Effective translation of rare disease diagnosis knowledge into therapeutic applications is achievable within a reasonable timeframe; where mutations are amenable to current antisense oligonucleotide technology. In our study, we identified five distinct types of abnormal splice-causing mutations in patients with rare genetic disorders and developed a tailored antisense oligonucleotide for each mutation type using phosphorodiamidate morpholino oligomers with or without octa-guanidine dendrimers and 2'-O-methoxyethyl phosphorothioate. We observed variations in treatment effects and efficiencies, influenced by both the chosen chemistry and the specific nature of the aberrant splicing patterns targeted for correction. Our study demonstrated the successful correction of all five different types of aberrant splicing. Our findings reveal that effective correction of aberrant splicing can depend on altering the chemical composition of oligonucleotides and suggest a fast, efficient, and feasible approach for developing personalized therapeutic interventions for genetic disorders within short time frames.

Department of Clinical Immunology and Allergology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Fondazione Fegato Area Science Park Basovizza 34149 Trieste Italy

Human Development and Health Faculty of Medicine University of Southampton Southampton UK

International Centre for Genetic Engineering and Biotechnology Padriciano 99 34149 Trieste Italy

Oxford Centre for Genomic Medicine Oxford University Hospitals NHS Foundation Trust Oxford UK

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Nguengang Wakap, S. et al. Estimating cumulative point prevalence of rare diseases: analysis of the Orphanet database. Eur. J. Hum. Genet.28, 165–173 (2019). 10.1038/s41431-019-0508-0 PubMed DOI PMC

Kohn, D. B., Chen, Y. Y. & Spencer, M. J. Successes and challenges in clinical gene therapy. Gene Therapy8, 1–9. 10.1038/s41434-023-00390-5 (2023). PubMed PMC

Tambuyzer, E. et al. Therapies for rare diseases: therapeutic modalities, progress and challenges ahead. Nat. Rev. Drug Discov.19, 93–111 (2019). 10.1038/s41573-019-0049-9 PubMed DOI

Dawkins, H. J. S. et al. Progress in Rare Diseases Research 2010–2016: An IRDiRC Perspective. Clin. Transl. Sci.11, 11–20 (2018). 10.1111/cts.12501 PubMed DOI PMC

The Lancet Diabetes Endocrinology. Spotlight on rare diseases. Lancet Diabetes Endocrinol.7, 75 (2019). PubMed

Douglas, A. G. L. & Baralle, D. Translating RNA splicing analysis into diagnosis and therapy. OBM Genet. 5. 10.21926/obm.genet.2101125 (2021).

Wai, H., Douglas, A. G. L. & Baralle, D. RNA splicing analysis in genomic medicine. Int. J. Biochem. Cell Biol.108, 61–71 (2019). 10.1016/j.biocel.2018.12.009 PubMed DOI

Newman, A. RNA splicing. Curr. Biol.8, R903–R905 (1998). 10.1016/S0960-9822(98)00005-0 PubMed DOI

Baralle, M. & Baralle, F. E. The splicing code. Biosystems164, 39–48 (2018). 10.1016/j.biosystems.2017.11.002 PubMed DOI

Wai, H. A. et al. Short amplicon reverse transcription-polymerase chain reaction detects aberrant splicing in genes with low expression in blood missed by ribonucleic acid sequencing analysis for clinical diagnosis. Hum. Mutat.43, 963–970 (2022). 10.1002/humu.24378 PubMed DOI PMC

Wai, H. A. et al. Blood RNA analysis can increase clinical diagnostic rate and resolve variants of uncertain significance. Genet. Mediclyol.22, 1005–1014 (2020).10.1038/s41436-020-0766-9 PubMed DOI PMC

Stephenson, M. L. & Zamecnik, P. C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc. Natl Acad. Sci. USA75, 285 (1978). 10.1073/pnas.75.1.285 PubMed DOI PMC

Crooke, S. T. Progress toward oligonucleotide therapeutics: pharmacodynamic properties. FASEB J.7, 533–539 (1993). 10.1096/fasebj.7.6.7682523 PubMed DOI

Crooke, S. T., Baker, B. F., Crooke, R. M. & Liang, X. H. Antisense technology: an overview and prospectus. Nat. Rev. Drug Discov.20, 427–453 (2021). 10.1038/s41573-021-00162-z PubMed DOI

Gleeson, J. G. et al. Personalized antisense oligonucleotides ‘for free, for life’—the n-Lorem Foundation. Nat. Med.29, 1302–1303 (2023). 10.1038/s41591-023-02335-2 PubMed DOI

Crooke, S. T., Witztum, J. L., Bennett, C. F. & Baker, B. F. RNA-targeted therapeutics. Cell Metab.27, 714–739 (2018). 10.1016/j.cmet.2018.03.004 PubMed DOI

