TSC1 exon 14, MLH1 exon 9), building minigenes and various ASOs (including branchpoint-targeting), they systematically compared the ability of different ASOs to rescue mis-splicing induced by different mutations. Results demonstrated that appropriate single ASOs (targeting adjacent 3’ss/5’ss/branchpoint) could significantly increase exon inclusion and correct aberrant splicing, regardless of mutation;
  • Additional molecular mechanism experiments with various PCR primer combinations confirmed that ASOs not only restore full-length mRNA expression but also modulate splicing kinetics, ultimately suppressing abnormal exon skipping.

IV. Key Research Findings and Contributions

  1. Large-Scale Experimental Splicing Function Assessment: For the first time, the splicing impact of over 30,000 naturally occurring and clinically observed exonic variants was systematically evaluated across 71 clinically actionable human disease genes.

  2. Identification of a Large Number of “Splicing-Disrupting” Variants: A total of 1,733 variants with significant impact on splicing (SDVs) were identified, with over 30% of the most extreme category labeled pathogenic or likely pathogenic in ClinVar.

  3. Global Map of “Splicing-Vulnerable Hotspot Exons”: Only around 8% of exons were categorized as “splicing mutation hotspots,” yet they bear the majority of SDVs. These hotspot exons are highly sensitive pharmacologically and mechanistically vulnerable, serving as primary targets for heterogeneous splicing abnormalities in hereditary diseases.

  4. ASO Correction Mechanism and Experimental Proof: Multiple cell experiments indicated that, regardless of mutation type, a well-designed ASO targeting the flanking splice sites of a hotspot exon restores normal inclusion—mechanistically, this is achieved by altering the competitive dynamics of abnormal splicing.

  5. Major Scientific and Practical Significance: A new “many-in-one” correction concept is proposed for the precision treatment of rare diseases—heterogeneous splicing abnormalities can be tackled using a single ASO for multiple mutation types and sites, improving accessibility of interventions, lowering the barriers to new drug development, and complementing the individualized, mutation-specific ASO approach at the population/exon level.

V. Summary of Research Highlights

  • Self-Built High-Throughput System: The MaPSy screening and analysis platform sets a technical paradigm for large-scale functional identification of splicing mutations, balancing systematics, throughput, and experimental physiological relevance.
  • Panoramic Distribution of Hotspot Exons with Abnormal Splicing: A combination of large-scale in-library and out-of-library experiments confirms that splicing mutations are not randomly distributed, providing a data foundation for gene diagnosis and target selection in rare diseases and cancers.
  • ASO “One-to-Many” Broad-Spectrum Application Scheme: With a single sequence design, multiple mutation types within the same exon can be corrected, theoretically expanding the applicability of antisense therapy across populations and mutation loci.
  • Mechanistic Innovation—Kinetic Regulation of Splicing: Rather than directly “repairing” the mutation, ASOs adjust competitive splicing around the exon region, overall enhancing exon inclusion and thus “bypassing” and remedying many pathogenic mutations.
  • Clinical Reference Example: For example, BRCA2 exon 13 includes more than 100 mutations in MyCode, and among them, 13 cancer patients—all of whom had significantly negative MaPSy splicing scores, theoretically suggesting that a single ASO could generally correct these splicing defects. This has major implications for germline cancer susceptibility screening and early warning.

VI. Conclusions, Prospects, and Limitations

This study not only deepens our understanding of the distribution of abnormal gene splicing and its molecular mechanisms but also offers an innovative correction tool for heterogeneous, clinically urgent conditions such as rare diseases or cancers, providing an important exploratory model for the entire field of gene therapies.

On a practical level, the single-ASO strategy proposed here offers several advantages: - Greatly enhances development efficiency, especially for rare variants or small patient populations in clinical translation; - Reduces the excessive fragmentation associated with individualized ASO development, facilitating drug standardization and industrialization; - Enriches the concept of precision medicine intervention at the “gene/exon level,” pushing forward new types of drug development.

In terms of limitations, the authors openly mention: - For missense mutations, only normal splicing is restored, but the underlying amino acid change may still yield dysfunctional protein and may require CRISPR or similar nucleic acid modification approaches for full correction; - Excessive ASO dosage or poor specificity may lead to multi-exon skipping and new phenotypes, mandating strict dosage and subject selection.

VII. Conclusion

This innovative and systematic study published in PNAS not only broadens the boundaries of exonic splicing abnormality and antisense therapy in molecular medicine, but also provides a full-spectrum experimental platform, molecular atlas, innovative ideas, and an entire translational pipeline paradigm. We look forward to its extension into more disease types, exon targets, and preclinical/clinical applications in the quest to achieve a true “gene-to-therapy” precision loop and bring feasible new options to patients with rare and variant diseases.