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What Are RNA Drugs?

Different classes of RNA therapies have been approved for clinical use while others are still under investigation. Here, we focus on mRNA-based drugs, oligonucleotide (oligo)-based drugs, and small molecules targeting RNA.. Behind each is a unique development process, but all involve rounds of experimental screening using different assays to assess activity, toxicity, specificity, mechanism of action, and optimal formulation. The results of these assays are used to decide which candidates go on to become clinical leads.

mRNA-based drugs

mRNA-based drugs comprise in vitro transcribed RNA which is then translated into protein in the patient’s cells.

  • mRNA vaccine:  mRNA encodes an antigen meant to trigger an immune response, whether against an infectious agent like a virus (as is the case with the Moderna and BioNTech/PfizerCOVID-19 vaccines) or against a tumor.
  • mRNA immunotherapies: mRNA encodes an immunomodulatory molecule like an antibody or cytokine.
  • protein replacement therapies: mRNA encodes a protein that is missing or deficient--an approach being explored for genetic diseases like Duchenne muscular dystrophy and cystic fibrosis.

One special challenge for mRNA therapies is formulation or delivery: naked mRNA is highly immunogenic and unstable. The most common solution is to encapsulate an mRNA in a lipid nanoparticle (LNP) made of charged lipids (which protect the mRNA and facilitate passage through cellular membranes) and “helper” lipids such as cholesterol and PEG-lipids (which stabilize the particle). Since there are many types of charged lipids with which unique mRNAs may interact differently, LNPs need to be systematically and empirically screened for the optimal mix of charged lipids and helper lipids, in terms of its packaging efficiency, delivery efficacy, and toxicity. Assays used to test these characteristics can include, for example, dye-based RNA quantification (RiboGreen encapsulation assay) and cell-based functional assays.

Oligo-based drugs (ASOs and siRNA)

This includes:

  • antisense oligonucleotides (ASO): short piece of single-stranded RNA designed to bind RNA targets to modulate function—for instance, by modulating mRNA splicing. This is the case for nusinersen, which blocks alternative splicing to treat spinal muscular atrophy (SMA), and tofersen, in clinical trials for ALS.
  • small interfering RNA (siRNA): short piece of double-stranded RNA that binds to mRNA via a special enzyme complex, to inhibit expression. Examples are patisiran and givosiran, which both treat hereditary conditions.

Like all modalities, oligo-based drugs also require in vitro testing, often at scale. Different sequences targeting the same gene may display varying on- and off-target activity. Also, as with mRNA-based drugs, stability and delivery are issues for ASOs and siRNAs, as these molecules tend to accumulate in the liver. To counter this, different sequences, chemical modifications, and conjugates can be screened for potency, stability, specificity, and uptake by the target cell type.  For example, the ASO tofersen was selected as one of the most potent hits in a screen of 2,000 candidate oligos with varying sequences and chemical modifications. Here, a common readout is qPCR or Quantigene-based measurement of gene expression, to directly test both on and off-target effects on mRNA transcript levels.

RNA-targeting Small Molecules

Traditionally, small molecule drug developers have gone after protein targets, but RNA targets are gaining attention. First, non-coding RNAs make up some 70% of the genome, vastly expanding the potential target landscape. Second, coding RNAs open a backdoor to targeting hard-to-drug proteins. Small molecules can interact with 3D RNA structures and modulate multiple RNA-associated mechanisms, ranging from translation to alternative splicing. In addition to qPCR, many of the cellular and biochemical assays and mass spectrometry methods adopted in high-throughput small molecule discovery apply, with minor tweaking for RNA-specific biology. For example, the small molecule SMA drugs risdiplam and branaplam were discovered in screens of 1.4 million and 200,000 compounds, respectively, using cellular reporter assays to detect alternative splicing.