Clinical Applications of Single-Stranded Oligonucleotides: Current Landscape of Approved and In-Development Therapeutics
Authors: Juergen Scharner, Isabel Aznarez
Journal: Molecular Therapy (2020)

Single-stranded oligonucleotides have been explored as a therapeutic modality for more than 20 years. Only during the last 5 years have single-stranded oligonucleotides become a modality of choice in the fields of precision medicine and targeted therapeutics. Recently, there have been a number of development efforts involving this modality that have led to treatments for genetic diseases that were once untreatable. This review highlights key applications of single-stranded oligonucleotides that function in a sequence-dependent manner when applied to modulate precursor (pre-)mRNA splicing, gene expression, and immune pathways. These applications have been used to address diseases that range from neurological to muscular to metabolic, as well as to develop vaccines. The wide range of applications denotes the versatility of single-stranded oligonucleotides as a robust therapeutic platform. The focus of this review is centered on approved single-stranded oligonucleotide therapies and the evolution of oligonucleotide therapeutics into novel applications currently in clinical development. Single-stranded oligonucleotides have been explored as a therapeutic modality for more than 20 years. Only during the last 5 years have single-stranded oligonucleotides become a modality of choice in the fields of precision medicine and targeted therapeutics. Recently, there have been a number of development efforts involving this modality that have led to treatments for genetic diseases that were once untreatable. This review highlights key applications of single-stranded oligonucleotides that function in a sequence-dependent manner when applied to modulate precursor (pre-)mRNA splicing, gene expression, and immune pathways. These applications have been used to address diseases that range from neurological to muscular to metabolic, as well as to develop vaccines. The wide range of applications denotes the versatility of single-stranded oligonucleotides as a robust therapeutic platform. The focus of this review is centered on approved single-stranded oligonucleotide therapies and the evolution of oligonucleotide therapeutics into novel applications currently in clinical development. Single-stranded oligonucleotides are synthetic, short, modified RNA or DNA molecules that function in either a sequence-dependent (antisense oligonucleotides [ASOs] and immune stimulatory oligonucleotides [ISOs]) or tertiary structure-dependent manner (e.g., aptamers1Zhu G. Chen X. Aptamer-based targeted therapy.Adv. Drug Deliv. Rev. 2018; 134: 65-78Crossref PubMed Scopus (103) Google Scholar). In this review, we focus our attention on oligonucleotide therapeutics whose activities are dictated by their sequences; ASOs bind to their target by Watson and Crick base-pairing,2Kole R. Krainer A.R. Altman S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides.Nat. Rev. Drug Discov. 2012; 11: 125-140Crossref PubMed Scopus (773) Google Scholar while ISOs are recognized and bound by proteins that detect specific motifs.3Koller E. Vincent T.M. Chappell A. De S. Manoharan M. Bennett C.F. Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes.Nucleic Acids Res. 2011; 39: 4795-4807Crossref PubMed Scopus (195) Google Scholar Single-stranded oligonucleotide therapeutics have evolved substantially since the use of DNA ASOs were first described in the late 1970s.4Stephenson M.L. Zamecnik P.C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide.Proc. Natl. Acad. Sci. USA. 1978; 75: 285-288Crossref PubMed Scopus (662) Google Scholar,5Zamecnik P.C. Stephenson M.L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide.Proc. Natl. Acad. Sci. USA. 1978; 75: 280-284Crossref PubMed Scopus (1403) Google Scholar The year 1998 saw the first ever approved ASO therapy, formivirsen, which consisted of a 21-mer DNA ASO with a phosphorothioate (PS) backbone (Figure 1).6Roehr B. Fomivirsen approved for CMV retinitis.J. Int. Assoc. Physicians AIDS Care. 1998; 4: 14-16PubMed Google Scholar Formivirsen, much like the first ASO applications, was an antiviral molecule, designed to target cytomegalovirus. It was not until 2013 that the second ASO drug, mipomersen, obtained US Food and Drug Administration (FDA) approval to treat homozygous familial hypercholesterolemia (HoFH) via RNase H-mediated degradation of apolipoprotein B (APOB).7Geary 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. 