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WO2023199261A1 - Molécule d'arn contenant des analogues de coiffe modifiés à l'extrémité 5', utilisation d'une molécule d'arn dans la synthèse in vitro de protéines ou de peptides, molécule d'arn destinée à être utilisée en médecine et utilisation d'analogues de coiffes modifiés pour le coiffage d'arn - Google Patents

Molécule d'arn contenant des analogues de coiffe modifiés à l'extrémité 5', utilisation d'une molécule d'arn dans la synthèse in vitro de protéines ou de peptides, molécule d'arn destinée à être utilisée en médecine et utilisation d'analogues de coiffes modifiés pour le coiffage d'arn Download PDF

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WO2023199261A1
WO2023199261A1 PCT/IB2023/053793 IB2023053793W WO2023199261A1 WO 2023199261 A1 WO2023199261 A1 WO 2023199261A1 IB 2023053793 W IB2023053793 W IB 2023053793W WO 2023199261 A1 WO2023199261 A1 WO 2023199261A1
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rna molecule
substituted
substituent
rna
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Marzena JANKOWSKA-ANYSZKA
Karolina PIECYK
Renata Grzela
Edward Darzynkiewicz
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Uniwersytet Warszawski
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Uniwersytet Warszawski
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression

Definitions

  • RNA molecule containing modified cap analogues at the 5’ terminus application of the RNA molecule in in vitro protein or peptide synthesis, a RNA molecule for use in medicine, use of modified cap analogues for RNA capping
  • the invention is related to RNA molecules containing novel caps of the 5’ mRNA terminus, an in vitro or cell culture method of protein or peptide synthesis, wherein said method involves translation of a RNA molecule, a RNA molecule for use in medicine and the use of new cap analogues in RNA capping.
  • RNA 2014 [3] discloses the methodology of producing monophosphate cap analogues containing N 2 -triazole and their biological evaluation as protein synthesis inhibitors.
  • Five cap analogues with a heterocyclic ring separated from m 7 -guanine by one to three carbon atoms and/or additionally substituted with various groups containing a benzene ring have been synthesised. All the obtained compounds proved to be effective translation inhibitors with IC50 similar to that of a m 7 GpppG dinucleotide triphosphate.
  • an example dinucleotide was made of the respective mononucleotide containing a benzyl-substituted 1,2,3-triazole and studied.
  • a technical issue the invention faces is to provide capped RNA molecules, which would be translated with high efficiency in cell-free systems or in cellular systems, whereby the caps should be preferentially incorporated in the RNA in the correct orientation.
  • capped RNA molecules should display preferable biological properties compared to the m 7 GpppG and ARCA caps used as a standard.
  • Another problem is the use of cap analogues in synthesis of RNA molecules modified such that their preferential incorporation into RNA in the correct orientation is ensured.
  • Capped RNA molecules should also display improved stability under enzymatic hydrolysis conditions.
  • the subject of the invention is a RNA molecule containing a compound defined by formula (II) at the 5’ terminus where n is 1 to 10; m is 1 to 2;
  • R 1 is selected from a group including a substituted aromatic ring, the aromatic ring being preferably phenyl, a substituted heteroaromatic ring, preferably a five-member heteroaromatic ring containing at least one nitrogen atom;
  • R 2 is selected from a group including a hydrogen atom, a substituted aromatic ring or an unsubstituted aromatic ring, with the aromatic ring preferably selected from a group including phenyl or naphthyl;
  • R 3 means a hydrogen atom or a methyl group
  • X 2 , X 4 , X 7 means: O-, S-, Se- or BH 3 ;
  • X 1 , X 3 , X 5 , X 6 means: O, S, NH or CH 2 ;
  • the base is selected from a group containing purine or pyrimidine derivatives.
