**Background:** Inborn errors of metabolism (IEMs) are a large group of rare genetic disorders caused by defects in enzymes, receptors, or transporters, leading to toxic metabolite accumulation or product deficiency. Despite traditional treatments like dietary manipulation, enzyme replacement, and transplantation, most IEMs lack effective therapies. Antisense oligonucleotides (ASOs) are synthetic single-stranded nucleic acids that bind to target RNA via Watson-Crick base pairing, modulating gene expression through RNase H-mediated degradation or steric hindrance. Splice-modulating ASOs, a type of steric blocker, can redirect aberrant splicing caused by mutations, restoring normal transcripts and functional protein. This review comprehensively analyzes the development of splice-switching ASOs for IEMs, covering mechanisms, chemical modifications, and rational design.
**Methods:** The authors conducted a narrative review of the literature, summarizing ASO technology and its application to IEMs. They describe ASO mechanisms (RNase H-dependent degradation and steric hindrance, including splice modulation), chemical modifications (phosphate linkage, sugar moiety, and backbone modifications), and design strategies (uniformly modified, gapmer, and mixmer designs). They then systematically review preclinical studies of splice-modulating ASOs targeting specific IEMs, including lysosomal storage diseases (Batten disease, Pompe disease, Fabry disease, Niemann-Pick type C, inclusion-cell disease, Hunter syndrome), organic acidemias (propionic acidemia, methylmalonic aciduria), congenital disorders of glycosylation, hereditary myopathy with lactic acidosis, 6-pyruvoyl-tetrahydropterin synthase deficiency, erythropoietic protoporphyria, complementation group cobalamin E type homocystinuria, and aromatic L-amino acid decarboxylase deficiency. Data are extracted from published studies, with emphasis on ASO design, target mutation, and outcomes in patient-derived cells or animal models.
**Key Results:** The review highlights several key findings. For Batten disease, milasen (TY777), a 22mer 2′-MOE-PS ASO, restored MFSD8 expression in patient fibroblasts, with RNA-seq showing a 34% increase in normal exon 6 splicing. Clinically, milasen reduced seizure count by 63% and seizure duration by 52%. In Pompe disease, 25mer PMOs (AMO 1, 2, 3) targeting the c.-32-13T>G mutation increased GAA activity by 70% and reduced glycogen accumulation. For Fabry disease, a 2′-OMe-PS 3′ss-SSO reduced pseudoexon inclusion by 40–73%. In propionic acidemia, ASO-29 (18mer 2′-MOE-PS) showed dose-dependent PCCA protein increase from 1 nM (onefold increase) to 25 nM (close to twofold increase). For PMM2-CDG, 25mer PMOs (AMOA and AMOB) at 20 µM increased PMM2 protein by 9–23% after 24 hours, with PMM activity reaching 50% of control after 48–72 hours. In hereditary myopathy with lactic acidosis, a 25mer PMO restored splicing to 100% wild-type levels, stable for 21 days. For PTPS deficiency, 25mer 2′-OMe-PS SSOs increased PTPS protein expression from 5% to 50% relative to untreated fibroblasts. In erythropoietic protoporphyria, V1-LNA-ASO (125 nM) reduced pseudoexon retention by 25%, normalizing FECH mRNA levels. For cblE homocystinuria, MTRR ESE-SSO restored correct splicing to control levels. In AADCD, ASO-D restored 41% of transcripts to the normal isoform.
**Clinical Implications:** Splice-modulating ASOs represent a promising individualized therapeutic strategy for IEMs, particularly those caused by mutations that create pseudoexons or disrupt splicing. The FDA permission for milasen (2018) as an N-of-1 therapy for Batten disease demonstrates clinical feasibility. However, most ASOs remain preclinical, with challenges including mutation specificity, delivery, and toxicity. The review suggests optimizing ASO design through novel chemistries (e.g., cEt, TMO, MsPA), mixmer designs, and cocktail therapies. Given the rarity of individual IEMs, ASOs offer a precision medicine approach, but further clinical translation is needed. The authors conclude that with continued optimization, more IEM-targeting ASOs will enter clinical trials, providing effective treatments for these devastating disorders.