Clinical advances of RNA therapeutics for treatment of neurological and neuromuscular diseases

Abstract

RNA therapeutics comprise a diverse group of oligonucleotide-based drugs such as antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs) that can be designed to selectively interact with drug targets currently undruggable with small molecule-based drugs or monoclonal antibodies. Furthermore, RNA-based therapeutics have the potential to modulate entire disease pathways, and thereby represent a new modality with unprecedented potential for generating disease-modifying drugs for a wide variety of human diseases, including central nervous system (CNS) disorders. Here, we describe different strategies for delivering RNA drugs to the CNS and review recent advances in clinical development of ASO drugs and siRNA-based therapeutics for the treatment of neurological diseases and neuromuscular disorders.Abbreviations 2'-MOE: 2'-O-(2-methoxyethyl); 2'-O-Me: 2'-O-methyl; 2'-F: 2'-fluoro; AD: Alzheimer's disease; ALS: Amyotrophic lateral sclerosis; ALSFRS-R: Revised Amyotrophic Lateral Sclerosis Functional Rating Scale; ARC: Antibody siRNA Conjugate; AS: Angelman Syndrome; ASGRP: Asialoglycoprotein receptor; ASO: Antisense oligonucleotide; AxD: Alexander Disease; BBB: Blood brain barrier; Bp: Basepair; CNM: Centronuclear myopathies; CNS: Central Nervous System; CPP: Cell-penetrating Peptide; CSF: Cerebrospinal fluid; DMD: Duchenne muscular dystrophy; DNA: Deoxyribonucleic acid; FAP: Familial amyloid polyneuropathy; FALS: Familial amyotrophic lateral sclerosis; FDA: The United States Food and Drug Administration; GalNAc: N-acetylgalactosamine; GoF: Gain of function; hATTR: Hereditary transthyretin amyloidosis; HD: Huntington's disease; HRQOL: health-related quality of life; ICV: Intracerebroventricular; IT: Intrathecal; LNA: Locked nucleic acid; LoF: Loss of function; mRNA: Messenger RNA; MS: Multiple Sclerosis; MSA: Multiple System Atrophy; NBE: New Biological Entity; NCE: New Chemical Entity; NHP: Nonhuman primate; nt: Nucleotide; PD: Parkinson's disease; PNP: Polyneuropathy; PNS: Peripheral nervous system; PS: Phosphorothioate; RISC: RNA-Induced Silencing Complex; RNA: Ribonucleic acid; RNAi: RNA interference; s.c.: Subcutaneous; siRNA: Small interfering RNA; SMA: Spinal muscular atrophy; SMN: Survival motor neuron; TTR: Transthyretin.

Keywords: CNS; RNA-based therapeutics; Small interfering RNA; antisense oligonucleotide; clinical trial; gene silencing; neurological disease; neuromuscular disorder.

Figure 1.

Common chemical modifications used in RNA-based therapeutics. Nuclease resistance of ASOs is improved by substituting the parent phosphodiester bonds with phosphorothioate (PS) linkages in which a sulphur atom replaces one of the non-bridging oxygen atoms in the phosphate group. In addition, PS modifications enhance plasma protein binding and reduce clearance of the ASOs, thereby enhancing the PK properties of PS-ASOs. Improved stability and increased binding affinity is achieved using nucleotides with sugar modifications, including different 2’-modified sugars such as the 2’-O-methyl (2’-O-Me), 2’-O-methoxyethyl (2’-MOE), or 2ʹ-fluoro (2’-F) modifications, or the bicyclic locked nucleic acid (LNA) modification, in which the ribose sugar is locked in a C3’-endo conformation by introduction of a 2’-O,4’-C methylene bridge. PMOs contain a backbone of hexagonal morpholine rings linked by phosphorodiamidate bonds.

Figure 2.

Schematic illustration depicting siRNA and ASO mechanisms of action. A: In the cytoplasm, siRNA triggers are incorporated into the RNA-induced silencing complex (RISC); a ribonucleoprotein complex consisting of Dicer, the RNA binding protein (TRBP), and the RNase Argonaute 2 (AGO2). Upon RISC loading, the strand with the less thermodynamically stable 5’-end is incorporated and guides the RISC to the complementary target mRNA. The mRNA target dissociates from the intact siRNA after AGO2 cleavage, freeing RISC to regenerate and cleave additional mRNA targets. B: ASOs modulate target mRNA expression either by steric blocking (mixmers) or recruitment of RNase H (gapmers). Gapmers are composed of a central DNA region flanked at both ends by chemically modified ribonucleotides and recruit RNase H, which recognizes and cleaves DNA:RNA heteroduplexes.

Figure 3.

Delivery of RNA therapeutics to the CNS. Schematic illustration depicting the delivery of RNA therapeutics to the CNS. A: Due to the inability to cross the blood-brain barrier, RNA drugs are currently delivered either by intrathecal (IT), intracerebroventricular (ICV), or intraparenchymal injections. B: RNA drugs penetrate and distribute in the brain parenchyma, where they will encounter the different cells of the brain. C: Cellular uptake of RNA drugs can either occur by clathrin- or caveolin-dependent endocytosis, macropinocytosis or other non-productive pathways. CL: Clathrin, CV: Caveolin, EE: Early endosome, LE: Late endosome, LY: lysosome, MA: Macropinocytosis, R: Receptor.

Figure 4.

Chemical compositions of nusinersen, eteplirsen, and inotersen. Nusinersen is an 18-nucleotide fully phosphorothioate (PS)-modified 2’-O-methoxyethyl (2’-MOE) ASO targeting survival motor neuron 2 (SMN2) for the treatment of spinal muscular atrophy (SMA). Eteplirsen is a 30-nucleotide PMO-based drug targeting exon 51 of the Dystrophin gene (DMD). Inotersen is a 20-mer gapmer ASO targeting transthyretin (TTR). The ASO is fully PS-modified with five 2ʹ-MOE-modified ribonucleotides at each terminus (i.e. 5–10-5 structure).