  • the RNA molecule contains a compound defined by formula (II) at the 5’ terminus where n is 1 to 8, preferably 1 to 4; m is 1 to 2;
  • R 1 is selected from a group including an aromatic ring, with the aromatic ring being preferably phenyl, the aromatic ring is preferably substituted with a halogen atom or an alkiloxy substitute, preferably a five-membered heteroaromatic ring containing at least one nitrogen atom, preferably the heteroaromatic ring is preferably substituted with a substituent selected from a group including the following substituent: alkiloxyaromatic, preferably 2,6-disubstituted with an alkoxy, phenyl or alkyl substituent;
  • R 2 is selected from a group including a hydrogen atom, an aromatic ring, wherein the aromatic ring is selected from a group containing phenyl or naphthyl;
  • R 3 means a hydrogen atom or a methyl group;
  • X 2 , X 4 , X 7 means: O or S-;
  • X 1 , X 3 , X 5 , X 6 means: O, S, NH or CH 2 ;
  • Base - is selected from a group including:
  • the RNA molecule contains at its S’ terminus a compound defined by the formula (II) where n is 1 to 4; m is 1 to 2;
  • R 1 is selected from a group including a phenyl ring substituted with a halogen atom or an alkyloxy substituent, a substituted five-membered heteroaromatic ring containing at least one nitrogen atom, wherein the heteroaromatic ring is substituted with a substituent selected from a group including the following substituent: alkyloxyaromatic, preferably 2,6-disubstituted with an alkoxy, phenyl or alkyl substituent;
  • R 2 is selected from a group including a hydrogen atom or phenyl
  • R 3 means a hydrogen atom or a methyl group
  • X 2 , X 4 , X 7 means O-, S-, Se- or BH 3 ;
  • X 1 , X 3 , X 5 , X 6 means O or S;
  • Base - is selected from a group including:
  • the RNA molecule contains at its 5’ terminus a compound defined by the formula (II) where n is 1 to 4; m is 1;
  • R 1 is selected from a group including a phenyl ring substituted with a halogen atom or an alkyloxy substituent, a substituted five-membered heteroaromatic ring containing at least one nitrogen atom, wherein the heteroaromatic ring is substituted with a substituent selected from a group including the following substituent: alkyloxyaromatic, preferably 2,6-disubstituted with an alkoxy, phenyl or alkyl substituent;
  • R 2 is selected from a group including a hydrogen atom or phenyl
  • R 3 means a hydrogen atom
  • X 2 , X 4 , X 7 means O- or S-;
  • X 1 , X 3 , X 5 , X 6 means O or S;
  • Base - is selected from a group including:
  • the RNA molecule contains at its 5’ terminus a compound defined by the formula (II) where n is 1 to 4; m is 1;
  • R 1 is selected from a group including a phenyl ring substituted with a halogen atom or an alkyloxy substituent, a substituted five-membered heteroaromatic ring containing at least one nitrogen atom, wherein the heteroaromatic ring is substituted with a substituent selected from a group including the following substituent: alkyloxyaromatic, preferably 2,6-disubstituted with an alkoxy, phenyl or alkyl substituent;
  • R 2 is selected from a group including a hydrogen atom or phenyl
  • R 3 means a hydrogen atom
  • X 2 , X 4 , X 7 means O ;
  • X 1 , X 3 , X 5 , X 6 means O;
  • Base - is selected from a group including:
  • the aromatic ring is substituted in the para position, preferably with a halogen atom or an alkyloxy substituent.
  • the five-membered heteroaromatic ring is selected from a group including 1,2,3-triazole, isoxazole, thiazole.
  • the heteroaromatic ring is preferably substituted in the 1-, 3- or 4- position.
  • the alkyloxyaromatic substituent is substituted in the 2- and 6- position with a methoxy substituent.
  • the halogen atom is selected from a group including a fluorine atom, a chlorine atom, a bromine atom or a iodine atom, preferably a chlorine atom.
  • R 1 is selected from a group including
  • the compound defined by formula (II) is selected from a group including:
  • the subject of the invention also includes the use of the RNA module defined in the first subject of the invention in in vitro synthesis of a protein or a peptide.
  • Another subject of the invention is a RNA molecule defined in the first subject of the invention, for use in medicine.
  • the invention also includes the use of cap analogues defined by formula (II) in RNA capping.
  • N 2 -modified cap analogues with formula (II) present very favourable biological properties compared to the m 7 GpppG and ARCA caps used as the standard. They show a much stronger ability to inhibit translation in RRL using ARCA-mRNA (ca. 15-4 stronger inhibition compared to the control cap m 7 GpppG).
