TY - JOUR
AU - Pereira, de Almeida, Luis
AB - Abstract Polyglutamine (polyQ) disorders are a group of nine neurodegenerative diseases that share a common genetic cause, which is an expansion of CAG repeats in the coding region of the causative genes that are otherwise unrelated. The trinucleotide expansion encodes for an expanded polyQ tract in the respective proteins, resulting in toxic gain-of-function and eventually in neurodegeneration. Currently, no disease-modifying therapies are available for this group of disorders. Nevertheless, given their monogenic nature, polyQ disorders are ideal candidates for therapies that target specifically the gene transcripts. Antisense oligonucleotides (ASOs) have been under intense investigation over recent years as gene silencing tools. ASOs are small synthetic single-stranded chains of nucleic acids that target specific RNA transcripts through several mechanisms. ASOs can reduce the levels of mutant proteins by breaking down the targeted transcript, inhibit mRNA translation or alter the maturation of the pre-mRNA via splicing correction. Over the years, chemical optimization of ASO molecules has allowed significant improvement of their pharmacological properties, which has in turn made this class of therapeutics a very promising strategy to treat a variety of neurodegenerative diseases. Indeed, preclinical and clinical strategies have been developed in recent years for some polyQ disorders using ASO therapeutics. The success of ASOs in several animal models, as well as encouraging results in the clinic for Huntington’s disease, points towards a promising future regarding the application of ASO-based therapies for polyQ disorders in humans, offering new opportunities to address unmet medical needs for this class of disorders. This review aims to present a brief overview of key chemical modifications, mechanisms of action and routes of administration that have been described for ASO-based therapies. Moreover, it presents a review of the most recent and relevant preclinical and clinical trials that have tested ASO therapeutics in polyQ disorders. antisense oligonucleotides, neurodegenerative diseases, polyglutamine disorders, preclinical studies, clinical trials Antisense oligonucleotide therapeutics in neurodegenerative diseases Several neurodegenerative diseases are caused by single gene mutations that lead to protein dysfunction and subsequent pathological cascade. Consequently, numerous therapeutic approaches aim at ameliorating the pathology by blocking protein misfolding and aggregation or by targeting pathways downstream of mutant protein dysfunction (reviewed in Scannevin, 2018). However, one arguably superior approach has been to directly target the source of the dysfunction, that is, target the genetic defect by altering the expression of aberrant RNA transcripts. Of the many ways to target RNA transcripts, RNA interference (RNAi) has been one of the most investigated mechanisms, opening new prospects for therapeutic progress for neurodegenerative diseases, including polyglutamine (polyQ) disorders. RNAi-based therapeutics have been employed using siRNAs, shRNAs and microRNAs, by delivering these therapeutic molecules naked/unmodified, complexed with carriers or encoded by viral vectors (reviewed in Matos et al., 2018). Other tools under investigation for targeting RNA expression are antisense oligonucleotides (ASOs), which are the main focus of this review. In 1978, Stephenson and Zamecnik introduced the use of ASOs for targeted gene inhibition. Their results showed that a 13-nucleotide long DNA molecule complementary to a portion of Rous sarcoma virus genome inhibited viral RNA translation (Stephenson and Zamecnik, 1978). Since then, while natural or unmodified synthetic oligonucleotides have very poor drug-like properties, a wide range of ASOs have been synthesized and optimized by modifying their backbone and sugar component. These modifications have massively improved the pharmacological properties of ASOs, including their stability, binding affinity, safety profile, as well as specificity (reviewed in Bennett and Swayze, 2010). ASOs are emerging as a class of therapeutics with high potential to treat diseases affecting the CNS. ASOs are synthetic single-stranded small chains of nucleic acids, typically 8–50 nucleotides in length. They are capable of hybridizing to RNA molecules via standard Watson-Crick base-pairing and thereby alter expression of the target RNA through different mechanisms. ASOs can be also designed in a way that will trigger the breakdown of the targeted transcript, interfere with the translation of the target transcript or alter the maturation of the transcript through splicing modulation (reviewed in Bennett and Swayze, 2010). Despite their great potential as therapeutics, the blood–brain barrier constitutes a major obstacle for the delivery of ASOs to the CNS (Agrawal et al., 1991). Indeed, very limited amounts of ASOs will penetrate the blood–brain barrier and reach the targeted brain cells after systemic administration. However, a number of studies have shown broad CNS distribution of ASOs after intracerebroventricular (ICV) injection (Chauhan, 2002; Casaca-Carreira et al., 2017) or intrathecal administration (Butler et al., 2005). For that reason, administration of ASOs directly into the CSF has been the method of choice to deliver ASOs to the CNS. Overall, ASO therapeutics offer new opportunities to address unmet medical needs for neurological disorders. Numerous preclinical and clinical efforts are currently being carried out to develop ASO therapeutics for neurological disorders, some of which have already provided great enthusiasm as they translated into the clinic (reviewed in Schoch and Miller, 2017). Indeed, validating the research progress and potential in recent years, two ASO drugs have been approved by the United States Food and Drug Administration (FDA). First, eteplirsen gained FDA approval for the treatment of Duchenne muscular dystrophy and, shortly after, nusinersen was approved for the treatment of spinal muscular atrophy. In this review, we will briefly discuss key chemical modifications, mechanisms of action and routes of administration that have been described and applied for ASO-based therapies. Finally, we will discuss current progress towards preclinical and clinical application of ASOs in neurodegenerative disorders, particularly in polyQ disorders. Understanding antisense oligonucleotide design and function ASO therapeutics encompass an extremely versatile strategy that can be applied to regulate gene expression through different functional mechanisms, which highly depends on the oligonucleotide sequence, its RNA versus DNA content and its chemical modifications. Structural and chemical modifications As mentioned, natural or unmodified oligonucleotides have very poor drug-like properties and have to be chemically optimized in order to improve their affinity, stability, potency and toxicity profiles. Indeed, early studies using unmodified ASOs revealed their limited clinical application due to their fast degradation rates by exo- and endonucleases, which cleave the phosphodiester bonds (Akhtar et al., 1991; Dagle et al., 1991; Eder et al., 1991). For instance, it was shown that unmodified oligodeoxynucleotides are rapidly degraded in the CSF by exonucleases following ICV administration in rats (Whitesell et al., 1993). To surmount such issues, over the past decades, a number of modifications to ASO backbone and sugar moieties have been implemented (Fig. 1 and Table 1). This resulted in an improvement of ASO stability and other pharmacological properties, increasing its potential as a therapeutic agent. Table 1 Examples of common antisense oligonucleotide chemical modifications, main properties and mechanisms of action Modification . Main properties . Mechanisms of action . References . Backbone modification Phosphodiester (PO) Naturally occurring RNase H degradation Akhtar et al., 1991; Dagle et al., 1991; Eder et al., 1991 Highly degraded by nucleases Phosphorothioate (PS) Enhanced nuclease resistance RNase H degradation Stein et al., 1988; Brown et al., 1994; Ogawa et al., 1995 Increased protein binding ability for cellular uptake Phophoroamidate (PA) Enhanced nuclease resistance Non-degrading RNA mechanisms Gryaznov et al., 1996; Hashizume et al., 2008 High affinity Phosphorodiamidate morpholino oligomers (PMO) High nuclease and protease resistance Non-degrading RNA mechanisms Hudziak et al., 1996; Kurreck, 2003; Alter et al., 2006 Increased affinity Peptide nucleic acids (PNA) High nuclease and protease resistance Non-degrading RNA mechanisms Demidov et al., 1994; Schwarz et al., 1999; Hu et al., 2009 High affinity Sugar modification 2′-O-methyl (2′-OMe) Enhanced nuclease resistance Non-degrading RNA mechanisms and RNase activity with gapmer design Lubinil et al., 1994; Tluk et al., 2009; Hamm et al., 2010 Increased target affinity Decreased toxicity 2′-O-methoxyl-ethyl (2′-MOE) Enhanced nuclease resistance Non-degrading RNA mechanisms and RNase activity with gapmer design Mckay et al., 1999; Henry et al., 2000; Geary et al., 2001 Increased target affinity Decreased toxicity Locked nucleic acids (LNA) Enhanced nuclease resistance Non-degrading RNA mechanisms and RNase activity with gapmer design Braasch et al., 2002; Frieden et al., 2003; Swayze et al., 2007 Potent target affinity Increased toxicity S-constrained-ethyl (cEt) High binding affinity Non-degrading RNA mechanisms and RNase activity with gapmer design Seth et al., 2009; Carroll et al., 2011 Improved toxic profiles Modification . Main properties . Mechanisms of action . References . Backbone modification Phosphodiester (PO) Naturally occurring RNase H degradation Akhtar et al., 1991; Dagle et al., 1991; Eder et al., 1991 Highly degraded by nucleases Phosphorothioate (PS) Enhanced nuclease resistance RNase H degradation Stein et al., 1988; Brown et al., 1994; Ogawa et al., 1995 Increased protein binding ability for cellular uptake Phophoroamidate (PA) Enhanced nuclease resistance Non-degrading RNA mechanisms Gryaznov et al., 1996; Hashizume et al., 2008 High affinity Phosphorodiamidate morpholino oligomers (PMO) High nuclease and protease resistance Non-degrading RNA mechanisms Hudziak et al., 1996; Kurreck, 2003; Alter et al., 2006 Increased affinity Peptide nucleic acids (PNA) High nuclease and protease resistance Non-degrading RNA mechanisms Demidov et al., 1994; Schwarz et al., 1999; Hu et al., 2009 High affinity Sugar modification 2′-O-methyl (2′-OMe) Enhanced nuclease resistance Non-degrading RNA mechanisms and RNase activity with gapmer design Lubinil et al., 1994; Tluk et al., 2009; Hamm et al., 2010 Increased target affinity Decreased toxicity 2′-O-methoxyl-ethyl (2′-MOE) Enhanced nuclease resistance Non-degrading RNA mechanisms and RNase activity with gapmer design Mckay et al., 1999; Henry et al., 2000; Geary et al., 2001 Increased target affinity Decreased toxicity Locked nucleic acids (LNA) Enhanced nuclease resistance Non-degrading RNA mechanisms and RNase activity with gapmer design Braasch et al., 2002; Frieden et al., 2003; Swayze et al., 2007 Potent target affinity Increased toxicity S-constrained-ethyl (cEt) High binding affinity Non-degrading RNA mechanisms and RNase activity with gapmer design Seth et al., 2009; Carroll et al., 2011 Improved toxic profiles Open in new tab Table 1 Examples of common antisense oligonucleotide chemical modifications, main properties and mechanisms of action Modification . Main properties . Mechanisms of action . References . Backbone modification Phosphodiester (PO) Naturally occurring RNase H degradation Akhtar et al., 1991; Dagle et al., 1991; Eder et al., 1991 Highly degraded by nucleases Phosphorothioate (PS) Enhanced nuclease resistance RNase H degradation Stein et al., 1988; Brown et al., 1994; Ogawa et al., 1995 Increased protein binding ability for cellular uptake Phophoroamidate (PA) Enhanced nuclease resistance Non-degrading RNA mechanisms Gryaznov et al., 1996; Hashizume et al., 2008 High affinity Phosphorodiamidate morpholino oligomers (PMO) High nuclease and protease resistance Non-degrading RNA mechanisms Hudziak et al., 1996; Kurreck, 2003; Alter et al., 2006 Increased affinity Peptide nucleic acids (PNA) High nuclease and protease resistance Non-degrading RNA mechanisms Demidov et al., 1994; Schwarz et al., 1999; Hu et al., 2009 High affinity Sugar modification 2′-O-methyl (2′-OMe) Enhanced nuclease resistance Non-degrading RNA mechanisms and RNase activity with gapmer design Lubinil et al., 1994; Tluk et al., 2009; Hamm et al., 2010 Increased target affinity Decreased toxicity 2′-O-methoxyl-ethyl (2′-MOE) Enhanced nuclease resistance Non-degrading RNA mechanisms and RNase activity with gapmer design Mckay et al., 1999; Henry et al., 2000; Geary et al., 2001 Increased target affinity Decreased toxicity Locked nucleic acids (LNA) Enhanced nuclease resistance Non-degrading RNA mechanisms and RNase activity with gapmer design Braasch et al., 2002; Frieden et al., 2003; Swayze et al., 2007 Potent target affinity Increased toxicity S-constrained-ethyl (cEt) High binding affinity Non-degrading RNA mechanisms and RNase activity with gapmer design Seth et al., 2009; Carroll et al., 2011 Improved toxic profiles Modification . Main properties . Mechanisms of action . References . Backbone modification Phosphodiester (PO) Naturally occurring RNase H degradation Akhtar et al., 1991; Dagle et al., 1991; Eder et al., 1991 Highly degraded by nucleases Phosphorothioate (PS) Enhanced nuclease resistance RNase H degradation Stein et al., 1988; Brown et al., 1994; Ogawa et al., 1995 Increased protein binding ability for cellular uptake Phophoroamidate (PA) Enhanced nuclease resistance Non-degrading RNA mechanisms Gryaznov et al., 1996; Hashizume et al., 2008 High affinity Phosphorodiamidate morpholino oligomers (PMO) High nuclease and protease resistance Non-degrading RNA mechanisms Hudziak et al., 1996; Kurreck, 2003; Alter et al., 2006 Increased affinity Peptide nucleic acids (PNA) High nuclease and protease resistance Non-degrading RNA mechanisms