RNAi-Based Therapeutics and Novel RNA Bioengineering Technologies

Abstract

RNA interference (RNAi) provides researchers with a versatile means to modulate target gene expression. The major forms of RNAi molecules, genome-derived microRNAs (miRNAs) and exogenous small interfering RNAs (siRNAs), converge into RNA-induced silencing complexes to achieve posttranscriptional gene regulation. RNAi has proven to be an adaptable and powerful therapeutic strategy where advancements in chemistry and pharmaceutics continue to bring RNAi-based drugs into the clinic. With four siRNA medications already approved by the US Food and Drug Administration (FDA), several RNAi-based therapeutics continue to advance to clinical trials with functions that closely resemble their endogenous counterparts. Although intended to enhance stability and improve efficacy, chemical modifications may increase risk of off-target effects by altering RNA structure, folding, and biologic activity away from their natural equivalents. Novel technologies in development today seek to use intact cells to yield true biologic RNAi agents that better represent the structures, stabilities, activities, and safety profiles of natural RNA molecules. In this review, we provide an examination of the mechanisms of action of endogenous miRNAs and exogenous siRNAs, the physiologic and pharmacokinetic barriers to therapeutic RNA delivery, and a summary of the chemical modifications and delivery platforms in use. We overview the pharmacology of the four FDA-approved siRNA medications (patisiran, givosiran, lumasiran, and inclisiran) as well as five siRNAs and several miRNA-based therapeutics currently in clinical trials. Furthermore, we discuss the direct expression and stable carrier-based, in vivo production of novel biologic RNAi agents for research and development. SIGNIFICANCE STATEMENT: In our review, we summarize the major concepts of RNA interference (RNAi), molecular mechanisms, and current state and challenges of RNAi drug development. We focus our discussion on the pharmacology of US Food and Drug Administration-approved RNAi medications and those siRNAs and miRNA-based therapeutics that entered the clinical investigations. Novel approaches to producing new true biological RNAi molecules for research and development are highlighted.

Graphical abstract

Fig. 1.

Overview of miRNA biogenesis and functions and siRNA mechanisms of action. (A) Intragenic or intergenic miRNA genes are transcribed by RNA polymerases II or III into primary miRNA (pri-miRNA; >1,000 nucleotides) transcripts in canonical pathway (black lines). (B) Pri-miRNAs are subjected to nuclear processing by the microprocessor Drosha-DGCR8 complex to release shorter precursor miRNAs (pre-miRNAs) (e.g., ∼65 nucleotides). (C) Noncanonical miRNA transcripts (e.g., mirtrons) are derived from the genome and subjected to RNA splicing to form pre-miRNAs independent on the microprocessor (orange lines). (D) Pre-miRNAs are exported into the cytoplasm via Exportin 5 transport complex. (E) In the cytoplasm, pre-miRNAs are processed by Dicer/TRBP complex to form miRNA duplexes (18–25 nucleotides). (F) Dicer-independent production of miRNA duplexes (yellow). (G) The guide strand (blue) of the miRNA duplex is selected and loaded into the RNA-induced silencing complex (RISC) to form the miRNA-RISC complex (miRISC) while the passenger strand (red) is degraded. (H) Functional miRNA binds to the 3′-untranslated region (3′UTR) of targeted mRNA to perform posttranscriptional gene regulation, either to accelerate mRNA cleavage or degradation or to repress translation. (I) Exogenous siRNAs can be introduced into cytoplasm through endocytosis or receptor-mediated uptake (green). (J) The antisense strand (blue) is selectively loaded into the RISC to form the siRNA-RISC complex (siRISC) while the sense strand (red) is degraded. (K) Functional siRNA typically acts on the protein coding sequence (CDS) of target transcript to cleave or initiate transcript degradation.

Fig. 2.

Common and specific actions of four FDA-approved siRNA medications in hepatocytes. (A) Patisiran is a double stranded siRNA drug (sense in red and antisense in blue) formulated in a lipid nanoparticle (LNP) decorated with polyethylene glycol (pegylation), and it is administered intravenously for the treatment of hereditary transthyretin-mediated amyloidosis. The LNP induces an opsonization-based immune response and is endocytosed by the hepatocyte prior to endosomal escape. (B) Givosiran, lumasiran, and inclisiran, in which the sense strand (red) is conjugated with three N-acetylgalactosamine (GalNAc) moieties, are administered subcutaneously for the treatments of acute hepatic porphyria, primary hyperoxaluria type 1, and heterozygous familial hypercholesterolemia, respectively. The GalNAc moieties are recognized by asialoglycoprotein receptor 1 (ASGR1), which is highly expressed on the surface of hepatocytes, to facilitate the uptake of siRNAs. (C) The antisense strand (blue) is preferably loaded into the RNA-induced silencing complex (RISC) to form the siRNA-RISC complex (siRISC) while the passenger strand (red) is degraded. (D) Givosiran-derived siRISC binds to the protein coding sequence (CDS) of target mRNA toward cleavage or degradation. (E) Patisiran-, lumasiran-, and inclisiran-derived siRISC interacts with the 3′-untranslated regions (3′UTRs) of target mRNAs to achieve gene silencing. (F) SiRNA drugs (givosiran, patisiran, lumasiran, and inclisiran) target specific mRNAs to achieve the control of their respective diseases. HAO1, hydroxyacid oxidase 1.

Fig. 3.

Novel biotechnologies to produce biologic RNAi agents. (A) Direct expression is one class of novel RNAi agent production. Two forms of direct expression make use of two bacterial strains, Rhodovulum sulfidophilum and an RNase III–deficient Corynebacterium glutamicum. The marine phototropic bacterium R. sulfidophilum has the capability to efflux oligonucleotides such as RNA but not RNases. These characteristics allow R. sulfidophilium to be transformed with an RNA expressing plasmids for direct expression of the target RNA followed by efflux and accumulation in RNase free culture media. Accumulated RNA in culture media can then be isolated and purified to attain the target RNA molecule. A novel strain of C. glutamicum contains a mutation in the RNase III gene (2256LΔrnc) and provides an RNase III ribonuclease–free bacterium for RNA accumulation. Target RNA may be directly expressed and accumulated in C. glutamicum free from RNase degradation for later isolation from cell lysate and purification. (B) The use of stable carriers constitutes the other class of novel RNAi agent production. Transfer RNA (tRNA) and ribosomal RNA (rRNA) can each function as stable RNA scaffolds. By retaining structures and sequences of these highly abundant RNA classes, target RNA is expected to exploit endogenous recognition and accumulate within bacteria. As most recombinant RNAs cannot be overexpressed with tRNA or rRNA scaffold, specific hybrid tRNA/pre-miRNA molecules showing high-level expression in E. coli have been identified, developed, and proven as unique carriers to effectively accommodate a wide variety of target RNAi molecules including RNA aptamers, miRNAs, siRNAs, or sRNAs along with their complimentary sequences for high-yield and large-scale production of biologic RNAi agents.