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Review
. 2019 Apr:196:91-104.
doi: 10.1016/j.pharmthera.2018.11.011. Epub 2018 Dec 4.

RNA therapy: Are we using the right molecules?

Ai-Ming Yu  1 , Chao Jian  2 , Allan H Yu  2 , Mei-Juan Tu  2
Affiliations
Review

RNA therapy: Are we using the right molecules?

Ai-Ming Yu et al. Pharmacol Ther. 2019 Apr.

Abstract

Small-molecule and protein/antibody drugs mainly act on genome-derived proteins to exert pharmacological effects. RNA based therapies hold the promise to expand the range of druggable targets from proteins to RNAs and the genome, as evidenced by several RNA drugs approved for clinical practice and many others under active trials. While chemo-engineered RNA mimics have found their success in marketed drugs and continue dominating basic research and drug development, these molecules are usually conjugated with extensive and various modifications. This makes them completely different from cellular RNAs transcribed from the genome that usually consist of unmodified ribonucleotides or just contain a few posttranscriptional modifications. The use of synthetic RNA mimics for RNA research and drug development is also in contrast with the ultimate success of protein research and therapy utilizing biologic or recombinant proteins produced and folded in living cells instead of polypeptides or proteins synthesized in vitro. Indeed, efforts have been made recently to develop RNA bioengineering technologies for cost-effective and large-scale production of biologic RNA molecules that may better capture the structures, functions, and safety profiles of natural RNAs. In this article, we provide an overview on RNA therapeutics for the treatment of human diseases via RNA interference mechanisms. By illustrating the structural differences between natural RNAs and chemo-engineered RNA mimics, we focus on discussion of a novel class of bioengineered/biologic RNA agents produced through fermentation and their potential applications to RNA research and drug development.

Keywords: Biotechnology; Cancer; RNAi; Therapy; miRNA; ncRNA.

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Conflict of interest statement

Conflict of interest statement

The authors declare no conflict of interests.

Figures

Fig. 1.
Fig. 1.. Expanding the range of druggable targets with RNA therapeutics.
Proteins derived from the genome remain as favorable targets for pharmacotherapy, whereas the majority of DNA sequences in the human genome are transcribed as non-protein coding transcripts. As current medications are mainly small molecules and proteins (e.g., antibodies) that act on proteins, RNA therapeutics hold great promise to expand druggable genome for the treatment of human diseases, including (1) RNA aptamers that block protein targets, (2) RNAs such as asRNAs, miRNAs, and siRNAs that target mRNAs or various forms of ncRNAs, and (3) gRNAs that directly edit gene sequences.
Fig. 2.
Fig. 2.. Cellular RNA interference pathway and manipulation with various types of RNA agents.
Endogenous RNAi pathway involves the initial production of pre-miRNA molecules (1)from longer pri-miRNAs transcribed from miRNA coding genes or directly excised out of introns in the nucleus. Pre-miRNAs are then exported into the cytoplasm and subsequently processed to miRNA duplexes. After unwinding from the duplexes and incorporation into the RISC complex, miRNAs and siRNAs recognize target transcripts via imperfect or perfect base-pairing, leading to RNA cleavage or translation inhibition. The use of synthetic dsRNAs (2), siRNAs (3), asRNAs (4), and miRNA mimics (5), which all consist of various types and degrees of chemical modifications, may exert target gene knockdown via cellular RNAi pathway. Bioengineered, single-stranded miRNA or siRNA or sRNA “prodrugs” (6) produced from bacteria fermentation represent a novel family of “natural” RNAi molecules.
Fig. 3.
Fig. 3.. Use of viral vectors or DNA plasmids for RNA interference.
DNA plasmids (1) and genetically-modified DNA viral vectors (e.g., adenovirus) (2) or RNA viral vectors (e.g., lentivirus) (3) may be employed for the production of target pre-miRNA or shRNA molecule (1) within the nucleus of host cell, before which the target coding sequence need be integrated into the host cell’s genome. Thus, transcribed shRNAs or pre-miRNAs (1) enter the cellular RNAi pathway; and the resultant siRNAs or miRNAs act on specific transcripts to exert target gene silencing.
Fig. 4.
Fig. 4.. Chemical structures of unmodified cellular nucleic acids and some postranscriptionally-modified nucleosides.
Different from DNA, an RNA molecule has a hydroxyl group at the 2’ position of each ribose, as well as the uracil base rather than thymine. Natural RNAs made in living cells are generally unmodified nucleic acids, and there is just a small fraction (e.g., < 3%) of ribonucleosides carrying posttranscriptional modifications which actually show a broad chemical diversity. Among these modifications, methylation is the most common form which may occur at both ribose and nucleobases. It is also noteworthy that substantial posttranscriptional modifications are present at the nucleobases.
Fig. 5.
Fig. 5.. Common chemical modifications used for the production of nucleic acid reagents including aptamers, ASOs, siRNAs, miRNA mimics, sgRNAs, tRNA fragments (tRFs), and other types of sRNAs.
Chemical modifications are mainly aimed at improving the pharmacokinetics properties of RNA reagents. These synthetic RNA mimics are characterized by the changes predominantly at the phosphate linkage and ribose vulnerable to RNase-mediated degradation, which is in contrast to cellular RNA modifications mainly occurring at the nucleobases (Fig. 4). Many RNA conjugates are also developed to enhance targeting, and the advanced modifications completely change the linkage moieties and just retain nucleobases for pairing.
Fig. 6.
Fig. 6.. The workflow for the production of biologic/bioengineered RNAi agent (BERA) where a target miRNA/siRNA/sRNA is assembled into a tRNA/pre-miRNA carrier.
After a target BERA is designed, corresponding coding sequence is cloned into a vector. Expression of target BERA in fermentation may be verified by RNA gel electrophoresis, and BERA can be purified to a high degree of homogeneity by using different methods (e.g., anion exchange FPLC). Purity of isolated BERA is determined by HPLC analysis and endotoxin pyrogen testing. These BERAs should better capture the properties of cellular RNAs and represent a novel class of RNAi agents for basic research (e.g., structural and functional studies) and drug development.
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