Demidov et al., 1994; Schwarz et al., 1999; Hu et al., 2009 High affinity Sugar modification 2′-O-methyl (2′-OMe) Enhanced nuclease resistance Non-degrading RNA mechanisms and RNase activity with gapmer design Lubinil et al., 1994; Tluk et al., 2009; Hamm et al., 2010 Increased target affinity Decreased toxicity 2′-O-methoxyl-ethyl (2′-MOE) Enhanced nuclease resistance Non-degrading RNA mechanisms and RNase activity with gapmer design Mckay et al., 1999; Henry et al., 2000; Geary et al., 2001 Increased target affinity Decreased toxicity Locked nucleic acids (LNA) Enhanced nuclease resistance Non-degrading RNA mechanisms and RNase activity with gapmer design Braasch et al., 2002; Frieden et al., 2003; Swayze et al., 2007 Potent target affinity Increased toxicity S-constrained-ethyl (cEt) High binding affinity Non-degrading RNA mechanisms and RNase activity with gapmer design Seth et al., 2009; Carroll et al., 2011 Improved toxic profiles Open in new tab Figure 1 Open in new tabDownload slide Commonly used chemical modifications to ASO backbone and sugar moieties. Schematic of unmodified DNA/RNA base pair (left). Decades of research have allowed the development of a number of chemical modifications to the backbone (top) and 2′ sugar modifications (bottom). These modifications have allowed an increase in nuclease resistance, target affinity and tolerability of ASO drugs. Figure 1 Open in new tabDownload slide Commonly used chemical modifications to ASO backbone and sugar moieties. Schematic of unmodified DNA/RNA base pair (left). Decades of research have allowed the development of a number of chemical modifications to the backbone (top) and 2′ sugar modifications (bottom). These modifications have allowed an increase in nuclease resistance, target affinity and tolerability of ASO drugs. One of the first and still widely used modifications aims at protecting the ASO from degradation by replacing the non-bridging oxygen atom of the phosphate groups with a sulphur atom, creating a phosphorothioate (PS) instead of a phosphodiester linkage (Fritz, 1966) (Fig. 1). This phosphorothioate linkage greatly enhances the resistance against nucleolytic degradation and thus the stability of the drug (Stein et al., 1988; Rifai et al., 1996). ASOs with this modification are more stable, having been reported to have a half-life up to 19 h in rat CSF and 9 h in human serum (Campbell et al., 1990) and did not induce overt non-specific toxic side effects after delivery in rodent brains (Liebsch et al., 1999). Importantly, the phosphorothioate linkage confers increased binding to plasma proteins, which may facilitate cellular uptake and provide additional protection from nuclease degradation (Brown et al., 1994; Watanabe et al., 2006), though non-specific protein binding may reduce therapeutic effects. Less favourable characteristics of phosphorothioate modifications relate to their potential cytotoxic effects at high concentrations either through increased protein binding, complement activation and/or destabilization of duplexes (Jaroszewski et al., 1996; Levin, 1999). Interestingly, converting phosphodiesters into phosphorothioates creates a chiral centre at every modified linkage along the oligonucleotide. As such, synthesizing a 20-mer oligonucleotide with 19 phosphorothioate linkages will produce a mixture of nearly half a million stereoisomers. Hence, controlling the chirality of each phosphorothioate linkage greatly increases the possibilities for lead optimization. Recently, it was demonstrated that controlling the stereochemistry of mipomersen, which is produced as a mixture of stereoisomers, could significantly improve its drug properties (Iwamoto et al., 2017). In addition to the backbone modifications that proved to increase stability, several modifications at the 2′-position of the sugar moiety can enhance ASO affinity and decrease their toxicity potential. Of these, the 2′-O-methyl (2′-OMe) and 2′-O-methoxyl-ethyl (2′-MOE) are most commonly used (Fig. 1), allowing stronger binding affinity to the target RNA (Lubinil et al., 1994; Freier and Altmann, 1997; Mckay et al., 1999) and enhanced resistance to nuclease degradation (Mckay et al., 1999; Geary et al., 2001). Moreover, decreased immune stimulation was reported after 2′-OMe (Tluk et al., 2009; Hamm et al., 2010) and 2′-MOE modifications (Henry et al., 2000). Notably, 2′-modifications reduced phosphorothioate backbone toxicity in the CNS (Peng Ho et al., 1998). Another sugar modification developed, similar to 2′-OMe, is termed locked nucleic acid (LNA), in which the 4′-carbon has been linked to the 2′-methyl group (Wengel, 1999) (Fig. 1). The main advantage of LNA ASOs is the increased nuclease resistance (Frieden et al., 2003) and improved affinity (Braasch et al., 2002). Compared with other 2′-sugar modifications, LNAs confer better potency and RNA binding affinity; however, they are associated with an increased toxicity potential (Swayze et al., 2007), which may limit its clinical use. Nevertheless, efforts to understand and solve toxicity are ongoing (Kasuya et al., 2016; Dieckmann et al., 2018) and it has been reported that some LNAs are well tolerated after administration to the rat brain (Wahlestedt et al., 2000). In addition, LNA analogues have been developed to tackle the toxicity problems (reviewed in DeVos and Miller, 2013). Other sugar modifications are the S-constrained-ethyl (cEt), in which the ethyl chain in the 2′-residue is constrained to 4′-position of the sugar ring (Seth et al., 2009) (Fig. 1). ASO drugs containing cEt modifications provide improved toxicity profile compared with LNAs (Seth et al., 2009) and increased potency compared with 2′-MOE ASOs (Carroll et al., 2011). Taken together, the second generation of ASO modifications provided a great improvement to the pharmacological properties of the antisense drugs. Particularly, the increase in affinity and stability, as well as the reduction in toxicity that the 2′-modifications conferred to phosphorothioate (PS)-ASOs (Henry et al., 2000; Geary et al., 2001). As ASO chemistry continued to evolve, other modifications were developed that comprised the third generation of ASO drugs. Other backbone modifications include the phosphoroamidate (PA) analogues, where 3′-oxygen is replaced by a 3′-amino group (Gryaznov and Chen, 1994) (Fig. 1). This phosphoroamidate linkage also confers increased nuclease resistance; however, it abrogates RNase H activity (Gryaznov et al., 1996). For that reason, PA-ASOs are more suited for non-degrading RNA applications, such as translation inhibition (Hashizume et al., 2008). Other oligonucleotide modifications include replacing the sugar phosphate backbone with a morpholine ring and the charged phosphodiester linkage with an uncharged phosphoroamidate linkage, termed phosphorodiamidate morpholino oligomers (PMO) (Fig. 1). PMOs showed to be highly resistant to nuclease and protease degradation (Hudziak et al., 1996). Similar to PA-ASOs, PMOs do not support RNase activity; however, they can be used to modify splicing (Alter et al., 2006) and inhibit translation (Sun et al., 2014). Peptide nucleic acids (PNAs) comprise a different class of oligonucleotide analogues that contain a peptide replacement for the sugar phosphate backbone to which the nucleobases are attached (Nielsen et al., 1991) (Fig. 1). These DNA analogues are still able to hybridize to complementary nucleic acids through Watson-Crick base-pairing (Egholm et al., 1993) and exhibit high resistance to nuclease and protease degradation (Demidov et al., 1994) and high affinity to RNA (Schwarz et al., 1999). PNAs exhibit poor cellular uptake, a characteristic that can be improved by using peptide conjugates and charged amino acids in the PNA backbone (reviewed in Bennett and Swayze, 2010). As illustrated in this section, ASOs are highly versatile tools that can be customized in a variety of ways to improve drug properties. This way of taking advantage of the combined benefits of different modifications, including improved nuclease resistance, target affinity and potency. Indeed, different chemical compositions can be used to achieve the desired application and mechanism of action (Table 1), as will be discussed below. A combination of different structures and chemistries in the nucleic acid bases can therefore dramatically improve the therapeutic potential of oligonucleotide-based therapies. Of note, for example, is the recent work from Khvorova and colleagues describing a novel divalent structure of highly chemically modified siRNAs, which contain chemical modifications common in ASO design such as phosphorothioate backbone and 2′-OMe (Alterman et al., 2019). Thus, research aiming at ASO chemistry design was and continues to be an important field of work for improving safety and pharmacological properties, particularly as more and more ASOs are moving towards clinical trials for several neurodegenerative disorders. Functional mechanisms Depending on the gene mutation and how the targeted transcript needs to be modulated, ASOs can be designed to act through a specific mechanism of action (Fig. 2 and Table 1). This is, however, highly dependent on their chemistry, binding sequence and target RNA molecule, as described below. Figure 2 Open in new tabDownload slide Functional mechanisms of ASOs and target mRNA fate. Left: Depending on their chemistry, sequence and target, ASOs can act through different mechanisms of action. After binding to their target RNA in the cell, ASOs can: recruit RNase H nuclease and induce degradation of the target mRNA molecule in the nucleus and cytoplasm (A); inhibit protein synthesis by blocking the translational machinery from assembly or running along the mRNA (B); modify splicing by inducing the inclusion or exclusion of specific exon(s) (C); modulate the mRNA molecule by interfering with 5′ cap formation or polyadenylation (D); and interfere with mRNA regulation by blocking the binding of microRNAs to their target (E). Figure 2 Open in new tabDownload slide Functional mechanisms of ASOs and target mRNA fate. Left: Depending on their chemistry, sequence and target, ASOs can act through different mechanisms of action. After binding to their target RNA in the cell, ASOs can: recruit RNase H nuclease and induce degradation of the target mRNA molecule in the nucleus and cytoplasm (A); inhibit protein synthesis by blocking the translational machinery from assembly or running along the mRNA (B); modify splicing by inducing the inclusion or exclusion of specific exon(s) (C); modulate the mRNA molecule by interfering with 5′ cap formation or polyadenylation (D); and interfere with mRNA regulation by blocking the binding of microRNAs to their target (E). Figure 3 Open in new tabDownload slide ASO-based therapies for Huntington’s disease and spinocerebellar ataxia type 3. Schematic representation of some of the ASO-based interventions that have been used in Huntington’s disease and SCA3 disease models over the years. For Huntington’s disease, allele-specific strategies have been used: (1) by targeting the expanded CAG repeats with ASOs that interfere with translation and result in reduction of mutant HTT protein, while maintaining normal levels of mRNA; or (2) by targeting a variety of SNPs in intronic and exonic regions tightly linked with the expanded mutation—represented here as an ASO targeting SNP located at exonic region of mRNA, which induces RNase H-mediated degradation of the mRNA and subsequently reduction of mutant HTT protein; non allele-specific approaches investigated have also used: (3) RNase-mediated degradation strategies by targeting HTT mRNA, ultimately resulting in reduction of wild-type and mutant HTT protein or (4) exon skipping strategies and ASOs that target disease-relevant exons that contain predicted cleavage sites and thereby resulting in the formation of HTT lacking disease-relevant exon(s). For SCA3, exon skipping strategies have been most studied using ASOs targeting: (5) exons containing cleavage sites; or (6 and 7) exon 10, containing the expanded CAG tract, which ultimately results in formation of a truncated ATXN3 protein lacking the disease-relevant exon(s); moreover, (8) ASOs targeting the 3′ UTR region of ATXN3 mRNA have been used to induce mRNA cleavage and ATXN3 protein reduction. Figure 3 Open in new tabDownload slide ASO-based therapies for Huntington’s disease and spinocerebellar ataxia type 3. Schematic representation of some of the ASO-based interventions that have been used in Huntington’s disease and SCA3 disease models over the years. For Huntington’s disease, allele-specific strategies have been used: (1) by targeting the expanded CAG repeats with ASOs that interfere with translation and result in reduction of mutant HTT protein, while maintaining normal levels of mRNA; or (2) by targeting a variety of SNPs in intronic and exonic regions tightly linked with the expanded mutation—represented here as an ASO targeting SNP located at exonic region of mRNA, which induces RNase H-mediated degradation of the mRNA and subsequently reduction of mutant HTT protein; non allele-specific approaches investigated have also used: (3) RNase-mediated degradation strategies by targeting HTT mRNA, ultimately resulting in reduction of wild-type and mutant HTT protein or (4) exon skipping strategies and ASOs that target disease-relevant exons that contain predicted cleavage sites and thereby resulting in the formation of HTT lacking disease-relevant exon(s). For SCA3, exon skipping strategies have been most studied using ASOs targeting: (5) exons containing cleavage sites; or (6 and 7) exon 10, containing the expanded CAG tract, which ultimately results in formation of a truncated ATXN3 protein lacking the disease-relevant exon(s); moreover, (8) ASOs targeting the 3′ UTR region of ATXN3 mRNA have been used to induce mRNA cleavage and ATXN3 protein reduction. Breakdown of RNA transcripts Degradation of dysfunctional mRNA will ultimately cause the reduction of the affected protein levels. This can have great beneficial effects in the context of several neurodegenerative diseases where the cause of the disorder can