Antisense Oligonucleotides: Translation from Mouse Models to Human Neurodegenerative Diseases
Authors: Kathleen M. Schoch, Timothy M. Miller
Journal: Neuron (2017)

Multiple neurodegenerative diseases are characterized by single-protein dysfunction and aggregation. Treatment strategies for these diseases have often targeted downstream pathways to ameliorate consequences of protein dysfunction; however, targeting the source of that dysfunction, the affected protein itself, seems most judicious to achieve a highly effective therapeutic outcome. Antisense oligonucleotides (ASOs) are small sequences of DNA able to target RNA transcripts, resulting in reduced or modified protein expression. ASOs are ideal candidates for the treatment of neurodegenerative diseases, given numerous advancements made to their chemical modifications and delivery methods. Successes achieved in both animal models and human clinical trials have proven ASOs both safe and effective. With proper considerations in mind regarding the human applicability of ASOs, we anticipate ongoing in vivo research and clinical trial development of ASOs for the treatment of neurodegenerative diseases. Multiple neurodegenerative diseases are characterized by single-protein dysfunction and aggregation. Treatment strategies for these diseases have often targeted downstream pathways to ameliorate consequences of protein dysfunction; however, targeting the source of that dysfunction, the affected protein itself, seems most judicious to achieve a highly effective therapeutic outcome. Antisense oligonucleotides (ASOs) are small sequences of DNA able to target RNA transcripts, resulting in reduced or modified protein expression. ASOs are ideal candidates for the treatment of neurodegenerative diseases, given numerous advancements made to their chemical modifications and delivery methods. Successes achieved in both animal models and human clinical trials have proven ASOs both safe and effective. With proper considerations in mind regarding the human applicability of ASOs, we anticipate ongoing in vivo research and clinical trial development of ASOs for the treatment of neurodegenerative diseases. Recent years have seen a re-emergence of antisense oligonucleotides (ASOs) as valuable tools to inform on disease mechanisms and as powerful therapeutics for disease intervention. Part of this recent success can be attributed to improvements made to the ASO structure and chemical modifications that have made ASOs more advantageous and efficient as potential strategies for the treatment of disease. ASOs can exert their gene-targeting effects through different mechanisms of action, making them amenable to a variety of molecular targets and disease processes. These advancements in ASO technology have been coupled with the surprising empiric finding that, despite being highly charged and large, ASOs distribute widely throughout the CNS when delivered to the cerebral spinal fluid (CSF). This characteristic has greatly enabled the application of ASOs as therapeutic strategies for CNS disorders, many of which currently have no adequate treatment. The first ASO to enter clinical trial, targeted against p53 in acute myelogenous leukemia, drew upon 20 years of chemical advancements prior to its introduction in 1993 (Bayever et al., 1993Bayever E. Iversen P.L. Bishop M.R. Sharp J.G. Tewary H.K. Arneson M.A. Pirruccello S.J. Ruddon R.W. Kessinger A. Zon G. et al.Systemic administration of a phosphorothioate oligonucleotide with a sequence complementary to p53 for acute myelogenous leukemia and myelodysplastic syndrome: initial results of a phase I trial.Antisense Res. Dev. 1993; 3: 383-390Crossref PubMed Google Scholar). This milestone was closely followed by the first market approval for the antisense therapeutic, fomivirsen, in 1998 for the treatment of cytomegalovirus retinitis in patients with immunodeficiency (Marwick, 1998Marwick C. First "antisense" drug will treat CMV retinitis.JAMA. 1998; 280: 871Crossref PubMed Scopus (116) Google Scholar). Since then, numerous ASOs have progressed to human clinical trial for diseases, including cancer, diabetes, and muscular dystrophy (Bennett and Swayze, 2010Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (600) Google Scholar), taking advantage of the various mechanisms and synthetic structures now available for the design of ASO therapies. Neurodegenerative diseases, specifically, are in imminent need of an effective therapy, and ASOs offer one possible strategy. Considering the remarkable clinical trial successes that, to date, have been achieved in diseases like spinal muscular atrophy and amyotrophic lateral sclerosis, researchers and patients are enthusiastic about ASO application to neurodegenerative diseases. Many neurodegenerative diseases collectively are characterized by the dysfunction or abnormal accumulation of toxic proteins. Therefore, strategies aimed at reducing levels of the toxic protein or creating