EP-4739778-A2 - RNA MOLECULE

Abstract

The present invention relates to RNA molecules, and particularly, although not exclusively, to methods for preparing RNA molecules, and to methods for translating RNA molecules into protein. The invention extends to improved methods for forming RNA by in vitro transcription, and to the resultant RNA molecules. Furthermore, the invention relates to novel methods for enhancing the expression and/or translation of RNA, i.e. protein expression, and to methods for improving the stability of an RNA molecule. The invention also involves reducing the activation of innate sensing, interferon generation and/or degradation of an RNA molecule in a host. The invention also incorporates the use of the RNA molecules in vaccines and other therapeutic pharmaceutical compositions, and their use in immunisation and therapy, such as RNAi, gene therapy, gene editing and protein replacement. In particular, the invention relates to locked nucleotides and locked nucleic acid (LNA)-RNA molecules, and the use or incorporation of such locked nucleotides and LNA-RNA molecules in any of the above applications.

Inventors

• SHATTOCK, ROBIN
• NOGAREDA, Laia

Assignees

• Imperial College Innovations Limited

Dates

Publication Date

20260513

Application Date

20240705

Claims (1)

1. Claims 1. One or more locked nucleotide, for use in: (i) enhancing the expression and/or translation of an RNA molecule comprising the one or more locked nucleotide; (ii) enhancing the stability of an RNA molecule comprising the one or more locked nucleotide; and/or (iii) reducing the activation of innate sensing, interferon generation and/or degradation of an RNA molecule comprising the one or more locked nucleotide. 2. One or more locked nucleotide, for use according to claim 1, wherein the RNA molecule is coding RNA, optionally for use in therapeutic applications and vaccine applications. 3. One or more locked nucleotide, for use according to claim 1, wherein the RNA molecule is non-coding RNA, optionally for use in RNAi applications. 4. One or more locked nucleotide, for use according to any preceding claim, wherein the RNA molecule is selected from a group consisting of: messenger RNA (mRNA), micro RNA (miRNA); short interfering RNA (siRNA); short hairpin RNA (shRNA); anti-sense RNA; RNA aptamers; self-amplifying RNA (saRNA); interference RNA (RNAi); non-coding RNA; circular RNA; and small RNA. 5. One or more locked nucleotide, for use according to any preceding claim, wherein the RNA molecule is self-amplifying RNA (saRNA). 6. One or more locked nucleotide, for use according to any preceding claim, wherein the RNA molecule is messenger RNA (mRNA). 7. One or more locked nucleotide, for use according to any preceding claim, wherein the RNA molecule comprises a cap, and the cap does not comprise the one or more locked nucleotide. 8. One or more locked nucleotide, for use according to any preceding claim, wherein the RNA molecule is: (i) at least 20, 21, 22 or 23 bases in length; (ii) at least 28, 29, 30, or 31 bases in (iv) at least 36, 37, 38 or 39 bases in length; (v) at least 46, 47, 48 or 49 bases in length; (vi) at least at least 50 bases, at least 60 bases, at least 75 bases, at least 100 bases, at least 200 bases, or at least 300 bases in length; or (vii) at least 400 bases, at least 500 bases, at least 600 bases, at least 700 bases, at least 800 bases, or at least 900 bases in length. 9. One or more locked nucleotide, for use according to any preceding claim, wherein the RNA molecule is: (i) at least 1000 bases, at least 2000 bases, at least 3000 bases, at least 4000 bases, or at least 5000 bases in length; or (ii), at least 6000 bases, at least 7000 bases, or at least 8000 bases in length. 