2015; 54: 133-146Crossref PubMed Scopus (106) Google Scholar Mipomersen represented a new generation of ASOs using new chemical modifications and design that led to more stable (increased nuclease resistance), safer (decreased pro-inflammatory response), and potent (increased RNA binding affinity) compounds with enhanced pharmacokinetic (PK) and pharmacodynamic (PD) properties (Figure 1). During the 15 years between the two approvals, the field of single-stranded oligonucleotide therapeutics matured, leading to an acceleration in the development of this therapeutic modality and an increase in the number of approved drugs. In the last 4 years, six new ASO therapies and one ISO therapy have obtained FDA or European Medicines Agency (EMA) approval. These therapies address a range of genetic diseases across neurological, metabolic/cardiovascular, and muscular conditions as well as potentiate the efficacy of a hepatitis B vaccine. The approved therapies utilize three major types of single-stranded oligonucleotide modalities, that is, gapmers, steric blocking, and CpGs, to modulate gene expression, precursor (pre-)mRNA splicing, and immune pathways. Target organs expanded beyond the eye and liver to the central nervous system (CNS) and muscle. Single-stranded oligonucleotides represent a broad-spectrum therapeutic platform that continues to evolve with innovations in applications, chemistry, and tissue targeting. Gapmers are synthetic, single-stranded, and typically 16–20 nt long with a central stretch of 8–10 DNA nt flanked on each side by a stretch or wing of 4–5 nt containing modifications in the sugar ring. These sugar modifications in the wings increase nuclease resistance protecting the ends of the ASO, avoid or reduce an immune response, and increase binding affinity. Commonly, gapmers contain PS bonds throughout to increase stability and plasma protein binding, which improves the PK properties of the gapmer ASO (Figure 1).8Geary R.S. Norris D. Yu R. Bennett C.F. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides.Adv. Drug Deliv. Rev. 2015; 87: 46-51Crossref PubMed Scopus (405) Google Scholar With their typical 4-8-4/5-10-5 (wing-DNA-wing) structure, gapmers bind in a sequence-specific manner by Watson and Crick base-pairing to their target RNA and form a stretch of double-stranded RNA-DNA hybrid. These RNA-DNA hybrids mimic endogenous RNA-DNA hybrids that occur naturally during DNA replication, which are recognized and disrupted via RNase H-mediated cleavage of the RNA strand. The cleavage then leads to the degradation of the RNA. Leveraging this endogenous mechanism, gapmers cause RNase H-mediated cleavage of the target pre-mRNA or mRNA at the location bound by the stretch of DNA. The target transcript is subsequently degraded, resulting in reductions of the target transcript and corresponding protein (Figure 2). As single-stranded ASOs, gapmers are taken up by cells in the CNS, eye, liver, kidney, adrenal glands, and lungs via a natural endocytic process, and they are subsequently released from endosomes into the cytoplasm, reaching the nucleus by a yet to be fully understood mechanism.3Koller E. Vincent T.M. Chappell A. De S. Manoharan M. Bennett C.F. Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes.Nucleic Acids Res. 2011; 39: 4795-4807Crossref PubMed Scopus (195) Google Scholar Gapmers can be used to reduce the level of any protein or specific protein isoforms, as well as toxic proteins that result from a gain-of-function or dominant negative mutation.9Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (891) Google Scholar (A) Expression of an example target gene from transcription through translation of a wild-type or toxic protein. (B) Gapmer ASO with the typical structure of modified chemistry in the wings to protect the ends from nucleases and an internal stretch of DNA that leads to the formation of the RNA-DNA hybrid when bound to the target transcript. In this example, the gapmer ASO binds to an exon in the pre-mRNA and mRNA and recruits RNase H that recognizes the RNA/DNA hybrid and cleaves the RNA. The cleavage triggers RNA degradation, leading to reduction of wild-type or toxic protein levels. Gapmer ASOs can be designed to target other transcript regions, e.g., introns. Even though RNase H is more abundant in the nucleus, RNase H-mediated cleavage of RNA-DNA hybrids can also occur in the cytoplasm. Mipomersen (Kynamro; Ionis Pharmaceuticals/Genzyme) was approved by the FDA in January 2013 to treat HoFH, a rare and serious hereditary condition with high risk for premature coronary heart disease associated with atherosclerosis.10Goldberg A.C. Hopkins P.N. Toth P.P. Ballantyne C.M. Rader D.J. Robinson J.G. Daniels S.R. Gidding S.S. de Ferranti S.D. Ito M.K. et al.National Lipid Association Expert Panel on Familial HypercholesterolemiaFamilial hypercholesterolemia: screening, diagnosis and management of pediatric and adult patients: clinical guidance from the National Lipid Association Expert Panel on Familial Hypercholesterolemia.J. Clin. Lipidol. 2011; 5: S1-S8Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar,11Rader D.J. Cohen J. Hobbs H.H. Monogenic hypercholesterolemia: new insights in pathogenesis and treatment.J. Clin. Invest. 2003; 111: 1795-1803Crossref PubMed Scopus (447) Google Scholar HoFH is caused by homozygous or compound heterozygous mutations in one of three genes (APOB, LDLR, or PCSK9), leading to severely elevated low-density lipoprotein cholesterol (LDL-C) levels.10Goldberg A.C. Hopkins P.N. Toth P.P. Ballantyne C.M. Rader D.J. Robinson J.G. Daniels S.R. Gidding S.S. de Ferranti S.D. Ito M.K. et al.National Lipid Association Expert Panel on Familial HypercholesterolemiaFamilial hypercholesterolemia: screening, diagnosis and management of pediatric and adult patients: clinical guidance from the National Lipid Association Expert Panel on Familial Hypercholesterolemia.J. Clin. Lipidol. 2011; 5: S1-S8Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar Mipomersen is a second-generation gapmer ASO with a 5-10-5 structure, 2′-O-methoxyethyl (2′MOE) modifications in the wings (2′MOE-DNA-2′MOE), and PS backbone (Figure 1). Mipomersen was designed to bind to the coding region of the APOB-100 mRNA (an isoform encoded by the APOB gene), and elicit RNase H-mediated degradation of the mRNA and reduce ApoB protein levels (see Figure 2B for the mechanism of action).12Crooke R.M. Graham M.J. Lemonidis K.M. Whipple C.P. Koo S. Perera R.J. An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis.J. Lipid Res. 2005; 46: 872-884Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar PK studies in preclinical species as well as humans showed that mipomersen has a 30-day half-life in plasma and target tissue (liver).7Geary 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. 2015; 54: 133-146Crossref PubMed Scopus (106) Google Scholar In patients with HoFH, mipomersen is administered systemically via weekly subcutaneous injections at a dose of 200 mg and is indicated as an adjunct therapy to lipid-lowering medications and diet to reduce LDL-C, ApoB, total cholesterol, and non-high-density lipoprotein cholesterol. In humans, PD studies showed a mipomersen-mediated reduction of ApoB protein of 46%, which resulted in a 47% decrease of LDL-C.7Geary 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. 