non-toxic modifications are attractive approaches for clinical development. ASOs and their gene-targeting effects provide a promising solution, and their successful translation from rodent models to human patients underscores their vast potential. ASOs are small, single-stranded sequences of DNA, 8–50 base pairs in length, composed of a phosphate backbone and sugar rings. The structure and sequence allows for specific, complementary matching with target RNA by Watson-Crick base pairing. ASO properties, including solubility, binding, potency, and stability, derive from various modifications to both the backbone and sugar rings. Backbone modifications both increase affinity to the RNA target and protect the ASO from nuclease degradation. One of the first and most commonly used modifications made to the phosphate backbone replaced the singly bonded oxygen group with a sulfur ion, creating a phosphorothioate (PS) linkage (Figure 1). This modification endows the ASO with enhanced stability and protection against nuclease degradation, increased binding to plasma proteins to maintain stable concentrations in serum, better cellular uptake, and the ability to recruit RNase H enzyme for target degradation (Bennett and Swayze, 2010Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (600) Google Scholar). A major disadvantage to the PS backbone modification is the potential inflammatory response generated at high concentrations (Crooke, 2007Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications, Second Edition. CRC Press, Boca Raton, FL2007Crossref Google Scholar) and prohibitive affinity for plasma protein binding (Geary et al., 2001Geary R.S. Watanabe T.A. Truong L. Freier S. Lesnik E.A. Sioufi N.B. Sasmor H. Manoharan M. Levin A.A. Pharmacokinetic properties of 2′-O-(2-methoxyethyl)-modified oligonucleotide analogs in rats.J. Pharmacol. Exp. Ther. 2001; 296: 890-897PubMed Google Scholar), which may produce adverse effects and other limitations in a clinical setting. Creating PS molecules and other ASOs with nuclease resistance and enhanced potency must be balanced against activation of the innate immune response. For instance, CpG oligodeoxynucleotides motifs (i.e., an unmethylated "C" followed by a "G" base separated by a phosphodiester linkage, common in particular base sequences) are potent immune activators (Younis et al., 2006Younis H.S. Vickers T. Levin A.A. Henry S.P. CpG and Non-CpG oligodeoxynucleotides induce differential proinflammatory gene expression profiles in liver and peripheral blood leukocytes in mice.J. Immunotoxicol. 2006; 3: 57-68Crossref PubMed Scopus (8) Google Scholar) and, therefore, are often avoided in designing ASOs for in vivo application. An exception might be when immune stimulation is a desired property, for example with immunotherapies (Vollmer and Krieg, 2009Vollmer J. Krieg A.M. Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists.Adv. Drug Deliv. Rev. 2009; 61: 195-204Crossref PubMed Scopus (304) Google Scholar). Yet, even with these considerations, it is likely that all ASOs are recognized as foreign DNA by toll-like receptors of the innate immune system. Much of the chemistry at the 2′ position has been focused on ways to stabilize the ASO without activating an immune response. In concert with the PS backbone modification, recent ASO pharmacology typically includes 2′ ribose sugar modifications (Figure 1). Of these, the 2′-O-methyl (2′-O-Me) and 2′-O-methoxyethyl (2′-MOE) are the most frequently used, enabling stronger binding affinity to target RNA and enhanced resistance to nuclease degradation (Bennett and Swayze, 2010Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (600) Google Scholar). These modifications may help to overcome the disadvantageous immunostimulatory properties of the PS backbone. Both 2′-O-Me and 2′-MOE design are reported to have a lowered pro-inflammatory response and be well tolerated compared to unmodified motifs (Henry et al., 2000Henry S. Stecker K. Brooks D. Monteith D. Conklin B. Bennett C.F. Chemically modified oligonucleotides exhibit decreased immune stimulation in mice.J. Pharmacol. Exp. Ther. 2000; 292: 468-479PubMed Google Scholar, Tluk et al., 2009Tluk S. Jurk M. Forsbach A. Weeratna R. Samulowitz U. Krieg A.M. Bauer S. Vollmer J. Sequences derived from self-RNA containing certain natural modifications act as suppressors of RNA-mediated inflammatory immune responses.Int. Immunol. 2009; 21: 607-619Crossref PubMed Scopus (22) Google Scholar). ASOs consisting of fully modified 2′ sugar moieties do not enable RNase H enzyme activity but instead support non-degrading ASO mechanisms, such as changes to alternative splicing or translational inhibition (Bennett and Swayze, 2010Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (600) Google Scholar). Activating RNase H and thus cleaving the target mRNA requires a creative pharmacologic manipulation to produce a chimeric or "gapmer" design, which takes advantage of the tolerability of 2′-O-Me or 2′-MOE sugar rings combined with the RNase-H-mediated degradation property of the PS backbone (Figure 1). A gapmer consists of 2′ modified sugar ring bases separated by a central region of unmodified nucleotides to enable target degradation without inducing overt inflammatory responses (Geary et al., 2015Geary R.S. Norris D. Yu R. Bennett C.F. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides.Adv. Drug Deliv. Rev. 2015; 87: 46-51Crossref PubMed Google Scholar). ASO chemistry continues to evolve, and modifications, including a morpholino backbone, peptide nucleic acid (PNA) chemistry, and locked nucleic acid (LNA) modifications, are becoming more prevalent in preclinical and clinical use (Figure 1). Both morpholinos and PNA designs afford highly stable RNA complementarity and, although they do not stimulate degradation of their target, they are useful for other mechanisms of action discussed below (Bennett and Swayze, 2010Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (600) Google Scholar). LNA sugar moieties are similar to the 2′-O-methyl structures but exhibit greater potency and can be designed to support RNase H activity. Although these modifications are advantageous for their potency and nuclease-resistant properties, problems associated with toxicity or poor penetration continue to be explored (McMahon et al., 2002McMahon B.M. Mays D. Lipsky J. Stewart J.A. Fauq A. Richelson E. Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration.Antisense Nucleic Acid Drug Dev. 2002; 12: 65-70Crossref PubMed Google Scholar, Swayze et al., 2007Swayze E.E. Siwkowski A.M. Wancewicz E.V. Migawa M.T. Wyrzykiewicz T.K. Hung G. Monia B.P. Bennett C.F. Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals.Nucleic Acids Res. 2007; 35: 687-700Crossref PubMed Scopus (169) Google Scholar). As various ASOs move closer toward clinical trial, ASO design remains an important avenue of development. Before ASOs can exert their gene-modulating effects, they must overcome membrane barriers and traffic to subcellular locations, typically the nucleus. These events can be viewed as limiting steps for ASO activity and underscore the importance of effective chemical designs to modulate both uptake and distribution at the cellular level. The proposed entry method of ASOs into cells involves association with high- and low-binding plasma proteins, internalization into lysosomal or endosomal compartments, and trafficking within the cell (Geary et al., 2015Geary R.S. Norris D. Yu R. Bennett C.F. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides.Adv. Drug Deliv. Rev. 2015; 87: 46-51Crossref PubMed Google Scholar), although the exact details of these processes are complex and may differ across cell types. A recent review of phosphorothioate ASO cellular uptake and intracellular trafficking summarizes the reported receptors, proteins, and pathways involved (Crooke, 2007Crooke S.T. Antisense Drug Technology: Principles, Strategies, and Applications, Second Edition. CRC Press, Boca Raton, FL2007Crossref Google Scholar). In brief, proteins that interact with ASOs at the cell surface and within intracellular organelles, in part, determine their subcellular trafficking and escape into the cytosol; however, many questions regarding specific protein interactions remain unanswered. Once inside cells, ASOs are able to travel between the cytosol and nucleus via proposed binding to cellular molecules (Lorenz et al., 2000Lorenz P. Misteli T. Baker B.F. Bennett C.F. Spector D.L. Nucleocytoplasmic shuttling: a novel in vivo property of antisense phosphorothioate oligodeoxynucleotides.Nucleic Acids Res. 2000; 28: 582-592Crossref PubMed Google Scholar). The single-stranded ASOs bind to their target RNA through Watson-Crick base pairing, which, depending on the target and ASO design, can trigger distinct mechanisms of action by which the ASO ultimately degrades or modifies its target. These mechanisms can be divided into several common categories, discussed below: RNase-mediated degradation; mRNA modification; or microRNA inhibition (Figure 2). RNase-mediated degradation is particularly advantageous in the context of many neurodegenerative disorders in which a distinct RNA/protein dysregulation or accumulation is recognized as the central cause of disease. It is not surprising, therefore, that RNase activation continues to be employed in preclinical ASO development and several clinical trials. Degradation of the mRNA target via this mechanism begins in the nucleus, where the DNA-RNA pairing of ASOs to their mRNA target triggers recruitment of the enzyme RNase H1 (Wu et al., 2004Wu H. Lima W.F. Zhang H. Fan A. Sun H. Crooke S.T. Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs.J. Biol. Chem. 2004; 279: 17181-17189Crossref PubMed Scopus (143) Google Scholar). Binding of the ASO to mRNA mimics the DNA-RNA binding that occurs during DNA replication, in which RNase H cleaves the RNA primer from newly synthesized DNA (Cerritelli and Crouch, 2009Cerritelli S.M. Crouch R.J. Ribonuclease H: the enzymes in eukaryotes.FEBS J. 2009; 276: 1494-1505Crossref PubMed Scopus (270) Google Scholar). For ASO therapeutics, destruction of the mRNA prohibits its translation into protein, yielding a reduction of the intended target. RNase H activation is facilitated by the PS backbone modification or gapmer ASO design. Because RNase H specifically cleaves RNA, the ASO itself avoids degradation and, therefore, is able to bind to additional target mRNA, enabling sustained mRNA and, in turn, protein reduction. Destruction of the cognate mRNA is the most common mechanism by which ASOs are used, yet several additional mechanisms can be employed to achieve protein lowering or manipulation (Bennett and Swayze, 2010Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (600) Google Scholar). Other possible mechanisms of ASO-mediated protein reduction include translational inhibition or altered RNA stability via RNA modification. RNA modification is a second, common mechanism by which ASOs can function. Acting within the nucleus, these ASOs pair with the target mRNA but, given their design, will not initiate the direct degradation of the mRNA. Instead, ASOs modify the mRNA at the 5′ cap or polyadenylation site to prevent mRNA translation or alter RNA stability, thereby lowering the intended mRNA and protein in a non-degrading fashion (Bennett and Swayze, 2010Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (600) Google Scholar). In addition, one surprisingly effective use of RNA-modifying ASOs has been to change alternative splicing patterns of mRNA. ASO binding to intron/exon junctions can affect recruitment and binding of splicing factors or destabilize splicing sites (Havens and Hastings, 2016Havens M.A. Hastings M.L. Splice-switching antisense oligonucleotides as therapeutic drugs.Nucleic Acids Res. 2016; 44: 6549-6563Crossref PubMed Scopus (45) Google Scholar). Such a strategy might be most beneficial for disorders with a known splicing defect to either restore protein function or to exclude mutated stretches of DNA. The degree of shift in splicing patterns has been striking in animals, as will be discussed below, and has advanced to successful clinical trial use. The most notable of these is the ASO design to promote the inclusion of exon 7 in the survival motor neuron (SMN1) gene, now US Food and Drug Administration (FDA)-approved for the treatment of spinal muscular atrophy (FDANewsRelease, 2016aFDANewsRelease (2016a). FDA approves first drug for spinal muscular atrophy. https://www.fda.gov/newsevents/newsroom/pressannouncements/ucm534611.htm.Google Scholar). ASOs can also act through binding of microRNA or other small RNA targets to inhibit their actions. MicroRNAs are sequences of approximately 22 base pairs in length and function in the regulation of gene expression through binding target mRNAs. ASOs act by binding these microRNA (miRNA) targets to inhibit their mRNA interaction by sequestration or degradation (Davis et al., 2006Davis S. Lollo B. Freier S. Esau C. Improved targeting of miRNA with antisense oligonucleotides.Nucleic Acids Res. 2006; 34: 2294-2304Crossref PubMed Scopus (267) Google Scholar, Davis et al., 2009Davis S. Propp S. Freier S.M. Jones L.E. Serra M.J. Kinberger G. Bhat B. Swayze E.E. Bennett C.F. Esau C. Potent inhibition of microRNA in vivo without degradation.Nucleic Acids Res. 2009; 37: 70-77Crossref PubMed Scopus (130) Google Scholar). Dysregulation of miRNAs has been identified in numerous disease states, including cancer, virology, and neurodegenerative disease, highlighting these small RNAs as potential targets for therapeutic intervention. This strategy is already being applied successfully in clinical trials for hepatitis C infection (Janssen et al., 2013Janssen H.L. Reesink H.W. Lawitz E.J. Zeuzem S. Rodriguez-Torres M. Patel K. van der Meer A.J. Patick A.K. Chen A. Zhou Y. et al.Treatment of HCV infection by targeting microRNA.N. Engl. J. Med. 2013; 368: 1685-1694Crossref PubMed Scopus (1048) Google Scholar). Although no miRNA-targeted ASO for neurodegenerative disorder is in clinical use, emerging knowledge on the roles of miRNA in disease continues to provide motivation for their future application to human patients. In order for ASOs to be effective, they must achieve delivery to affected regions or cells at appropriate concentrations. In addition, they must be stable and maintain efficacy over time for feasible treatment throughout the course of disease. Diseases of the CNS often pose problems that limit efficient and effective drug delivery. Most notably, the blood brain barrier restricts entry of certain molecules based on size, charge, or solubility (Pardridge, 1998Pardridge W.M. CNS drug design based on principles of blood-brain barrier transport.J. 