be traced to a specific RNA/protein gain of toxic function. In the case of ASOs, this mechanism occurs via recruitment of RNase H enzyme after oligonucleotide binding to the target mRNA (Wu et al., 2004) (Fig. 2A). The DNA/RNA heteroduplex forms a substrate for RNase H, resulting in cleavage of the mRNA strand and release of the intact DNA (reviewed in Cerritelli and Crouch, 2009). As the ASO DNA molecule is left intact, it can further hybridize with additional target mRNA molecules, thus providing a rather long duration of action. Importantly, PS-ASOs are able to efficiently recruit RNase H, which promotes the degradation of the target mRNA (Wu et al., 2004), allowing the use of such chemically modified drugs in disorders where downregulation of target mRNA is necessary. In contrast, ASOs containing fully modified 2′-sugar moieties are unable to recruit and activate RNase H-mediated degradation of the target mRNA (reviewed in Bennett and Swayze, 2010). Hence, this type of ASO is only used in other non-degrading RNA applications. To activate RNase H-mediated degradation, at least five consecutive 2′-unmodified nucleotides are required (Monia et al., 1993; Wu et al., 1999). Thus, in order to take advantage of the beneficial effects of 2′-sugar rings modifications and, at the same time, allow target mRNA degradation, a gapmer design was developed. The gapmer is comprised of a central region of 2′-unmodified nucleotides that is flanked by nucleotides with 2′-modified sugar moieties. In this way, the external 2′-modified nucleotides increase affinity and stability and the central region allows for the recruitment of RNase H (Monia et al., 1993). It has also been shown that the rate of ASO activity is related to RNase H levels (Wu et al., 2004) and that RNase H-dependent ASO activity is present in both the nucleus and the cytoplasm (Cazenave et al., 1994; Liang et al., 2017) (Fig. 2A). Accordingly, given that the levels of RNase H are higher in the nucleus, the degradation rate of the target mRNAs is greater for nuclear retained mRNAs than cytoplasmic mRNAs (Vickers and Crooke, 2015). RNase H ability to function in both cellular compartments allows for targeting of sequences located in both intronic and exonic regions (Fig. 2A), which allows for targeting of previously inaccessible drug targets. Moreover, activation of RNase H is highly dependent on the structure and sequence of the DNA/RNA heteroduplex (Lima et al., 2007; Kiełpiński et al., 2017). This specificity may allow for selective RNase H-mediated cleavage of the dysfunctional mRNA, keeping normal levels of wild-type mRNA. The selective modulation can be achieved, for instance, by targeting a single nucleotide polymorphism (SNP) associated with the disorder (Carroll et al., 2011) or specifically target expanded trinucleotide repeats and structural differences between wild-type and mutant mRNAs (Hu et al., 2009; Evers et al., 2011) (Fig. 2A2). This is particularly relevant in disorders where the wild-type protein has important or still unknown cellular functions, which is the case in many neurodegenerative diseases. Translational inhibition As mentioned above, ASOs fully modified at the 2′-position do not support RNase H activity. Instead, RNase H-independent ASOs can be used in a wide range of applications where mRNA breakdown is not strictly required. One functional mechanism of ASO drugs that reduces the levels of a dysfunctional protein, apart from recruitment of cellular nucleases, is translational inhibition (Fig. 2B). The binding of non-degrading ASOs to mRNA can block the translational machinery from assembly or running along the mRNA (Johansson et al., 1994; Bennett and Swayze, 2010), preventing translation or elongation of the target mRNA. Though, this type of strategy has not been a main effort for ASO-based therapies. Splicing modulation Other applications apart from breakdown or translational inhibition of RNA transcripts are being pursued and show promising results. Thus far the most used alternative has been splice-modulating ASOs. These ASOs have 2′-sugar modifications in all nucleotides preventing RNase H recruitment. Splice-modulating ASOs can be designed to regulate the splicing machinery at specific exon-intron junctions, by forcing the machinery to either skip exon(s) containing a mutation, include an essential exon that is abnormally excluded, or shift the ratio between isoforms (Fig. 2C). ASOs interfere in this process by disrupting splicing sites or binding of splicing factors. This leads to the exclusion or inclusion of specific exons, resulting in alteration of the targeted mRNA sequence and respective protein (reviewed in Havens and Hastings, 2016). Interestingly, it was shown that exon-skipping ASOs can also lead to mRNA reduction through the nonsense-mediated decay pathway by inducing premature stop codons (Ward et al., 2014). Splice-modifying ASOs may be highly beneficial in disorders with splicing defects (Hua et al., 2008, 2010; Jearawiriyapaisarn et al., 2008; Peacey et al., 2012) or where specific exons contain disease-causing mutations (Evers et al., 2013, 2014). Other strategies Other potential applications of ASO-based therapies may be modulation of mRNA maturation. ASOs can be designed to target and interfere with 3′-polyadenylation site or 5′-cap formation (Fig. 2D), in this way blocking mRNA translation or altering mRNA stability (Baker et al., 1999; Vickers et al., 2001; Bennett and Swayze, 2010). Another exciting application is the use of ASOs against microRNAs. In this way, ASOs can be developed to target endogenous microRNAs and inhibit its function (Fig. 2E). Binding of ASOs to the microRNA blocks its ability to bind to their own target mRNAs, thereby increasing the levels of the target molecule (Stenvang et al., 2012; Jan et al., 2015). This approach is particularly interesting as the role of microRNAs in neurodegenerative disorders is increasingly more evident (reviewed in Junn and Mouradian, 2012). The characteristics of the different ASO functional mechanisms are key considerations to take into account for effective ASO design and application and, once again, highlight the versatility of these therapeutic agents. Cellular uptake and intracellular trafficking of antisense oligonucleotides To be effective, ASOs need to overcome the membrane barrier of the cells and in some cases of organelles to act in the targeted RNA molecule. The mechanistic aspects of cellular entry of ASOs are still largely unknown; however, efforts have been made to understand ASO uptake and improve therapy design. Cellular uptake and intracellular trafficking of ASO molecules have been thoroughly reviewed elsewhere (Geary et al., 2015; Juliano and Carver, 2015; Crooke et al., 2017), so in this section we aim to provide a brief overview of these mechanisms. Cellular uptake of ASO drugs can be highly influenced by its chemical components, which is thought to be related with different protein binding affinities. Indeed, phosphorothioate backbone modification can enhance protein binding (Brown et al., 1994; Liang et al., 2015), which in turn influences cellular uptake. Overall, efficient cellular entry of ASOs appears to be a two-step process. The first step is adsorption to the cell membrane surface, which is rapid and appears to be a saturable process. Accordingly, the presence of a competitive ASO molecule during the adsorption process can alter ASO activity (Geary et al., 2009; Koller et al., 2011). These results suggest that there is competition for binding of ASOs to specific proteins associated with the cell membrane during the adsorption process. Importantly, data suggest the presence of several pathways of ASO uptake in cells, with some leading to productive ASO activity, while others result in non-productive uptake (reviewed in Crooke et al., 2017). A wide variety of cell surface proteins have been associated with productive ASO cellular uptake, including integrins (Alam et al., 2008), G protein-coupled receptors (Ming et al., 2010), scavenger receptors (Miller et al., 2016), and epidermal growth factor receptor (Wang et al., 2018). Of note, modest modifications to ASO chemistry, such as phosphorothioate modifications to the backbone and 2′-sugar modifications, can greatly influence ASO interaction with proteins involved in productive uptake and in this way improve therapeutic performance (Liang et al., 2016). Following ASO association with membrane proteins, internalization occurs through the endocytic pathway. Once internalized, ASOs then need to be released into the cytosol or nucleus in order to reach their target RNAs. After being released into these cell compartments, ASOs can bind to multiple intracellular proteins that will dictate the fate of the ASO drug inside the cell (Liang et al., 2014, 2015, 2016; Wang et al., 2016). Importantly, although a variety of intracellular proteins have been identified that bind to PS-ASOs, many have no effect on oligonucleotide activity, however some proteins are able to enhance or reduce ASO activity (reviewed in Crooke et al., 2017). Thus, identification of intracellular proteins, as well as cell-surface proteins that mediate uptake of ASOs in productive and non-productive processes is of great importance to better inform ASO design and make these therapeutic tools more efficient. Antisense oligonucleotide delivery and routes of administration to the CNS For ASOs to wield their therapeutic effect, they need to be delivered to the organism through an appropriate route in order to travel to the target tissues and cells. Different strategies are available for ASO delivery into the organism (reviewed in Juliano, 2016). Thus, it is important to take into account the characteristics of the disorder and the available delivery approaches to correctly choose the appropriate method of administration of the therapeutic drug. ASO-based therapeutics must also be able to reach the affected tissues or cells at appropriate concentrations. In the context of CNS disorders, the impermeability of the blood–brain barrier to large molecules, including ASOs, constitutes a major challenge (Agrawal et al., 1991). In this section we review several strategies of ASO delivery to the CNS, which have shown varying degrees of success. They include a less invasive strategy via the systemic route and a more direct approach via delivery to the CSF. Systemic delivery There is growing interest in systemic delivery of ASOs to the CNS as this route of administration is significantly less invasive, and therefore a preferable method for clinical applications. Notably, some studies have shown that ASOs can be delivered to the brain in effective doses via the systemic route (Banks et al., 2001; Farr et al., 2014). However, the intravenous dose must be ∼100 times higher than the ICV dose (Banks et al., 2001), which greatly increases the risk of toxicity. Therefore, presently this approach does not appear to be an appropriate delivery method of ASOs to the CNS. Different strategies are being investigated to overcome this issue, such as the use of receptor-mediated endocytosis/transcytosis or cell penetrating peptides (CPP). For instance, one potential approach is to link nanoparticles to transferrin receptor ligands or anti-receptor antibodies, which allows receptor-mediated endocytosis. It was reported that ASOs can be loaded into nanoparticles conjugated with anti-transferrin receptor-1 antibody, which improves CNS entry through the blood–brain barrier (Kozlu et al., 2014). However, in vivo studies demonstrating the efficacy of this strategy have yet to be reported. Liposome-mediated delivery of oligonucleotides has also been developed using a novel liposome formulation (DCL64). Intravenous administration of these novel lipid particles mixed with oligonucleotides resulted in interaction with low-density lipoprotein receptors involved in brain microvascular endothelial cell endocytosis/transcytosis with subsequent accumulation in Purkinje cells of mouse cerebellum (Ashizawa et al., 2019). Another interesting strategy is CPP-mediated delivery of ASOs to the CNS. One study reported that systemically delivered PMOs tagged with arginine-rich CPPs reached the brain, with wide uptake throughout the rodent brain (Du et al., 2011). More recently, another study reported that intravenous administration of peptide bound PMOs (Pip6A-PMO) efficiently reached the CNS, resulting in a dramatic phenotypic improvement in a spinal muscular atrophy mouse model (Hammond et al., 2016). Other peptides, such as angubindin-1, derived from a toxin of Clostridium perfringens, enabled the delivery of ASOs across the blood–brain barrier into the mouse CNS through intravenous administration by modulating tight junction permeability (Zeniya et al., 2018). Interestingly, nano-sized extracellular vesicles (exosomes) have been emerging as delivery systems that can be loaded with nucleic acids and engineered to target the CNS (reviewed in Rufino-Ramos et al., 2017). Alvarez-Erviti and colleagues reported that exosomes presenting the rabies virus glycoprotein (RVG) at the surface and loaded with siRNAs crossed the blood–brain barrier after intravenous administration, causing reduction of target mRNA in an Alzheimer’s disease mouse model (Alvarez-Erviti et al., 2011). A similar approach was also described for Parkinson’s disease, in which siRNA-RVG-exosomes were delivered intravenously and reached the mouse brain, resulting in widespread reduction of α-synuclein mRNA and protein levels (Cooper et al., 2014). Although not yet described, ASOs could in theory be loaded into RVG exosomes and be systemically delivered in the context of several neurodegenerative disorders that would benefit from ASO-based therapies. Together, these novel combinations could open a promising line of research for ASO systemic delivery into the CNS and treatment of neurological disorders. Direct delivery To circumvent the blood–brain barrier, therapeutic drugs can be directly delivered to the CSF, whose circulation throughout the CNS will ensure delivery to the regions in need of therapy. Although more invasive, this approach allows the administration