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Antisense technology is now beginning to deliver on its promise to treat diseases by targeting RNA. Nine single-stranded antisense oligonucleotide (ASO) drugs representing four chemical classes, two mechanisms of action and four routes of administration have been approved for commercial use, including the first RNA-targeted drug to be a major commercial success, nusinersen. Although all the approved drugs are for use in patients with rare diseases, many of the ASOs in late- and middle-stage clinical development are intended to treat patients with very common diseases. ASOs in development are showing substantial improvements in potency and performance based on advances in medicinal chemistry, understanding of molecular mechanisms and targeted delivery. Moreover, the ASOs in development include additional mechanisms of action and routes of administration such as aerosol and oral formulations. Here, we describe the key technological advances that have enabled this progress and discuss recent clinical trials that illustrate the impact of these advances on the performance of ASOs in a wide range of therapeutic applications. We also consider strategic issues such as target selection and provide perspectives on the future of the field.Antisense technology is now beginning to deliver on its promise to treat diseases by targeting RNA. Here, Crooke and colleagues describe the key technological advances that have enabled this progress and discuss recent clinical trials that illustrate the impact of these advances on the performance of antisense oligonucleotides in a wide range of therapeutic applications.

The growing understanding of RNA functions and their crucial roles in diseases promotes the application of various RNAs to selectively function on hitherto “undruggable” proteins, transcripts and genes, thus potentially broadening the therapeutic targets. Several RNA-based medications have been approved for clinical use, while others are still under investigation or preclinical trials. Various techniques have been explored to promote RNA intracellular trafficking and metabolic stability, despite significant challenges in developing RNA-based therapeutics. In this review, the mechanisms of action, challenges, solutions, and clinical application of RNA-based therapeutics have been comprehensively summarized.

Technologies that recruit and direct the activity of endogenous RNA-editing enzymes to specific cellular RNAs have therapeutic potential, but translating them from cell culture into animal models has been challenging. Here we describe short, chemically modified oligonucleotides called AIMers that direct efficient and specific A-to-I editing of endogenous transcripts by endogenous adenosine deaminases acting on RNA (ADAR) enzymes, including the ubiquitously and constitutively expressed ADAR1 p110 isoform. We show that fully chemically modified AIMers with chimeric backbones containing stereopure phosphorothioate and nitrogen-containing linkages based on phosphoryl guanidine enhanced potency and editing efficiency 100-fold compared with those with uniformly phosphorothioate-modified backbones in vitro. In vivo, AIMers targeted to hepatocytes with N -acetylgalactosamine achieve up to 50% editing with no bystander editing of the endogenous ACTB transcript in non-human primate liver, with editing persisting for at least one month. These results support further investigation of the therapeutic potential of stereopure AIMers. RNA in non-human primate liver is efficiently edited using short stereopure oligonucleotides.

Refinements in the chemistries employed in oligonucleotide therapeutics have galvanized clinical progress. The complex interplay between chemical modifications and integration into sequence architecture is discussed in the context of antisense and small-interfering RNA drugs. After nearly 40 years of development, oligonucleotide therapeutics are nearing meaningful clinical productivity. One of the key advantages of oligonucleotide drugs is that their delivery and potency are derived primarily from the chemical structure of the oligonucleotide whereas their target is defined by the base sequence. Thus, as oligonucleotides with a particular chemical design show appropriate distribution and safety profiles for clinical gene silencing in a particular tissue, this will open the door to the rapid development of additional drugs targeting other disease-associated genes in the same tissue. To achieve clinical productivity, the chemical architecture of the oligonucleotide needs to be optimized with a combination of sugar, backbone, nucleobase, and 3′- and 5′-terminal modifications. A portfolio of chemistries can be used to confer drug-like properties onto the oligonucleotide as a whole, with minor chemical changes often translating into major improvements in clinical efficacy. One outstanding challenge in oligonucleotide chemical development is the optimization of chemical architectures to ensure long-term safety. There are multiple designs that enable effective targeting of the liver, but a second challenge is to develop architectures that enable robust clinical efficacy in additional tissues.

Antisense oligonucleotides (ASOs) modified with phosphorothioate (PS) linkages and different 2′ modifications can be used either as drugs (e.g., to treat homozygous familial hypercholesterolemia and spinal muscular atrophy) or as research tools to alter gene expression. PS-ASOs can enter cells without additional modification or formulation and can be designed to mediate sequence-specific cleavage of different types of RNA (including mRNA and non-coding RNA) targeted by endogenous RNase H1. Although PS-ASOs function in both the cytoplasm and nucleus, localization to different subcellular regions can affect their therapeutic potency. Cellular uptake and intracellular distribution of PS ASOs are mediated by protein interactions. The main proteins involved in these processes have been identified, and intracellular sites in which PS ASOs are active, or inactive, cataloged.

The molecular mechanisms of toxicity of chemically modified phosphorothioate antisense oligonucleotides (PS-ASOs) are not fully understood. Here, we report that toxic gapmer PS-ASOs containing modifications such as constrained ethyl (cEt), locked nucleic acid (LNA) and 2′- O -methoxyethyl (2′-MOE) bind many cellular proteins with high avidity, altering their function, localization and stability. We show that RNase H1–dependent delocalization of paraspeckle proteins to nucleoli is an early event in PS-ASO toxicity, followed by nucleolar stress, p53 activation and apoptotic cell death. Introduction of a single 2′- O -methyl (2′-OMe) modification at gap position 2 reduced protein-binding, substantially decreasing hepatotoxicity and improving the therapeutic index with minimal impairment of antisense activity. We validated the ability of this modification to generally mitigate PS-ASO toxicity with more than 300 sequences. Our findings will guide the design of PS-ASOs with optimal therapeutic profiles. Chemical modification of PS-ASO therapeutics reduces binding to cellular proteins and decreases toxic side-effects.