2015; 54: 133-146Crossref PubMed Scopus (106) Google Scholar During clinical trials, safety concerns of hepatotoxicity as determined by increased levels of transaminases were observed leading to a black label warning for mipomersen.13Genzyme Kynamro: full prescribing information.2013https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/203568s000lbl.pdfGoogle Scholar Because of the risk of hepatotoxicity, mipomersen is available only through a restricted program under a risk evaluation and mitigation strategy. Other adverse effects include injection site reaction, fever, flu-like symptoms, and fatigue.14Genzyme Kynamro® (mipomersen sodium) injection prescribing information.2013https://www.accessdata.fda.gov/drugsatfda_docs/label/2019/203568s011lbl.pdfGoogle Scholar Mipomersen was not approved by the EMA based on safety concerns. In 2016, Ionis and Kastle Therapeutics acquired the rights to mipomersen, and in May 2018 the marketing of the drug was discontinued. Inotersen (Tegsedi; Ionis Pharmaceuticals/Akcea) was approved by the FDA and EMA in 2018 for the treatment of patients with hereditary amyloid transthyretin (ATTR) amyloidosis, a rare systemic disorder that is characterized by progressive peripheral polyneuropathy, cardiomyopathy, nephropathy, and gastrointestinal dysfunction. The disorder is caused by dominant-negative mutations in the TTR gene that lead to destabilization of the tetrameric TTR protein complex, causing aggregation of monomers into extracellular amyloid deposits.15Sekijima Y. Transthyretin (ATTR) amyloidosis: clinical spectrum, molecular pathogenesis and disease-modifying treatments.J. Neurol. Neurosurg. Psychiatry. 2015; 86: 1036-1043Crossref PubMed Scopus (135) Google Scholar The design and mechanism of action of inotersen are analogous to mipomersen, and it was designed to bind and elicit RNase H-mediated degradation of mutant and wild-type TTR transcripts, resulting in decreased mutant and wild-type TTR proteins (see Figure 2B for the mechanism of action).16Ackermann E.J. Guo S. Benson M.D. Booten S. Freier S. Hughes S.G. Kim T.W. Jesse Kwoh T. Matson J. Norris D. et al.Suppressing transthyretin production in mice, monkeys and humans using 2nd-generation antisense oligonucleotides.Amyloid. 2016; 23: 148-157Crossref PubMed Scopus (80) Google Scholar Analyses of preclinical species and healthy human volunteers dosed with inotersen demonstrated a more than 80% reduction of plasma TTR protein levels, which correlated with a reduction in the TTR transcript in the liver (target organ) of preclinical species.16Ackermann E.J. Guo S. Benson M.D. Booten S. Freier S. Hughes S.G. Kim T.W. Jesse Kwoh T. Matson J. Norris D. et al.Suppressing transthyretin production in mice, monkeys and humans using 2nd-generation antisense oligonucleotides.Amyloid. 2016; 23: 148-157Crossref PubMed Scopus (80) Google Scholar In patients with ATTR amyloidosis, inotersen is administered via weekly subcutaneous injection of a recommended dose of 284 mg to treat the polyneuropathy phenotype. Clinical data indicated that inotersen improved the course of the neurological disease as well as the quality of life of ATTR amyloidosis patients. During clinical trials, safety concerns included thrombocytopenia (fatal in some patients) and glomerulonephritis, which occurred independently in 3% of patients.17Benson M.D. Waddington-Cruz M. Berk J.L. Polydefkis M. Dyck P.J. Wang A.K. Planté-Bordeneuve V. Barroso F.A. Merlini G. Obici L. et al.Inotersen treatment for patients with hereditary transthyretin amyloidosis.N. Engl. J. Med. 2018; 379: 22-31Crossref PubMed Scopus (530) Google Scholar Inotersen is the first approved example of a gapmer ASO that directly targets the root cause of a disease. Volanesorsen (Waylivra; Ionis Pharmaceuticals/Akcea) received a conditional market authorization in Europe issued by the EMA in 2019 for the treatment of familial chylomicronemia syndrome (FCS), which is characterized by 10- to 100-fold increased levels of triglycerides compared to the healthy population, which leads to hypertriglyceridemia and recurrent episodes of pancreatitis. The elevated triglycerides result from reduced or lack of lipoprotein lipase activity caused predominantly by recessive mutations in the LPL gene.18Johansen C.T. Kathiresan S. Hegele R.A. Genetic