Antisense oligonucleotides

Oligonucleotide phosphate linkage modifications

Modifications to the oligonucleotide phosphate linkages predominantly assist in nuclease avoidance. In the phosphorodiamidate morpholino (PMO), the phosphodiester (PO) linkages in the oligonucleotide backbone are replaced with nonionic phosphorodiamidate linkages leading to resistance to PO.¹² Other ASO types have PS modifications that result in resistance to a broad spectrum of nucleases, support RNase H activity, and increase protein binding, which also improves tissue uptake.^10,11

Morpholinos

Morpholinos are oligonucleotides with unique modifications to the ribose sugar that lead to greater target affinity and facilitate nuclease avoidance.¹² This modification reduces oligonucleotide-protein interactions.¹² Eteplirsen, a 30-bp morpholino-based ASO drug for the treatment of Duchenne muscular dystrophy (DMD), represents the only FDA-approved morpholino therapy for a neurodegenerative (or neuromuscular) disease.

Methoxyethyl oligonucleotides

A chemical modification included in most so-called second-generation ASOs is 2′ O-methoxyethyl (MOE). MOE ASOs have an MOE modification at the 2′-position of the ribose sugar. This change enables enhanced binding affinity to the target mRNA and is considerably less toxic than the 2′-O methyl (OMe) modification. In addition, MOE is sufficiently nuclease resistant that some MOE nucleotides can be synthesized with normal PO linkages so that a mix of PO and PS linkages can be used to fine tune the pharmacokinetics of the ASO. This can facilitate more rapid distribution into tissue while keeping the terminal elimination rate slow.¹³ MOE modifications also reduce plasma protein binding, which seem to shift ASOs away from hepatic metabolism and toward the kidney for excretion in urine.¹³ 2′-MOE PS ASOs delivered to the CSF can have biological half-lives exceeding 6 months.^50,58

Constrained nucleic acids

Nucleotides that are covalently modified to limit conformation are referred to as constrained or locked. Nucleic acids are considered “locked” when they have a methylene bridge connection made between 2′-oxygen and the 4′-carbon of the ribose sugar molecule (figure 2). This bond effectively locks the base into a conformation predominantly characterizing the RNA ribose sugar and prevents the conformation characteristic of the deoxyribose sugar.¹⁴ The benefit of locked nucleic acids (LNAs) is that they can produce both increased target specificity and reduced recognition by nucleases. LNAs can hybridize to both DNAs and RNAs forming highly stable double-helix duplexes.¹⁴ LNAs can be incorporated into siRNAs¹⁵ and gapmer ASOs supporting RNAse H activity.¹⁶ LNA ASOs can be more potent in vivo than their 2′-MOE analogs.¹⁷

LNAs have been associated with increased liver toxicity¹⁸ but can be used in combination with unmodified bases to reduce toxicity while improving ASO efficacy.^19,20 To get around hepatotoxicities associated with LNA chemistry, chemists have created a sort of LNA/MOE hybrid by adding a methyl-bridge characteristic of LNAs to MOE oligonucleotides. The result is ASOs with reduced liver toxicity and increased potency.²¹ There are multiple types of constrained ethyl (cET) and MOE oligonucleotides (S-cEt, R-cEt, S-cMOE, and R-cMOE), where S and R refer to the left and right chiral structures, respectively. ASOs with these structures hybridize to target RNAs with affinities like their corresponding LNAs and have improved liver toxicity and increased resistance to nuclease degradation compared with the LNA chemistry.

Stereopure PS ASOs

PS ASOs are usually stereorandom with regard to chiral PS centers, each of which has 2 distinct stereochemical configurations, making 2¹⁹ stereoisoforms possible for a 20mer ASO with 19 linkages. Although it is recognized that the 2 stereoisomers (Rp and Sp) differ in their binding affinity and susceptibility to degradation,^22,–,24 there are tradeoffs between the two, and it is still debated whether stereopure isomers can be more potent than stereorandom ASOs. Studies in cell culture have failed to show any potency differences between stereopure and stereorandom ASOs. On the other hand, 1 study reported that ASOs with repeated left-left-right (or SSR) chiral PS centers optimized ASO recognition by RNAse H and resulted in greater potency in vivo.²² Therapeutic development of stereopure ASOs is the strategy used at WAVE Life Sciences.

