The Splice of Life: How Alternative Splicing Is Reshaping the Therapeutic Landscape Across Rare Diseases and Oncology
核心洞察
Alternative splicing generates vast transcriptomic diversity, with approximately 95% of multiexon genes undergoing alternative splicing across major human tissues, creating both therapeutic opportunities and pathogenic vulnerabilities.
Small-molecule splicing modulators have achieved clinical success, highlighted by risdiplam (Evrysdi), the first FDA-approved orally administered splicing modifier for spinal muscular atrophy (搜索), and the SF3B1 (搜索)-targeting agent H3B-8800 in myeloid neoplasms.
Antisense oligonucleotides including nusinersen (Spinraza) for SMA and multiple PMO-based exon-skipping therapies for Duchenne muscular dystrophy (搜索) demonstrate the clinical viability of splice-switching approaches.
The coordinated interplay of cis-acting elements, trans-acting factors, and epigenetic dynamics that governs alternative splicing has emerged as one of the most fertile frontiers in pharmaceutical development. Once viewed primarily through the lens of basic transcriptomics, splicing modulation now underpins approved therapies for neuromuscular disorders and is generating a robust pipeline of candidates in oncology, where dysregulated splicing drives both tumorigenesis and immune evasion.
The Regulatory Architecture of Isoform Diversity
Alternative splicing represents the predominant contributor to transcript diversity, generating isoforms that vary in exon and intron composition from the same gene. The majority of introns—U2-type—are bound by 5′ and 3′ splice sites featuring the core dinucleotides GU and AG, respectively, and are processed by the major spliceosome composed of the snRNPs U1, U2, U4, U5, and U6. Minor introns (U12-type), accounting for less than 0.4% to 1% of introns in human genes, feature less efficient AU-AC splice site motifs and are excised by the minor spliceosome.
Beyond spliceosome-mediated cleavage, a large portion of transcript variability emerges at the transcriptional level through alternative promoters, transcription start sites, and alternative cleavage and polyadenylation sites. More than half of human genes encode two or more canonical polyadenylation signals, with differential usage associated with cell and tissue types, developmental stage, and disease. These mechanisms are regulated through a dense network of cis-regulatory elements—including exonic and intronic splicing enhancers and silencers—and trans-acting factors such as serine-arginine-rich proteins, hnRNPs, and RNA-binding proteins.
When Splicing Goes Awry: Pathogenic Mechanisms
A large portion of disease-associated mutations exert their primary functional impact through disruption of RNA splicing rather than direct alterations to protein-coding sequences. These variants primarily occur within cis-regulatory elements, destroying 5′ or 3′ splice boundaries and leading to exon skipping or intron retention. A more complex class involves activation of cryptic splice sites, creating de novo splice site consensus sequences that disrupt normal regulation.
Such defects have been documented across a wide range of human diseases. In Duchenne muscular dystrophy (搜索), mutations targeting canonical splice junctions of the dystrophin gene lead to exon skipping and disruption of the open reading frame. In β-thalassemia, the IVS1-110G>A variant prevalent in the Middle East and Mediterranean generates a novel splice acceptor within the first intron of HBB, while the IVS2-654C>T variant common in East Asian populations activates a de novo 5′ splice site. In Hutchinson-Gilford progeria syndrome, a synonymous point mutation in exon 11 of the LMNA gene activates a cryptic 5′ splice donor site, producing the truncated progerin protein.
Spinal muscular atrophy (搜索) exemplifies disruption of auxiliary splicing regulatory elements. A single nucleotide substitution (c.840C>T) in exon 7 of SMN2 (搜索) disrupts an exonic splicing enhancer and creates an exonic splicing silencer, reducing SRSF1 binding while recruiting hnRNP A1/A2 and promoting exon 7 skipping. The resulting protein isoform lacks an essential C-terminal site necessary for SMN protein stability and self-oligomerization.
Systemic trans-acting dysregulation also drives disease. Mutations in core spliceosomal components cause spliceosomopathies such as retinitis pigmentosa, frequently driven by mutations in PRPF8, PRPF31, PRPF3, and SNRNP200. In oncology, recurrent somatic mutations in SF3B1 (搜索), U2AF1, and SRSF2 drive a large portion of myelodysplastic syndromes (搜索) and are associated with myeloid leukemias. The most prevalent MDS mutation occurs in SF3B1, causing aberrant recognition of alternative 3′ splice sites approximately 20 nucleotides upstream of the canonical site.
Small-Molecule Splicing Modulators: From Natural Products to Approved Drugs
Early splicing-targeting strategies focused on natural products that inhibit functional components of the spliceosome. In 2008, O'Brien et al. reported the discovery of isoginkgetin, a natural biflavonoid from Ginkgo biloba that inhibits pre-mRNA splicing by disrupting recruitment of the U4/U5/U6 tri-snRNP. The antitumor drug E7107, another natural product, blocks spliceosome assembly and was evaluated in a phase I study in patients with advanced solid tumors.
A landmark achievement came in 2020 when risdiplam (Evrysdi) became the first FDA-approved, orally administered small-molecule splicing modulator for treating SMA. It functions by targeting a weak secondary structure at the 5′ splice site of SMN2 (搜索) pre-mRNA, stabilizing its interaction with the spliceosome and overcoming an inhibitory stem-loop structure to ensure inclusion of exon 7.