determinants of plasma triglycerides.J. Lipid Res. 2011; 52: 189-206Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar To address the lack of LPL activity, volanesorsen was designed to reduce the levels of APOC3 mRNA and apolipoprotein C (ApoC)-III protein to decrease plasma triglycerides via an LPL-independent mechanism (see Figure 2B for the mechanism of action). Volanesorsen is another second-generation gapmer ASO that targets a primarily liver-expressed mRNA. Preclinical studies in rodents and non-human primates showed a significant reduction of ApoC-III and triglyceride plasma levels. Similar results were obtained from the administration of volanesorsen to healthy human subjects, demonstrating a good translation between preclinical species and human clinical studies.19Graham M.J. Lee R.G. Bell 3rd, T.A. Fu W. Mullick A.E. Alexander V.J. Singleton W. Viney N. Geary R. Su J. et al.Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans.Circ. Res. 2013; 112: 1479-1490Crossref PubMed Scopus (257) Google Scholar In patients suffering from FCS, volanesorsen treatment consists of a once weekly subcutaneous dose of 300 mg in combination with dietary restrictions. Clinical data indicated that 77% of patients treated with volanesorsen have significantly reduced levels of triglyceride in plasma, reaching values lower than 750 mg/dL, well below the 880 mg/dL required to reduce the risk of pancreatitis. In these patients, dietary restriction alone was not sufficient to reduce cholesterol levels. Similar to the other two systemically administered, approved second-generation gapmer ASOs, thrombocytopenia and site of injection reactions were common adverse events.20Witztum J.L. Gaudet D. Freedman S.D. Alexander V.J. Digenio A. Williams K.R. Yang Q. Hughes S.G. Geary R.S. Arca M. et al.Volanesorsen and triglyceride levels in familial chylomicronemia syndrome.N. Engl. J. Med. 2019; 381: 531-542Crossref PubMed Scopus (140) Google Scholar Steric-blocking ASOs are synthetic, fully modified, single-stranded oligonucleotides that typically range from 15 to 30 nt in length and contain chemical modifications in the sugar as well as the backbone (Figure 1). Many of the chemical modifications are similar to gapmer ASOs and are also intended to increase nuclease resistance, reduce the immune response, and increase binding affinity. Unlike gapmers, steric-blocking ASOs do not elicit RNase H-mediated degradation of the target RNA, but rather bind to the target RNA via sequence-specific Watson and Crick base-pairing and either hinder the binding of trans-acting factors (e.g., small nuclear RNA [snRNA], microRNA [miRNA], long non-coding RNA [lncRNA], or RNA-binding proteins) to their cognate sequence or prevent the formation of RNA secondary structures.2Kole R. Krainer A.R. Altman S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides.Nat. Rev. Drug Discov. 2012; 11: 125-140Crossref PubMed Scopus (773) Google Scholar,9Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (891) Google Scholar Their first application dates to mid-1980s in which an ASO was designed to target a 3′ splice site in the herpes simplex virus 1 pre-mRNA preventing splicing and functioning as an antiviral agent.21Smith C.C. Aurelian L. Reddy M.P. Miller P.S. Ts'o P.O. Antiviral effect of an oligo(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5.Proc. Natl. Acad. Sci. USA. 1986; 83: 2787-2791Crossref PubMed Scopus (261) Google Scholar Since then, steric blocking ASOs have been used to correct mutation-driven splicing defects and modulate mRNA stability and protein translation.22Lundin K.E. Gissberg O. Smith C.I. Oligonucleotide therapies: the past and the present.Hum. Gene Ther. 2015; 26: 475-485Crossref PubMed Scopus (164) Google Scholar Steric-blocking ASOs are thought to be taken up by cells in the CNS, eye, liver, kidney, adrenal glands, lungs, and, to a much lesser extent, to the muscle (see below for a specific example) via the same mechanism as gapmer ASOs.3Koller E. Vincent T.M. Chappell A. De S. Manoharan M. Bennett C.F. Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes.Nucleic Acids Res. 2011; 39: 4795-4807Crossref PubMed Scopus (195) Google Scholar Nusinersen (Spinraza; Ionis Pharmaceuticals/Biogen) was approved by the FDA in December 2016 and the EMA in 2017 for the treatment of spinal muscular atrophy (SMA), a rare neuromuscular disorder that is the most common genetic cause of infant mortality. SMA is caused by recessive mutations in the survival motor neuron 1 (SMN1) gene, and the disease severity is mitigated by a copy gene, SMN2, which produces low levels of normal SMN protein.23Feldkötter M. Schwarzer V. Wirth R. Wienker T.F. Wirth B. Quantitative analyses of SMN1 and SMN2 based on real-time LightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy.Am. J. Hum. Genet. 2002; 70: 358-368Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar SMN2 exon 7 carries a single nucleotide change with respect to SMN1 that severely decreases its inclusion in the final mRNA, leading to an unstable SMN protein (Figure 3A).24Monani U.R. Lorson C.L. Parsons D.W. Prior T.W. Androphy E.J. Burghes A.H. McPherson J.D. A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2.Hum. Mol. Genet. 1999; 8: 1177-1183Crossref PubMed Scopus (681) Google Scholar Nusinersen is an 18-mer, fully modified 2′MOE-PS (Figure 1) that was designed to bind to position +10 of intron 7 of the SMN1/2 pre-mRNA and block access of the splicing repressor hnRNPA1 to its cognate intronic splicing silencer (Figure 3B).25Hua Y. Vickers T.A. Okunola H.L. Bennett C.F. Krainer A.R. Antisense masking of an hnRNP A1/A2 intronic splicing silencer corrects SMN2 splicing in transgenic mice.Am. J. Hum. Genet. 2008; 82: 834-848Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar This results in increased levels of SMN2 exon 7 inclusion, leading to correctly spliced mRNA and increased levels of SMN protein, which compensates for the loss of SMN1 (Figure 3B).26Hua Y. Sahashi K. Rigo F. Hung G. Horev G. Bennett C.F. Krainer A.R. Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model.Nature. 2011; 478: 123-126Crossref PubMed Scopus (480) Google Scholar,27Passini M.A. Bu J. Richards A.M. Kinnecom C. Sardi S.P. Stanek L.M. Hua Y. Rigo F. Matson J. Hung G. et al.Antisense oligonucleotides delivered to the mouse CNS ameliorate symptoms of severe spinal muscular atrophy.Sci. Transl. Med. 2011; 3: 72ra18Crossref PubMed Scopus (350) Google Scholar (A) Region of SMN2 pre-mRNA containing exons 6, 7, and 8. SMN2 exon 7 carries a silent single nucleotide change with respect to SMN1 that causes exon 7 skipping, which leads to an unstable SMN protein. Only 10% of SMN2 pre-mRNA is properly spliced, resulting in an insufficient level of functional SMN protein to compensate for the loss of the SMN1 gene. (B) Nusinersen binding to intron 7 of the SMN2 pre-mRNA and promoting exon 7 inclusion, which leads to increased levels of SMN protein and improved motor neuron function. Preclinical studies in mice indicated that nusinersen has a long-lasting effect on SMN2 splicing in the brain with continued maximal effect through the 6-month observational period following a single intracerobroventricular injection. In addition, intrathecal (i.t.) injection in non-human primates showed a widespread distribution throughout the spinal cord where motor neurons (target cell) reside.28Rigo F. Chun S.J. Norris D.A. Hung G. Lee S. Matson J. Fey R.A. Gaus H. Hua Y. Grundy J.S. et al.Pharmacology of a central nervous system delivered 2′-O-methoxyethyl-modified survival of motor neuron splicing oligonucleotide in mice and nonhuman primates.J. Pharmacol. Exp. Ther. 2014; 350: 46-55Crossref PubMed Scopus (155) Google Scholar PK studies in patients injected i.t. with up to a 9-mg dose of nusinersen showed consistent results with preclinical species and indicated a prolonged half-life of 4–6 months in cerebrospinal fluid (CSF).29Chiriboga C.A. Swoboda K.J. Darras B.T. Iannaccone S.T. Montes J. De Vivo D.C. Norris D.A. Bennett C.F. Bishop K.M. Results from a phase 1 study of nusinersen (ISIS-SMNRx) in children with spinal muscular atrophy.Neurology. 