Peptide nucleic acid

The peptide nucleic acid (PNA) has a peptide in the position of the ribose sugar. PNAs have typically been used for modulating transcription and other applications not supporting RNase H activity.²⁵ But, PNA gapmers have been shown to support RNase H cleavage of target mRNAs.²⁶ However, a PNA ASO targeting exon skipping in the DMD gene was poorly effective compared with an ASO with the 2′-OMe modification.²⁷

5′-methylcytosine modification

Methylation of cytosines at the 5′ position is one way to increase ASO specificity. For example, all cytosines in an ASO against SOD1 included the 5′-methylcytosine modification.⁵ This inclusion can enhance base pairing by modifying the hydrophobic nature of the ASO. On the other hand, 5′-methylcytosine-thymidine repeats can increase cytotoxicity,²⁸ and CpG motifs can stimulate immunoreactivity that can be at least partially alleviated by including 5′-methylcytosine.^29,30

Gapmers, mixed chemistry, and target fate

Gapmer ASOs have “wings” on either side of LNA or MOE modified bases flanking a tract of unmodified bases with PS linkages usually throughout the ASO backbone. Mixed chemistry is intended to maximize resistance to nucleases and minimize toxicity while supporting RNAse H activity.³¹ The target mRNAs cleaved by RNAse H will be degraded in either the nucleus or the cytoplasm by the exosome complex and XRN exonucleases³² (figure 1). Lead optimization may include a screen for optimal structure activity relationship. For PS ASOs, this may combine cET, LNA, MOE, cMOE, and 5′-methylcytosine chemistries. Tracts of 4 guanosines should be avoided because they can result in complex ASO structures.³⁰ For gapmer ASOs, this may include unmodified bases. Splice-switching oligonucleotides (SSOs) will exclude a gap feature as RNase H activity that could lead to target degradation is unwanted.

Spinocerebellar ataxias

Three Spinocerebellar ataxias (SCAs), all caused by DNA CAG repeat expansion encoding a polyglutamine (polyQ) repeat, have been investigated as diseases targetable by ASO treatment in mouse models. SCA2 is caused by CAG repeat expansion in exon 1 of the ATXN2 gene.³³ The phenotypes in 2 SCA2 mouse models have been well characterized at the morphologic, physiologic, and transcriptome levels.^34,–,36 ATXN2 mutation is also associated with a substantial rise in the expression of the stress granule protein Staufen1 (STAU1) and production of ATXN2/STAU1/TIA1–positive stress granules.³⁷ After screening for ASOs targeting ATXN2 in a human cell line, we took the best ASOs reducing expression to in vivo testing by injection into the lateral ventricle of the mouse. These ASOs had a gapmer design. Several ASOs induced a glial or microglial response, a common off-target effect of some ASOs, and were not further evaluated.

The top lead ASO was designated ASO7 and lowered ATXN2 expression in SCA2 mouse cerebella by >60% for up to 13 weeks, without activating markers of gliosis. When injected into symptomatic mice, ASO7 improved motor function and restored proteomic and physiologic Purkinje cell abnormalities.^36,38 The study provided proof of concept of lowering of ATXN2 for treatment of ATXN2-related diseases and the impetus for identifying more efficacious SCA2 ASO for use in humans.

SCA3 (Machado-Joseph disease) is caused by a CAG repeat expansion in exon 10 of the ATXN3 gene. Different strategies have been used to develop SCA3 ASOs. This has included allele-specific ASOs antisense to the expanded CAG repeat that sterically block translation,³⁹ splice-switching MOE ASOs to exclude exon 10 encoding the expanded repeat,⁴⁰ and MOE gapmer ASOs targeting wild-type and mutant ATXN3 alleles^41,42 (figure 1).

SCA7 is characterized by cerebellar ataxia and progressive cone-rod dystrophy and caused by CAG repeat expansion in the ATXN7 gene. ASO therapies are ideally suited for treating the eye, as they can easily be injected intravitreally. Although no longer in use, 2 decades ago, fomivirsen became the first ever FDA-approved ASO drug, injected into the vitreous of the eye for cytomegalovirus retinitis.⁴³ Niu et al.⁴⁴ developed proof-of-concept data in mice for the use of an ASO therapy for blindness in SCA7. Of note, the ASO therapy was effective in mice with symptomatic eye disease.

Huntington disease

HD is caused by a CAG repeat expansion in an encoded region of the HTT gene. PolyQ expansion in huntingtin causes a gain of toxic function. Development of ASOs targeting HTT has followed different approaches. One targets both mutant and nonmutant alleles, whereas another approach is allele specific whereby ASOs were screened that target single nucleotide polymorphisms in HTT in linkage disequilibrium with expanded CAG repeats.^45,46 This strategy may potentially target >75% of HD mutation carriers.⁴⁷ Currently, 3 ASOs targeting HTT are in clinical trials (table).

