Antisense oligonucleotides (ASOs) are primarily high molecular weight compounds with nucleotide base pairs capable of binding to mRNA and impeding protein translation or splicing [1]. ASOs represent a new era of gene therapy (transcript-targeted therapy) for the treatment of genetic disorders that are otherwise impossible to treat [2]. Rare diseases often have fewer treatment options due to their lower frequency, improper diagnosis, and limited knowledge about their pathological basis. Gene therapy shows promise in treating rare diseases, although clinical challenges must be overcome to realize its therapeutic benefits fully. Current research on ASOs and FDA approval of drugs like Nusinersen, Eteplirsen, and Inotersen shed new light on the potential of this approach. However, the molecular mechanisms involved in the pathophysiology of rare diseases are so complex that the nonselective action of ASOs can cause more harm than benefit. Therefore, ASO therapeutics can be a miracle or a mirage unless they are rigorously tested and approved. This paper emphasizes the importance of ASOs in the treatment of rare diseases by weighing their advantages and disadvantages and citing recent studies conducted with ASOs.

ASOs play a prominent role in the treatment of genetic diseases, and a “miracle” approach was once seen as just a mirage. A thorough literature review, analysis of ongoing clinical trials, and revisiting the approvals of drug regulatory agencies reaffirmed the promising benefits of the ASOs.

An autosomal dominant disease called hereditary transthyretin amyloidosis (hATTR) occurs due to mutations (single nucleotide substitution) in the TTR gene on chromosome 18q12.1. hATTR is a progressive condition with a poor prognosis and a higher mortality rate, indicating that current approaches are ineffective in their management. Transcript-targeted therapy using ASO inotersen can reduce TTR production by interfering with the mRNA responsible for its turnover. The clinical trial results showed that the patients receiving inotersen had improved their quality of life. However, the study also indicated adverse effects such as glomerulonephritis, blood dyscrasias, and death [17].

Familial chylomicronemia is an autosomal recessive disorder caused by the mutation of the lipoprotein lipase gene present on chromosome 19q13.32. Volanesorsen, a second-generation ASO, is found to be effective in reversing this condition by specifically targeting apolipoprotein C1 (APOC2) mRNA. However, the therapeutic benefits of this study are outweighed by adverse drug reactions, especially the high reduction of thrombocytes [18].

Non-arteritic ischemic optic neuropathy (NAION), characterized by ischemia of the posterior ciliary arteries, is responsible for optic disc compartment syndrome and blindness. Chemically modified ASO called QPI-007 (developed by Quark Pharmaceuticals) can interfere with Caspase-2 expression and prevent NAION. QPI-007 produced a significant neuroprotective effect in animal and preclinical studies [19].

Familial hypercholesterolemia (FH) is a genetic disorder characterized by an increase in blood LDL level (>190 mg/dL). An experimental ASO, Mipomersen, can inhibit Apo B-mRNA and check the translation [20]. A similar ASO Inclisiran developed by Novartis is approved for the treatment of HHF. The therapeutic benefits of Mipomersen are similar to those of inclisiran and can be promising in managing FH with fewer adverse effects [21].

Amyotrophic lateral sclerosis (ALS) caused by the SOD1 mutation is a highly debilitating condition with a poor prognosis. Tofersen-elastic experimental ASO therapy is currently in clinical trials for the treatment of ALS. Tofersen exerts its action through RNase H-dependent degradation of the SOD1 protein, which helps counter the effects of ALS. However, the initial findings of the clinical trial revealed an increased risk of Cerebrospinal Fluid (CSF) pleocytosis [22].

Spinal muscular atrophy (SMA) occurs due to the loss of lower motor neurons and is caused by a mutation in the survival motor neuron (SMN2) gene that encodes the SMN2 protein. A second-generation Nusinseren ASO can inhibit the SMN2 gene splicing and block the intronic splicing silencer. In early clinical trials, Nusinseren improved neurological function and life expectancy, and therefore, it could be a miraculous treatment if adverse effects are ruled out [23].

Milasen is another promising ASO that has been experimentally used to treat a rare neurodegenerative disease that can lead to death in adolescence. Milasen inhibits the expression of MFSD8 and combats the disease by preventing neurodegeneration. Milasen is one of the finest examples of personalized medicine approved by the FDA [24]. Custirsen is another ASO that can negatively regulate the production of BCL2 and NfkB proteins that help the apoptosis of cancerous cells. It binds to clusterin mRNA and prevents tumor cell proliferation of tumor cells [25].

