Amplifying gene expression with RNA-targeted therapeutics

Abstract

Many diseases are caused by insufficient expression of mutated genes and would benefit from increased expression of the corresponding protein. However, in drug development, it has been historically easier to develop drugs with inhibitory or antagonistic effects. Protein replacement and gene therapy can achieve the goal of increased protein expression but have limitations. Recent discoveries of the extensive regulatory networks formed by non-coding RNAs offer alternative targets and strategies to amplify the production of a specific protein. In addition to RNA-targeting small molecules, new nucleic acid-based therapeutic modalities that allow highly specific modulation of RNA-based regulatory networks are being developed. Such approaches can directly target the stability of mRNAs or modulate non-coding RNA-mediated regulation of transcription and translation. This Review highlights emerging RNA-targeted therapeutics for gene activation, focusing on opportunities and challenges for translation to the clinic.

Fig. 1. Overview of strategies to increase protein production.

Currently, several strategies are approved or in development to increase protein production. a, In protein-replacement therapy, recombinant proteins are administered to replace a mutant variant or to supplement for a deficient variant in a patient. b, For gene therapy, viral vectors are used to deliver cDNA encoding functional proteins. c, For gene editing, specific mutations are corrected in situ by targeted DNA-editing or RNA-editing constructs (for example, transcription activator-like effector nucleases, zinc finger nucleases, CRISPR–Cas, base editors). d, For mRNA delivery, functional mRNA is delivered to cells to increase protein levels using various delivery modalities such as lipid nanoparticles (LNPs). e, RNA-targeted approaches include nucleic acid-based therapeutic (NBT) and small-molecule strategies. NBTs, such as antisense oligonucleotides, natural antisense transcript-specific oligonucleotides (AntagoNATs), small activating RNAs and microRNA blockers (antagomirs), modulate non-coding RNA-mediated regulation of transcription and translation through various mechanisms involving, for example, RNAse H, RNA-induced silencing complex-mediated RNA interference and steric blocking. Small molecules directly target RNA–protein interactions or recruit endogenous enzymes to target RNA, leading to protein upregulation.

Fig. 2. Biology of protein upregulation.

Physical interaction of distal enhancer elements with active gene promoters via chromosomal looping recruits transcription factors, chromatin modifiers, Mediator complex and RNA polymerase II (Pol II), leading to transcription activation. Bidirectional transcription from the promoter region and at enhancers gives rise to non-coding promoter RNAs (pRNAs) and enhancer RNAs (eRNAs), respectively. Natural antisense transcripts (NATs) transcribed from the antisense strand of protein-coding loci, trans-acting long non-coding RNA (lncRNA), pRNAs and eRNAs can scaffold epigenetic modifiers and transcription regulatory proteins at the target gene locus. Alternatively, they can pair directly with complementary regions in mRNA, inducing distinct stimulatory and inhibitory effects on transcription. Nascent pre-mRNA undergoes co-transcriptional and post-transcriptional modification (PTM) such as 5′-capping, 3′-end processing and polyadenylation, alternative splicing, in situ introduction of N⁶-methyladenosine, 5-methylcytosine, N¹-methyladenosine, pseudouridine and 2′-O-methyl modifications and deamination of adenosine to inosine by adenosine deaminases acting on RNA. The resulting mature mRNA isoforms are exported to the cytoplasm through nuclear pores. Translation of mRNA by the ribosome begins at a translation initiation site (TIS) and is regulated by proteins recruited by mRNA structural elements such as an internal ribosome entry site (IRES), and NATs and lncRNAs targeting untranslated regions (UTRs). miRNAs targeting unique sequences in 3′ UTRs can induce mRNA degradation through the RNA interference (RNAi) mechanism. Short upstream open reading frames (uORFs) within the 5′ UTR compete for the ribosome with the productive open reading frame (ORF) that gives rise to the functional full-length protein, thus slowing down its translation. Non-productive transcripts with inclusion of toxic exons characterized by the presence of dysfunctional termination codons are degraded by the nonsense-mediated decay (NMD) pathway. A, activator; R, repressor; Sp, splice-promoting or inhibiting factors.

Fig. 3. NBTs that increase mRNA abundance.

a, Therapeutic mRNA transcribed in vitro and encapsulated in lipid nanoparticles (LNPs) is introduced into cells and initiates translation. Targeting molecules (TMs) that bind specific cell surface receptors can be added for cell type-specific delivery. b, Small activating RNAs (saRNAs) are double-stranded synthetic RNAs, formed by guide (red) and passenger (yellow) strands encapsulated in LNPs (known as SMARTICLEs), which are delivered to cells. saRNAs are initially recognized by double-stranded RNA loading factors, followed by argonaute 2 (AGO2) protein binding. The passenger strand of the saRNA is discarded, and a complex consisting of the guide saRNA strand, AGO2 and heterogeneous nuclear ribonucleoproteins (hnRNPs) is imported into the nucleus, where it binds directly to DNA and participates in the RNA-induced transcriptional activation (RITA) complex. RITA interacts with RNA polymerase II (RNA Pol II) to initiate transcription. c, Enhancer RNA (eRNAs), bidirectionally transcribed from an enhancer region, interact with proteins such as BRD4, CREB-binding protein (CBP) or NELF to maintain chromatin in an active state and initiate RNA Pol II pause release. Transcriptional activation of some proteins involves interaction of eRNAs and enhancer-associated natural antisense transcripts (NATs) with the Mediator complex and cohesin. Nucleic acid-based therapeutics (NBTs) mimicking eRNAs or inducing their expression can facilitate transcriptional regulation by blocking eRNA interaction with inhibitory factors. d, NAT scaffold transcriptional repressors at target gene loci. NBT treatment blocks NAT interaction with repressors and/or chromosomes and accelerates transcription. e, NBTs targeting NATs involved in silencing imprinted alleles can derepress transcription of target mRNA. f, NBTs that interact with splice factors that promote or inhibit splicing, or with their binding sites on pre-mRNA, can modulate splice factor binding and/or activity. g, NBTs designed to bind to natural miRNAs (antagomirs) serve as decoys to prevent microRNA (miRNA) binding to mRNAs. Alternatively, NBTs can be designed to function as miRNA mimics (promirs). ASO, antisense oligonucleotide; lncRNA, long non-coding RNA; pRNA, promoter RNA.

Fig. 4. NBTs and small molecules targeting mRNA translation.

a, Upstream open reading frame (uORF)-targeting nucleic acid-based therapeutics (NBTs) increase protein expression through blocking uORF translation and thus redirecting the ribosome to the main start site. b, Other regulatory secondary structures in untranslated regions (UTRs) can be targeted. NBTs targeting stem-loop and other secondary structures within 5′ UTRs that inhibit translation can increase protein expression by hybridizing them and releasing inhibitory factors (IFs) that recognize them or by removing obstacles for binding or movement of ribosomes and translation-enhancing factors. c, NBTs targeting polyadenylation (polyA) signals can preferentially prevent expression of mutated or toxic isoforms and redirect expression towards beneficial isoform ratios. d, Small molecules can enable premature termination codon (PTC) readthrough. Upon encountering a PTC, ribosomes recruit proteins such as eukaryotic translation termination factor 1 (eRF1) and eRF3, facilitating premature termination of translation. Small molecules interact with ribosomes to enable recruitment of near-cognate tRNAs and facilitate readthrough, allowing translation of full-length protein. RNA Pol II, RNA polymerase II; TSS, translation start site.