Amyotrophic lateral sclerosis

ATXN2 as a target for ALS: Although mutations in several genes cause ALS, a number of studies point to ATXN2 as a therapeutic target for ALS. Over the past decade, it has become well established that intermediate CAG repeat expansions in the ATXN2 gene increase the risk of ALS.^48,49 Intertwined with this discovery was the finding that reducing ATXN2 expression improved transactive response DNA-binding protein 43 (TDP-43) toxicity in both yeast and flies.⁴⁹ When endogenous Atxn2 was reduced in TDP-43 transgenic mice by crossing with Atxn2 knockout mice, survival was significantly improved, TDP-43–positive stress granules were eliminated in motor neurons, and gait scores improved.⁵⁰ Similarly, the survival of TDP-43 transgenic mice was improved by treating with an ASO targeting the Atxn2 gene.⁵⁰ The effect of targeting ATXN2 on TDP-43 aggregations might be explained by related effects on STAU1.³⁷

Alzheimer disease and tauopathies

Parkinson disease

Reduction of LRRK2 abundance is predicted to be therapeutic for Parkinson disease (PD). This is supported by observations that disease-causing mutations in LRRK2 are associated with elevated α-synuclein expression.⁶¹ Targeting Lrrk2 in wild-type mice was well tolerated supporting that PD is not associated with LRRK2 loss of function.⁶² MOE-gamper ASOs reducing LRRK2 in mice treated with α-synuclein preformed fibirls were also associated with reduced aggregations of phospho(S129)-α-synuclein.⁶³ Some effort has also been made to develop an ASO therapeutic targeting α-synuclein for PD.⁶⁴

Spinal muscular atrophy and nusinersen

SMA is caused by loss-of-function mutations in the SMN1 gene resulting in the loss of the survival motor neuron (SMN) protein. SMA severity is inversely correlated with copy number of the homologous SMN2 gene. The cDNA encoded by SMN2 is identical to that encoded by SMN1 except for lack of exon 7. The ASO drug nusinersen is an SSO that functions by blocking an intronic splicing silencer element in the SMN2 intron 7 preventing the spliceosome from excluding exon 7 (figure 1). The result of nusinersen action is expression of the functional, full-length SMN protein from the SMN2 gene. In Smn1^−/−; SMN2^+/+ mice, SSOs that restore SMA2 exon 7 splicing also restored tail and ear necrosis phenotypes.⁶⁵ Nusinersin was well tolerated in patients with SMA^1,66,67 and was approved by the FDA for use in humans for the treatment of SMA in December of 2016.²⁷

Duchenne muscular dystrophy and eteplirsen

DMD is caused by mutation in the DMD gene encoding dystrophin, which is one of the largest gene in the human genome. Most of the DMD mutations causing DMD result in premature dystrophin truncations. The predominant ASO strategy for treating DMD is employment of ASOs to exclude exons resulting in DMD proteins with partially restored functions. Eteplirsen, an ASO that has a PMO oligomer structure, interacts with the DMD pre-mRNA at exon 51 resulting in exon 51 exclusion. As illustrated in figure 1, it is used in DMD patients who have a deletion including exons 49 and 50 resulting in truncated dystrophin; exclusion of exon 51 restores the reading frame from exon 48 to exon 52, partially restoring DMD function.⁶⁸ Approved by the FDA in 2016 for the treatment of DMD, eteplirsen can be used to treat approximately 14% of DMD cases,⁶⁹ and efforts are ongoing to develop additional ASOs targeting other exons to treat DMD cases caused by other mutations in the dystrophin gene.⁶⁸ Wave Life Sciences has also initiated a phase 1 clinical trial for testing a stereopure ASO for DMD exon 51 exon skipping.

Familial amyloid polyneuropathy and inotersen

Missense mutation in the TTR gene encoding transthyretin is the cause of hereditary transthyretin amyloidosis (ATTR). TTR mutations result in transthyretin misfolding and progressive accumulation of amyloid deposition in many tissues resulting in polyneuropathy, multiorgan dysfunction, and cardiomyopathy. Multiorgan failure and cardiac arrest pose a greatest risk of death for ATTR patients. Lowering the total expression of TTR at its source in the liver is an effective strategy for ATTR, which is achieved by systemic treatment using the 2′-MOE ASO drug inotersen,³ now approved by the FDA for FAP. Inotersen is treated systemically with delivery made by weekly subcutaneous injections. In the brain, transthyretin is produced by the choroid plexus, and TTR mutations cause leptomeningeal amyloidosis that potentially could also be treated by ASO therapy targeting TTR expression.⁷⁰