Therapeutics Current t rends in RNA- based t hera peutic devel op ment Cellular RNAs play crucial roles during disease progression and represent a diverse and largely untapped class of biomolecules that can be exploited for drug development. By Dr Xiaoqiu Wu NA species include messenger RNAs l Antisense RNAs, or RNA interference (RNAi) and Dr Andrew P. (mRNAs) that are translated into proteins, via miRNAs and siRNAs, to partially or complete - Turnbull Rlong non-coding RNAs including transfer ly turn off gene expression. RNAs (tRNAs) and ribosomal RNAs (rRNAs), and l RNA aptamers, or ‘chemical antibodies’, which small non-coding RNAs such as micro RNAs bind to specific molecular targets and can act as (miRNAs) and small interfering RNAs (siRNAs). drug carriers to deliver small-molecule chemother - Exploiting RNA species as therapeutic agents offers apeutics, siRNAs, miRNAs or nanoparticles into new opportunities for drug developers, and the pos - targeted tissues. sibility to develop agents against ‘undruggable’ genes and gene products (for a comprehensive review on These efforts have led to the therapeutic poten - RNA-targeted therapeutics, please refer to reference tial of RNA drugs being realised 2, with the RNA 1). Furthermore, new screening tools now make it aptamer – pegaptanib (brand name Macugen) – easier to target disease-associated RNA sequences. representing the first FDA approval for an RNA- However, developing RNA-based therapeutics is not based drug in 2004. Since then, two antisense without its challenges since RNA is inherently unsta - RNAs – nusinersen (Spinraza) and eteplirsen ble and prone to degradation by active and abundant (Exondys 51) – and one siRNA drug – patisiran ribonucleases (RNases), is potentially immunogenic (Onpattro) – have gained FDA approval ( Table and may require a delivery vehicle for efficient and 1). As of July 2018, 69 companies have mRNA, specific transport to target cells and across the lipid antisense RNA, RNAi or RNA aptamer therapeu - bilayer. These development hurdles have largely been tics in clinical development with 315 ongoing overcome by chemically modifying RNA to enhance clinical trials (data provided by GlobalData Plc; its stability, and by employing synthetic carriers such https://www.globaldata.com/ ). Furthermore, sev - as lipid nanoparticle (LNP) or polymer-based eral strategic collaborations and partnerships nanoparticle (PNP) systems for RNA drug delivery. have been forged between big Pharma and RNA drug development efforts have primarily Biotech companies to leverage proprietary tech - focused on four modalities: nology platforms. For example, Arbutus Biopharma Corporation, which has proprietary l mRNA vaccines for cancer and infectious disease. LNP and ligand-conjugate delivery technologies, l In vitro transcribed (IVT) mRNAs to replace or recently entered into an agreement with Roivant supplement proteins. Sciences to launch Genevant Sciences. New 16 Drug Discovery World Fall 2018 Therapeutics Table 1: RNA-based therapeutics in clinical development. RNA drugs in five or more clinical trials as of July 2018 are tabulated. Data provided by GlobalData Plc Cl ass Co mpany Dr ug Th erapeutic In dication Cl inical De velopment (Bra nd Nam e) Area Tri als Sta ge (! 5) mR NA Ar gos Ro capuldencel-T On cology Mu scle Invasive Bladder Cancer (MI BC); Non- 9 Ph ase II Th erapeutics Inc Sm all Cell Lu ng Cancer; Renal Cell Carcinoma AGS -004 In fe ctious Hu ma n Immu nodeficiency Vi rus (HI V) 6 Ph ase II Dis ease In fe ctions (A ID S) eT heR NA Tr iMix-ba sed On cology Me lanoma; Mu ltiple My eloma (Kahler Disease) 6 Ph ase II Im munothera pies im munoth erapy NV Bo ehringer BI -1361849 On cology No n-Sm all Cell Lu ng Cancer 5 Ph ase II In gelheim GmbH An tisense Bi ogen Inc Nu sinersen Ce ntral Ner vous Sp inal Muscular Atrophy (SM A) 13 Ma rketed RNA (S pinra za) Sys tem Ak cea Vo lanesorsen Me tabolic Fa milial Ch ylomicronemia (Type I 13 Pr e-re gistra tion Th erapeutics Inc so dium Dis orders Hy perlipoproteinemi a) Sa repta Et eplirsen Ge netic Du chenne Muscular Dy strophy 12 Ma rketed Th erapeutics Inc (E xondys 51) Dis orders An tisense AT L1102 Ce ntral Ner vous Re lapsing Re mitting Mu ltiple Sclerosis (RRMS ); 8 Ph ase II Th erapeutics Lt d Sys tem Se condary Pr ogressive Multiple Sc lerosis (S PMS) Ph armaxis Lt d AS M-8 Re spiratory Al lergic As thma 6 Ph ase II si RNA Th e Medicines In clisira n Ca rdiovascular At herosclerosis; Ca rdiovascular Disease 11 Ph ase III Co mpany Met ab olic Ho mo zygous Fami lial Dis orders Al nylam Pa tisiran Me tabolic He reditary transthyretin-me diated amy loidosis 8 Ma rketed Ph armaceuticals (O npattro ) Dis orders (h ATTR) In c RX i RX I-109 De rma tology Hy pertrophic Scars 8 Ph ase II Ph armaceuticals Co rp Opht ha lmol ogy We t (Neovascular/Exudative) Ma cular De generation Al nylam