Hua, Y. et al. Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model. Genes Dev.24, 1634 (2010). 10.1101/gad.1941310 PubMed DOI PMC

Finkel, R. S. et al. Nusinersen versus Sham control in infantile-onset spinal muscular atrophy. N. Engl. J. Med.377, 1723–1732 (2017). 10.1056/NEJMoa1702752 PubMed DOI

Charleston, J. S. et al. Eteplirsen treatment for Duchenne muscular dystrophy. Neurology90, e2146–e2154 (2018). 10.1212/WNL.0000000000005680 PubMed DOI

Kim, J. et al. Patient-customized oligonucleotide therapy for a rare genetic disease. N. Engl. J. Med.381, 1644–1652 (2019). 10.1056/NEJMoa1813279 PubMed DOI PMC

Spiekerkoetter, U. et al. Genomic newborn screening: are we entering a new era of screening? J. Inherit. Metab. Dis.46, 778–795 (2023). 10.1002/jimd.12650 PubMed DOI

Ceyhan-Birsoy, O. et al. Interpretation of genomic sequencing results in healthy and Ill newborns: results from the BabySeq Project. Am. J. Hum. Genet.104, 76–93 (2019). 10.1016/j.ajhg.2018.11.016 PubMed DOI PMC

Kim, J. et al. A framework for individualized splice-switching oligonucleotide therapy. Nature619, 828–836 (2023). 10.1038/s41586-023-06277-0 PubMed DOI PMC

Holmes-Hampton, G. P. et al. Use of antisense oligonucleotides to correct the splicing error in ISCU myopathy patient cell lines. Hum. Mol. Genet.25, 5178 (2016). PubMed PMC

Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc.8, 2281–2308 (2013). 10.1038/nprot.2013.143 PubMed DOI PMC

Hwang, G. H. et al. CRISPR-sub: Analysis of DNA substitution mutations caused by CRISPR-Cas9 in human cells. Comput. Struct. Biotechnol. J.18, 1686–1694 (2020). 10.1016/j.csbj.2020.06.026 PubMed DOI PMC

Fukushima, S. et al. Dual fluorescence splicing reporter minigene identifies an antisense oligonucleotide to skip exon v8 of the CD44 Gene. Int J. Mol. Sci.21, 1–16 (2020).10.3390/ijms21239136 PubMed DOI PMC

Preußner, M. et al. ASO targeting RBM3 temperature-controlled poison exon splicing prevents neurodegeneration in vivo. EMBO Mol. Med.15, e17157 (2023). 10.15252/emmm.202217157 PubMed DOI PMC

Enkhjargal, S. et al. Antisense oligonucleotide induced pseudoexon skipping and restoration of functional protein for Fukuyama muscular dystrophy caused by a deep-intronic variant. Hum. Mol. Genet.32, 1301–1312 (2023). 10.1093/hmg/ddac286 PubMed DOI

Feng, P. et al. Rescue of mis-splicing of a common SLC26A4 mutant associated with sensorineural hearing loss by antisense oligonucleotides. 10.1016/j.omtn.2022.03.015 (2022). PubMed PMC

Sakaguchi, N. & Suyama, M. Pervasive occurrence of splice-site-creating mutations and their possible involvement in genetic disorders. npj Genom. Med.7, 1–13 (2022). 10.1038/s41525-022-00294-0 PubMed DOI PMC

Crooke, S. T. Meeting the needs of patients with ultrarare diseases. Trends Mol. Med28, 87–96 (2022). 10.1016/j.molmed.2021.12.002 PubMed DOI

Liang, X. H., Sun, H., Shen, W. & Crooke, S. T. Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages. Nucleic Acids Res.43, 2927–2945 (2015). 10.1093/nar/gkv143 PubMed DOI PMC

Cirak, S. et al. Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet378, 595–605 (2011). 10.1016/S0140-6736(11)60756-3 PubMed DOI PMC

Frank, D. E. et al. Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy. Neurology94, e2270–e2282 (2020). 10.1212/WNL.0000000000009233 PubMed DOI PMC

Geary, R. S., Baker, B. F. & Crooke, S. T. Clinical and preclinical pharmacokinetics and pharmacodynamics of mipomersen (kynamro®): a second-generation antisense oligonucleotide inhibitor of apolipoprotein B. Clin. Pharmacokinet.54, 133–146 (2015). 10.1007/s40262-014-0224-4 PubMed DOI PMC

Zanardi, T. A. et al. Chronic toxicity assessment of 2’-O-methoxyethyl antisense oligonucleotides in mice. Nucleic Acid Ther.28, 233–241 (2018). 10.1089/nat.2017.0706 PubMed DOI

Ferguson, D. P., Dangott, L. J. & Lightfoot, J. T. Lessons learned from vivo-morpholinos: How to avoid vivo-morpholino toxicity. Biotechniques56, 251–256 (2014). 10.2144/000114167 PubMed DOI PMC