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RNA-based and RNA-targeted therapeutics

RNA-based and RNA-targeted therapeutics

RNA-based therapeutics include RNA and oligonucleotide therapeutics that target RNA and proteins.

RNA therapeutics include RNA molecules that encode proteins, RNA molecules that target nucleic acids (either DNA or RNA) and RNA molecules that target proteins. Those that target nucleic acids include single-stranded antisense oligonucleotides (ASOs) and double-stranded molecules known to operate through the RNA interference (RNAi) pathway. RNA therapeutics that target proteins are called RNA aptamers.

ASOs are short stretches or modified DNA of 13-25 nucleotides which act by preventing mRNA from being translated into protein through blocking start of translation or tagging mRNA for degradation. An ASO may alter mRNA splicing, a maturation process necessary for translation to occur. RNAi based therapies use double-stranded molecules of small interfering RNAs (siRNAs) of 21-23 nucleotides or microRNAs, which prevent protein translation through degradation of mRNA. Double stranded molecules used in RNAi based therapies are more difficult to gain entry into cells compared with single stranded ASOs.

RNA aptamers are designed to bind a specific site on a specific protein to affect its function. RNA aptamers have rapid action and reversibility.

Noncoding RNA (ncRNA)

Messenger RNA (mRNA) are the RNA molecules that code for proteins. After gene transcription, coding RNAs undergo processing steps such as splicing before forming a mature mRNA molecules. Some noncoding RNAs (ncRNAs) perform enzymatic catalysis during splicing and translation and others regulate gene expression. Small ncRNA (sncRNA), also called small RNA (sRNA) includes microRNA (miRNA) which can suppress translation, degrade mRNA and silence gene loci through epigenetics. sncRNA have been discovered that originate from small nucleolar RNA (snoRNA), tRNA and rRNA and act as miRNA or modulate cellular processes like ribosome biogenesis. Long ncRNA (lncRNA) are more than 200 nucleotides long and function in regulating gene expression in the nucleus at the level of transcription and epigenetics or in the cytosol control mRNA turnover and regulation. Expression of ncRNAs is tissue-specific as they regulate development and physiology. Illnesses such as cardiovascular disease, diabetes, neuronal degenerative disease and cancer have shown abnormal expression of miRNAs and lncRNAs.

lncRNAs are more often localised to the nucleus where they function in chromatin conformation, which indirectly impacts gene expression, as well as directly regulating gene expression through R-loops, interference with RNA polymerase machineries and transcription of the lncRNA locus. A subset of lncRNAs are exported to the cytoplasm where they regulate mRNA turnover, translation and modification of proteins.

miRNA and RNA interference (RNAi)

miRNAs are 18-25 nucleotides in length and double stranded. The initial miRNA transcript is usually several kilobases in length and is called a primary miRNA (pri-miRNA). It contains a reverse-complement base pair segment forming a double stranded RNA hairpin loop. The pri-miRNA is processed to the pre-miRNA form by ribonuclease cleavage at the stem of the hairpin structure to form a hairpin intermediate of about 70-100 nucleotides. After transport to the cytoplasm, the pre-miRNA is further processed by the ribonuclease Dicer to generate the mature double stranded miRNA. One strand is degraded and the other strand called the guide strand is incorporated into an RNA-induced silencing complex (RISC) that targets degradation or translational repression of the mRNA targeted by the miRNA guide strand.

RNA-based and oligonucleotide-based therapeutics

RNA therapeutics include mRNA molecules that encode proteins, RNA molecules and oligonucleotides that target nucleic acids (either DNA or RNA) and RNA molecules and oligonucleotides that target proteins. Those that target nucleic acids include single-stranded antisense oligonucleotides (ASOs) and double-stranded siRNA molecules known to operate through the RNA interference (RNAi) pathway. An antisense molecule is complementary to the coding strand of DNA or RNA produced by the target gene. RNA therapeutics may target RNA molecules by binding complimentary sequences. In addition, RNAs can fold on themselves into complex 3D structures due to short stretches of complementary bases within their sequences and also by noncanonical hydrogen bonds formed by Hoogsteen base-pairing and hydrogen bonds between ribose-phosphate backbone moieties. These tertiary RNA structures can generate aptamers that recognize small-molecule ligands, nucleic acids or proteins.


