Recent advances in genetic testing have accelerated the genetic diagnosis of rare diseases; however, the development of therapeutics has progressed comparatively slowly [1]. Currently only 5% of rare genetic disorders have approved treatments and 15% are undergoing clinical trials [2]. Considering that 80% of rare genetic disorders have neurological involvement, early treatment is crucial for most of these patients, given the rapidly progressive and fatal nature of the diseases, especially in pediatric patients with a developmental time frame [3].

Monogenic disorders account for 80% of rare genetic disorders [4]. Thus, gene-based treatment has become more of an interest in the clinical and research fields. Gene-based therapies involve both DNA- and RNA-directed oligonucleotide treatments. DNA-directed approaches include gene transfer via viral or non-viral vectors and gene-editing techniques such as CRISPR-Cas9, base editing, and prime editing. These methods enable precise genetic modifications and potentially correct disease-causing mutations at the DNA level. However, gene transfer requires a viral vector that can activate the immune system. Gene expression depends on the stability of the episomal forms. These effects may wane, particularly in rapidly dividing tissues. In contrast, RNA-directed therapies do not require a viral vector and messenger RNA (mRNA) is continuously replenished, ensuring stability and sustained expression. RNA-targeting drugs, including antisense oligonucleotides (ASOs), small interfering RNA (siRNA), mRNA therapy, and RNA editing, also exhibit high selectivity owing to their nucleotide sequence specificity, effectively targeting the intended sites while maintaining stability.

In this review, we discuss the basic mechanisms and latest clinical applications of ASOs in rare neurogenetic disorders. ASOs have gained considerable attention due to the clinical success of nusinersen (Spinraza), an ASO that modifies pre-mRNA splicing of SMN2 to promote increased production of full-length SMN protein [5]. This breakthrough has encouraged further development of ASOs for rare genetic disorders. Furthermore, the first personalized ASO was proven successful in a female child with Batten disease, which opened a new paradigm for n-of-1 treatment in the world of oligonucleotide therapeutics. The significance of ASOs was underscored by Kim et al. [6], who reported that ASOs show therapeutic potential in approximately 9-15% of rare genetic diseases, highlighting their growing impact in precision medicine regarding rare neurogenetic disorders.

ASOs typically comprise 12-25 nucleotides and attach to complementary RNA sequences via Watson–Crick base pairing. ASOs can target both protein-coding and non-coding RNA to modulate protein expression. Their small size (4-10 kDa), single-stranded structure, and hydrophilic properties enable spontaneous cellular uptake without the need for carriers, similar to that of small-molecule drugs. ASOs can penetrate the brain, spinal cord, and retina without chemical modifications; however, modifications can enhance their delivery to the liver or muscles. Among gene-based therapies, ASOs are notable for their minimal off-target and side effects combined with the benefit of chemically stable modifications [7].

The mechanisms of action primarily involve RNA cleavage and blockage, shown in detail in Fig. 1 [8,9]. RNA cleavage is facilitated by the recruitment of RNase H, an enzyme that specifically degrades the RNA strand of a DNA-RNA hybrid. ASOs, which are designed as gapmers, contain a central DNA gap flanked by chemically modified RNA segments that enhance their stability and binding affinity. The DNA region of the gapmer hybridizes with the target RNA, forming a duplex that activates RNase H, leading to the selective degradation of the target RNA. This mechanism may be used to knock down target transcripts and downregulate mRNA expression (Fig. 1A).

In contrast, RNA blockage occurs through steric hindrance or modulation of RNA splicing. ASOs can interact with pre-mRNAs to influence splicing events, including the skipping of exons, to potentially correct splicing defects or alter the production of specific protein isoforms (Figs. 1B and C).

Furthermore, ASOs could be used to increase the production of proteins from the wild-type (WT) transcript by upregulating the WT allele via targeted augmentation of nuclear gene output (TANGO), which augments the targeted area of the WT allele to induce greater production of functional proteins [10]. An ASO can be created to skip poison exons, target antisense transcripts (ASTs), and untranslated regions, including upstream open reading frames (uORFs) (Fig. 1D). Poison exons are conserved sequences that, when included in mRNA, lead to the early termination of translation. The use of ASOs to skip these exons boosts the generation of functional transcripts, thereby increasing the protein expression. ASTs are non-coding RNAs that regulate gene expression via RNA interference or masking. ASOs may inhibit the expression of AST to increase the protein expression. uORFs are defined as translation sites found within the 5’ untranslated region of mRNAs, positioned before the main protein-coding sequence. ASOs can both enhance or inhibit protein expression by blocking or skipping exons containing uORFs [11].