of lower doses when compared with systemic deliveries, minimizing the risk of toxicity. In fact, the most promising results for ASO-based therapies have been through direct delivery of ASO drugs into the CSF, which include intrathecal or ICV administration. Indeed, ASOs present widespread distribution throughout the brain and spinal cord after ICV or intrathecal injection in rodents models, as well as non-human primates (Smith et al., 2006; Passini et al., 2011; Kordasiewicz et al., 2012; Rigo et al., 2014; Casaca-Carreira et al., 2017; Schoch and Miller, 2017). Nevertheless, studies in non-human primates have shown that after intrathecal administration of ASOs, the cerebellum and deeper structures such as the striatum, do not present significant reduction of target mRNA levels (Kordasiewicz et al., 2012), which may suggest that lower concentrations of ASOs are achieved in those regions. More potent, better brain-penetrating, or higher therapeutic doses of ASOs may be necessary for effective targeting of these regions. In spite of this, intrathecal ASO administration has been successfully implemented in human clinical trials for amyotrophic lateral sclerosis and spinal muscular atrophy, without major side effects (Miller et al., 2013; Chiriboga et al., 2016). Thus, intrathecal delivery may be particularly effective for disorders affecting the spinal cord. Interestingly, data from rodents demonstrated that bolus ICV injection appear to provide better distribution and exposures of ASOs in the CNS than ICV infusions (Southwell et al., 2012). An issue that remains is the need to do a lumbar puncture every 3–4 months to perform the intrathecal readministration, with the associated discomfort and risks. Alternative approaches, such as the injection into an Ommaya reservoir cannulating the lateral ventricle may be envisioned in the future. In summary, multiple delivery approaches are currently under investigation in preclinical models, with the hope to improve distribution and exposure to relevant CNS structures. Less invasive and safer methods, such as systemic delivery of ASOs with the help of carriers, may be a successful approach in the future. However, as there is an urgent need for therapies to treat neurodegenerative disorders, it is likely that direct delivery of ASOs, particularly intrathecal administration, will be the primary method of choice for the immediate years. Antisense oligonucleotide therapeutics in polyglutamine disorders PolyQ disorders are a group of rare nine neurodegenerative diseases that include Huntington’s disease, spinocerebellar ataxias (SCA 1, 2, 3, 6, 7 and 17), spinal and bulbar muscular atrophy, and dentatorubral-pallidoluysian atrophy (reviewed in Lieberman et al., 2019). This family of disorders is characterized by an expansion of CAG repeats in the coding sequence of the causative gene, which encodes for an expanded polyQ tract. Other shared features include the intergenerational instability of the trinucleotide repeats and the correlation of number of unstable CAG repeats with age of onset and severity of symptoms. Furthermore, expanded polyQ tracts are thought to lead to cellular toxicity and tend to form insoluble protein aggregates that constitute a neuropathological hallmark for polyQ diseases (Orr, 2012; Saudou and Humbert, 2016). Whether these protein aggregates are neurotoxic or a protective mechanism is still a matter of discussion (Orr, 2012), but evidence shows that fragments and soluble oligomers correlate with increased cytotoxicity (Sánchez et al., 2003; Takahashi et al., 2008; Leitman et al., 2013). Despite the shared features, distinct cell populations and brain regions are differently affected in polyQ diseases, underlying the variability in clinical manifestations (reviewed in Lieberman et al., 2019). Although the genetic origin of polyQ disorders has been clearly identified for several decades, the neuropathological mechanisms underlying neurodegeneration in selective brain regions are still not fully understood. Moreover, no treatment for these progressive and fatal disorders is currently available. However, research efforts are ongoing to uncover the molecular and cellular pathways mediating disease. Today it is known from different studies that mutant polyQ proteins are implicated in the disruption of a wide range of cellular processes, including autophagy, proteasomal protein degradation, transcription, mitochondrial function, neuroinflammation, axonal transport, glutamate transport, and synaptic transmission (reviewed in Spada and Taylor, 2010). Based on the current knowledge of disease mechanisms, different studies have been developed in recent years in the hope of finding a therapy. These include use of pharmacological compounds, stem cell-based therapies and gene therapies (reviewed in Paulson et al., 2017; Buijsen et al., 2019). Given that polyQ disorders all stem from a single genetic mutation, a strong rationale can be made for the prevention of mutant protein synthesis, this way interrupting the pathogenic cascade in a more upstream stage. Indeed, preclinical studies using several gene therapy approaches have provided encouraging results over recent years, including ASO drugs (reviewed in Matos et al., 2018; Buijsen et al., 2019). This section will focus on preclinical studies that have tested promising strategies of ASO therapeutics in some polyQ disorders and resulted in therapeutic benefit in disease models (Tables 2 and 3). Additionally, ongoing efforts for clinical trials using ASOs will be discussed in Huntington’s disease, which is as yet the only polyQ disorder for which ASOs are being investigated in the clinic (Table 4). Table 2 Description of preclinical studies using non-allele specific antisense oligonucleotides for polyQ disorders Disease . ASO chemistry . Mechanism . Model . Administration . Molecular effect . Phenotypic effect . Reference . (gene) . HD (HTT) PS-2′-MOE gapmer RNase H-mediated degradation BACHD, YAC128 and R6/2 mice ICV Sustained reduction of HTT levels Reversal of motor deficits and anxiety Kordasiewicz et al., 2012 infusion Rhesus monkey IT Prevention of brain loss and increase survival infusion PS-2′-MOE gapmer RNase H-mediated degradation YAC128 mice ICV infusion Reduction of HTT Amelioration of motor and psychiatric deficits Stanek et al., 2013 Improvement of transcriptional patterns PS-2′-OMe Exon skipping HD patient-derived fibroblasts Transfection HTT mRNA lacking exon 12 NA Evers et al., 2014 C57BL/6J mice Intrastriatal injection HTT resistant to caspase-6 cleavage PS-2′-OMe Exon skipping YAC128 mice ICV infusion HTT mRNA lacking exon 12 NA Casaca-Carreira et al., 2016 SCA1 (ATXN1) 2′-MOE gapmer RNase H-mediated degradation SCA166Q/2Q and SCA1154Q/2Q ICV bolus injection Reduction of ATXN1 mRNA and protein levels Improvement of motor deficits and survival Friedrich et al., 2018 SCA2 (ATXN2) 2′-MOE gapmer RNase H-mediated degradation SCA2 patient-derived fibroblasts Transfection Reduction of ATXN2 mRNA and protein levels Improvement of motor phenotype Scoles et al., 2017 ATXN2-Q127 and BAC-Q72 mice ICV bolus injection Restoration of Purkinje cell firing rate SCA3 (ATXN3) PS-2′-OMe Exon skipping SCA3 patient-derived fibroblasts Transfection ATXN3 lacking exon 9 and 10 NA Evers et al., 2013 C57BL/6J mice ICV bolus injection Retention of ubiquitin binding ability in modified ATXN3 PS-2′-OMe Exon skipping SCA3 patient-derived fibroblasts Transfection ATXN3 lacking exon 8 and 9 NA Toonen et al., 2016 PS-2′-MOE gapmer RNase H-mediated degradation SCA3 patient-derived fibroblasts and HEPG2 cell line Transfection Efficient silencing of ATXN3 in cell cultures and MJD84.2 mice but not in MJDQ135 mice NA Moore et al., 2017 Hemizygous MJD84.2 and MJDQ135 mice ICV bolus injection Reduction of high molecular weight ATXN3 aggregates; Decreased nuclear localization of ATXN3 PS-2′-MOE gapmer RNase H-mediated degradation MJD84.2/84.2 homozygous mice ICV bolus injection Efficient silencing of ATXN3 Rescue of motor deficits; McLoughlin et al., 2018 Reduction of high molecular weight aggregates Prevention of ATXN3 nuclear localization Restoration of Purkinje cell firing rate Partial rescue of transcriptional changes SCA7 (ATXN7) PS-cEt gapmer RNase H-mediated degradation Knockin SCA7 mice Intravitreal injection Efficient silencing of ATXN7 Amelioration of retinal degeneration and visual function Niu et al., 2018 Reduction of protein aggregation SBMA (AR) cEt gapmer RNase H-mediated degradation AR113Q and BAC fxAR121 mice Subcutaneous Reduction of AR in skeletal muscle Amelioration of disease phenotype Lieberman et al., 2014 Improvement in survival PS-cEt/2′-MOE gapmer RNase H-mediated degradation AR97Q mice ICV bolus injection Reduction of AR in spinal cord Amelioration of motor dysfunction and neuropathological features Sahashi et al., 2015 Reduction of AR nuclear accumulation in motor neurons Disease . ASO chemistry . Mechanism . Model . Administration . Molecular effect . Phenotypic effect . Reference . (gene) . HD (HTT) PS-2′-MOE gapmer RNase H-mediated degradation BACHD, YAC128 and R6/2 mice ICV Sustained reduction of HTT levels Reversal of motor deficits and anxiety Kordasiewicz et al., 2012 infusion Rhesus monkey IT Prevention of brain loss and increase survival infusion PS-2′-MOE gapmer RNase H-mediated degradation YAC128 mice ICV infusion Reduction of HTT Amelioration of motor and psychiatric deficits Stanek et al., 2013 Improvement of transcriptional patterns PS-2′-OMe Exon skipping HD patient-derived fibroblasts Transfection HTT mRNA lacking exon 12 NA Evers et al., 2014 C57BL/6J mice Intrastriatal injection HTT resistant to caspase-6 cleavage PS-2′-OMe Exon skipping YAC128 mice ICV infusion HTT mRNA lacking exon 12 NA Casaca-Carreira et al., 2016 SCA1 (ATXN1) 2′-MOE gapmer RNase H-mediated degradation SCA166Q/2Q and SCA1154Q/2Q ICV bolus injection Reduction of ATXN1 mRNA and protein levels Improvement of motor deficits and survival Friedrich et al., 2018 SCA2 (ATXN2) 2′-MOE gapmer RNase H-mediated degradation SCA2 patient-derived fibroblasts Transfection Reduction of ATXN2 mRNA and protein levels Improvement of motor phenotype Scoles et al., 2017 ATXN2-Q127 and BAC-Q72 mice ICV bolus injection Restoration of Purkinje cell firing rate SCA3 (ATXN3) PS-2′-OMe Exon skipping SCA3 patient-derived fibroblasts Transfection ATXN3 lacking exon 9 and 10 NA Evers et al., 2013 C57BL/6J mice ICV bolus injection Retention of ubiquitin binding ability in modified ATXN3 PS-2′-OMe Exon skipping SCA3 patient-derived fibroblasts Transfection ATXN3 lacking exon 8 and 9 NA Toonen et al., 2016 PS-2′-MOE gapmer RNase H-mediated degradation SCA3 patient-derived fibroblasts and HEPG2 cell line Transfection Efficient silencing of ATXN3 in cell cultures and MJD84.2 mice but not in MJDQ135 mice NA Moore et al., 2017 Hemizygous MJD84.2 and MJDQ135 mice ICV bolus injection Reduction of high molecular weight ATXN3 aggregates; Decreased nuclear localization of ATXN3 PS-2′-MOE gapmer RNase H-mediated degradation MJD84.2/84.2 homozygous mice ICV bolus injection Efficient silencing of ATXN3 Rescue of motor deficits; McLoughlin et al., 2018 Reduction of high molecular weight aggregates Prevention of ATXN3 nuclear localization Restoration of Purkinje cell firing rate Partial rescue of transcriptional changes SCA7 (ATXN7) PS-cEt gapmer RNase H-mediated degradation Knockin SCA7 mice Intravitreal injection Efficient silencing of ATXN7 Amelioration of retinal degeneration and visual function Niu et al., 2018 Reduction of protein aggregation SBMA (AR) cEt gapmer RNase H-mediated degradation AR113Q and BAC fxAR121 mice Subcutaneous Reduction of AR in skeletal muscle Amelioration of disease phenotype Lieberman et al., 2014 Improvement in survival PS-cEt/2′-MOE gapmer RNase H-mediated degradation AR97Q mice ICV bolus injection Reduction of AR in spinal cord Amelioration of motor dysfunction and neuropathological features Sahashi et al., 2015 Reduction of AR nuclear accumulation in motor neurons AR = androgen receptor; ATXN = ataxin; cEt = S-constrained-ethyl; HTT = huntingtin; HD = Huntington’s disease; IP = intraparenchymal; IT = intrathecal; NA = not assessed; NHP = non-human primates; SBMA = spinal and bulbar muscular atrophy. Open in new tab Table 2 Description of preclinical studies using non-allele specific antisense oligonucleotides for polyQ disorders Disease . ASO chemistry . Mechanism . Model . Administration . Molecular effect . Phenotypic effect . Reference . (gene) . HD (HTT) PS-2′-MOE gapmer RNase H-mediated degradation BACHD, YAC128 and R6/2 mice ICV Sustained reduction of HTT levels Reversal of motor deficits and anxiety Kordasiewicz et al., 2012 infusion Rhesus monkey IT Prevention of brain loss and increase survival infusion PS-2′-MOE gapmer RNase H-mediated degradation YAC128 mice ICV infusion Reduction of HTT Amelioration of motor and psychiatric deficits Stanek et al., 2013 Improvement of transcriptional patterns PS-2′-OMe Exon skipping HD patient-derived fibroblasts Transfection HTT mRNA lacking exon 12 NA Evers et al., 2014 C57BL/6J mice Intrastriatal injection HTT resistant to caspase-6 cleavage PS-2′-OMe Exon skipping YAC128 mice ICV infusion HTT mRNA lacking exon 12 NA Casaca-Carreira et al., 2016 SCA1 (ATXN1) 2′-MOE gapmer RNase H-mediated degradation SCA166Q/2Q and SCA1154Q/2Q ICV bolus injection Reduction of ATXN1 mRNA and protein levels Improvement of motor deficits and survival Friedrich et al., 2018 SCA2 (ATXN2) 2′-MOE gapmer RNase H-mediated degradation SCA2 patient-derived fibroblasts Transfection Reduction of ATXN2 mRNA and protein levels Improvement of motor phenotype Scoles et al., 2017 ATXN2-Q127 and BAC-Q72 mice ICV bolus injection Restoration of Purkinje cell firing rate SCA3 (ATXN3) PS-2′-OMe Exon skipping SCA3 patient-derived fibroblasts Transfection ATXN3 lacking exon 9 and 10 NA Evers et al., 2013 C57BL/6J mice ICV bolus injection Retention of ubiquitin binding ability in modified ATXN3 PS-2′-OMe Exon skipping SCA3 patient-derived fibroblasts Transfection ATXN3 lacking exon 8 and 9 NA Toonen et al., 2016 PS-2′-MOE gapmer RNase H-mediated degradation SCA3 patient-derived