Rett syndrome is a genetic disorder caused by mutations in the MECP2 gene, and potential treatments include gene therapy and gene editing. However, the main challenge is to find the correct dosage of the wild-type MECP2 gene, as too much could cause dysfunction in wild-type cells. CRISPR-Cas9 has been proven to edit genes in neurons of living animals, but studies have not yet investigated its effectiveness in reversing the symptoms of Rett syndrome. Another research approach is to use antisense oligonucleotides to silence the different alleles of the MECP2 gene in mice and human cells, which has been shown to reverse molecular, behavioral, and synaptic defects. The challenge with this approach is to get the dosage right, as too low could cause Rett syndrome. Another option is to use the chemical fostriecin to inhibit protein phosphatase 2A, which reduces MECP2 in the central nervous system and improves motor abnormalities in mice with MECP2 duplication syndrome. This approach has the advantage of fine-tuning the dose so that the amounts of MECP2 do not drop below a threshold that would cause deficiency symptoms [26].

Ataxia telangiectasia (AT) is a genetic disorder that affects the nervous system and causes a progressive deterioration of motor skills. It is caused by mutations in the ATM gene located on chromosome 11. The ATM protein, primarily found in the nucleus, is important for controlling cell cycle checkpoints, DNA repair, and responses to stress, and it plays a role as a tumor suppressor. The disorder usually appears in early childhood and affects approximately 1 in 40,000 to 1 in 100,000 individuals. Many mutations in AT are related to splicing, which can result in the deletion of part of an exon or the insertion of an intron, producing a shortened, non-functional ATM protein. A new mutation in the ATM gene that causes a splicing defect was identified in a study by Cavalieri and colleagues [27]. The study aimed to correct a genetic defect in intron 11 of a gene known as ATM, leading to a disorder called Ataxia-Telangiectasia (AT). The researchers used PMO to mask a cryptic splice site in the gene, resulting in an increase of 26% in correctly spliced mRNA in the patient’s cells. They also used a similar oligonucleotide called vivomorpholino, which led to an exon skipping efficiency of 50% - 95%, depending on the concentration used. However, these concentrations also had a cytotoxic effect on patient cells and reduced the functional protein activity to 50% of wild-type levels. The researchers suggest that even minimal rescue of functional ATM protein levels could provide therapeutic benefits to AT patients, as patients with 5% - 20% functional ATM have a mild phenotype, and ATM heterozygotes with 40% - 50% of normal ATM protein levels do not show any signs of disease.

The use of antisense oligonucleotides (ASOs), specifically phosphodimethyloluene (PMOs), is used as a treatment for Congenital Disorders of Glycosylation (CDG). CDG is a group of genetic disorders caused by gene mutations involved in glycoprotein synthesizing. These disorders lead to multiorgan failure, including neurologic deficits such as cognitive impairment, ataxia, pigmentary retinal degeneration, and neuropathy. The treatment targets a specific mutation, c.640-15479C > T, which activates a cryptic 5’ splice site in intron 7 of the PMM2 gene. To correct this splicing defect, Vega et al. [28] used PMOs to target the 5’ or 3’ cryptic splice sites of the pseudoexon in intron 7. Using the Endo-Porter reagent, PMOs were transfected into patient fibroblasts carrying the c.640-15479C > T mutation. The treatment completely restored correctly spliced mRNA and increased PMM2 protein levels from 9% to 23% of the amount detected in the control cell line. The enzymatic activity was also restored to almost 50% of that of the control fibroblasts. The use of ASOs, specifically PMOs, shows promise as a treatment for rare genetic disorders by targeting specific mutations.

FTDP-17, or Frontotemporal Dementia and Parkinsonism linked to chromosome 17, is a rare neurodegenerative disorder caused by mutations in the MAPT gene located on chromosome 17q21. The disorder manifests through cognitive impairment, changes in behavior and personality, and motor symptoms. More than 100 families with 44 different mutations in the MAPT gene have been identified throughout the world, and the symptoms and onset age of FTDP-17 can vary greatly between families and even within families carrying the same mutation. The pathological hallmarks of the disease are the aggregation of tau protein in neurofibrillary tangles (NFTs) in neurons and/or glial cells. The mechanisms underlying the disorder are related to changes in the proportion of tau isoforms or the ability of tau to bind to microtubules. Currently, there is no cure for FTDP-17, and treatment is only symptomatic and supportive, with a variable rate of progression and life expectancy [29].