10. One or more locked nucleotide, for use according to any preceding claim, wherein the one or more locked nucleotide comprises a bridge between the 2’ and 4’ positions in the ribose sugar, optionally wherein the bridge displaces the hydrogen groups from the 2’OH of the ribose and from the 4’ position of the ribose. 11. One or more locked nucleotide, for use according to any preceding claim, wherein the one or more locked nucleotide is represented herein by formula [I]: wherein Base is a nucleobase; L is a C 1-5 alkylene; X 1 is O, CH2, CO, NR 5 , Se, S, SO or SO2; X 2 is absent, or is CO, O, NR 5 , Se, S, SO or SO2; one of R 1 and R 2 is a phosphate linker, a modified phosphate linker or a 5 to 10 membered heteroarylene, wherein the phosphate linker, the modified phosphate linker or the 5 to 10 membered heteroarylene is a linker between the rest of the locked nucleotide of formula [I] and an attachment point to a first adjacent nucleotide in the RNA molecule, and the other of R 1 and bond to a second adjacent nucleotide in the RNA molecule; R 3 and R 4 are independently H or C 1-6 alkyl; and . or more for use according to any preceding claim, wherein tthe one or more locked nucleotide is represented herein as formula [Ia]: 13. One or more locked use any wherein the nucleobase is a primary nucleobase or a modified nucleobase, optionally wherein the primary nucleobase is a purine or a pyrimidine; and/or the modified nucleobase has the structure of a primary nucleobase with one or more modifications, which is selected from: - replacing a hydrogen and/or an NH2 group in the primary nucleobase with an optionally substituted C 1-6 alkyl, an optionally substituted C 2-6 alkenyl, an optionally substituted C2-6 alkynyl, OR 15 , NR 15 R 16 , COOR 15 , CONR 15 R 16 or a halogen; - replacing a double bond with a single bond; - replacing a CH in the structure with an N; - replacing an N in the structure with a CR 15 ; - replacing an O in the structure with an NR 15 , S, Se or CR 15 R 16 ; and/or - the nucleobase is attached to the rest of the locked nucleotide of formula [I] at a different point to the point where the primary nucleobase would typically be attached; wherein R 15 and R 16 are independently H, an optionally substituted C1-6 alkyl, an optionally substituted C 1-6 alkenyl, an optionally substituted C 1-6 alkynyl or an optionally substituted C6-12 aryl, or R 15 and R 16 together with the atom to which they are bonded for an optionally substituted 3 to 10 membered cycloalkyl or an optionally substituted 3 to 10 membered heterocyclyl. 14. One or more locked nucleotide, for use according to any preceding claim, a bond, R 3 is H, R 4 is H, X 1 is O, X 2 is absent and L is CH2, adenine, cytosine, 5-methyl-cytosine, guanine, uracil or 15. One or more locked nucleotide, for use according to any preceding claim, wherein the use comprises using one or more locked nucleotide selected from a group consisting of: locked adenine; locked cytosine; 5-methyl-cytosine; locked guanine; locked uracil and locked thymine. 16. One or more locked nucleotide, for use according to any preceding claim, wherein the RNA molecule further comprises one or more modified nucleotide comprising a 2’-substituted group in which the OH group normally at the 2’ position is replaced with a halogen (preferably fluorine), an optionally substituted aromatic group, a NH2, a N3, a H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, wherein in each instance the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR, CN, NR2 or SR, wherein R is H or C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl. 17. One or more locked nucleotide, for use according to claim 16, wherein the one or more modified nucleotide is represented herein by formula [II]: wherein Base is a nucleobase; one of R 6 and R 8 is a phosphate a or a membered heteroarylene, wherein the phosphate linker, the modified phosphate linker or the 5 to 10 membered heteroarylene is a linker between the rest of the locked