The idea to use RNA oligos as a therapeutic has been around since the late 1990s, however there is now an explosion of renewed interest. The Wall Street Journal‘s exclusive billion dollar club includes the RNA therapy companies Moderna and CureVac along side the likes of Uber and Airbnb.

Some of the more memorable funding announcements and deals of late include:

  • DARPA’s  €33.1M sponsorship of a CureVac, Sanofi Pasteur, and In-Cell-Art collaboration in 2011
  • AstraZeneca’s $28M collaboration with Regulus Therapeutics in April 2012
  • AstraZeneca’s $240M deal with Moderna Therapeutics in March 2013
  • DARPA’s $25M grant to Moderna Therapeutics in October 2013
  • Sanofi’s $700M deal with Alnylam Pharmaceuticals in January 2014
  • Roche’s $250M purchase of Santaris in August 2014
  • Riboxx’s €600K crowdfunding round in September 2014
  • BioMarin’s $840M purchase of Prosensa in November 2014
  • Cystic Fibrosis Foundation Therapeutics $15M funding of Shire
  • Moderna’s $450M January 2015 funding round.
  • Janssen’s agreement to pay Isis Pharma $35 million upfront for RNA-targeted technology
  • Bill and Melinda Gates Foundation’s €46M funding of CureVac in March 2015
  • Silence Therapeutics raised £38.9M by selling two “tranches” of new shares in April 2015
  • Horizon2020 €2.5M  funding of the project TRIGGDRUG in June 2015

RNA Therapy:  What is it? 

Using RNA molecules directly as a therapy rather than delivering DNA or protein into a cell has many potential advantages. While DNA as a therapy comes with the complications of delivering a long sequence (i.e. including promoter) into the nucleus and the potential for genomic integration, RNA molecules can be produced as a pure substance, chemically or in vitro under GMP conditions and need only be delivered to the cytosol. RNA molecules could be used for RNA interference (RNAi) to suppress a gene or just the disease-causing mRNA variant of a gene or RNA molecules could be functional mRNAs encoding proteins for use in applications such as vaccine delivery or replacement therapy. The very exciting field of genome editing with CRISPR/Cas9 is closely tied to RNA therapy as this technology requires delivery of the Cas9 protein together with sgRNA into cells. The therapeutic potential of CRISPR/Cas9 therefore seems to rely on RNA therapy becoming a viable therapy.

RNA-comparedto-DNA_thymineAndUracilCorrectedImage source

So what has changed? The rebirth of RNA Therapy

But haven’t we seen all of this before? In 2007, for example Roche spent $274M to license Alnylam’s RNAi intellectual property only to shut down their RNA operations in 2011. A recent (and cleverly titled) review of the RNA therapy field entitled Evading innate immunity in nonviral mRNA delivery: don’t shoot the messenger gives a nice summary of some of the major innovations in the field that have made this rebirth possible. The key points highlighted are that:

  1. In vitro transcribed mRNAs activate the innate immune response including toll-like receptors (e.g. TLR3, TLR7) and RIG-I like receptors. These pattern recognition receptors (PRRs) presumably interpret the RNA as foreign/viral and lead to activation of NF-κB and interferon pathways and therapeutically dangerous cytokine storms. Indeed some companies are betting on this phenomenon and are proposing mRNA as a vaccine that is its own adjuvant.
  2. RNAs produced with various combinations of chemically-modified nucleosides (pseudouridine5-methylcytidine, N6-methyladenosine, 5-methyluridine, or 2-thioruridine) instead of (or in addition to) the standard A,U,C,G can reduce this innate immune response, and increase mRNA stability and translation efficiency. Much of this work was originally published by Katalin Karikó and Michael Kormann.
  3. In vitro RNA primary transcripts carry a 5′ triphosphate (pppRNA), and in vitro capping, even with ARCA, is not 100% efficient. pppRNA will activate the RIG-I and PKR pathways. Often it is sufficient to dephosphorylate pppRNA with RNA 5′ Polyphosphatase to prevent this innate immune response.

pseudouridineStructure of Pseudouridine-5′-Triphosphate

Some of the work highlighted in the review is that of Derrick Rossi’s group demonstrating the ability of chemically-synthesized RNA made with pseudouridine and 5-methylcytidine to be used for reprogramming of human cells to pluripotent without activating the innate immune response. Another interesting paper highlighting much of these innovations has been published by CureVac. The company indicates that in addition to use of modified nucleosides pseudouridine and 5-methylcytidine, optimization of sequence is also important. Clearly different companies will make their bets on different strategies/technologies/patents in this exciting new market of modified RNA therapy.

5'_cap_structuremRNA cap structure. Image source.

How to get started in RNA therapy?

Whether you are interested in making a functional siRNA, an expressing mRNA or an sgRNA for CRISPR/Cas9, you might be able to discover the perfect combinations of modifications to give your molecule an advantage over other strategies. An easy entry point is the vast collection of catalog mRNAs carrying pseudouridine and 5-methylcytidine modifications commercially available. These mRNAs have sequence optimized 5′ and 3′ untranslated regions (UTRs) are capped and polyadenylated and encode commonly expressed proteins as induced pluripotent stem cell programming factors (Oct4Klf4SOX2c-MycLin28), reporter genes (EGFPYFPbeta-galluciferasemCherry and hAAT), EPO, and Cas9 carrying a nuclear localization signal. This NLS-Cas9 5meC/Ψ mRNA, similar to NLS-Cre recombinase mRNA will be translated into a protein targeted to the nucleus of cells for genome editing. Other catalog mRNAs available or at least their encoded proteins (TRP-2 and gp100) are intended for the opposite effect: to induce an immune response.

Once initial experimental setup is established to optimize transfection efficiency, for example, custom RNA synthesis can be performed by the precise same standards as were used to produce these catalog modified mRNAs. The modified nucleic acid experts at TriLink Biotech have even established an easy path to GMP production of chemically modified oligonucleotides for use in early stage clinical trials, so the R&D discoveries made can be efficiently translated into therapeutics for clinical trial use.