In oncology, H3B-8800, an orally available small-molecule splicing modulator that targets mutated SF3B1 (搜索) to selectively kill cancer cells, has been evaluated in phase I studies. Steensma et al. reported a first-in-human dose escalation study of H3B-8800 in myeloid neoplasms, and Foran et al. subsequently conducted a dose-expansion study in lower-risk myelodysplastic syndrome. Other small-molecule strategies include indisulam, which targets RBM39 for degradation via recruitment to DCAF15, and PRMT5 inhibitors such as JNJ-64619178 and PF-06939999, which have entered clinical testing in splicing-dysregulated malignancies.
Antisense Oligonucleotides: A Clinically Proven Platform
Splice-switching antisense oligonucleotides represent the most clinically validated therapeutic strategy for selective targeting of pathogenic splicing. The first generation of naked ASOs were highly unstable in vivo, but foundational optimization through phosphorothioate backbone modification, combined with 2′ sugar modifications and locked nucleic acid chemistry, resulted in higher binding affinity and specificity.
Nusinersen (Spinraza), an 18-mer 2′-MOE-PS oligonucleotide, binds to an intronic splicing silencer (ISS-N1) in SMN2 (搜索), forcing inclusion of exon 7 to produce stable, full-length SMN protein. In the DMD space, charge-neutral phosphorodiamidate morpholinos (PMOs) have yielded multiple approved therapies: eteplirsen (Exondys 51) for exon 51 skipping, casimersen (Amondys 45) for exon 45 skipping, and both golodirsen (Vyondys 53) and viltolarsen (Viltepso) for exon 53 skipping.
In cancer, SSOs have been applied to rewire BCL2L1 isoform usage, redirecting splicing from the antiapoptotic BCL-xL (搜索) to the pro-apoptotic BCL-xS, demonstrating robust antitumor effects. Additional applications include SSO targeting of HER2 exon 15 in breast cancer and LNA-based oligonucleotides targeting androgen receptor expression in prostate cancer.
CRISPR-Based and Emerging Modalities
CRISPR/Cas-based technology has evolved into a precise tool for transcriptomic engineering. Yue et al. successfully rewired expression of long- or short-splicing isoforms of the mouse Xist gene by modifying the 5′ splice site. Du et al. reported CRISPR Artificial Splicing Factors (CASFx) that enhanced inclusion of SMN2 (搜索) exon 7 in SMA patient fibroblasts. Yuan et al. developed a platform using CRISPR-guided cytidine deaminase to convert guanines to adenines at splice sites, correcting aberrant splicing.
Recent advances in RNA-targeting technologies have expanded the toolkit. CRISPR-Csm (Type III) systems enable programmable, RNA-guided, RNA-targeting tools for precise knockdown without permanent genome editing. The CREST platform enables simultaneous alternative splicing modulation and RNA base editing while reducing off-target editing by nearly 99%. CasRx/dCasRx-based platforms regulate alternative splicing by recruiting splicing effectors or sterically modulating splice site recognition.
PROTAC-mediated targeted degradation offers a transformative strategy to selectively eliminate aberrant splicing factors and protein isoforms. Qiu et al. demonstrated that SIAIS361034 selectively degrades BCL-xL (搜索) and inhibits tumor growth with low platelet toxicity compared with conventional inhibitors. Ghidini et al. introduced RNA-PROTACs for targeting RNA-binding proteins by employing small RNA mimics as targeting groups conjugated with E3-recruiting peptides.
Splicing-Derived Neoantigens and Immunotherapy
The intersection of alternative splicing and tumor immunology has opened new therapeutic avenues. Kwok et al. reported that tumour-wide RNA splicing aberrations generate actionable public neoantigens. Pan et al. developed IRIS, a computational platform for discovery of cancer immunotherapy targets arising from pre-mRNA alternative splicing. Li et al. introduced SNAF for splicing neoantigen discovery, revealing shared targets for cancer immunotherapy.
Splicing modulation can also enhance antitumor immunity. Lu et al. demonstrated that pharmacologic modulation of RNA splicing enhances anti-tumor immunity. Bowling et al. showed that spliceosome-targeted therapies trigger an antiviral immune response in triple-negative breast cancer. Matsushima et al. reported that chemical induction of splice-neoantigens attenuates tumor growth in a preclinical model of colorectal cancer.
Soluble immune checkpoint isoforms generated through alternative splicing further illustrate the clinical relevance. Soluble PD-L1 variants mediate resistance to PD-L1 blockade therapy in non-small cell lung cancer, as demonstrated by Gong et al. A splicing isoform of PD-1 promotes tumor progression as a potential immune checkpoint, as reported by Wang et al. Ding et al. showed that lactate modulates RNA splicing to promote CTLA-4 expression in tumor-infiltrating regulatory T cells.
Technological Advances Driving Discovery
The advent of long-read RNA-sequencing technologies, pioneered by Pacific Biosciences and Oxford Nanopore Technologies, has enabled direct, isoform-resolved transcriptome profiling. These platforms have led to identification of previously unappreciated classes of splicing defects, including multiexon skipping, exonic intron creation, intronic polyadenylation, and combinatorial splicing defects. Direct linkages between genetic variants and splice isoforms established from long-read data have enabled diagnosis of multiple rare diseases.
Emerging single-cell and spatial long-read RNA-sequencing technologies have advanced discovery of isoform usage shifts across cell types, cell states, and spatial niches. Pan et al. reported a comprehensive cell-level isoform atlas of the adult human heart and heart failure, revealing hundreds of cell type-specific and disease-associated isoform usage-shifting events. In cancer, identification of hundreds of tumor cell-enriched isoforms offers a rich source of candidate targets for selective tumor cell killing.