RNA Drugs


Small RNA: MicroRNA (miRNA) Therapeutics

MicroRNA (miRNA) is a single-stranded small RNA of 21-25 nt, which is cleaved from a 70 nt single-stranded, intramolecular hairpin RNA precursor e.g. by the cytosolic DICER  nuclease. The primarily double-stranded cleavage product is finally processed to a single-stranded microRNA (miRNA) molecule, which seems to exhibit both post-transcriptional gene silencing, and activation. For example, the microRNA-122 activates Hepatitis C RNA genome translation, and is therefore selected as a target to develop antagomirs (Santaris Pharma A/S, Regulus Pharmaceuticals Inc, Merck & Co, …) for hepatitis C treatment.

MicroRNA research is currently focussed on the discovery of organ-specific expression profiles by next generation sequencing techniques. Some Biotech companies licenced microRNA (miRNA) molecules for the in vitro and in vivo therapeutic application from academic R&D, the current pipeline of microRNA (miRNA) targets both as a mimic or an antagomir can be found here.

There´s also a recent post on Antagomirs, which you can find on our microRNA blog.


Small RNA: Short Interfering RNA (siRNA)

Small interfering RNA (siRNA) is a double-stranded RNA of 19-23 nt, which is cleaved from a short hairpin RNA (shRNA) by the cytosolic DICER endonuclease. Some Biotech companies filed IP on the use of siRNA molecules as a chemical  compound for therapeutic applications:

1. Silence Therapeutics, formerly known as Atugen AG before the company was merged with SR Pharma. A strong IP on the company´s propietary 2´-O-methyl modified small interfering RNA (siRNA) molecules generated revenues e.g. from Quark Pharmaceuticals Inc. Silence Therapeutics currently runs a Phase 1 study on cancer patients with liposomal formulated siRNA targeting PKN3.

2. Alnylam Pharmaceuticals


Small RNA: Piwi-Interacting RNA (piRNA)

Small piwi-interacting RNAs (piRNAs) are internally expressed small RNAs, which are a little bit longer than siRNA or microRNA (miRNA): up to 31 nt rather than 23 nt and below. At the same time, piRNAs represent the largest class of small RNA expressed in vivo for post-transcriptional gene silencing. In mammals, piRNAs are most-predominantely found in the testis, piRNAs also appear to have a major role in spermatogenesis. The bioactivity of piRNAs is again mediated by members of the Argonaute protein family, whose PIWI domain interacts with these small RNAs.

The typical small piwi-interacting RNA (piRNA) bear a 2´-O-methyl modification at its 3´-end, preventing the tailoring or trimming of small RNA after binding to the ribonuclease Ago2 (1). Scientists of New England Biolabs (NEB) demonstrated this 3´-end modification also helps the piRNA to escape from commonly used cloning techniques like poly-(A)-tailing, or RNA ligase mediated adaptor addition (2).

To our knowledge so far, no Biotech company is thinking of an therapeutic agent targeting a piRNA, neither as a mimic nor as an antagomir.


(1) Ameres SL et al (2010), Science 328: 1543-9.
(2) Munafó DB & Robb GB (2010), RNA 16: 2537-52.


Small RNA: Short Hairpin RNA (shRNA)

Small hairpin ribonucleic acid (shRNA) is an alternative to the chemically synthetized small interfering RNA (siRNA) therapeutic approach. These small RNA molecules utilizes basically the same machinery for post-transcriptional gene silencing like double-stranded siRNA. A small single-stranded RNA molecule forms an intra-molecular double-stranded hairpin, which is cleaved by intracellular nucleases, e.g. like DICER, leading to a double-stranded siRNA ready for RISK-mediated transkript knockdown. Some Biotech companies filed IP on the in vitro and in vivo expression of short hairpin RNA (shRNA) for therapeutic applications:

1. Marina Biotech utilizes non-pathogenic Escherichia coli vectors for the oral delivery of shRNA (short hairpin RNA) for mRNA knockdown of the beta-catenin mRNA in epithelial cells. Marina Biotech is currently recruiting patients for a clinical Phase 1b/2a study (1).

2. Australian Benitec Ltd uses lentiviral vectors to introduce short hairpin RNA (shRNA) genes into the host genome, which should lead to a stable expression of shRNA in the target cell/organ (2).


(1) Marina Biotech Press Release, dated April 20, 2011.
The company was formerly known as MDRNA Inc., and was renamed to Marina in 2010.
(2) Gu W et al (2011), Cancer Gene Therapy 18: 219-27.


In Vitro Messenger RNA Therapeutics for Gene Transfer

Vaccination via Messenger RNA
CureVac GmbH, Tübingen, Germany

Gene Transfer via Messenger RNA

Another biotechnology company (Ethris GmbH, Germany) applied naked, chemically stabilized transcripts to mouse models (1). The corresponding patent basically reflects the same data (2). Biostability seems to be enhanced by 2-thiouridine and 5-methylcytidine incorporation into the in vitro transcripts. 2-thiouridine is a naturally occuring nucleoside with enhancing hybridization properties. It was recently applied to siRNA synthesis for post-transcriptional gene silencing.

Animal treatment material (ATM) was produced by in vitro transcription, and varied in length from 0.7 to 2.4 kb (endotoxin testing was not performed, or at least it was not published).

The following doses were shown to be effective in mouse models (1):
- 1.5 mg/kg (single 0.05 mg intramuscularly injection of naked RNA);
- 0.3-0.6 mg/kg (single or twice weekly 0.01 or 0.02 mg intratracheally microspray of naked RNA);
- 0.14 mg/kg (single 0.005 mg intravenously bolus injection of liposomal complexed RNA)

In vivo pharmacokinetic analysis of the transcript was not performed so far.

In vivo pharmacodynamic analysis comprised:
- quantification of translation product encoded by the transcript drug (ELISA, Western);
- immune response (interferon-gamma, interleukin-12, interferon-alpha ELISA)

The authors applied an interesting immunoprecipitation assay to detect transcripts bound to a couple of Toll-like receptors, but they forgot to mention how the quantitative RT-qPCR assay was performed (1). Typically Nature editors do not pay too much attention to the completeness of the methods section. Lucky we are there is at least the patent ([2], see section [0124] for details on RT-qPCR). This also means that the transcript´s chemical modification claimed in the patent (2-thiouridine and 5-methylcytidine) obviously can be reverse transcribed, e.g. for pharmacokinetic analysis.

(1) Kormann MSD et al (2011), Nature Biotech 29(2): 154-7.
(2) Rudolph C and Kormann MSD (2011), Patent WO 2011012316 (A2).




RNA Aptamer Therapeutics

coming soon, please be patient

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