When ASOs bind to target RNA they may function through occupancy-only mechanisms or by occupancy-mediated degradation. Occupancy-only effects include alterations in RNA processing, inhibition of translation, enhancement of translation and obstructing interactions between the target RNA and certain proteins. ASOs may target splicing mechanisms to cause exon skipping, cryptic splicing restoration or change the levels of alternate gene splicing. ASOs that work through degradation are usually single-stranded with a central DNA gapmer region. A DNA gapmer is a region of modified DNA bases that induce RNase H cleavage. Hybridization of ASO gapmers to a target RNA forms a DNA:RNA deteroduplex, creating a substrate for cleavage by RNase H1. Some chemical modifications can optimize ASOs to promote cleavage through the Ago2 complex. The following are classes of ASOs:

  • Double stranded siRNA
  • miRNA mimics
  • Antagomirs are ASOs complementary to mature miRNAs and counteract miRNAs implicated in disease pathogenesis
  • Peptide nucleic acids (PNA), DNA/RNA analogues in which the sugar-phosphate backbone is replaced by N-2-aminoethylglycine repeating units.
  • Janus bases are used to make two-sided analogs to DNA and RNA that can bind both DNA and RNA (Neubase Therapeutics)
ASO chemical modifications

ASOs are chemically modified to improve stability. Oligonucleotide analogs allow potential ASO drugs to avoid degradation in biological fluids. Other modifications improve binding affinity with target.

  • Phosphorothioate (PS-ASO), modification to backbone where sulfur atom replaces oxygen atom, reduces hydrophilicity, stabilizes, enhances protein binding, improves cell uptake and intracellular distribution
  • 2′-O-methoxyethyl (MOE), a chemical modification of the gapmer which increases the binding of ASOs to their target
  • Methylphosphonate, non-anionic internucleotide linkages
  • Phosphoramidate, non-anionic internucleotide linkages
  • Phosphorodiamidate morpholino (PMO), non-anionic internucleotide linkages
  • Peptide nucleic acids (PNA), non-anionic internucleotide linkages. Synthetic mimics of DNA or RNA where sugar-phosphate backbone is replaced by repeating units of N-2 aminoethylglycine units linked by amide bonds
  • 5-methyl-2′-deoxycytidine (5-MedC), heterocyclic modification to improve binding affinity
  • Sugar modifications: Oligoribonucleotides, 2′-O-Alkyl Sugars, bridged nucleic acids (BNA) such as locked nucleic acids (LNA)

siRNAs are double stranded, containing a sense and an antisense strand, where the antisense strand is the pharmacologically active moiety and the sense strand acts to help deliver the antisense strand and facilitate interactions with the intracellular RNA endonuclease, Argonaute 2 (Ago2). In mammals Ago2 is the protein within the RNA-induced silencing complex (RISC) that degrades target mRNA. Ago2 contains an RNase H domain and cleaves RNA in an RNA-RNA duplex, not a DNA-RNA duplex. The Ago2 complex of proteins facilitates the hybridization of the antisense strand to the target RNA with an 8 nucleotide sequence of Watson-Crick hybridization that targets the mRNA to be degraded. Since Ago2 is mainly localized to the cytoplasm, siRNAs are effective at targeting cytoplasmic RNAs.

Delivery and targeting
  • Conjugation of GalNac, an amino sugar moiety that targets a receptor on the surface of hepatocytes can be added to siRNA and ASO therapeutics to cause efficient liver targeting and cellular uptake
  • Intrathecal injection (for central nervous system)
  • Nanoparticles to target lungs
  • Cationic lipid nanoparticles (for siRNA)
  • Conjugation of siRNAs with antibodies that recognize specific surface receptors to target muscle or cancer cells
  • Exosomes
  • Lipid nanoparticles (LNPs) accumulate in the liver when systemically delivered
  • Lipid-anchored polyethylene glycol (PEG)
  • LNPs conjugated to antibodies for targeted delivery
  • Polymeric carriers such as polyethylenimine (PEI), polysaccharides such as chitosan and inulin (INU), and polyaminoacids such as α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) and poly-l-lysine (PLL)
  • Targeted RNAi Molecule, or TRiMTM, platform utilizes ligand-mediated delivery for tissue-specific targeting (Arrowhead Pharmaceuticals)
  • SomaLogic
miRNA mimics (miR-Mimic)

miRNAs normally function to control the expression of genes and have tissue-specific expression. Altered miRNA expression profiles are associated with diseased states. Therefore, miRNAs can serve as diagnostic biomarkers and therapeutic targets. Therapeutic strategies that aim to restore normal miRNA expression either block the expression of a disease-associated signature miRNA or substitute for the loss of expression of the miRNA through the use of synthetic miRNAs, also known as miRNA mimics.

miRNA inhibitors

AntagomiRs are chemically modified oligonucleotides that bind to microRNAs to inhibit their ability to silence their target mRNA transcripts.

Sponge RNAs are small synthetic RNAs that have the same ‘seed region’ sequence and bind multiple microRNAs.

mRNA production and delivery

mRNA-based therapeutic designs strive to reduce immongenicity or activation of innate immune responses and increase stability in the presence of RNases in the body.