fibroblasts and HEPG2 cell line Transfection Efficient silencing of ATXN3 in cell cultures and MJD84.2 mice but not in MJDQ135 mice NA Moore et al., 2017 Hemizygous MJD84.2 and MJDQ135 mice ICV bolus injection Reduction of high molecular weight ATXN3 aggregates; Decreased nuclear localization of ATXN3 PS-2′-MOE gapmer RNase H-mediated degradation MJD84.2/84.2 homozygous mice ICV bolus injection Efficient silencing of ATXN3 Rescue of motor deficits; McLoughlin et al., 2018 Reduction of high molecular weight aggregates Prevention of ATXN3 nuclear localization Restoration of Purkinje cell firing rate Partial rescue of transcriptional changes SCA7 (ATXN7) PS-cEt gapmer RNase H-mediated degradation Knockin SCA7 mice Intravitreal injection Efficient silencing of ATXN7 Amelioration of retinal degeneration and visual function Niu et al., 2018 Reduction of protein aggregation SBMA (AR) cEt gapmer RNase H-mediated degradation AR113Q and BAC fxAR121 mice Subcutaneous Reduction of AR in skeletal muscle Amelioration of disease phenotype Lieberman et al., 2014 Improvement in survival PS-cEt/2′-MOE gapmer RNase H-mediated degradation AR97Q mice ICV bolus injection Reduction of AR in spinal cord Amelioration of motor dysfunction and neuropathological features Sahashi et al., 2015 Reduction of AR nuclear accumulation in motor neurons Disease . ASO chemistry . Mechanism . Model . Administration . Molecular effect . Phenotypic effect . Reference . (gene) . HD (HTT) PS-2′-MOE gapmer RNase H-mediated degradation BACHD, YAC128 and R6/2 mice ICV Sustained reduction of HTT levels Reversal of motor deficits and anxiety Kordasiewicz et al., 2012 infusion Rhesus monkey IT Prevention of brain loss and increase survival infusion PS-2′-MOE gapmer RNase H-mediated degradation YAC128 mice ICV infusion Reduction of HTT Amelioration of motor and psychiatric deficits Stanek et al., 2013 Improvement of transcriptional patterns PS-2′-OMe Exon skipping HD patient-derived fibroblasts Transfection HTT mRNA lacking exon 12 NA Evers et al., 2014 C57BL/6J mice Intrastriatal injection HTT resistant to caspase-6 cleavage PS-2′-OMe Exon skipping YAC128 mice ICV infusion HTT mRNA lacking exon 12 NA Casaca-Carreira et al., 2016 SCA1 (ATXN1) 2′-MOE gapmer RNase H-mediated degradation SCA166Q/2Q and SCA1154Q/2Q ICV bolus injection Reduction of ATXN1 mRNA and protein levels Improvement of motor deficits and survival Friedrich et al., 2018 SCA2 (ATXN2) 2′-MOE gapmer RNase H-mediated degradation SCA2 patient-derived fibroblasts Transfection Reduction of ATXN2 mRNA and protein levels Improvement of motor phenotype Scoles et al., 2017 ATXN2-Q127 and BAC-Q72 mice ICV bolus injection Restoration of Purkinje cell firing rate SCA3 (ATXN3) PS-2′-OMe Exon skipping SCA3 patient-derived fibroblasts Transfection ATXN3 lacking exon 9 and 10 NA Evers et al., 2013 C57BL/6J mice ICV bolus injection Retention of ubiquitin binding ability in modified ATXN3 PS-2′-OMe Exon skipping SCA3 patient-derived fibroblasts Transfection ATXN3 lacking exon 8 and 9 NA Toonen et al., 2016 PS-2′-MOE gapmer RNase H-mediated degradation SCA3 patient-derived fibroblasts and HEPG2 cell line Transfection Efficient silencing of ATXN3 in cell cultures and MJD84.2 mice but not in MJDQ135 mice NA Moore et al., 2017 Hemizygous MJD84.2 and MJDQ135 mice ICV bolus injection Reduction of high molecular weight ATXN3 aggregates; Decreased nuclear localization of ATXN3 PS-2′-MOE gapmer RNase H-mediated degradation MJD84.2/84.2 homozygous mice ICV bolus injection Efficient silencing of ATXN3 Rescue of motor deficits; McLoughlin et al., 2018 Reduction of high molecular weight aggregates Prevention of ATXN3 nuclear localization Restoration of Purkinje cell firing rate Partial rescue of transcriptional changes SCA7 (ATXN7) PS-cEt gapmer RNase H-mediated degradation Knockin SCA7 mice Intravitreal injection Efficient silencing of ATXN7 Amelioration of retinal degeneration and visual function Niu et al., 2018 Reduction of protein aggregation SBMA (AR) cEt gapmer RNase H-mediated degradation AR113Q and BAC fxAR121 mice Subcutaneous Reduction of AR in skeletal muscle Amelioration of disease phenotype Lieberman et al., 2014 Improvement in survival PS-cEt/2′-MOE gapmer RNase H-mediated degradation AR97Q mice ICV bolus injection Reduction of AR in spinal cord Amelioration of motor dysfunction and neuropathological features Sahashi et al., 2015 Reduction of AR nuclear accumulation in motor neurons AR = androgen receptor; ATXN = ataxin; cEt = S-constrained-ethyl; HTT = huntingtin; HD = Huntington’s disease; IP = intraparenchymal; IT = intrathecal; NA = not assessed; NHP = non-human primates; SBMA = spinal and bulbar muscular atrophy. Open in new tab Table 3 Description of preclinical studies using allele specific antisense oligonucleotides for polyQ disorders Disease (gene) . Allele specificity . ASO chemistry . Mechanism . Model . Administration . Molecular effect . Phenotypic effect . Reference . HD (HTT) SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation HD patient derived fibroblasts; HD mice primary neurons Electroporation in fibroblasts; Free uptake in primary neurons Potent and selective reduction of mtHTT NA Carroll et al., 2011 BACHD mice IP bolus injection SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Hu97/18 mice ICV bolus injection Potent and highly selective reduction of mtHTT NA Østergaard et al., 2013 CAG repeats PMO Translation inhibition HD patient-derived fibroblasts; STHdhQ111/111 cells Transfection Selective decrease of mtHTT Amelioration of depressed phenotype Sun et al., 2014 N171-82Q and HdhQ7/Q150 mice ICV bolus injection Reduction of mtHTT neurotoxicity SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Primary neurons from Hu97/18 mice Free uptake Potent and highly selective reduction of mtHTT NA Skotte et al., 2014 SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Hu97/18 mice ICV bolus injection Potent and selective reduction of mtHTT NA Southwell et al., 2014 CAG repeats LNA Blockage of expanded CAG repeats in mRNA HD patient-derived fibroblasts Transfection Preferential binding to mtHTT mRNA Attenuation of motor deficits Rué et al., 2016 No change in the expression of HTT mRNA and protein R6/2 mice IP bolus injection Recovery of striatal neuronal markers CAG repeats PS-2′-OMe Translation inhibition R6/2 and Q175 mice ICV infusion Sustained reduction of HTT levels Improvement in motor phenotype Datson et al., 2017 Reduction of HTT protein aggregates Improvement of metabolite profile Increase in brain volume Increase in striatal neuronal markers SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Hu97/18 mice ICV bolus injection Sustained and potent reduction of mtHTT levels in mice Rescue of cognitive and psychiatric deficits; Southwell et al., 2018 Cynomolgus monkeys IT bolus injection Prevention of DARPP-32 loss; Decrease in HTT mRNA levels in cortical and limbic systems in NHP Prevention of brain atrophy SCA1 (ATXN1) CAG repeats PS-2′-OMe Translation inhibition SCA1154Q/2Q ICV infusion Suppression of mutant ATXN1 protein levels NA Kourkouta et al., 2019 SCA3 (ATXN3) SNP PS-2′-MOE Exon skipping SCA3 patient-derived fibroblasts Transfection Both alleles ATXN3 lacking exon 10 NA Toonen et al., 2017 Reduction of insoluble ATXN3 MJD84.2 hemizygous mice ICV bolus injection Prevention of ATXN3 nuclear localization in the substantia nigra CAG repeats PS-2′-OMe Translation inhibition and exon skipping SCA3 patient-derived fibroblasts Transfection Suppression of mutant and wild-type ATXN3 protein levels NA Kourkouta et al., 2019 MJD84.2 hemizygous mice ICV infusion Formation of ATXN3 protein isoform lacking polyQ tract Disease (gene) . Allele specificity . ASO chemistry . Mechanism . Model . Administration . Molecular effect . Phenotypic effect . Reference . HD (HTT) SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation HD patient derived fibroblasts; HD mice primary neurons Electroporation in fibroblasts; Free uptake in primary neurons Potent and selective reduction of mtHTT NA Carroll et al., 2011 BACHD mice IP bolus injection SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Hu97/18 mice ICV bolus injection Potent and highly selective reduction of mtHTT NA Østergaard et al., 2013 CAG repeats PMO Translation inhibition HD patient-derived fibroblasts; STHdhQ111/111 cells Transfection Selective decrease of mtHTT Amelioration of depressed phenotype Sun et al., 2014 N171-82Q and HdhQ7/Q150 mice ICV bolus injection Reduction of mtHTT neurotoxicity SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Primary neurons from Hu97/18 mice Free uptake Potent and highly selective reduction of mtHTT NA Skotte et al., 2014 SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Hu97/18 mice ICV bolus injection Potent and selective reduction of mtHTT NA Southwell et al., 2014 CAG repeats LNA Blockage of expanded CAG repeats in mRNA HD patient-derived fibroblasts Transfection Preferential binding to mtHTT mRNA Attenuation of motor deficits Rué et al., 2016 No change in the expression of HTT mRNA and protein R6/2 mice IP bolus injection Recovery of striatal neuronal markers CAG repeats PS-2′-OMe Translation inhibition R6/2 and Q175 mice ICV infusion Sustained reduction of HTT levels Improvement in motor phenotype Datson et al., 2017 Reduction of HTT protein aggregates Improvement of metabolite profile Increase in brain volume Increase in striatal neuronal markers SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Hu97/18 mice ICV bolus injection Sustained and potent reduction of mtHTT levels in mice Rescue of cognitive and psychiatric deficits; Southwell et al., 2018 Cynomolgus monkeys IT bolus injection Prevention of DARPP-32 loss; Decrease in HTT mRNA levels in cortical and limbic systems in NHP Prevention of brain atrophy SCA1 (ATXN1) CAG repeats PS-2′-OMe Translation inhibition SCA1154Q/2Q ICV infusion Suppression of mutant ATXN1 protein levels NA Kourkouta et al., 2019 SCA3 (ATXN3) SNP PS-2′-MOE Exon skipping SCA3 patient-derived fibroblasts Transfection Both alleles ATXN3 lacking exon 10 NA Toonen et al., 2017 Reduction of insoluble ATXN3 MJD84.2 hemizygous mice ICV bolus injection Prevention of ATXN3 nuclear localization in the substantia nigra CAG repeats PS-2′-OMe Translation inhibition and exon skipping SCA3 patient-derived fibroblasts Transfection Suppression of mutant and wild-type ATXN3 protein levels NA Kourkouta et al., 2019 MJD84.2 hemizygous mice ICV infusion Formation of ATXN3 protein isoform lacking polyQ tract ATXN1 = ataxin-1; ATXN3 = ataxin-3; cEt = S-constrained-ethyl; HTT = huntingtin; HD = Huntington’s disease; IP = intraparenchymal; IT = intrathecal; mtHTT = mutant huntingtin; NA = not assessed; NHP = non-human primates. Open in new tab Table 3 Description of preclinical studies using allele specific antisense oligonucleotides for polyQ disorders Disease (gene) . Allele specificity . ASO chemistry . Mechanism . Model . Administration . Molecular effect . Phenotypic effect . Reference . HD (HTT) SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation HD patient derived fibroblasts; HD mice primary neurons Electroporation in fibroblasts; Free uptake in primary neurons Potent and selective reduction of mtHTT NA Carroll et al., 2011 BACHD mice IP bolus injection SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Hu97/18 mice ICV bolus injection Potent and highly selective reduction of mtHTT NA Østergaard et al., 2013 CAG repeats PMO Translation inhibition HD patient-derived fibroblasts; STHdhQ111/111 cells Transfection Selective decrease of mtHTT Amelioration of depressed phenotype Sun et al., 2014 N171-82Q and HdhQ7/Q150 mice ICV bolus injection Reduction of mtHTT neurotoxicity SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Primary neurons from Hu97/18 mice Free uptake Potent and highly selective reduction of mtHTT NA Skotte et al., 2014 SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Hu97/18 mice ICV bolus injection Potent and selective reduction of mtHTT NA Southwell et al., 2014 CAG repeats LNA Blockage of expanded CAG repeats in mRNA HD patient-derived fibroblasts Transfection Preferential binding to mtHTT mRNA Attenuation of motor deficits Rué et al., 2016 No change in the expression of HTT mRNA and protein R6/2 mice IP bolus injection Recovery of striatal neuronal markers CAG repeats PS-2′-OMe Translation inhibition R6/2 and Q175 mice ICV infusion Sustained reduction of HTT levels Improvement in motor phenotype Datson et al., 2017 Reduction of HTT protein aggregates Improvement of metabolite profile Increase in brain volume Increase in striatal neuronal markers SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Hu97/18 mice ICV bolus injection Sustained and potent reduction of mtHTT levels in mice Rescue of cognitive and psychiatric deficits; Southwell et al., 2018 Cynomolgus monkeys IT bolus injection Prevention of DARPP-32 loss; Decrease in HTT mRNA levels in cortical and limbic systems in NHP Prevention of brain atrophy SCA1 (ATXN1) CAG repeats PS-2′-OMe Translation inhibition SCA1154Q/2Q ICV infusion Suppression of mutant ATXN1 protein levels NA Kourkouta et al., 2019 SCA3 (ATXN3) SNP PS-2′-MOE Exon skipping SCA3 patient-derived fibroblasts Transfection Both alleles ATXN3 lacking exon 10 NA Toonen et al., 2017 Reduction of insoluble ATXN3 MJD84.2 hemizygous mice ICV bolus injection Prevention of ATXN3 nuclear localization in the substantia nigra CAG repeats PS-2′-OMe Translation inhibition and exon skipping SCA3 patient-derived fibroblasts Transfection Suppression of mutant and wild-type ATXN3 protein levels NA Kourkouta et al., 2019 MJD84.2 hemizygous mice ICV infusion Formation of ATXN3 protein isoform lacking polyQ tract Disease (gene) . Allele specificity . ASO chemistry . Mechanism . Model . Administration . Molecular effect . Phenotypic effect . Reference . HD (HTT) SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation HD patient derived fibroblasts; HD mice primary neurons Electroporation in fibroblasts; Free uptake in primary neurons Potent and selective reduction of mtHTT NA Carroll et al., 2011 BACHD mice IP bolus injection SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Hu97/18 