Tau is a protein in the central and peripheral nervous systems and plays a role in the formation and stability of microtubules, as well as in processes such as neurogenesis and axonal maintenance. The MAPT gene on chromosome 17 produces tau, and its expression can be regulated by alternative splicing of exons. In the human brain, specific exons are excluded or included in the final protein, leading to tau’s microtubule-binding domain variations. Kalbfuss and colleagues [30] researched the E10 5’ splice site, which Peacey and colleagues [34] later extended. They targeted this site with a bipartite 2’OMe-PS AON and compared its efficiency in inducing E10 skipping to that of Kalbfuss’ continuous 5’ splice site-targeting 2’OMe-PS oligonucleotide [31]. In addition, researchers have developed a type of ASO called bipartite 2’OMe-PS, designed to interact with specific regions of a gene known as exon ten and intron ten. This interaction results in a reduction in a specific form of the protein tau known as 4R and an increase in another form known as 3R. When tested in different forms of the minigene, the ASO reduced the expression of 4R tau and increased the expression of 3R tau. The effects of AON on endogenous tau mRNA were also tested in HEK 293 cells, resulting in a 42% reduction in E10 inclusion.

Niemann-Pick disease Type C (NPC) is a genetic disorder caused by mutations in the NPC1 or NPC2 genes and affects about 1 in 150,000 people. The disease is linked to a significant decrease in life expectancy and causes progressive neurological deterioration, psychiatric symptoms, and liver problems. Symptoms can vary greatly depending on the age of onset and can include muscle weakness, developmental delays, cognitive impairment, and psychosis. Mutations cause NPC in the NPC1 gene, many of which are missense mutations. However, some splicing mutations have also been identified. For example, a mutation in intron 9 of the NPC1 gene creates a pseudoexon, leading to abnormal splicing. Researchers have used a PMO (phosphorodiamidate morpholino oligomer) to correct this mutation by targeting the cryptic 5’ splice site and blocking the access of the splicing machinery to the premRNA. Delivering the treatment to patient fibroblasts via the Endo-Porter system restores normal splicing. The PMO successfully eliminated aberrant splicing after 48 hours of treatment at a concentration of 10 mM [32]. The PMO was able to eliminate aberrant splicing after 48 hours of treatment at a concentration of 10 mM [32].

Neurofibromatosis type 1 (NFT 1) is a genetic disorder passed down through families and is characterized by certain physical traits and an increased risk of certain types of tumors. It affects about 1 in 3000 people and often affects young children [33]. About 44% of these mutations are splicing mutations. Researchers have used a specific antisense oligonucleotide (PMO) to restore normal splicing in primary cell lines derived from NFT1 patients with deep intronic mutations. PMOs were designed to target the mutant 5’ cryptic splice sites and delivered via an Endo-porter reagent. Treatment with PMO effectively restored normal splicing at the mRNA level for the three mutations studied in different cell lines, and it was found that the PMOs had an immediate effect on fibroblasts that lasted for several days. The researchers also found that PMOs positively affected neurofibromin function by reducing Ras-GTP levels, which is consistent with the restoration of neurofibromin function [34].

Neurofibromatosis type 2 (NFT 2) is a genetic disorder that causes tumors in the nervous system and ocular abnormalities. It affects approximately 1 in 33,000 newborns and is caused by mutations in the NF2 gene, which is responsible for producing the tumor suppressor protein Merlin. The presence of tumors such as bilateral vestibular nerve schwannomas and other types of tumors such as meningiomas, ependymomas, and tumors in other cranial, spinal, and peripheral nerves characterizes this disorder. A recent study by Castellanos et al. found a patient with a deep intronic mutation in the NF2 gene, which leads to the inclusion of a pseudoexon and results in a truncated Merlin protein. The study used PMO, a therapeutic approach, to target the deep intronic mutation and inhibit the inclusion of the pseudoexon in patient-derived fibroblasts, which increased Merlin protein levels [35].

Megalencephalic leukoencephalopathy with subcortical cyst type 1 (MLC1) is a rare genetic disorder that affects the brain and is caused by mutations in the MLC1 gene. The main symptoms of this disease include a large head, motor delays and disability, seizures, and cognitive decline. The MLC1 protein is found mainly in the plasma membrane of glial cells and neurons. Recent research has found a specific mutation in the MLC1 gene that reduces certain transcripts. The troublesome exon was completely removed when this mutation was targeted in a patient’s cells with PMO therapy [36].