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Intrathecal ASO delivery has already been implemented in human clinical trials for amyotrophic lateral sclerosis and spinal muscular atrophy with safe and tolerable results (Chiriboga et al., 2016Chiriboga C.A. Swoboda K.J. Darras B.T. Iannaccone S.T. Montes J. De Vivo D.C. Norris D.A. Bennett C.F. Bishop K.M. Results from a phase 1 study of nusinersen (ISIS-SMN(Rx)) in children with spinal muscular atrophy.Neurology. 2016; 86: 890-897Crossref PubMed Scopus (129) Google Scholar, Miller et al., 2013Miller T.M. Pestronk A. David W. Rothstein J. Simpson E. Appel S.H. Andres P.L. Mahoney K. Allred P. Alexander K. et al.An antisense oligonucleotide against SOD1 delivered intrathecally for patients with SOD1 familial amyotrophic lateral sclerosis: a phase 1, randomised, first-in-man study.Lancet Neurol. 2013; 12: 435-442Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). The adequate distribution achieved with intrathecal delivery and the growing clinical experience with ASO delivery by this method will likely lead to an increasing number of clinical studies being performed with an intrathecal delivery route. Intraventricular delivery in humans remains open to further study and may have some practical advantages in patients with anatomic abnormalities, such as severe scoliosis, that make intrathecal delivery more challenging. Intraventricular or intrathecal injections are relatively invasive compared with orally delivered medications, yet thus far have been well tolerated. Ongoing research is focused on developing novel ASO delivery methods for the CNS, including oral, subcutaneous, and intranasal routes. Success with intrathecal delivery programs, including current and potential commercialization of some ASO candidates, is likely to spark innovative approaches to make a successful therapy even easier to administer. On the other hand, with the urgent need for neurodegenerative disease therapies, intrathecal delivery will likely continue as a standard method. One general concern often raised regarding ASO application for human diseases is the possibility of adverse side effects. ASO toxicities and off-target effects are known to be both sequence and chemistry dependent, and thus, each ASO molecule must be considered independently and with a complete toxicology evaluation. However, there are some general points to consider. The first consideration is on-target toxicity as a result of lowering levels of a total protein. The effects of gene deletion in rodents provide a useful first-pass evaluation of the potential toxicities resulting from protein reduction, but these models have the potential to be over-interpreted in both directions. Whereas protein reduction from birth—as in knockout mouse experiments—may be deleterious, lowering this same protein in the adult—as is proposed in many ASO programs—may be very well tolerated. Conversely, protein reduction from birth may be assumed safe, but this conclusion may be misguided by the known caveat for potential compensation of gene deletion in mice. ASO administration with substantial lowering of mRNA and protein in diseased and age-matched rodents and in non-human primates may provide the best reassurance of safety for an ASO approach. Yet, ASO studies in non-human primates are typically in a relatively small number of animals and for limited periods of time. Thus, the broad array of potential problems associated with lowering protein levels remains unknown and is still challenging to predict in the adult human. For many neurodegenerative diseases, this small theoretical risk is dwarfed by the ongoing, known hazard of the disease-causing protein, yet ASOs will continue to be evaluated for their on-target toxicity in preclinical and clinical testing. Second, concerns for "off-target" toxicity focus on binding to other mRNAs and immune activation. For human clinical candidates, each ASO is screened in silico against the human genome and "hits" with one, two, or three mismatches are empirically tested for ASO-mediated changes of these mRNAs (Kamola et al., 2015Kamola P.J. Kitson J.D. Turner G. Maratou K. Eriksson S. Panjwani A. Warnock L.C. Douillard Guilloux G.A. Moores K. Koppe E.L. et al.In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization.Nucleic Acids Res. 2015; 43: 8638-8650Crossref PubMed Scopus (21) Google Scholar, Monia et al., 1992Monia B.P. Johnston J.F. Ecker D.J. Zounes M.A. Lima W.F. Freier S.M. Selective inhibition of mutant Ha-ras mRNA expression by antisense oligonucleotides.J. Biol. Chem. 1992; 267: 19954-19962Abstract Full Text PDF PubMed Google Scholar). Thus, ASOs with potential overlap can be routinely screened against. In vivo, the ASO is recognized as foreign DNA by toll-like receptors and likely causes "off-target" activation of the immune system (Agrawal and Kandimalla, 2004Agrawal S. Kandimalla E.R. Role of Toll-like receptors in antisense and siRNA [corrected].Nat. Biotechnol. 2004; 22: 1533-1537Crossref PubMed Scopus (90) Google Scholar). This response has been largely mitigated by sc