mice ICV bolus injection Potent and highly selective reduction of mtHTT NA Østergaard et al., 2013 CAG repeats PMO Translation inhibition HD patient-derived fibroblasts; STHdhQ111/111 cells Transfection Selective decrease of mtHTT Amelioration of depressed phenotype Sun et al., 2014 N171-82Q and HdhQ7/Q150 mice ICV bolus injection Reduction of mtHTT neurotoxicity SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Primary neurons from Hu97/18 mice Free uptake Potent and highly selective reduction of mtHTT NA Skotte et al., 2014 SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Hu97/18 mice ICV bolus injection Potent and selective reduction of mtHTT NA Southwell et al., 2014 CAG repeats LNA Blockage of expanded CAG repeats in mRNA HD patient-derived fibroblasts Transfection Preferential binding to mtHTT mRNA Attenuation of motor deficits Rué et al., 2016 No change in the expression of HTT mRNA and protein R6/2 mice IP bolus injection Recovery of striatal neuronal markers CAG repeats PS-2′-OMe Translation inhibition R6/2 and Q175 mice ICV infusion Sustained reduction of HTT levels Improvement in motor phenotype Datson et al., 2017 Reduction of HTT protein aggregates Improvement of metabolite profile Increase in brain volume Increase in striatal neuronal markers SNP PS-2′-MOE gapmer and PS-cEt gapmer RNase H-mediated degradation Hu97/18 mice ICV bolus injection Sustained and potent reduction of mtHTT levels in mice Rescue of cognitive and psychiatric deficits; Southwell et al., 2018 Cynomolgus monkeys IT bolus injection Prevention of DARPP-32 loss; Decrease in HTT mRNA levels in cortical and limbic systems in NHP Prevention of brain atrophy SCA1 (ATXN1) CAG repeats PS-2′-OMe Translation inhibition SCA1154Q/2Q ICV infusion Suppression of mutant ATXN1 protein levels NA Kourkouta et al., 2019 SCA3 (ATXN3) SNP PS-2′-MOE Exon skipping SCA3 patient-derived fibroblasts Transfection Both alleles ATXN3 lacking exon 10 NA Toonen et al., 2017 Reduction of insoluble ATXN3 MJD84.2 hemizygous mice ICV bolus injection Prevention of ATXN3 nuclear localization in the substantia nigra CAG repeats PS-2′-OMe Translation inhibition and exon skipping SCA3 patient-derived fibroblasts Transfection Suppression of mutant and wild-type ATXN3 protein levels NA Kourkouta et al., 2019 MJD84.2 hemizygous mice ICV infusion Formation of ATXN3 protein isoform lacking polyQ tract ATXN1 = ataxin-1; ATXN3 = ataxin-3; cEt = S-constrained-ethyl; HTT = huntingtin; HD = Huntington’s disease; IP = intraparenchymal; IT = intrathecal; mtHTT = mutant huntingtin; NA = not assessed; NHP = non-human primates. Open in new tab Table 4 Clinical trials using antisense oligonucleotides for polyQ disorders Disease . ASO . Target gene . Target SNP . Mechanism . Administration . Trial phase . Study design . Recent status . Sponsor . Identifier . drug . HD ISIS 443139 HTT NT Non-allele specific ASO Intrathecal Phase 1/2 Randomized, double-blind, placebo-controlled study Completed Ionis Pharmaceuticals Inc. NCT02519036 2′-MOE-PS ISIS 443139/ RO7234292 HTT NT Non-allele specific ASO Intrathecal Phase 2 Open-label extension study Active Ionis Pharmaceuticals Inc. NCT03342053 2′-MOE-PS ISIS 443139/ RO7234292 HTT NT Non-allele specific ASO Intrathecal Phase 3 Randomized, double-blind, placebo-controlled study Recruiting Hoffmann-La Roche NCT03761849 2′-MOE-PS WVE-120101 HTT SNP1 Allele-specific ASO Intrathecal Phase 1/2 Randomized, double-blind, placebo-controlled study Recruiting Wave Life Sciences Ltd. NCT03225833 Stereopure rs362307 WVE-120102 HTT SNP2 Allele-specific ASO Intrathecal Phase 1/2 Randomized, double-blind, placebo-controlled study Recruiting Wave Life Sciences Ltd. NCT03225846 rs362331 Stereopure Disease . ASO . Target gene . Target SNP . Mechanism . Administration . Trial phase . Study design . Recent status . Sponsor . Identifier . drug . HD ISIS 443139 HTT NT Non-allele specific ASO Intrathecal Phase 1/2 Randomized, double-blind, placebo-controlled study Completed Ionis Pharmaceuticals Inc. NCT02519036 2′-MOE-PS ISIS 443139/ RO7234292 HTT NT Non-allele specific ASO Intrathecal Phase 2 Open-label extension study Active Ionis Pharmaceuticals Inc. NCT03342053 2′-MOE-PS ISIS 443139/ RO7234292 HTT NT Non-allele specific ASO Intrathecal Phase 3 Randomized, double-blind, placebo-controlled study Recruiting Hoffmann-La Roche NCT03761849 2′-MOE-PS WVE-120101 HTT SNP1 Allele-specific ASO Intrathecal Phase 1/2 Randomized, double-blind, placebo-controlled study Recruiting Wave Life Sciences Ltd. NCT03225833 Stereopure rs362307 WVE-120102 HTT SNP2 Allele-specific ASO Intrathecal Phase 1/2 Randomized, double-blind, placebo-controlled study Recruiting Wave Life Sciences Ltd. NCT03225846 rs362331 Stereopure HD = Huntington’s disease; HTT = huntingtin; NT = not targeted; PS = phosphorothioate. Open in new tab Table 4 Clinical trials using antisense oligonucleotides for polyQ disorders Disease . ASO . Target gene . Target SNP . Mechanism . Administration . Trial phase . Study design . Recent status . Sponsor . Identifier . drug . HD ISIS 443139 HTT NT Non-allele specific ASO Intrathecal Phase 1/2 Randomized, double-blind, placebo-controlled study Completed Ionis Pharmaceuticals Inc. NCT02519036 2′-MOE-PS ISIS 443139/ RO7234292 HTT NT Non-allele specific ASO Intrathecal Phase 2 Open-label extension study Active Ionis Pharmaceuticals Inc. NCT03342053 2′-MOE-PS ISIS 443139/ RO7234292 HTT NT Non-allele specific ASO Intrathecal Phase 3 Randomized, double-blind, placebo-controlled study Recruiting Hoffmann-La Roche NCT03761849 2′-MOE-PS WVE-120101 HTT SNP1 Allele-specific ASO Intrathecal Phase 1/2 Randomized, double-blind, placebo-controlled study Recruiting Wave Life Sciences Ltd. NCT03225833 Stereopure rs362307 WVE-120102 HTT SNP2 Allele-specific ASO Intrathecal Phase 1/2 Randomized, double-blind, placebo-controlled study Recruiting Wave Life Sciences Ltd. NCT03225846 rs362331 Stereopure Disease . ASO . Target gene . Target SNP . Mechanism . Administration . Trial phase . Study design . Recent status . Sponsor . Identifier . drug . HD ISIS 443139 HTT NT Non-allele specific ASO Intrathecal Phase 1/2 Randomized, double-blind, placebo-controlled study Completed Ionis Pharmaceuticals Inc. NCT02519036 2′-MOE-PS ISIS 443139/ RO7234292 HTT NT Non-allele specific ASO Intrathecal Phase 2 Open-label extension study Active Ionis Pharmaceuticals Inc. NCT03342053 2′-MOE-PS ISIS 443139/ RO7234292 HTT NT Non-allele specific ASO Intrathecal Phase 3 Randomized, double-blind, placebo-controlled study Recruiting Hoffmann-La Roche NCT03761849 2′-MOE-PS WVE-120101 HTT SNP1 Allele-specific ASO Intrathecal Phase 1/2 Randomized, double-blind, placebo-controlled study Recruiting Wave Life Sciences Ltd. NCT03225833 Stereopure rs362307 WVE-120102 HTT SNP2 Allele-specific ASO Intrathecal Phase 1/2 Randomized, double-blind, placebo-controlled study Recruiting Wave Life Sciences Ltd. NCT03225846 rs362331 Stereopure HD = Huntington’s disease; HTT = huntingtin; NT = not targeted; PS = phosphorothioate. Open in new tab Preclinical studies using antisense oligonucleotide therapeutics for polyQ disorders Huntington’s disease The most prevalent polyQ disorder—Huntington’s disease—is an autosomal dominantly-inherited neurodegenerative disorder caused by more than 36 CAG repeats within exon 1 of the huntingtin gene (HTT) (The Huntington’s Disease Collaborative Research Group, 1993). The CAG expansion is translated into a stretched polyQ tract within the HTT protein and results in a toxic gain-of-function. Evidence supports that toxicity of the expanded polyQ tract is a primary disease mechanism in Huntington’s disease (reviewed in Bates et al., 2015). Moreover, it has been shown that mutant HTT mRNA may also present detrimental function (Mykowska et al., 2011; Bañez-Coronel et al., 2012; Krauß et al., 2013), which suggests that both mutant mRNA and protein toxic gain-of-function contribute to disease pathogenesis. Among the polyQ family of disorders, Huntington’s disease has attracted the most attention for ASO therapeutics and several studies using chemically-modified ASOs have already shown promising results in different Huntington’s disease animal models (Tables 2 and 3). As shown in Table 3, most studies using ASOs for Huntington’s disease use an allele-specific strategy and the majority induce RNase H-mediated degradation of HTT mRNA. However, some studies have also reported the use of mechanisms different from RNase H-dependent degradation with positive results. Non-allele specific approaches Kordasiewicz et al., (2012) reported the use of ASOs as a non-allele specific strategy against HTT mRNA in several animal models. This study reported that transient delivery of PS-2′-MOE gapmer ASOs targeting HTT mRNA through infusion into the CSF, resulted in sustained reduction of mRNA and protein levels of HTT in several brain areas affected by the disorder. This strategy resulted in the sustained improvement of disease phenotype for several months observed in BACHD and YAC128 mice, as well as the increase in survival and prevention of brain loss observed in R6/2 Huntington’s disease mouse model (Kordasiewicz et al., 2012). Remarkably, phenotypic amelioration in the BACHD mouse model was observed after restoration of mutant HTT to its initial level, suggesting that the therapeutic effects of ASO treatment persist for longer than target reduction. Moreover, it was shown that intrathecal ASO infusion into the larger brain of rhesus monkeys resulted in sustained reduction of HTT mRNA in most brain and spinal cord regions, including areas affected by Huntington’s disease (Kordasiewicz et al., 2012). Further work demonstrated that phenotypic improvement observed in YAC128 mice after ASO ICV infusion was associated with significant correction of transcriptional profiles (Stanek et al., 2013), which has been shown to be altered in the brains of Huntington’s disease patients, as well as in the YAC128 mouse model (Becanovic et al., 2010; Stanek et al., 2013). This finding supports the use of transcript levels of striatal genes as early biomarkers for evaluating therapeutic interventions (Stanek et al., 2013). Another interesting non-allele-specific approach for Huntington’s disease involves the use of exon skipping ASOs to remove protein regions targeted by proteases (Table 2). It has been reported that HTT aggregates contain truncated protein fragments of varying sizes (Lunkes et al., 2002; Ratovitski et al., 2009; Landles et al., 2010) and evidence suggests that proteolysis of the mutant protein can contribute to Huntington’s disease pathogenesis (reviewed in Bates et al., 2015). Thus, an approach that reduces the formation of cleaved fragments could be beneficial. Indeed, in vivo studies have shown that mutation of specific amino acids in caspase-6 cleavage site of mutant HTT resulted in improvement of disease phenotype in Huntington’s disease mouse models (Graham et al., 2006; Pouladi et al., 2009). Accordingly, efforts have been made to test this strategy using ASO therapeutics. Evers and colleagues (2014) reported that PS-2′-OMe ASOs transfected into patient-derived fibroblasts can induce partial exon 12 skip in human HTT, which contains the caspase-6 cleavage motifs. The main advantage of such an approach is that HTT mRNA and protein levels are unaltered. Indeed, this exon skipping approach resulted in the formation of a shorter HTT protein lacking the caspase cleavage sites in wild-type mice while maintaining total protein levels. More recently, this exon skipping strategy was tested in the YAC128 Huntington’s disease mouse model (Casaca-Carreira et al., 2016). In this work, it was demonstrated that ICV infusion of 2′-OMe ASOs with phosphorothioate backbone resulted in exon 12 skip in several brain regions affected by Huntington’s disease, including the striatum and cortex (Casaca-Carreira et al., 2016). Together, these proof-of-concept studies show the potential of protein modification with exon-skipping ASOs as a therapeutic intervention for Huntington’s disease. Allele-specific strategies Studies have shown that reduction of both wild-type and mutant HTT allele of up to 75% appears to be well tolerated in Huntington’s disease rodent models and non-human primates (Boudreau et al., 2009; McBride et al., 2011; Kordasiewicz et al., 2012). Nevertheless, suppression of HTT in the adult brain may not be desirable as wild-type HTT has numerous functions in the brain. Wild-type HTT protein has been shown to play important roles in different cellular processes including transcription, apoptosis, calcium homeostasis, axonal transport, endocytosis, and synaptic transmission (reviewed in Zuccato et al., 2010). Ablation of the mouse Huntington’s disease gene homologue (Hdh) was found to be lethal during embryonic development (Zeitlin et al., 1995) and inactivation of this gene at embryonic Day 15.5 or postnatal Day 5 resulted in progressive neurodegeneration (Dragatsis et al., 2000). Although studies have shown that lowering of both HTT alleles can be well tolerated, long-term assessment of HTT suppression is still needed to ensure safety. Particularly as Huntington’s disease therapy in humans would likely be required for decades. Together, this provides a logical rationale for the use of ASOs that specifically target mutant HTT mRNA. In recent years, many allele-specific ASOs have been designed to reduce mutant HTT while maintaining normal levels of wild-type HTT protein (Table 3). These include ASOs targeting the expanded CAG repeats (Sun et al., 2014; Rué et al., 2016; Datson et al., 2017). However, this approach could result in non-selective HTT suppression, as the wild-type allele also contains CAG repeats, albeit in smaller numbers, or non-specific reduction of other genes containing CAG repeats. Alternatively, SNPs that have been linked to the CAG expansion can be targeted (Carroll et al., 2011; Østergaard et al., 2013; Skotte et al., 2014; Southwell et al., 2014, 2018). Sun and colleagues (2014), for instance, reported the use of PMOs that selectively inhibit the translation of mutant HTT mRNA by targeting the CAG repeats of the mutant