EP-4103577-B1 - NOVEL MRNA 5'-END CAP ANALOGS, RNA MOLECULE INCORPORATING THE SAME, USES THEREOF AND METHOD OF SYNTHESIZING RNA MOLECULE OR PEPTIDE

Inventors

• WARMINSKI, Macin
• SIKORSKI, Pawel
• KOWALSKA, JOANNA
• JEMIELITY, JACEK

Dates

Publication Date

20260506

Application Date

20210212

Claims (11)

1. A compound of formula I: wherein: R 1 , R 2 , R 3 and R 4 are selected from the group consisting of: H, CH 3 and alkyl, where R substituents with different numbers may be the same or different, n is 0 or 1, R 5 is selected from the group consisting of: aryl, alkylaryl, preferably benzyl, 1-naphthylmethyl or 2-naphtylmethyl, substituted benzyl, preferably mono- or di-substituted, preferably with a substituent selected from the group consisting of: chlorine, fluorine, bromine, iodine, methyl, alkyl, nitro group, carboxyl group, azide group, amine group, hydroxyl group or a combination thereof, substituted naphthylmethyl, wherein aryl is an unsaturated, ring, aromatic or heteroaromatic hydrocarbon substituent, preferably having from 6 to 10 carbon atoms, wherein alkylaryl is an unsaturated hydrocarbon substituent constructed from an alkyl and aryl portion linked together, Base, and Base 2 are independently selected from the group consisting of: X 1 , X 3 , are selected from a group including: O, S, Se, whereby substituents X with different numbers may be the same or different, X 2 , X 4 and X 5 are selected from the group consisting of: O, S, Se, BH 3 , wherein X substituents with different numbers can be the same or different, X 6 is selected from the group consisting of: O, CH 2 , CF 2 , CCl 2 ,wherein preferably R 5 is benzyl, monosubstituted benzyl, disubstituted benzyl, 1-napthylmethyl or 2-naphtylmethyl, X 1 , X 4 , X 5 , X 6 are O, X 2 , X 3 are O or S, R 3 , R 4 are H.
2. A compound according to claim 1, wherein said compound is selected from the group consisting of: compound m 7 Gppp Bn6 A m pG of formula: compound m 7 Gppp 2MeBn6 A m pG of formula: compound m 7 Gppp 3MeBn6 A m pG of formula: compound m 7 Gppp 4MeBn6 A m pG of formula: compound m 7 Gppp 4FBn6 A m pG of formula: compound m 7 Gppp 3,4diFBn6 A m pG of formula: compound m 7 Gppp 1Naphm6 A m pG of formula: compound m 7 Gppp 2Naphm6 A m pG of formula: compound m 7 Gppsp Bn6 A m pG of formula: compound m 7 Gppp 5'S,Bn6 A m pG of formula: compound m 7 Gppp Bn6 A m pGpG of formula:
3. A compound according to claim 1, wherein said compound consists essentially of a single stereoisomer or comprises a mixture of at least two stereoisomers, the first diastereomer and the second diastereomer, wherein the diastereomers are otherwise identical, except that they have different stereochemical configurations around the stereogenic phosphorus atom, wherein a stereogenic phosphorus atom is attached to a sulfur atom, selenium atom, or borane group.
4. An RNA molecule whose 5' end incorporates a compound as defined in any one of claims 1-3.
5. A method for synthesizing an RNA molecule as defined in claim 4 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a compound as defined in claims 1-3, and a polynucleotide template in the presence of RNA polymerase, under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; wherein some of the RNA copies will incorporate the compound as defined in any of claims 1-3 to make an RNA molecule as defined in claim 4.
6. An in vitro protein or peptide synthesis method, said method comprising translating the RNA molecule according to claim 4, in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.
7. A method for synthesizing a protein or peptide in a living cell in-vitro, said method comprising introducing an RNA molecule as defined in claim 4 into a cell, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.
8. A method of purifying a molecule as defined in claim 4, wherein said method comprises using a chromatographic method, preferably a reversed-phase HPLC method, whereby the column is equilibrated, a sample containing a molecule as defined in claim 4 is introduced onto the chromatographic column, the components of the sample are separated in a buffered aqueous solution/organic solvent system, the fractions containing the molecule as defined in claim 4 are collected, wherein mRNA molecules defined in claim 4 are separated from other RNA molecules that do not have at the 5' end structures as defined in claims 1-3.
9. The use of a compound as defined in any one of claims 1-3 in in-vitro synthesis of RNA molecules.
10. The use of an RNA molecule as defined in claim 4 in in-vitro synthesis of protein or peptide.
11. A compound as defined in claims 1-3 or the RNA molecule as defined in claim 4 for use in medicine, pharmacy or diagnostics.