nucleotide of formula [I] and an attachment point to a first adjacent nucleotide in the RNA molecule, and the other of R 6 and R 8 is a bond to a second adjacent nucleotide in the RNA molecule; R 7 and R 10 are independently H or C1-6 alkyl; R 9 is a halogen (preferably fluorine), an substituted aromatic group, a NH2, a N3, a H, an optionally substituted O-alkyl, O-alkenyl or O-alkynyl group, or an optionally substituted alkyl, alkenyl or alkynyl group, wherein in each instance the aromatic group, alkyl, alkenyl or alkynyl is optionally substituted with halogen, oxo, OR 11 , CN, NR 11 R 12 or SR 11 ; and R 11 and R 12 are independently H or C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl. 18. One or more locked nucleotide, for use according to either claim 16 or 17, wherein the one or more modified nucleotide is represented herein by formula [IIa]: [IIa] 19. One or more locked nucleotide, for use according to any one of claims 16-18, wherein R 9 is a halogen, optionally chl or bromine, and preferably wherein R 9 is fluorine. 20. One or more locked nucleotide, for use according to any one of claims 16-19, wherein R 9 is Me or OMe, preferably OMe. 21. One or more locked nucleotide, for use according to any one of claims 16-20, wherein the use comprises using at least one modified nucleotide selected from a group consisting of: a modified adenine; a modified cytosine; a modified guanine; a modified uracil and/or a modified thymine. 22. One or more locked nucleotide, for use according to any preceding claim, wherein the use or method of the invention comprises using a combination of: (i) one or more locked nucleotide represented by formula [I]; and (ii) one or more modified nucleotide represented by formula [II]. 23. One or more locked nucleotide, for use according to any preceding claim, wherein the use comprises using a combination of: (i) one or more locked nucleotide by formula [I], wherein R 1 is , R 2 is a bond, R 3 is H, R 4 is H, X 1 is O, X 2 is absent and L is CH2, and the base is adenine, cytosine, 5-methyl-cytosine, guanine, uracil or thymine; and (ii) one or more modified nucleotide represented by formula [II], wherein R 6 is , R 7 is hydrogen, R 8 is a bond, R 9 is a halogen (preferably fluorine), R 10 is hydrogen, and the base is adenine, cytosine, 5-methyl-cytosine, guanine, uracil or thymine. 24. One or more locked nucleotide, for use according to any preceding claim, wherein (i) at least 0.01%, 0.02%, 0.03%, 0.04% or 0.05% of the constituent nucleotides in the resultant LNA-RNA molecule comprise locked nucleotides; (ii) at least 0.06%, 0.07%, 0.08%, 0.09% or 0.10% of the constituent nucleotides in the resultant LNA-RNA molecule comprise locked nucleotides; or (iii) at least 0.15%, 0.2%, 0.3%, 0.4% or 0.5% of the constituent nucleotides in the resultant LNA-RNA molecule comprise locked nucleotides, optionally wherein the locked nucleotides comprise locked adenine, locked cytosine, locked guanine, locked thymine and/or locked uracil. 25. A method of preparing a locked nucleic acid RNA (LNA-RNA) molecule, wherein the method comprises contacting, in the presence of at least 20mM magnesium ions, (i) a template nucleic acid sequence, (ii) an RNA polymerase, and (iii) a plurality of nucleotide triphosphates (NTPs), one or more of which comprises a locked nucleotide, wherein the RNA polymerase transcribes the template nucleic acid sequence to form a LNA-RNA molecule comprising at least 25 nucleotides. 26. Use of 20mM magnesium ions in a transcription reaction to prepare a locked nucleic acid RNA (LNA-RNA) molecule comprising at least 25 nucleotides. 