allele. This study reported improvement of mutant HTT-associated toxicity in neuronal cultures and significant reduction of mutant HTT in Huntington’s disease patient-derived fibroblasts, as well as in the N174-82Q transgenic mouse model 2 weeks post-ICV injection. Moreover, ICV injection of CAG-targeting PMOs resulted in selective reduction of mutant HTT levels and amelioration of depressive-like phenotype in the HdhQ7/Q150 Huntington’s disease mouse model (Sun et al., 2014). Importantly, these authors also observed unwanted off-target effects in other transcripts that contain CAG repeats in Huntington’s disease cell lines, which illustrate the limitations of repeat-targeting ASOs. Nevertheless, other CAG-targeting ASOs, with phosphorothioate backbone and 2′-OMe modifications, led to significant and sustained reduction of mutant HTT throughout the brain of Huntington’s disease mice, while no reduction in protein levels of endogenous mouse genes with CAG repeats was observed (Datson et al., 2017). Importantly, these authors reported that CAG-ASO treated mice showed motor performance improvement. Together these findings show that ASOs targeting CAG repeats may be an attractive approach for allele-specific silencing in polyQ disorders and selectivity may be highly dependent on ASO chemistry. Recently, another study investigated the potential of CAG-targeting ASOs using LNA-ASOs (Rué et al., 2016). In this work, a novel strategy was implemented in which LNA-ASOs bind CAG repeats and prevent its availability, without altering HTT levels. In theory, this allows for blockage of the harmful effects of CAG expansion in HTT mRNA, while maintaining HTT protein levels and function. Remarkably, the authors observed a recovery of neuronal markers, whose expression is decreased in Huntington’s disease striatum, and an improvement in the motor phenotype in R6/2 Huntington’s disease mouse model after intrastriatal injection of LNA-ASOs, for at least 5 weeks post-treatment. Importantly, mutant HTT mRNA and HTT protein levels were not reduced, neither in neuronal cell models nor in vivo after CAG-targeting LNA delivery, suggesting that neuroprotection can be promoted by simply blocking the detrimental effects of expanded HTT mRNA (Rué et al., 2016). Although this strategy has not yet been validated by others, this study highlights the versatility of ASO-based therapies. Other studies have also used an allele-specific approach by targeting SNPs tightly linked to CAG expansion in Huntington’s disease patients (Carroll et al., 2011; Østergaard et al., 2013; Skotte et al., 2014; Southwell et al., 2014, 2018) (Table 3). For instance, Carroll et al. (2011) showed that PS-2′-MOE gapmer ASOs targeting specific SNPs results in potent and selective silencing of targeted alleles in Huntington’s disease fibroblasts and primary neurons from BACHD mice, which express the targeted SNP. Remarkably, SNP-targeting ASOs delivered intrastriatally resulted in potent silencing of HTT in BACHD mice striatum but not in YAC18 mice, which did not express the targeted SNP variant (Carroll et al., 2011), validating the selectivity of this ASO-based therapy in vivo. In addition, this work compared the potency of MOE-modified ASOs with ASOs containing cEt-modified oligonucleotides and showed that the latter presented increased potency, while sparing selectivity, in neuronal cultures (Carroll et al., 2011). Further work from the same group confirmed chimeric cEt-ASOs increased silencing potency (Skotte et al., 2014; Southwell et al., 2014), as well as increased duration of action, showing mutant HTT reduction up to 36 weeks post-injection in the humanized Huntington’s disease mouse model Hu97/18 (Southwell et al., 2014). Given that Huntington’s disease is characterized not only by motor dysfunction but also cognitive decline and psychiatric disturbances (reviewed in Ross and Tabrizi, 2011), an ideal therapy for Huntington’s disease patients would modify all disease-mediated impairments. Most preclinical studies evaluating therapeutic potential have mainly focused on motor phenotype. Recently, Southwell and colleagues (2018) investigated the potential of SNP-targeting ASOs to alter psychiatric and cognitive deficits in Huntington’s disease using a humanized Huntington’s disease mouse model. This work demonstrated that early (before disease phenotype onset) and late (after disease phenotype onset) intervention resulted in potent and selective reduction of mutant HTT for up to 46 weeks. The sustained suppression of mutant HTT rescued learning deficits during both time points of intervention, as well as improved anxiety behaviour of Hu97/18 mice after ASO treatment. Remarkably, intrathecal administration of ASOs targeting non-human primate HTT into cynomolgus monkeys resulted in reduction of HTT mRNA levels in brain areas implicated in cognitive and psychiatric function, including cortical and limbic regions (Southwell et al., 2018). Together, these studies show that allele-specific approaches through SNP-targeting ASOs are very promising in the context of Huntington’s disease. However, this strategy requires the development of multiple drugs targeting a different SNP to use an allele-specific therapy for all Huntington’s disease patients, as a myriad of SNPs have been linked to Huntington’s disease populations. Moreover, a strategy that specifically targets SNPs in order to silence solely the mutant allele cannot be implemented in patients that are homozygous for the targeted SNP variant. Nevertheless, several studies have been developed in Huntington’s disease to identify and evaluate therapeutically relevant SNPs, as well as to optimize design of ASOs to greatly enhance drug properties (Carroll et al., 2011; Østergaard et al., 2013; Skotte et al., 2014; Southwell et al., 2014). Spinocerebellar ataxia type 1 SCA1 is a progressive fatal neurodegenerative disorder characterized by a CAG trinucleotide repeat expansion located in exon 8 of the ataxin-1 gene (ATXN1) (Orr et al., 1993). As in other polyQ disorders, the primary pathogenic mechanism in SCA1 has been shown to be related with toxic gain-of-function of mutant ATXN1 (Zoghbi and Orr, 2009). Theoretically, halting ATXN1 expression would be a beneficial therapeutic approach. Indeed, studies using RNAi strategies demonstrated efficient reduction of ATXN1 levels and dampening of phenotypic alterations (Xia et al., 2004; Keiser et al., 2014). Following this rationale, ICV injection of ATXN1-targeting gapmer ASOs resulted in efficient downregulation of ATXN1 mRNA and protein levels in SCA1 knockin mice (Friedrich et al., 2018) (Table 2). The same study demonstrated that early stage administration of ATXN1 ASOs resulted in improvement of motor deficits and survival of SCA1 mouse model. Moreover, ASO treatment rescued transcriptional deregulation and magnetic resonance spectroscopy-based neurochemical abnormalities in affected brain tissue (Friedrich et al., 2018). This study consisted of a non-allele specific approach that targeted both the wild-type and expanded form of ATXN1. Importantly, mice lacking ATXN1 display behavioural abnormalities related to learning deficits (Matilla et al., 1998), which may present a challenge for the implementation of SCA1 non-allele specific therapies. More recently, CAG repeat-targeting ASOs were tested in SCA1 patient-derived fibroblasts and in the SCA1154Q/2Q mouse model (Kourkouta et al., 2019). The same group previously demonstrated the therapeutic potential of CAG-targeting PS-2′-OMe for Huntington’s disease, which were hypothesized to act through translational interference (Datson et al., 2017). Weekly ICV infusions of CAG-targeting ASOs resulted in widespread reduction of mutant ATXN1 throughout different brain regions affected in SCA1. Though this strategy is in theory allele-specific, specificity was not confirmed because wild-type ATXN1 protein could not be detected by western blot analysis (Kourkouta et al., 2019). Surprisingly, the CAG-targeting ASOs induced exon 8 skipping in SCA1 patient-derived fibroblasts and in the SCA1 mouse model, but only at low levels. Spinocerebellar ataxia type 2 SCA2 is caused by a dominant mutation in the ataxin-2 gene (ATXN2) characterized by an expansion of CAG repeats (Pulst et al., 1996). This mutation confers an abnormally long and toxic polyQ stretch in ATXN2 protein that eventually results in neuronal dysfunction and death (reviewed in Scoles and Pulst, 2018). Given that a single gene is the sole cause of the disorder, ASO therapeutics that reduce ATXN2 transcript levels are a promising strategy for SCA2. Importantly, no SNPs associated with the SCA2 mutation have been identified (Pulst, 2016). Although Atxn2 knockout mice exhibit a specific set of mild behavioural alterations, no morphological abnormalities in the CNS or effects on hippocampal function and survival were reported in these mice (Huynh et al., 2009). For those reasons, it appears that ASOs targeting both the wild-type and mutant ATXN2 mRNA may be a viable therapy for SCA2. However, further preclinical studies are needed to assess long-term safety. Scoles and co-workers (2017) reported that ICV delivery of lead PS-2′-MOE gapmer into two SCA2 mouse models resulted in significant reduction of ATXN2 mRNA, with improvement in rotarod performance in ATXN2-Q127 mice and no toxicity. Other studies have shown that mutant ATXN2 is associated with abnormal firing rate of Purkinje cells that precedes disease phenotype (Hansen et al., 2013) and translational dysregulation (Dansithong et al., 2015). In accordance, ATXN2 suppression restored protein expression and Purkinje cell firing frequency, which may underlie the amelioration of motor behaviour (Scoles et al., 2017). Intermediate-length polyQ expansions in ATXN2 are also implicated in amyotrophic lateral sclerosis pathogenesis due to abnormal interactions between ATXN2 and TDP-43 (Elden et al., 2010). Given that the ATXN2 polyQ length contributes to the risk of amyotrophic lateral sclerosis, the ATXN2 gene is also being targeted as a therapeutic strategy for this disorder. Of note, reduction of ATXN2 after ASO administration extended lifespan and dampened motor deficits of TDP-43 transgenic mice (Becker et al., 2017). Spinocerebellar ataxia type 3 SCA3, also known as Machado-Joseph disease, is the most common dominantly inherited spinocerebellar ataxia and is caused by a toxic gain-of-function mutation leading to a stretched polyQ domain of the ataxin-3 protein (ATXN3) (Takiyama et al., 1993; Kawaguchi et al., 1994). The biological functions of ATXN3 are still largely unknown, however it is thought to be involved in deubiquitination and proteasomal protein degradation. ATXN3 protein contains an N-terminal domain with deubiquitinating catalytic activity, termed Josephin domain, and a C-terminal containing three ubiquitin-interaction motifs (UIMs) (reviewed in Matos et al., 2011). The mutant protein displays toxic properties that result in cellular dysfunction and eventually lead to neurodegeneration in several brain regions (reviewed in Paulson, 2012). Various studies have evaluated different types of ASO-based therapies for the treatment of SCA3 (Tables 2 and 3). Among these, most efforts have been based on splicing modulation of the ATXN3 mRNA. Non-allele specific suppression of ATXN3 appears to be well tolerated in rodents (Alves et al., 2010). However, given the deubiquitinating function and other roles of ATXN3, long-term silencing of the ATXN3 gene may not be the most favourable approach. With this rationale, different strategies have been developed to induce skipping of disease-relevant exons (Evers et al., 2013; Toonen et al., 2016, 2017). For instance, Evers and colleagues (2013) evaluated the potential of fully modified PS-2′-OMe ASOs to induce skipping of the exon containing toxic polyQ repeats, i.e. exon 10. In this study, treatment of SCA3 patient-derived fibroblasts with splice modifying ASOs resulted in the formation of an ATXN3 protein lacking exon 9 and 10, while maintaining normal levels of ATXN3 protein. Importantly, the protein lacking exons 9 and 10 retained ubiquitin binding ability (Evers et al., 2013). In more recent work from the same group, a similar exon skipping strategy, but using fully modified 2′-MOE ASOs with phosphorothioate backbone, resulted in ATXN3 lacking exon 10 in fibroblasts derived from SCA3 patients, as well as in the hemizygous MJD84.2 mouse model (Toonen et al., 2017). Surprisingly, although one of the tested ASOs targeted an SNP associated with the mutant allele, no allelic selectivity was observed. Nevertheless, repeated ICV injections in MJD84.2 mice were tolerated and resulted in effective protein modification throughout the brain for up to 2.5 months and reduction of nuclear ATXN3 aggregates in the substantia nigra (Toonen et al., 2017). Importantly, functional testing of the modified protein demonstrated that truncated ATXN3 retained ubiquitin binding and cleavage abilities (Toonen et al., 2017). The main advantage of this exon skipping approach is the removal of the toxic polyQ stretch from ATXN3, while preventing potential deleterious effects of loss of ATXN3 function. Nonetheless, future studies are needed to assess whether this strategy could be sufficiently efficient to ameliorate behavioural deficits in SCA3 animal models. Other studies have also attempted allele-specific approaches for SCA3 by targeting the causative mutation, i.e. the expanded CAG repeats. Building on the work carried out in Huntington’s disease (Datson et al., 2017), Kourkouta and colleagues (2019) used PS-2′-OMe ASOs to specifically target the expanded CAG tract in SCA3 patient-derived fibroblasts and the MJD84.2 mouse model. Six weekly ICV infusions of CAG-targeting ASOs resulted in a significant reduction of ATXN3 protein levels in the cerebellum, brainstem and hippocampus of MJD84.2 mice. Interestingly, the CAG-targeting ASOs also induced skipping of the mutation-containing exon 10 in SCA3 patient-derived fibroblasts and to a lesser extent in the MJD84.2 mouse brain (Kourkouta et al., 2019). Skipping of ATXN3 exon 10 resulted in a truncated isoform