Description

Technical field This invention relates to novel mRNA 5'-end cap analogs, an RNA molecule incorporating the same, uses thereof and a method of synthesizing the RNA molecule in vitro, as well as a method of synthesizing a protein or peptide in vitro or in cells, said method comprising translating the RNA molecule. THE STATE OF ART The 7-methylguanosine cap (m7G) present at the 5' end of eukaryotic mRNA plays an important role in numerous fundamental cellular processes, mainly by protecting mRNA from premature degradation and by serving as a molecular marker for proteins involved in mRNA transport and translation. [1] Therefore, chemical 5' cap modifications pave the way for the design of molecular tools for the selective modulation of cap-dependent processes and, consequently, mRNA metabolism.[2] The presence of 5' cap is needed to control mRNA and its effective translation under normal conditions. Chemically synthesized m7GpppG cap mRNA analogs are used as in vitro transcription reagents for capped mRNA. [3] In vitro transcribed (IVT) 5'-capped mRNAs are useful tools for studying translation, transport and processing of mRNAs and are an emerging class of promising therapeutic molecules. IVT mRNA finds application in the expression of proteins in eukaryotic cells, extracts, cell cultures and even in whole organisms. Finally, IVT RNA has recently attracted considerable attention as a tool for safe exogenous protein delivery for anticancer vaccination, antiviral vaccination, and gene replacement therapy [4]. The synthesis of 5'-capped mRNA using cap analogues can be achieved by in vitro transcription.[3] In this method, called co-transcriptional capping, RNA synthesis is performed by RNA polymerase on a DNA matrix in the presence of all 4 ribonucleoside 5'-triphosphates (NTPs: ATP, GTP, CTP, UTP) and a dinucleotide cap such as m7GpppG. The DNA template is designed so that the first transcribed nucleotide is G. Polymerase initiates transcription using GTP or m7GpppG, thereby incorporating one of the nucleotides at the 5' end of the resulting RNA. To increase the percentage of cap analog incorporation (capping efficiency), GTP concentration is reduced relative to other NTPs, and dinucleotide cap concentration is increased (4-10 fold over GTP). Unfortunately, even when using high dinucleotide cap excess over GTP, the capping efficiency is less than 100% and rarely exceeds 90%. Uncapped mRNAs are much less stable and translationally active than capped mRNAs and, moreover, can lead to induction of an unwanted immune response in cells leading to a reduction in translation efficiency also for capped mRNAs [11]. A way to remove uncapped mRNAs is to treat the post-transcriptional mixture with appropriate enzymes (e.g., a mixture of 5'-polyphosphatase and 5'-exonuclease) that degrade uncapped RNA and leave the capped mRNA intact. Another limitation of the mRNA capping method using dinucleotides is the fact that the dinucleotide cap can be reversely incorporated, leading to "Gpppm7G-capped" RNAs that are translationally inactive. This problem was solved by the discovery of 'anti-reverse cap analogs' (ARCAs), which are modified at the 2'- or 3'- positions of 7-methylguanosine (usually by replacing one of the OH groups with an OCH3 group) to block reverse incorporation. [5,6] It has already been shown that the co-transcriptional capping method enables the incorporation of various modified cap structures at the 5' mRNA end. These modified cap structures can be molecular label carriers or give mRNA molecules new properties such as increased translation efficiency and stability. Especially preferred cap analogues are among those modified in the triphosphate bridge. [7] It has been shown that substitution of even one atom in a 5', 5'-triphosphate bridge can significantly affect mRNA properties. For example, substitution of a single atom in the β position of the cap oligophosphate bridge, depicted as β-S-ARCA, resulted in a significant increase in mRNA translation efficiency in vitro and in vivo, [8,9] while substitution of a single O atom with a CH2 group at the α-β position resulted in a reduction in translation efficiency. [10] These dramatically different biological effects of different single atom substitutions within the cap indicate for the high sensitivity of the translational machinery to oligophosphate chain modifications and suggest that this is an important area for further exploration. Moreover, it has been disclosed [22] that replacement of the methyl-group of m7G in the dinucleotide cap analogue derivative m7GpppG with a benzyl-group results in higher translation efficiency of mRNA with such 5'-caps. The objective of the invention is to provide new mRNA 5' end cap analogs that will enable higher transcription efficiency of the mRNA capped with them and a higher level of expression of proteins encoded by such mRNA than those obtained with prior art mRNA 5' end cap analogs. A particular objective of the invention is