Pelizaeus-Merzbacher disease (PMD) is a rare genetic disorder that affects the nervous system and is passed down through families. It is caused by mutations in the PLP1 gene, which produces the proteolipid protein (PLP), which plays an essential role in the development of myelin in the central nervous system. Symptoms typically appear in early childhood, including nystagmus and hypotonia, ranging from mild to severe. A recent study found a mutation in the PLP1 gene that leads to the introduction of regulatory motifs that affect splicing, resulting in a loss of the significant PLP transcript. A therapeutic approach using PMO was used to target the mutation, and the results showed an increase in the ratio of PLP/(DM20 + PLP) mRNA. However, it remains to be seen whether the recovered PLP protein is functional [37].

There are no established guidelines for testing the safety of antisense oligonucleotides (ASOs) outside clinical trials. Since these compounds are chemically synthesized, the safety testing guidelines for small-molecule drugs are typically used. Repeat-dose toxicity studies are generally required in rodents and non-rodents, using a model relevant to the compound’s pharmacology. Nonhuman primates are the preferred model for these studies, but the Göttingen Minipig is a suitable alternative due to its similar safety profile. The genome of the Göttingen Minipig has been sequenced, allowing the development of homologous ASOs that cross-react in swine and evaluate adverse effects related to the pharmacological target [38]. However, only a small percentage of ASOs have been tested in minipigs for non-rodent toxicity studies.

ASOs are also being explored to treat pediatric diseases, such as neuromuscular diseases and retinopathy of prematurity, so more repeat-dose toxicity studies may be required in juvenile animals before starting pediatric clinical trials [39]. The juvenile minipig is a promising model for these studies due to its similarity to humans, technical feasibility, and welfare considerations. However, data on the pharmacodynamic, toxicological, and metabolic profiles of ASOs in juvenile minipigs are currently limited. To determine whether the juvenile Göttingen Minipig is a viable model for pediatric evaluation of ASOs, more information is needed about the ontogeny of the key nucleases that play a role in the metabolism and pharmacologic activity of ASOs, as well as an understanding of the functional immaturity of these nucleases.

A high level of complementarity between the oligonucleotide and mRNA is necessary to target an mRNA with an antisense oligonucleotide successfully. However, this can be difficult to achieve due to secondary or tertiary RNA structures or proteins bound to the RNA. Therefore, various methods have been developed to identify the best binding sites, such as using algorithms and thermodynamic properties to predict the structure of the RNA, measuring the melting temperatures or free energy of the oligonucleotide/RNA duplexes, or using combinatorial oligonucleotides to directly locate the hybridization sites within the RNA through techniques such as RNase H cleavage, microarrays, or mass spectrometry. These methods can be complex but have the potential to pinpoint optimal binding sites for antisense oligonucleotides [5].

Antisense oligonucleotides (ASOs) have shown promising potential in the treatment of many rare diseases. While challenges remain to be addressed, such as off-target effects and toxicity, technological advances are helping to increase specificity and reduce toxicity, making ASO therapy a more effective and safe treatment option for patients. One of the most exciting future perspectives for ASOs is the treatment of neurological disorders. ASOs have a remarkable ability to enter the central nervous system (CNS) after intrathecal delivery and have not shown an immune reaction at therapeutically relevant doses. ASOs are a promising option for treating disorders such as spinal muscular atrophy (SMA) and Huntington’s disease. Following guidelines when using antisense oligonucleotides is important to ensure they are used appropriately. Guidelines for using ASOs may change as new oligonucleotides and related molecules are developed. However, following these guidelines and continuing to research how these molecules work will allow the full potential of this technology to be reached. Another important area of focus is the development of chemically modified ASOs that are more stable and have a longer half-life. It is crucial to ensure that the therapeutic payload reaches its target in the body and has the desired effect. Overall, the future of ASO therapy looks bright. With continued research and development, we can expect to see more effective and safe treatments for rare diseases in the near future.

Rare diseases often have poor clinical outcomes, are difficult to diagnose, and lack effective treatment options due to their complexity and rarity. Developing new therapeutic agents is crucial for improving these conditions’ management, treatment, and symptom alleviation. Gene therapy, particularly ASOs, offers a promising approach to addressing rare diseases. Although ASOs have potential off-target effects, their high customizability and targeted action make them valuable tools for developing effective treatments. Robust methodologies and precise combinations are essential for maximizing their therapeutic benefits. By utilizing ASOs in gene therapy, researchers can strive to develop more effective treatments for patients with rare diseases.