27. A method according to claim 25 or a use according to claim 26, wherein the method comprises the use of: (i) at least 25mM magnesium ions, at magnesium ions, at least 35mM magnesium ions, at least 40mM magnesium ions, or at least 5omM magnesium ions; (ii) at least 60mM magnesium ions, at least 70mM magnesium ions; (iii) at least 75mM magnesium ions, at least 80mM magnesium ions, at least 85mM magnesium ions, at least 90mM magnesium ions, or at least 100mM magnesium ions; or (iv) less than 120mM magnesium ions, less than 100mM magnesium ions, less than 90mM magnesium ions, less than 80mM magnesium ions, or less than 75mM magnesium ions. 28. A method or use according to any one of claims 25-27, wherein the magnesium ions are provided as magnesium acetate, magnesium chloride, magnesium citrate, magnesium sulphate, magnesium gluconate, or magnesium lactate. 29. A method or use according to any one of claims 25-28, wherein the magnesium ions are provided as magnesium acetate. 30. A method or use according to any one of claims 25-29, wherein the method comprises the use of an RNA polymerase, which is selected from a group consisting of: T7; T3; SP6; KP34; Syn5; or other DNA-dependent RNA polymerase; or a mutated variant of any of these RNA polymerases. 31. A method or use according to any one of claims 25-30, wherein the plurality of nucleotide triphosphates (NTPs) are selected from the group consisting of ATP, GTP, CTP, TTP and/or UTP. 32. A method or use according to any one of claims 25-31, wherein the method comprises the use of the plurality of nucleotide triphosphates at a concentration of: (i) at least 1mM, 2mM, 3mM or 4mM; (ii) at least 5mM, 6mM or 7mM; or (iii) at least 8mM, 9mM, 10mM. 33. A method or use according to any one of claims 25-32, wherein the template nucleic acid sequence comprises DNA, optionally a vector, preferably a plasmid. 34. A method or use according to of claims 25-33, wherein the template nucleic acid sequence encodes an antigen which is derived from a tumour, virus, a bacteria, a mycoplasma, a fungus, an animal, a plant, an alga, a parasite, or a protozoan, or other organism which causes a disease in a subject, preferably a human or animal. 35. A method or use according to any one of claims 25-34, wherein the template nucleic acid encodes a therapeutic protein, which is derived from an animal or a human, and which treats, prevents or ameliorates disease in a subject, preferably a human or animal subject. 36. A method or use according to any one of claims 25-35, wherein the template nucleic acid encodes an innate inhibitor protein, which counteracts the innate immune response in a subject administered with a vaccine comprising the RNA molecule. 37. An RNA molecule obtained or obtainable by the method according to any one of claims 25-36. 38. An RNA molecule comprising at least 25 nucleotides, wherein one or more of the nucleotides is a locked nucleotide. 39. An RNA molecule according to claim 38, wherein the RNA molecule is as defined in any one of claims 4-9 and/or the locked nucleotide is as defined in any one of claims 10-24. 40. A pharmaceutical composition comprising the RNA molecule according to any one of claims 37-39, and a pharmaceutically acceptable vehicle. 41. A method of preparing the pharmaceutical composition according to claim 40, the method comprising contacting the RNA molecule according to any one of claims 37- 39and a pharmaceutically acceptable vehicle. 42. The RNA molecule according to any one of claims 37-39, or the pharmaceutical composition according to claim 40, for use as a medicament. 43. The RNA molecule according to of claims 37-39, or the pharmaceutical composition according to claim 40, for use in treating, preventing or ameliorating a disease in a subject. 44. A vaccine composition comprising the RNA molecule according to any one of claims 37-39, or the pharmaceutical composition according to claim 40. 45. The RNA molecule according to any one of claims 37-39, the pharmaceutical composition according to claim 40, or the vaccine according to claim 44, for use in stimulating an immune response in a subject.