of ATXN3 lacking the polyQ tract and the C-terminus, similarly to the results obtained by Toonen et al. (2017). Nevertheless, it is important to note that silencing of both mutant and wild-type ATXN3 allele was observed in SCA3 patient-derived fibroblasts after treatment with the CAG-targeting ASOs (Kourkouta et al., 2019). Cleavage of mutant ATXN3 has been correlated with neurotoxicity, a mechanism known as the toxic fragment hypothesis. Several studies have focused on the proteolytic cleavage of ATXN3 and its potential as a therapeutic target. For instance, inhibition of calpain-mediated cleavage has been shown to reduce ATXN3 aggregation and neurodegeneration in the mouse brain (Simões et al., 2014). More recently, a study by Toonen et al. (2016) evaluated the ability of ASOs to reduce proteolytic cleavage of ATXN3. For that, removal of exons 8 and 9, which contain predicted cleavage sites, was induced by using ASOs targeting splicing sites in SCA3 patient-derived fibroblasts. As expected, the modified ATXN3 protein was not cleaved when incubated with caspases. However, ATNX3 lacking exon 8 and 9 was shown to be unable to bind to the poly-ubiquitin chains, which may affect deubiquitinating function, suggesting that this exon skipping therapeutic strategy may not be a viable option for SCA3 (Toonen et al., 2016). A non-allele specific approach has also been used to induce RNase H-mediated degradation of ATXN3 in vivo (Moore et al., 2017). ICV bolus injection of ASO drug with gapmer design led to widespread ASO delivery to the CNS and effective ATXN3 reduction in multiple brain regions, as well as marked reduction of oligomeric high molecular weight ATXN3, in MJD84.2 mice, but not in MJDQ135 mice (Moore et al., 2017). Lack of efficacy of the tested ASO drugs in the MJDQ135 mouse model may result from reduced targeting capacity for the cDNA ATXN3 transgene of the MJDQ135 mouse model compared with the full-length transgene of the MJD84.2 model (Moore et al., 2017). Further work has been conducted to evaluate the therapeutic benefit of ASO-mediated ATXN3 suppression in a more severe SCA3 mouse model, the homozygous MJD84.2/84.2 mouse model (McLoughlin et al., 2018). McLoughlin and colleagues demonstrated that a single ICV bolus injection of ASOs targeting ATXN3 transcript resulted in robust and sustained reduction of ATXN3 mRNA for up to 22 weeks. Furthermore, protein levels and high molecular weight ATXN3 aggregates were significantly reduced for several weeks post-injection (McLoughlin et al., 2018). ATXN3 silencing was followed by great improvement in neuropathological features in this homozygous MJD84.2 mouse model (McLoughlin et al., 2018). Importantly, an additional ASO injection at 21 weeks of age, allowed, not only the rescue of motor coordination and balance, but also the restoration of Purkinje cells’ firing rate (McLoughlin et al., 2018), which has been shown to be altered in the MJD84.2 mouse model (Shakkottai et al., 2011). Although this is a promising therapeutic approach, it is still unknown whether long-term silencing of both mutant and non-mutant ATXN3 alleles in the human brain will be well tolerated. Spinocerebellar ataxia type 7 SCA7 is an autosomal dominant neurodegenerative disease caused by an expansion of unstable CAG repeats in the ataxin-7 gene (ATXN7), which encodes an expanded polyQ tract (Trottier et al., 1995; David et al., 1998). Similar to other SCA disorders, SCA7 is characterized by cerebellar degeneration. However, this particular SCA can be distinguished clinically by the occurrence of retinal degeneration, resulting in visual impairment (reviewed in Karam and Trottier, 2018). Recently, an ASO-based therapy has shown promise as an effective treatment for SCA7 unusual form of retinal degeneration, known as cone-rod dystrophy. Selection of a lead sequence of ASOs targeting ATXN7 was followed by intravitreal injection in SCA7 knockin mouse model, with effective ATXN7 silencing of both wild-type and mutant alleles, as well as reduction of aggregated forms (Niu et al., 2018). Importantly, improvement in many phenotypical features, such as visual function and retinal degeneration, were observed. These were observed even when administration of ATXN7 ASOs was applied after retinal symptoms developed in the SCA7 mouse model (Niu et al., 2018). This proof-of-concept study illustrates the therapeutic potential of ASOs to treat ocular degeneration in SCA7 through intravitreal injection. This administration route is a much safer approach compared with ICV and/or intrathecal injections, and therefore a much easier method to use in the clinic. Treatment of the cerebellar syndrome and other brain regions affected in SCA7 would require the implementation of ASO interventions similar to the ones used in SCA1, SCA2 and SCA3. Spinal and bulbar muscular atrophy SBMA is an X-linked inherited neuromuscular disorder that is characterized by a CAG repeat expansion in exon 1 of the androgen receptor gene (AR) that leads to degeneration of lower motor neurons and skeletal muscle cells (Spada et al., 1991; Cortes and Spada, 2018). Mutant AR protein leads to several cellular dysfunctions such as altered axonal transport (Katsuno et al., 2006; Kemp et al., 2011), transcription dysregulation (Lieberman et al., 2002; Katsuno et al., 2006) and mitochondrial abnormalities (Lieberman et al., 2002). Given that this disorder is linked to a single gain-of-toxic function mutation, targeting of the affected AR transcript using ASOs holds promise as a therapeutic intervention. As both skeletal muscle cells and motor neurons are affected in SBMA, strategies that target both peripheral and CNS mutant AR protein have been developed (Table 2) (Lieberman et al., 2014; Sahashi et al., 2015). Lieberman and collaborators (2014) demonstrated that subcutaneous administration of AR-targeting ASOs resulted in significant suppression of AR mRNA and protein levels in the muscle but not in the CNS, confirming the peripheral selectivity of this approach. In this same study, ASO treatment improved the skeletal muscle pathology and increased survival in two different SBMA mouse models (Lieberman et al., 2014). Taken together, these results suggest that targeting AR at the periphery may be a promising therapeutic approach for SBMA. Another study demonstrated that targeting of AR transcripts in the CNS may also be a promising therapeutic approach for disease amelioration (Sahashi et al., 2015). In this study, ASO ICV injection in a SBMA mouse model resulted in sustained AR mRNA suppression, as well as AR protein reduction in the spinal cord for at least 5 weeks and 8 weeks post-injection, respectively. Moreover, amelioration of motor neuron pathology as well as improved neurogenic disease phenotypes in the skeletal muscle and neuromuscular junctions was also reported (Sahashi et al., 2015). Clinical trials using antisense oligonucleotide therapeutics for Huntington’s disease Based on the success of several preclinical studies, five clinical trials have been developed for Huntington’s disease using ASOs (Table 4). In all clinical studies, intrathecal administration has been the elected method of ASO administration, which as previously mentioned has been a standard method for drug delivery to the CNS for several neurodegenerative disorders (Miller et al., 2013; Chiriboga et al., 2016; Rinaldi and Wood, 2018). Following the promising results from preclinical studies in a variety of Huntington’s disease rodent models and non-human primates (Tables 2 and 3), a phase 1/2 clinical trial was carried out from 2015 to 2017. This trial aimed at investigating the safety and pharmacokinetics of a non-selective gapmer ASO targeting HTT mRNA in early stage Huntington’s disease patients (NCT02519036). The findings from this study suggest good tolerability and no adverse effects associated with treatment were observed. Importantly, a significant and dose-dependent reduction of mutant HTT in the CSF was reported (Tabrizi et al., 2019). An open-label extension study (NCT03342053) to assess the safety and pharmacology profiles of the above-mentioned ASO in Huntington’s disease patients who completed the phase 1/2 trial has since been launched. Moreover, a new phase 3 trial involving 660 participants has also begun recruitment (NCT03761849). Two other phase 1/2 clinical trials were recently launched to investigate the safety and tolerability profiles of two separate stereopure ASOs, WVE-120101 (NCT03225833) and WVE-120102 (NCT03225846), which selectively target mutant HTT transcripts. Each ASO targets a specific SNP associated with the mutant allele. This way preventing the reduction of the wild-type HTT and potential side effects associated with a reduction of the normal protein. Conclusions Given their monogenic nature, polyQ disorders are ideally suited for therapies that specifically target the causative gene transcripts and modulate its expression. Thus, although delivery of ASOs to the CNS remains a challenge, this class of therapeutics provides a unique approach to target the root cause of these diseases. In any case, ASOs are able to widely distribute throughout the brain and spinal cord following intrathecal administration. Moreover, reports demonstrate the presence of ASOs in the brain and spinal cord for several months after intrathecal delivery (Rigo et al., 2014; Schoch and Miller, 2017). However, although long-lasting, this therapeutic approach is not permanent, offering the possibility of termination of treatment. In contrast, RNAi-based therapeutics encoded by viral vectors, such as lentiviral or adeno-associated viral vectors, can result in permanent transgene expression. This can be highly beneficial as patients could require only a single treatment; however, it is not without risk given that a therapeutic commitment is made. Interestingly, recently reported divalent siRNAs with chemical modifications similar to the ones used in ASOs, reached multiple brain regions after a single injection and enabled sustained silencing of HTT in rodent and non-human primate brains for several months (Alterman et al., 2019). This approach could greatly affect the frequency of treatment delivery to only a few times a year, contrary to the monthly delivery of ASOs to Huntington’s disease patients being currently used (Tabrizi et al., 2019). Nevertheless, this novel divalent siRNA still needs to be tested in disease models to assess its effects in disease neuropathology and phenotype. Moreover, another potential limitation of RNAi is the possibility of saturation of the cellular machinery involved in the processing of RNAi-mediated molecules. The recent FDA approval of two ASO therapies for the treatment of Duchenne muscular dystrophy and spinal muscular atrophy inspired great hope and further strengthened the case of ASO therapeutics for neurological disorders. Preclinical studies in the context of polyQ diseases have also provided encouraging results for ASO therapeutics (Tables 2 and 3), particularly in Huntington’s disease, and point towards a promising future. This is nicely illustrated by the fact that several ASO programmes have transitioned into clinical trials for Huntington’s disease (Table 4). Promising studies in SCA3 have also been launched (Tables 2 and 3). Though more data from preclinical studies in SCA3 are necessary, great efforts are underway to develop an ASO programme in this polyQ disorder. Biodistribution and tolerability in larger brains can be informed by the findings of other ASO therapeutics studies (Kordasiewicz et al., 2012; Southwell et al., 2018). Taking this into account and given the recent success of Huntington’s disease clinical trial (Tabrizi et al., 2019), translation into a trial in SCA3 patients may be foreseeable in the near future. Moreover, given the overlap of ATXN2 role in the pathogenesis of SCA2 and amyotrophic lateral sclerosis, development of tolerable and efficient lead ASOs targeting mutant mRNA is of great importance. It is likely that SCA2 will follow trials in amyotrophic lateral sclerosis. Ongoing medical chemistry efforts are greatly expanding the possibilities for optimization of ASOs, either by improving their delivery to targeted cells or by improving their overall pharmacology and safety profiles. While the majority of efforts for this therapeutic option in polyQ disorders started a few years after RNAi strategies were carried out, ASOs will likely be translated into clinic first or already have for the case of Huntington’s disease. Altogether, the recent success of ASO therapeutics in the clinic as well as in preclinical models suggest that this class of therapeutics might become standard treatments for neurodegenerative disorders in the near future. Funding Our group is supported by the European Regional Development Fund through the Regional Operational Program Center 2020, Competitiveness Factors Operational Program (COMPETE 2020) and National Funds through Foundation for Science and Technology (FCT): BrainHealth2020 projects (CENTRO-01-0145-FEDER-000008), ViraVector (CENTRO-01-0145-FEDER-022095), CortaCAGs (POCI-01-0145-FEDER-016719), SpreadSilencing POCI-01-0145-FEDER-029716, Imagene POCI-01-0145-FEDER-016807, CancelStem POCI-01-0145-FEDER-016390, as well as PD/BD/114171/2016 (to I.M.M.), SFRH/BD/51673/2011 (to S.M.L.), SFRH/BPD/87552/2012 and PTDC/MED-NEU/32309/2017 (to S.p.D.), and the Association Française contre les Myopathies -Téléthon no. 21163 and the SynSpread, European SCA3/MJD Initiative and ModelPolyQ under the EU Joint Program, the last two co-funded by the European Union H2020 program, GA No. 643417; by National Ataxia Foundation, the American Portuguese Biomedical Research Fund and the Richard Chin and Lily Lock Machado-Joseph Disease Research Fund. 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TI - Antisense oligonucleotide therapeutics in neurodegenerative diseases: the case of polyglutamine disorders
JF - Brain
DO - 10.1093/brain/awz328
DA - 2020-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/antisense-oligonucleotide-therapeutics-in-neurodegenerative-diseases-Irn0caxgGO
SP - 407
VL - 143
IS - 2
DP - DeepDyve
ER -