2016; 86: 890-897Crossref PubMed Scopus (358) Google Scholar PK/PD analysis in autopsy tissue demonstrated broad distribution of the drug in the spinal cord and brain regions, as well as increases in SMN protein in the spinal cord.30Finkel R.S. Chiriboga C.A. Vajsar J. Day J.W. Montes J. De Vivo D.C. Yamashita M. Rigo F. Hung G. Schneider E. et al.Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, open-label, dose-escalation study.Lancet. 2016; 388: 3017-3026Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar Clinical trials have successfully demonstrated efficacy in infants and older children treated with nusinersen shown by an increase in motor function and survival.30Finkel R.S. Chiriboga C.A. Vajsar J. Day J.W. Montes J. De Vivo D.C. Yamashita M. Rigo F. Hung G. Schneider E. et al.Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, open-label, dose-escalation study.Lancet. 2016; 388: 3017-3026Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar Nusinersen is approved to treat infants through adult SMA patients and is administered to patients directly into the CSF via i.t. injection by lumbar puncture at a dose of 12 mg in 5 mL every 4 months after an initial loading dose of 12 mg administered monthly for the first 4 months.31US Food and Drug AdministrationFDA approves first drug for spinal muscular dystrophy.2016https://www.fda.gov/news-events/press-announcements/fda-approves-first-drug-spinal-muscular-atrophyGoogle Scholar Adverse reactions include lower respiratory infection, upper respiratory infection, and constipation. The label has warnings for thrombocytopenia and coagulation abnormalities as well as renal toxicity. While these warnings relate to the class of ASOs, they have not been observed for nusinersen.30Finkel R.S. Chiriboga C.A. Vajsar J. Day J.W. Montes J. De Vivo D.C. Yamashita M. Rigo F. Hung G. Schneider E. et al.Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, open-label, dose-escalation study.Lancet. 2016; 388: 3017-3026Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar In addition to the approved patient population, nusinersen has been further tested in pre-symptomatic infants with mutations in SMN1 and two to three copies of SMN2 in the NURTURE trial, and the results underscore the proven efficacy of the treatment and the importance of neonatal diagnosis.32De Vivo D.C. Bertini E. Swoboda K.J. Hwu W.L. Crawford T.O. Finkel R.S. Kirschner J. Kuntz N.L. Parsons J.A. Ryan M.M. et al.NURTURE Study GroupNusinersen initiated in infants during the presymptomatic stage of spinal muscular atrophy: Interim efficacy and safety results from the phase 2 NURTURE study.Neuromuscul. Disord. 2019; 29: 842-856Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar Eteplirsen (Exondys 51; Sarepta Therapeutics) received an accelerated approval by the FDA in 2016 for the treatment of Duchenne muscular dystrophy (DMD), which is caused by complete loss-of-function mutations in the X-linked DMD gene and predominantly affects males. DMD is a fatal disease due to loss of muscle function caused by the lack of dystrophin protein (Figure 4A). Loss of function mutations in the DMD gene that result in partially functional albeit truncated dystrophin protein (e.g., in-frame deletions) lead to a milder disease presentation known as Becker muscular dystrophy.33Koenig M. Beggs A.H. Moyer M. Scherpf S. Heindrich K. Bettecken T. Meng G. Müller C.R. Lindlöf M. Kaariainen H. et al.The molecular basis for Duchenne versus Becker muscular dystrophy: correlation of severity with type of deletion.Am. J. Hum. Genet. 1989; 45: 498-506PubMed Google Scholar This genotype-phenotype correlation provided the rationale to skip specific DMD exons to avoid deleterious nonsense and frameshift mutations that cause DMD to restore the frame at the transcript level and produce a Becker-like truncated, partially functional dystrophin.34Aartsma-Rus A. Bremmer-Bout M. Janson A.A. den Dunnen J.T. van Ommen G.J. van Deutekom J.C. Targeted exon skipping as a potential gene correction the