Therapeutics
Chemical modification mRNAs can be stabilised by incorporating natural- ly-occurring modified nucleosides including pseu- douridine, which represents one of the most abun- dant post-transcriptional RNA modifications, and the more recently identified 5’-methyl-cytidine triphosphate (m5CTP), N6-methyl-adenosine-5’- triphosphate (m6ATP), 2-thio-uridine triphosphate (s2UTP), N6-methyladenosine (m6A), and N6,2-O-
dimethyladenosine (m6Am)5,6. In addition, a 5’cap, optimised 3’ poly(A) tail, and 5’- or 3’- untranslated regions can be added or the mRNA can be codon optimised to improve translational efficiency. Modified mRNAs can reduce immunogenicity and increase protein expression levels compared with unmodified mRNA. The most common chemical modifications that have been incorporated to enhance the stability of RNAi and antisense RNA drugs are phosphorothioate RNA backbone modi- fications and ribose modifications including 2’-O- methyl, 2’-fluoro and 2’-O-methylethyl substitu- tions7. These modifications enhance the stability of the RNA drug and provide protection from nucle- ase degradation. Furthermore, the new chemistries confer drug-like properties to RNA, reduce immune stimulation, maximise on-target potency and pro- long the duration of the drug.
Delivery RNA-based therapeutics must be delivered to the target cell and enter the cell to be active8 (Figure 1). Overcoming delivery of RNAs across the lipid bilayer and into cells remains a major challenge3. Furthermore, once internalised, the endocytic pathway – a major cellular active uptake mecha- nism for agents too large to permeate passively – leads to entrapment in the endosome and subse- quent degradation in the lysosome. For example, only 0.1% to 2% of siRNAs evade degradation and reach the RNAi machinery in the cytosol. New in vivo RNA delivery technologies including LNP or PNP systems and the use of aptamer or antibody conjugation have overcome some of the challenges associated with delivery of RNA-based therapeu- tics, with the selection of the delivery system depending on the therapeutics properties, type of target cell and desired delivery route. For example, LNPs tend to end up in the liver, which has been exploited at Alnylam Pharmaceuticals, Dicerna Pharmaceuticals and Arrowhead Pharmaceuticals Inc by attaching N-acetylgalactosamine (GalNAc) to siRNAs to specifically target the hepatic asialo- glycoprotein receptor on liver cells and trigger internalisation. Improving endosome escape is another key step. The most common approaches
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are to use endosomolytic agents such as fusogenic peptides and polymers to enhance endosomal escape of siRNAs7.
Classes of RNA-based therapeutics RNA-based therapies can be classified according to their mechanism of action and include single- stranded mRNAs and antisense RNAs, double- stranded miRNAs and siRNAs, and RNA aptamers (Figure 1). RNA-based therapeutics range in size from thousands of bases for mRNAs down to 8-50 nucleotides for antisense RNAs and 20-25 base pairs for miRNAs and siRNAs.
mRNA IVT mRNA is single-stranded and comprises struc- tural features in common with native mRNA, with its bioavailability being determined by RNase degradation, delivery and cytosolic translocation. IVT mRNAs usually incorporate chemically modi- fied nucleosides such as pseudouridine, which reduce immunogenicity and increase its transla- tional efficiency9. Furthermore, the development of improved formulations, for example the use of LNPs and PNPs, protect IVT mRNAs from RNases and facilitate cellular uptake (Figure 1). IVT mRNA can potentially be used to transient-
ly express proteins to prevent or alter a disease state, with mRNA drugs being developed for can- cer immunotherapies and infectious disease, pro- tein-replacement and regenerative medicine9. mRNA-based protein replacement therapies are used to replace proteins in vivo that are not expressed/expressed at a low level or are non-func- tional using IVT mRNA. mRNA cancer immunotherapy agents are at advanced stages of development (Table 1), with first in man trials under way for mRNA vaccines including Rocapuldencel-T (Argos Therapeutics Inc) and BI- 1361849 (Boehringer Ingelheim GmbH).
Antisense RNA Most current antisense RNAs have been developed from sequences complementary to the target mRNA, and are introduced into cells to reduce or modify expression of the protein upon binding to mRNA to alleviate the symptoms of the disease. Sequence-specific antisense RNAs inhibit gene expression by altering mRNA splicing, arresting mRNA translation and inducing mRNA degrada- tion by ribonucleases (RNase H). Previously, natural antisense RNAs were evaluated for gene silencing, however, their inherent instability led to the develop- ment of modified antisense RNAs that are either more nuclease resistant but still activate RNase H or
Drug Discovery World Fall 2018
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