Recent advancements in the chemical modification of ASOs have resulted in three generations. The first generation consists of a phosphorothioate (PS) backbone, which involves substituting the non-bridging oxygen in the phosphate backbone with sulfur. Second-generation modifications include 2‘-O-modifications such as 2‘-O-methoxyethyl (2‘-MOE), 2‘-O-methyl (2‘-OMe), and 2‘-fluoro (2‘-F), which improve RNA affinity and metabolic stability. Third-generation modifications include the locked nucleic acid (LNA) method. LNA modifications lock the ribose sugar in a specific conformation, which significantly enhances binding affinity and stability. Furthermore, emerging strategies include conjugating ASOs with targeting ligands such as GalNAc, which enhance tissue delivery and minimize side effects [12,13]. These modifications enhance ASO stability, binding affinity, and cellular uptake, while reducing nuclease susceptibility, thereby prolonging its half-life in the bloodstream and tissues [9,14]. Therefore, ASOs achieve high specificity and efficacy in targeting and modulating gene expression, making them promising therapeutic options for a wide range of genetic disorders.

As of April 2024, 19 oligonucleotide therapies have been approved by the New Drug Application (NDA) of the United States Food and Drug Administration (US FDA) and the European Medicines Agency (EMA) for the treatment of rare genetic disorders [12,15]. Eight of the 19 oligonucleotide therapies were designated as ASOs for rare neurogenetic disorders (Table 1). Furthermore, several n-of-1 personalized ASOs have been granted approval under Investigational New Drug (IND) status (Table 2).

1. Spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder, the majority of which is caused by a homozygous deletion of the survival motor neuron 1 (SMN1) gene, leading to the progressive degeneration of motor neurons, muscle weakness, and atrophy. The SMN2 gene partially compensates for SMN1 function (approximately 10-15%) but contains a splicing defect in exon 7. Nusinersen (Spinraza), an 18-mer 2’-MOE-modified ASO, modulates splicing in this region to increase the SMN protein levels [16]. Approved by the FDA in 2016, it is administered intrathecally every four months. Nusinersen has demonstrated therapeutic efficacy in pediatric and adult populations with SMA, improving motor function and stabilizing disease progression, particularly when initiated early [17,18]. Significant gains were observed in both ambulant and non-ambulant patients, with improvements in the Hammersmith Functional Motor Scale Expanded, Revised Upper Limb Module, and 6-Minute Walk Test, regardless of the SMA type. Real-world data have confirmed its effectiveness in maintaining function and managing disease progression across diverse patient populations [18,19].

2. Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD), an X-linked recessive disorder caused by mutations in the dystrophin gene (Xp21), results in progressive skeletal muscle degeneration and weakness. Approximately 65% of patients have exonic deletions, 5-10% have exonic duplications, and the remainder are mainly nonsense and indel mutations. Four ASOs, using phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping, have been approved: Eteplirsen (Exondys 51, exon 51, FDA 2016) [20], Golodirsen (Vyondys 53, exon 53, FDA 2019) [21], Viltolarsen (Viltepso, exon 53, FDA 2020) [22], and Casimersen (Amondys 45, exon 45, FDA 2021) [23]. The ASOs are administered weekly via intravenous injections. All four ASOs have shown modest improvements by increasing dystrophin levels and slowing disease progression. Clinical outcomes vary with a limited impact on long-term ambulation and mixed results regarding the ability to preserve motor function in patients [24,25]. Observational studies, such as the EVOLVE trial, have highlighted favorable safety profiles; however, ongoing research is needed to confirm their long-term efficacy and functional benefits [26].

3. Hereditary transthyretin-mediated amyloidosis

Hereditary transthyretin-mediated amyloidosis (hATTR), an autosomal dominant disorder resulting from mutations in the TTR gene on chromosome 18, leads to the accumulation of abnormal transthyretin protein as amyloid that affects nerves and various tissues. Inotersen (Tegsedi), a knockdown 2‘-MOE gapmer ASO, cleaves mRNA coding for the transthyretin protein via RNAse H. Inotersen received FDA approval in 2018 [27]. Subsequently, Eplontersen (Wainua), a gapmer ASO with the same sequence as inotersen but with enhanced potency (30-50-fold) through chemical modifications (phosphodiester linkage and GalNAc ligand conjugation), gained FDA approval in 2023 [28]. Both inotersen and eplontersen show promise in slowing neuropathy progression and improving patient outcomes [29]. Eplontersen, administered monthly, offers a more favorable safety profile than inotersen, which requires weekly injections and has a higher risk of thrombocytopenia, which is manageable through monitoring. Both treatments demonstrated efficacy over placebo, with eplontersen’s targeted liver mechanism allowing for smaller doses, resulting in fewer side effects and comparable efficacy in reducing disease progression [28].

4. Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) selectively affects the motor neurons and progressively destroys the brain, brainstem, and spinal cord. Approximately 5-10% of ALS cases have a genetic etiology, with approximately 20% linked to mutations in SOD1. Tofersen (Qalsody), an 2‘-MOE gapmer ASO, cleaves SOD1 mRNA via RNase H, thereby reducing abnormal SOD1 protein production. Administered intrathecally monthly, tofersen received FDA approval in 2023 [30]. Tofersen has shown the potential to slow disease progression, although its effect on functional outcomes did not reach statistical significance in the 28-week phase 3 VALOR trial [31]. A longer follow-up in the open-label extension indicated that patients who started treatment earlier experienced a slower decline in respiratory function and motor abilities than those who began treatment later [32]. These findings emphasize the importance of early intervention; however, further studies are required to confirm the long-term clinical benefits of tofersen.

The advent of personalized ASOs has provided a breakthrough for patients with neurogenetic disorders who lack effective treatment options. A notable case involved a child with Batten disease who received a splice-modulating ASO, resulting in marked clinical improvement and demonstrating the potential for individualized genetic therapies. This case set a precedent and inspired further global initiatives. Leading efforts in personalized ASO development include programs such as N=1 Collaborative, n-Lorem, Dutch Center for RNA Therapeutics (DCRT), and 1 Mutation 1 Medicine (1M1M). These organizations have pioneered customized therapies for ultra-rare conditions, with several impactful cases documented. Anecdotally, more than 20 projects involving patient-customized ASOs are ongoing internationally, some of which are listed in Table 2 [6,33-41]. This progress highlights a promising shift toward precision medicine and the transformation of care for patients with otherwise untreatable genetic diseases.

1. Batten disease

Milasen was the first personalized ASO therapy targeting a specific mutation in Mila, a six-year-old with Batten disease [33]. Batten disease leads to vision loss, seizures, limb paralysis, and death. Mila’s condition was caused by a SINE-VNTR-Alu (SVA) retrotransposon insertion in the MFSD8 (CLN7) gene, altering transcript splicing [42]. An 18-mer 2’-MOE-modified ASO was designed to sterically block this SVA region, designed in a similar sequence to the well-known Nusinersen. The efficacy and toxicity were validated in vitro and in vivo. Milasen received fast-track approval from the US FDA for IND status, leading to the publication of draft guidelines on ASOs for fatal rare genetic diseases. The process from diagnosis to the first drug administration took approximately 15 months. Mila’s seizures, which occurred up to 30 times a day before treatment, decreased to less than 10 times a day. Unfortunately, Mila died because of late treatment; however, the development of personalized therapies and clinical application potential of ASOs have been clearly demonstrated.

2. Ataxia telangiectasia

Following the success of Milasen, the second personalized ASO therapy, Atipeksen, was developed for Ipek, who was diagnosed with ataxia telangiectasia (AT) at age 1 after newborn screening revealed low T-cell receptor excision circles [6]. AT, caused by mutations in the ATM gene, leads to progressive neurodegeneration, motor impairment, and eventually death in adolescence [43]. Exome sequencing results revealed c.8585-13_8598del27 and c.7865C>T mutations in the ATM gene. The c.7865C>T variant created a novel splice site in exon 56, resulting in mis-splicing, with an out-of-frame truncation of 64 nucleotides. An ASO with PS/2’-MOE chemical modification (Atipeksen) was designed to block this splice site. The patient, initiated on Atipeksen at 2.5 years, prior to the natural onset of AT symptoms, remains asymptomatic at 6 years of age, following 4 years of treatment [34,35].

3. KIF1A-associated disorder

Following the two splice-modulating n-of-1 ASOs, the first knockdown gapmer ASO was successful in a patient with KIF1A-associated disorder (KAND). KAND is associated with brain and optic nerve atrophy, intractable epilepsy, intellectual disabilities, and other neuropathies. The 9-year-old patient had a de novo pathogenic variant of KIF1A, c.914C>T (p.Pro305Leu), found in trans with the alternate allele for a common single-nucleotide polymorphism in intron 37 of 48 of KIF1A, which was detected using long-read sequencing. A 20-mer ASO with PS/2’-MOE chemical modification, nL-KIF1-001, was designed to bind in cis with the pathogenic variant. The ASO was administered intrathecally every 3 months. With treatment, her seizure frequency decreased considerably, and her electroencephalogram showed improvement. The patient showed an increased ability to walk and improved cognitive function.

