RNA-Based Therapeutics: From Antisense Oligonucleotides to miRNAs

Abstract

The first therapeutic nucleic acid, a DNA oligonucleotide, was approved for clinical use in 1998. Twenty years later, in 2018, the first therapeutic RNA-based oligonucleotide was United States Food and Drug Administration (FDA) approved. This promises to be a rapidly expanding market, as many emerging biopharmaceutical companies are developing RNA interference (RNAi)-based, and RNA-based antisense oligonucleotide therapies. However, miRNA therapeutics are noticeably absent. miRNAs are regulatory RNAs that regulate gene expression. In disease states, the expression of many miRNAs is measurably altered. The potential of miRNAs as therapies and therapeutic targets has long been discussed and in the context of a wide variety of infections and diseases. Despite the great number of studies identifying miRNAs as potential therapeutic targets, only a handful of miRNA-targeting drugs (mimics or inhibitors) have entered clinical trials. In this review, we will discuss whether the investment in finding potential miRNA therapeutic targets has yielded feasible and practicable results, the benefits and obstacles of miRNAs as therapeutic targets, and the potential future of the field.

Keywords: RNAi; Rna therapeutics; antisense RNA; drug delivery; miRNA.

Figure 1

Mechanisms of RNA-based therapeutics that are dependent on the endogenous microRNA (miRNA) pathway. (A) miRNAs are encoded in the genome, often in the intron of protein-coding genes. The transcript, produced by RNA polymerase II, containing the miRNA forms a characteristic stem-loop structure which is processed in the nucleus by an RNases III enzyme, Drosha, to form an RNA hairpin (approx. 70 nucleotides) called the pre-miRNA. Pre-miRNA moves into the cytoplasm via exportin-5 (XPO-5), where it is further processed by Dicer, producing a double-stranded miRNA–miRNA* duplex. One strand of this duplex is loaded onto an Argonaute (Ago protein) to from the RNA-induced silencing complex (RISC). The other strand of the duplex (the passenger strand) is degraded. RISC is guided by the loaded miRNA strand which imperfectly binds to complementary sites commonly found in the 3’ untranslated region (UTR) of target mRNAs. RISC inhibits the translation of the bound mRNA and can cause deadenylation and degradation of the targeted transcript. Therapeutic miRNA mimics (B) are synthesized as miRNA duplexes. Upon entry into the cell, one strand binds to an endogenous Ago protein forming RISC, while the passenger strand degrades. The synthesized miRNA acts as a guide, directing the RISC to the therapeutic target, and inhibiting its translation. (C) AntagomiRs are single-stranded, synthesized, modified RNA molecules which are complementary to an endogenous miRNA. Upon entry into the cell, the antagomiR will bind to its target miRNA, preventing the miRNA from being loaded onto an Ago protein and forming RISC. (D) Once therapeutic siRNA duplexes enter the cell, one strand is loaded onto an Ago2 protein forming RISC. RISC is directed to the target mRNA by the loaded siRNA which binds with 100% complementarity to its target, Ago2 then cleaves the transcript. (E) DNA plasmids designed to encode short hairpin (sh) RNA enter the cell nucleus, where they are transcribed, producing an RNA with a characteristic stem-loop structure, allowing the RNA to enter the endogenous miRNA biogenesis pathway.

Figure 2

Common Delivery Methods for RNA-based Therapeutics. (A) RNA-based therapeutics are often encapsulated, or attached on the surface of, nanoparticles to aid delivery of the drug into the cell. These nanoparticles are often modified with moieties such as cholesterol or polyethylene glycol (PEG) which aid uptake of the nanoparticle via the cell membrane. Some nanoparticles are directed to particular cells by the addition of a targeting moiety, often a ligand for a cell surface receptor specifically expressed on the target cell. Commonly, the nanoparticle enters the cell via endocytosis, forming an endosome, which, after environmental changes (e.g., lowered pH), degrades, releasing the RNA therapeutic into the cell. (B) Alternatively, some RNA therapeutics are directly conjugated to moieties to aid their transport across the cell membrane, e.g., cholesterol (C) Synthesized RNA therapeutics can be chemically modified to increase their stability and binding affinity and decrease their toxicity. LNA: locked nucleic acid (2′4′-methylene; 2′OMe: 2′-O-methyl; 2′MOE: 2′-O-methoxyethyl.

Figure 3

Mechanism of approved therapeutics. (A) Nusinersen regulates splicing of the Survival Motor Neuron (SMN) 2 gene to treat patients with spinal muscular atrophy (SMA). Due to weak splice site, masked by the binding of hnRNP, the SMN2 gene usually produces a truncated transcript lacking exon 7, which, when translated, produces a non-functional and unstable protein (SMN2∆7). Nusinersen (Spinraza^TM, Biogen) is an antisense oligonucleotide (ASO) therapy that binds, via complementarity, to SMN2 pre-mRNA, displacing hnRNP, exposing the splice site and increasing the inclusion of exon 7, forming a full-length, mature SMN2 transcript. Once translated, this produces a full-length, functional SMN protein, which improves patient’s motor neuron function and slows disease progression. (B) Patisiran (Onpattro) reduces the production of transthyrethin (TTR) protein to reduce the formation of amyloid fibrils in hereditary transthyretin-mediated (hATTR) amyloidosis. Mutations in the TTR gene causes misfolding of the TTR protein, the misfolded protein aggregates into amyloid fibrils. Patisiran is a synthesized siRNA therapy, which is 100% complementary to a specific sequence in the 3′ UTR of the TTR mRNA. Once Patisiran enters the cell, one strand of the short interfering RNA (siRNA) duplex is loaded onto an Ago2 protein, forming RISC. RISC binds to the TTR transcript, which is subsequently cleaved by Ago2, therefore reducing TTR protein production, preventing further amyloidosis and improving patient’s quality of life.