  • RNA capped in the IVT reaction (in vitro transcription) using compounds 1-6 and 7-15 show much better translation-related properties in a cell-free system for protein production made of rabbit reticulocytes (translation yields ca.
  • RNA capped using compounds 1-6 were also tested in HEK293 cells, where they showed the total protein expression by ca. 1.5-3 times higher compared to the standard m 7 GpppG-RNA; for comparison, in the case of ARCA 3’ caps this ratio is 1.46). All compounds were preferentially incorporated into the RNA in the correct orientation, and compounds 4 and 12 were incorporated similar to ARCA, only in the correct orientation.
  • the characteristic feature of the invention is the use of dinucleotide cap analogues with formula (II) in the N 2 position of the first base, displaying significantly improved properties compared to m 7 GpppG and ARCA commonly used in RNA capping reactions in RNA synthesis.
  • a trinucleotide i.e. 12-14
  • the available polymerases do not incorporate dinucleotides in the capping reaction, where adenosine methylated at the sugar is the second nucleoside.
  • RNA ribonucleic acid
  • a base should be understood as any substituted or unsubstituted purine or pyrimidine derivative.
  • the base is selected from a group including adenine, guanine, xanthine, N 6 -(A 2 -isopentenyl)adenine, acycloguanosine, hypoxanthine, uracil, cytosine, N 4 - methylcytosine, 6-carboxyuracil, 2-thiocytosine or barbituric acid, or their substituted derivatives.
  • Example embodiments of the invention are presented in the figures, where:
  • Fig. 1 presents the efficacy of mRNA translation in HEK293 cells, including Fig. 1A presenting the translation level as a relative luciferase activity after normalisation to protein concentration, and Fig. 1B presents the level of total luciferase expression presented as the surface area under the curve, normalised to the value of expression obtained for m7GpppG-mRNA, wherein both graphs present the average values ⁇ SD ( Standard Deviation) for at least two independent experiments, each one including triplicates;
  • ⁇ SD Standard Deviation
  • Fig. 2 illustrates a comparison of IC50 values (concentration inhibiting the protein biosynthesis in 50%) of the cap analogues 1 -6 and of the control compound m 7 GpppG;
  • Fig. 3 presents the hydrolysis of a 5' mRNA capped with N 2 -modified cap analogues, using the enzymes human Nudt 16 (panels 3A) and human Dcp2 (panel 3B);
  • Fig. 4 presents the hydrolysis level of a cap incorporated into mRNA using hNudt16.
  • Example 1 General synthetic procedure for dinucleotide, tri- or tetraphosphate cap analogues modified at the N 2 position
  • the reaction mixture was separated on a DEAE-Sephadex A-25 column, in a 0-1,0 M linear TEAB gradient (the total solution volume was 2 L).
  • Dinucleotide cap analogues were purified using high-performance liquid chromatography (HPLC) in a reversed phase system.
  • the dinucleotide (pAmpG) synthesis was performed in a solution, using phosphotriester chemistry.
  • the conjugation reaction was performed using 1 eq. of 5'-O-DMT-2'-O-methyl-3'-O-phosphoramide and 1 eq. of protected guanosine in the presence of 0.40 M 5-(benzylthio)-1-H-tetrazole in acetonitrile.
  • the reaction was performed over 4 hours at room temperature, under argon atmosphere. After 4h, the mixture was cooled to 4°C and 0.1 M iodine in pyridine was added and mixed for 1 hour at room temperature.
  • the reaction mixture was extracted with dichloromethane and washed with brine.
  • the obtained organic layer was dried, evaporated and purified using flash chromatography on silica gel, using gradient elution (0— >5% methanol in dichloromethane).
  • the purified compound was dissolved in 20% aqueous TFA solution and mixed at RT for 4 hours. The mixture was evaporated under vacuum and evaporated 6 times with methanol.
  • the raw nucleotide was crystallised from diethyl ether. The precipitate was filtered, washed with diethyl ether and dried in a vacuum dessicator over phosphorus pentaoxide. In the last stage, the dinucleotide was phosphorylated at the 5'-OH position using the standard Yoshikawa method, according to the previously described protocols ([2], [3]).