  • In vitro transcription of mRNA with optimized cap structure, optimized 5’ untranslated region (UTR), codon optimized coding sequence, optimized 3’ UTR and repeated adenine nucleotides (polyA tail) enable efficient translation of mRNA and improved stability
  • HPLC purification and incorporation of modified nucleosides such as 1-methylpseudouridine (m1ψ), increase translation efficiency and make it less immunogenic
  • Lipid nanoparticle (LNP)-formulated mRNA (mRNA LNPs can cause immune related as well as cellular toxicities due to accumulation of lipids in the liver)
  • mRNA electroporation
  • ex vivo mRNA-electroporated dendritic cells (mRNA-DCs)
Commercially available mRNA tranfection reagents
  • jetMESSENGER (Polyplus-transfection)
mRNA-based immunotherapy

Immunotherapies aim to manipulate immune responses to direct the immune system to fight cancer or to reestablish immune tolerance in the case of autoimmune diseases. The use of mRNA coding for the production of antigens offers a similar advantage to using plasmid DNA in terms of cost compared with antigens administered as recombinant proteins. However since mRNA has higher transfection efficiency and does not need to enter the nucleus, it could be more effectively delivered quiescent cells and also potentially be safer due to lack of genomic integration.

Gene editing

mRNA is used to transiently express enzymes required for gene editing such as zinc finger nucleases (ZFNs), Transcripton Activator-like Effector Nucleases (TALENs) and Cas cutting enzyme in CRISPR/Cas systems.

Passive immunization with monoclonal antibodies (mAb)

Passive immunization is when manufactured antibodies or antibodies derived from another individual provide protection from an infectious disease. Passive immunization can provide rapid protective immunity, which could be advantageous to block the spread of a virus in a pandemic. Delivery of mRNA encoding antibodies for passive immunization is a less costly alternative to the manufacturing of recombinant antibodies.

  • Neutralizing antibody against HIV delivered by encoding the light and heavy chains of VRC01, formulated in LNPs and delivered systemically in mice
  • Rabies (animal study)
  • Botulism (animal study)
  • Anti-tumor therapy (animal study)

RiboMABs are bispecific antibodies, bispecific T cell engaging antibodies or BiTEs of which blinatumamab is an example. Blinatumomab is used to treat acute lymphoblastic leukemia and is a bispecific antibody that targets CD19 and CD3ε and aims to cluster T cells to lymphoma cells. mRNA encoding RiboMAB formulated in nanoparticles are injected intravenously. The liver cells produce and secrete antibodies into the circulation.

RNA therapeutic products

Approved RNA therapeutics

Medical condition
Product name
Type of RNA therapeutic

Familial chylomicronemia syndrome (FCS), hypertriglyceridemia and familial partial lipodystrophy (FPL)

Volanesorsen (Waylivra®) (ASO)

Subcutaneous injection



Acute Hepatic Porphyrias

Givlaari, givosiran (RNAi, siRNA), targets aminolevulinic acid synthase 1 (ALAS1)

Subcutaneous injections

U.S., E.U.


Age-related macular degeneration

Pegaptanib (Macugen), RNA aptamer against VEGF

Injection into vitreous body of eye


RNA aptamer

Citomegalovirus retinitis

Formivirsen (ASO), Vitravene

Injection into vitreous body of eye

Marketing authorization withdrawn (U.S., E.U.)


Duchenne muscular dystrophy

VYONDYS 53™ (golodirsen)


phosphorodiamidate morpholino oligomer (PMO)

RNA therapeutics in clinical trials

Medical condition

Acute kidney injury following cardiac surgery (preventative)

Teprasiran (previously QPI-1002), siRNA targeting p53

Phase II

Acute Nonarteritic Anterior Ischemic Optic Neuropathy (NAION)

QPI-1007, siRNA targeting Caspase 2

Alpha-1 antitrypsin deficiency (AATD) associated liver disease


Arrowhead Pharmaceuticals

Phase II/III

Alpha-1 antitrypsin deficiency associated liver disease



Amyotrophic Lateral Sclerosis (ALS, Lou Gehrig's disease)

ASO against C9orf72

The ALS Association, Don Cleveland, Richard Smith, Timothy Miller


RNA therapeutics in development

Medical condition
Product type

Respiratory syncytial virus (RSV)

mRNA encoded antibody

Georgia Institute of Technology and Emory University

B-cell lymphomas and leukemias

mRNA encoded antibody against CD20

Botulinum toxin

mRNA encoded antibody



hTERT targeted therapy, ASO, PNA

Neubase Therapeutics

Cystic fibrosis

ARO-ENaC, triggers RNAi to reduce production of epithelial sodium channel alpha subunit (αENaC)

Personalized RNA therapeutics

Personalized ASO therapies are designed single patients with a rare genetic mutations. ASO-based therapeutics have frequently been associated with FDA N-of-1 trial requests.