There are many emerging ASOs in the process of moving from the laboratory bench to the bedside, offering hope for patients with previously untreatable neurogenetic disorders. Table 3 summarizes the ASOs currently being administered to patients and those undergoing clinical trials. Before reaching this stage, these ASOs had already demonstrated therapeutic potential in patient-derived cell lines and in vivo models, thereby establishing a foundation for clinical translation. We have thoroughly reviewed the following three exemplary neurogenetic disorders targeted by ASOs that are currently undergoing active clinical trials.

1. Dravet syndrome: SCN1A

Dravet syndrome is characterized by seizures during early childhood and is often accompanied by developmental disorders [44]. More than 80% of cases are associated with SCN1A gene mutations (haploinsufficiency). Through whole-genome sequencing, a poison exon near intron 20 of the SCN1A gene was discovered, which leads to the inefficient formation of productive SCN1A mRNA transcripts [45]. Therefore, inducing efficient mRNA formation using the TANGO technology is expected to overcome haploinsufficiency and yield therapeutic effects [46]. The phase 1/2a and open label extension study data of STK-001 shows that a total of 81 patients were treated with the ASO, and patients have shown substantial reductions in convulsive seizure frequency (43 to 85% reduction from baseline) and improvements in multiple measures of cognition and behavior (NCT04442295, Stoke Therapeutics) [47].

2. SCN2A developmental and epileptic encephalopathy

Mutations in SCN2A result in diverse phenotypes. Gain-of-function (GoF) mutations cause severe developmental and epileptic encephalopathy (DEE), whereas loss-of-function (LoF) variants lead to milder epilepsy, intellectual disability, and autism. Thus, balancing the GoF and LoF phenotypes is challenging because oversuppression can exacerbate LoF symptoms, necessitating careful dose optimization to maintain therapeutic efficacy without introducing cognitive or behavioral side effects. Elsunersen (PRAX-222), an ASO targeting GoF mutations, has shown promise for reducing seizures in pre-clinical models [48]. In a phase 2 trial, four patients receiving monthly intrathecal elsunersen experienced a 44% reduction in seizures (NCT05737784, Praxis Precision Medicines) [49]. These results highlight the potential of ASO therapies in precision medicine for the management of complex neurogenetic disorders.

3. Angelman syndrome: UBE3A antisense transcript

Angelman syndrome manifests with severe developmental delay, epilepsy, and other symptoms. This is due to the loss of function of the maternally inherited UBE3A gene through genomic imprinting. UBE3A AST (UBE3A-AS) silences the paternal UBE3A allele. An ASO (ION582 and GTX-102) was made to inhibit UBE3A-AS, unsilencing the paternal UBE3A allele and increasing UBE3A production in the brain. A phase 1/2 open-label study of ION582 has shown to improve overall symptoms in 97% of the participants, measured by the Symptoms of Angelman Syndrome–Clinician Global Impression-Change (NCT05127226, Ionis) [50]. Interim phase 1/2 data of GTX-102 also showed clinical improvement in multiple domains compared to the natural history data (NCT04259281, Ultragenyx Pharmaceutical) [51].

ASOs have redefined the landscape of rare neurogenetic disorders by delivering targeted therapies that address their root causes at the molecular level. Their impact is exemplified by breakthrough treatments, such as nusinersen and personalized ASOs, which show how precision interventions can alter disease trajectories and enhance the quality of life. The integration of evolving diagnostics with precision therapeutics will not only enable early detection, but also allow timely, individualized treatment to prevent irreversible progression. As advancements in delivery systems and chemical modifications continue to enhance ASO efficacy, these therapies are poised to become a foundational pillar of precision medicine, transforming the care for patients with previously untreatable genetic diseases.

We acknowledge the Medical Illustration & Design (MID) team, a member of the Medical Research Support Services of Yonsei University College of Medicine, for providing excellent support with the medical illustration.

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: RS-2024-00405260 and RS-2024-00439002).

Conception and design: HJS, HCK. Acquisition of data: HJS, AK, JYO. Analysis and interpretation of data: HJS, AK, JYO. Drafting the article: HJS. Critical revision of the article: AK, HCK. Final approval of the version to be published: HCK.