Key Points The human transcriptome is the product of a coordinated series of transcriptional, co-transcriptional and post-transcriptional regulatory events. RNA splicing is a key regulatory step in gene expression that allows a limited genome to express an impressive diversity of coding and non-coding RNAs. RNA mis-splicing causes a large array of human diseases due to hereditary and somatic mutations. Mis-splicing may result from mutations to RNA cis -regulatory elements, core spliceosomal components or trans -acting regulatory factors. Mutations in some genes, such as lamin A ( LMNA ), cause multiple types of diseases, from muscular dystrophy to premature ageing syndromes. A key small nuclear RNA (snRNA) component of the minor spliceosome functions as a stress-activated switch to control expression levels of genes containing minor introns. Some splicing factors linked to diseases, such as amyotrophic lateral sclerosis, contain low-complexity regions with prion-like domains that are susceptible to abnormal aggregation. Splicing modulatory therapeutic strategies have been developed that target a range of diseases, including muscular dystrophies and motor neuron diseases, and are currently being tested in clinical trials. Complex and intricate RNA splicing mechanisms are crucial for gene regulation and for maximizing proteomic diversity. This Review discusses how alterations to splicing mechanisms — such as mutations in pre-mRNAs, or mutations and dysregulation of core spliceosome proteins and other RNA-binding proteins — results in diverse molecular consequences and various diseases. Opportunities for therapeutic correction of these defects are also explored. The human transcriptome is composed of a vast RNA population that undergoes further diversification by splicing. Detecting specific splice sites in this large sequence pool is the responsibility of the major and minor spliceosomes in collaboration with numerous splicing factors. This complexity makes splicing susceptible to sequence polymorphisms and deleterious mutations. Indeed, RNA mis-splicing underlies a growing number of human diseases with substantial societal consequences. Here, we provide an overview of RNA splicing mechanisms followed by a discussion of disease-associated errors, with an emphasis on recently described mutations that have provided new insights into splicing regulation. We also discuss emerging strategies for splicing-modulating therapy.

RNA base editing represents a promising alternative to genome editing. Recent approaches harness the endogenous RNA-editing enzyme adenosine deaminase acting on RNA (ADAR) to circumvent problems caused by ectopic expression of engineered editing enzymes, but suffer from sequence restriction, lack of efficiency and bystander editing. Here we present in silico-optimized CLUSTER guide RNAs that bind their target messenger RNAs in a multivalent fashion, achieve editing with high precision and efficiency and enable targeting of sequences that were not accessible using previous gRNA designs. CLUSTER gRNAs can be genetically encoded and delivered using viruses, and are active in a wide range of cell lines. In cell culture, CLUSTER gRNAs achieve on-target editing of endogenous transcripts with yields of up to 45% without bystander editing. In vivo, CLUSTER gRNAs delivered to mouse liver by hydrodynamic tail vein injection edited reporter constructs at rates of up to 10%. The CLUSTER approach opens avenues for drug development in the field of RNA base editing. Optimized guide RNAs improve RNA editing with endogenous enzymes.

While most monogenic diseases are caused by loss or reduction of protein function, the need for technologies that can selectively increase levels of protein in native tissues remains. Here we demonstrate that antisense-mediated modulation of pre-mRNA splicing can increase endogenous expression of full-length protein by preventing naturally occurring non-productive alternative splicing and promoting generation of productive mRNA. Bioinformatics analysis of RNA sequencing data identifies non-productive splicing events in 7,757 protein-coding human genes, of which 1,246 are disease-associated. Antisense oligonucleotides targeting multiple types of non-productive splicing events lead to increases in productive mRNA and protein in a dose-dependent manner in vitro. Moreover, intracerebroventricular injection of two antisense oligonucleotides in wild-type mice leads to a dose-dependent increase in productive mRNA and protein in the brain. The targeting of natural non-productive alternative splicing to upregulate expression from wild-type or hypomorphic alleles provides a unique approach to treating genetic diseases. Restoration of normal gene expression is one way to treat monogenic disorders. Here the authors target naturally occurring non-productive alternative splicing using antisense oligonucleotides to promote the production of functional proteins.

Efforts to use RNA-cleaving DNA enzymes (DNAzymes) as gene-silencing agents in therapeutic applications have stalled due to their low efficacy in clinical trials. Here we report a xeno-nucleic-acid-modified version of the classic DNAzyme 10–23 that achieves multiple-turnover activity under cellular conditions and resists nuclease digestion. The new reagent, X10–23, overcomes the problem of product inhibition, which limited previous 10–23 designs, using molecular chemotypes with DNA, 2′-fluoroarabino nucleic acid and α-l-threofuranosyl nucleic acid backbone architectures that balance the effects of enhanced biological stability with RNA hybridization and divalent metal ion coordination. In cultured mammalian cells, X10–23 facilitates persistent gene silencing by efficiently degrading exogenous and endogenous messenger RNA transcripts. Together, these results demonstrate that new molecular chemotypes can improve the activity and stability of DNAzymes, and may provide a new route for nucleic acid enzymes to reach the clinic.RNA-cleaving DNA enzymes (DNAzymes) have the potential to function as therapeutic agents by silencing the expression of disease-associated proteins. Xeno-nucleic acids were used to improve the catalytic activity and biological stability of a highly evolved DNAzyme known as 10–23. The enzyme exhibits a robust multiple-turnover activity in cultured mammalian cells.