  • Milasen, a splice modulating ASO used in an N-of-1 study to treat a child with the fatal neurodegenerative condition, Batten disease (Boston Children's Hospital, Timothy Yu)
  • ASO for a rare form of ALS caused by a mutation in the gene FUS (Ionis Pharmaceuticals and Neil Shneider, the Eleanor and Lou Gehrig ALS Center at Columbia Medical Center)
Small RNA biomarkers
Small molecules that target RNA

RIBOTAC (ribonuclease-targeting chimeras) is a druglike molecule that binds specific RNA that is combined with an RNA-degrading enzyme to target the miRNA-96 oncogene (Matthew Disney, Scripps Research Institute, Florida)

Therapeutic RNA editing

RNA editing enzymes bind to RNAs and alter their sequence. In RNA editing therapeutics these enzymes are targeted at specific RNAs. Adenosine deaminases acting on RNA (ADARs) occur naturally inside human cells but some therapeutic strategies aim to add additional ADARs. Other strategies include engineering guide RNAs with chemical modifications that attract ADARs already in the cell to the editing site.

  • RNA-Guided Adenosine Deaminases
  • ADARs change adenosine to inosine
  • LEAPER (leveraging endogenous ADAR for programmable editing of RNA) uses short engineered ADAR-recruiting RNAs (arRNAs) to recruit ADAR1 and ADAR2 to change a specific adenosine to inosine
  • RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing) uses chemosynthetic antisense oligonucleotides to recruit endogenous ADARs
  • ADARs to edit the mRNA for a gene encoding the sodium channel Nav1.7, which controls how pain signals are transmitted to the brain
  • APOBEC are enzymes that change cytosine to uridine
  • Pseudouridylation does not change the sequence of the mRNA or encoded protein but increases stability of the RNA
RNA editing companies

Transcriptomics is the study of the complete set of RNA transcripts produced by the genome, called the transcriptome, under certain conditions or in a specific cell or tissue type. Transcriptomics research can be used to discover RNA-based biomarkers and potential drugs that target RNA or are themselves based on RNA molecules.

Microarray technology

Microarray technology, also called GeneChip, DNA/RNA chip or BioChip, is the fixation of up to millions of nucleic acid probe molecules of known sequence onto a support that is hybridized with labelled sample molecules which may be RNA or cDNA. The microarray is scanned by a laser and analysed by computer software. Microarrays can be used to analyze gene expression in cell culture or tissue samples and may detect gene expression changes associated with disease conditions or drug treatments. Microarrays cannot be used to discovery of new transcripts, it analyses the levels of the known transcripts represented by probes on the chip.


RNA-seq uses next generation sequencing (NGS) to sequence transcripts. RNA-seq can qualitatively and quantitatively analyse mRNA, microRNA, small interfering RNA and long noncoding RNA. The technology can characterise different RNA isoforms that result from alternative splicing.

Single-cell RNA sequencing (scRNA-seq)

Single-cell RNA sequencing (scRNA-seq) is the sequencing of a transcriptome at the level of a single-cell. Differences in gene expression between individual cells can identify rare cell populations, such as diverse immune cell populations in healthy and diseased states. The identification of rare cell populations has implications in drug resistance and relapse in cancer treatment. scRNA-seq is used to delineate cell lineage relationships in development and differentiation with applications in stem cell technology and regenerative medicine. scRNA-seq can be used to analyse the response of individual cell types to treatments and conditions.

scRNA-seq methods:

  • CEL-seq2
  • Drop-seq
  • MARS-seq
  • SCRB-seq
  • Smart-seq
  • Smart-seq2
RNA modifications (epitranscriptomics)

RNA undergoes various modifications by the coordinataed actions of writer proteins (RNA-modifying enzymes), reader proteins (RNA-bining proteins (RBPs)) and eraser proteins. Endogenous or exogenous chemical damage can also cause RNA modifications. Some modifications are reversible and others are irreversible. RNA modifications can affect molecular processes such as pre-mRNA splicing, RNA export, mRNA translation and RNA degradation and contribute to the overall cellular transcriptome and proteome. Some modifications that affect RNA stability include N6-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), 8-oxo-7,8-dihydroguanosine (8-oxoG), pseudouridine (Ψ), 5-methylcytidine (m5C), and N4-acetylcytidine (ac4C).

  • MODOMICS database
  • RBS-Seq is a modification of RNA bisulfite sequencing for detection of m5C, Ψ, and m1A at single-base resolution transcriptome-wide


Spatial transcriptomics

Spatial transcriptomics methods characterize gene expression profiles while retaining spatial tissue context. Fluorescence in situ hybridization (FISH)-based methods label transcripts in tissue sections enabling single-cell and subcellular location to be visualized. Single-cell RNA sequencing (scRNA-seq) methods dissociate cells first and link transcriptomes back to their original locations.

  • microarray-based spatial transcriptomics
  • sequential FISH (seqFISH)

RNA therapeutics companies


Further Resources



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