Description

RNA molecule The present invention relates to RNA molecules, and particularly, although not exclusively, to methods for preparing RNA molecules, and to methods for translating RNA molecules into protein. The invention extends to improved methods for forming RNA by in vitro transcription, and to the resultant RNA molecules. Furthermore, the invention relates to novel methods for enhancing the expression and/or translation of RNA, i.e. protein expression, and to methods for improving the stability of an RNA molecule. The invention also involves reducing the activation of innate sensing, interferon generation and/or degradation of an RNA molecule in a host. The invention also incorporates the use of the RNA molecules in vaccines and other therapeutic pharmaceutical compositions, and their use in immunisation and therapy, such as RNAi, gene therapy, gene editing and protein replacement. In particular, the invention relates to locked nucleotides and locked nucleic acid (LNA)-RNA molecules, and the use or incorporation of such locked nucleotides and LNA-RNA molecules in any of the above applications. Locked nucleotides and corresponding locked nucleic acids (LNA) are a modified version of RNA nucleotides where the ribose moiety is modified by a bridge between the 2’ oxygen and 4’ carbon, displacing the hydrogen group from the 2’OH of ribose. First synthesised in the late 1990’s [1,2], this locks the ribose in a 3’-endo (North) conformation, reducing the conformational flexibility of the ribose, and increasing the local organization of the phosphate backbone. As a result, the ribose ring is "locked" in an ideal conformation for Watson-Crick binding. For locked RNA improved hydrophobic interactions between bases increases base staking, an unwinding of the LNA helix relative to RNA, widening of the major groove with an enlarged helical pitch [3-5]. For each incorporated LNA monomer in an oligonucleotide, the melting temperature (Tm) of a duplex increases by 2–8°C, stabilising double stranded LNA- RNA sequences [6, 7]. Such changes in structure likely influence recognition of RNA binding proteins associated with innate RNA recognition. The first LNA compounds, introduced in the late 1990s, are bicyclic nucleotide analogues in which the furanose ring is modified by the introduction of a methylene group linking the 2’-oxygen and the 4’-carbon (2’-O,4’-methylene-d-ribofuranosyl nucleotides) [2,9]. Subsequent derivatives of LNA, include 2’-O,4’-aminoethylene bridged nucleic, 2’-O,4’-C-ethylene-bridged nucleic acid (ENA), 2’-O,4’-C- methylenoxymethylene-bridged nucleic 2'-N-methanesulfonyl-2'-amino-locked nucleic acid (see Figure 1) [8]. When used to make nucleic acid sequences, these are referred to as “locked nucleic acid” (LNA) or “bridged nucleic acid” (BNA), and sometimes inaccessible RNA [9]. The abbreviation LNA is used interchangeably to indicate locked nucleic acid nucleotides (LNA-NTs), and Locked RNA (LNA-RNA) or Locked DNA (LNA-DNA). Locked NTs and LNAs have been widely used to stabilise short oligonucleotides sequences generated by synthetic synthesis [9]. The inclusion of LNA-NTs is reported to increase oligonucleotide stability, resistance to ribonucleases and reduce innate recognition by cellular RNA binding proteins. The improved stability is thought to be mediated by the locking of the 2’ oxygen, removing its nucleophilic potential to hydrolyse the phosphate linkage between RNA nucleotides [10,11]. The increased resistance to ribonucleases and reduced innate recognition may be mediated by changes in NT/oligonucleotide structure reducing recognition by ribonuclease and innate sensing proteins [3-5, 12, 13]. In many extracellular fluids, RNA degradation is primarily mediated by endoribonucleases that cleave RNAs at certain single-stranded dinucleotide motifs (UA/UA, CA/UG) [14] likely exposed by random thermal fluctuations. LNA modifications of these motifs can effectively enhance RNA nuclease resistance [15-20] while wider LNA modification may enhance nuclease resistance in a sequence independent manner as enhancement of thermostability will make exposure of single stranded motifs to RNase less likely [21]. LNA nucleotides are often used together with other RNA and DNA NTs when synthesised as short oligonucleotides as “miximers” or “gapmers” to fine tune anti-sense function [9]. Although it has not been previously possible to generate LNA-RNA sequences >100 NTs, it can be assumed that LNA-NTs would provide similar properties to longer RNA sequences could they be generated. RNA is composed by the nucleotides ATP, CTP, GTP and UTP, however, a wide range of natural and synthetic modified nucleotides exist [29]. TTP is associated with DNA but has also been used in the generation of locked oligonucleotides. Furthermore, 5meCTP is often used in locked oligonucleotides as it is thought to improve complex stability due to enhanced stacking [30]. Importantly, however, LNAs have never been assessed for their abil