Fi tusiran Haem at ological He mo philia A; He mo philia B 7 Ph ase III Ph armaceuticals Dis orders In c Ce mdisiran Ga strointestina At ypical He mo lytic Ur emi c Syndrome 7 Ph ase II (N ondiarrh ea- As sociated He mo lytic Ur emi c Sy ndrome) Haem at ological Pa roxysmal Nocturnal Haem oglobinuria Dis orders Qu ark QP I-1002 Ge nito Uri nary Ac ute Renal Failure (AR F) (Ac ute Ki dney 6 Ph ase III Ph armaceuticals Sys tem And In jury ) In c Sex Hor mone s Immu nology Ki dney Transplant Rejection Br istol-My ers BM S-986263 Ga strointestinal Li ver Fi brosis 6 Ph ase II Sq uibb Co Pf izer Inc PF -655 Me tabolic Di abetic Macular Edema 5 Ph ase II Dis orders Opht ha lmol ogy We t (Neovascular/Exudative) Ma cular De generation RNA Pf izer/Valeant Pe gaptanib Op hthalmo logy Ag e-Rel at ed Macu lar Deg en er at ion (A MD) 38 Ma rketed Ph armaceuticals (M acugen) Apt amer In tern ational In c Op hthotech Corp Av acincaptad Op hthalmo logy Dr y (Atr ophic ) Macu lar Deg en er at ion 11 Ph ase III pe gol sodi um (Z imura ) No xxon Pharma Ol aptesed pegol On cology B-Cel l Chr oni c Lym phoc yt ic Leu kem ia 11 Ph ase II AG Em apticap pegol On cology So ld Tum our 5 Ph ase II Met ab olic Dia betic Nep hropat hy Dis orders modalities to target RNA are also being devel - Development hurdles oped including the application of CRISPR-Cas9 Despite the potential of RNA therapeutics, efficient genome editing technology and the development ! and safe delivery remains a significant challenge. ! of selective small-molecule modulators of RNA or There are a number of significant issues that need to RNA-modifying enzymes. The global RNA drugs be overcome in their development: instability and market is forecast to exceed $10 billion by 2024 immunogenicity; rapid clearance from the blood by (based on an analysis carried out using the the kidneys and liver scavenger receptors; cellular GlobalData Plc database), highlighting the signif - uptake and endosomal escape 3. These hurdles can icant commercial potential of this emerging class be overcome by chemically modifying RNA and by of therapeutics. using improved synthetic delivery carriers 4. Drug Discovery World Fall 2018 17 Therapeutics Chemical modification are to use endosomolytic agents such as fusogenic mRNAs can be stabilised by incorporating natural - peptides and polymers to enhance endosomal ly-occurring modified nucleosides including pseu - escape of siRNAs 7. douridine, which represents one of the most abun - dant post-transcriptional RNA modifications, and Classes of RNA-based therapeutics the more recently identified 5’-methyl-cytidine RNA-based therapies can be classified according to triphosphate (m 5CTP), N 6-methyl-adenosine-5’- their mechanism of action and include single- triphosphate (m 6ATP), 2-thio-uridine triphosphate stranded mRNAs and antisense RNAs, double- (s2UTP), N 6-methyladenosine (m 6A), and N 6,2-O- stranded miRNAs and siRNAs, and RNA 6 5,6 dimethyladenosine (m Am) . In addition, a 5’cap, aptamers ( Figure 1 ). RNA-based therapeutics optimised 3’ poly(A) tail, and 5’- or 3’- untranslated range in size from thousands of bases for mRNAs regions can be added or the mRNA can be codon down to 8-50 nucleotides for antisense RNAs and optimised to improve translational efficiency. 20-25 base pairs for miRNAs and siRNAs. Modified mRNAs can reduce immunogenicity and increase protein expression levels compared with mRNA unmodified mRNA. The most common chemical IVT mRNA is single-stranded and comprises struc - modifications that have been incorporated to tural features in common with native mRNA, with enhance the stability of RNAi and antisense RNA its bioavailability being determined by RNase drugs are phosphorothioate RNA backbone modi - degradation, delivery and cytosolic translocation. fications and ribose modifications including 2’-O- IVT mRNAs usually incorporate chemically modi - methyl, 2’-fluoro and 2’-O-methylethyl substitu - fied nucleosides such as pseudouridine, which tions 7. These modifications enhance the stability of reduce immunogenicity and increase its transla - the RNA drug and provide protection from nucle - tional efficiency 9. Furthermore, the development ase degradation. Furthermore, the new chemistries of improved formulations, for example the use of confer drug-like properties to RNA, reduce immune LNPs and PNPs, protect IVT mRNAs from stimulation, maximise on-target potency and pro - RNases and facilitate cellular uptake ( Figure 1 ). long the duration of the drug. IVT mRNA can potentially be used to transient - ly express proteins to prevent or alter a disease Delivery state, with mRNA drugs being developed for can - RNA-based therapeutics must be delivered to the cer immunotherapies and infectious disease, pro - target cell and enter the cell to be active 8 (Figure tein-replacement and regenerative medicine 9.