  • the obtained product had its protection removed with ammonia, was evaporated and purified using ion exchange chromatography on DEAE-Sephadex (A-25, HCO 3 - form) using a linear gradient of triethylammonium bicarbonate (TEAB), pH 7.5 in water. The fractions containing the desired product have been combined, evaporated and lyophilised to obtain the TEA salt of the product as white powder.
  • TEAB triethylammonium bicarbonate
  • the product was isolated using ion exchange chromatography on DEAE Sephadex (gradient elution using 0-1.2 M TEAB) and purified using semi-preparative RP HPLC (SUPELCOSILTM LC-18-DB, gradient elution 0-50% MeOH in 0.05 M ammonium acetate buffer pH 5,9), to obtain the ammonium salt of trinucleotide cap analogues after evaporation and repeated lyophilisation.
  • the reaction yield varied between 10 and 40%.
  • a PCR product containing a sequence encoding firefly luciferase and the SP6 promotor sequence (for cap analogues: no. 1, 2, 3, 4, 5, 6) or T7 (for cap analogues: no. 4, 7, 8, 9, 10, 11, 12, 13, 14, 15) was used as the dsDNA template for the in vitro transcription (IVT) reaction.
  • the PCR product had been purified using a NucleoSpin® Gel PCR Clean-up kit (Macherey-Nagel) before being added to the reaction.
  • the transcription reaction contained: the transcription buffer, 25 ng/ ⁇ L of the dsDNA matrix, 0.5 mM ATP/CTP/UTP, 0.1 mN GTP, 0.5 mM of the dinucleotide cap analogue, 0.5 U/ ⁇ L of the Ribolock ribonuclease inhibitor and 1 U/ ⁇ L of the RNA SP6 polymerase (Thermo Fisher Scientific) or the RNA T7 polymerase (Thermo Fisher Scientific).
  • the IVT reaction mixture was incubated for 1 h at 37°C, followed by an addition of 0.025 U/ ⁇ L DNaze I (Thermo Scientific) which was incubated with the IVT reaction mixture for 20 min at 37°C in order to remove the DNA template.
  • the reaction mixture was purified using NucleoSpin® RNA Clean-Up (Macherey-Nagel) according to the instructions of the manufacturer. Transcript integrity was checked using non-denaturing 1% agarose gel and the concentration was determined using spectrophotometry.
  • mRNA for experiments in HEK293 cells was obtained in an in vitro transcription reaction (IVT) using dsDNA of the PCR product containing the sequence coding firefly luciferase as a template and the SP6 promotor sequence (for cap analogues: no. 1, 2, 3, 4, 5, 6) or of the T7 promotor (for cap analogues: no. 4, 7, 8, 9, 10, 11, 12, 13, 14, 15).
  • IVTT in vitro transcription reaction
  • the PCR product had been purified using a NucleoSpin® Gel PCR Clean-up kit (Macherey-Nagel).
  • the newly synthesised cap analogues according to the invention were added to the transcription reaction, in a molar ratio cap:GTP 5:1, which enabled the cap to be incorporated during the synthesis of the RNA product.
  • the transcription reaction included: the transcription buffer, 25 ng/ ⁇ L of the dsDNA template, 0.5 mM ATP/CTP, 0.5 mM 4*UTP, 0.1 mM GTP, 0.5 mM of the dinucleotide cap analogue, 0.5 U/ ⁇ L of the Ribolock ribonuclease inhibitor and 1 U/ ⁇ L of the RNA SP6 polymerase (Thermo Fisher Scientific) or 2.5 U/ ⁇ L of the RNA T7 High polymerase (NEB).
  • the IVT reaction mixture was incubated for 1 h at 37°C for the SP6 polymerase or 2 - 4 h at 50°C for the T7 High polymerase, followed by addition of 0.025 U/ ⁇ L DNaze I (Thermo Scientific) and incubated with the reaction mixture for 20 min at 37°C in order to remove the DNA template.
  • the poliA tail was added to the transcripts in a reaction including the poliA buffer, 1 mM ATP, 0.1 U/ ⁇ L of the poliA (NEB) polymerase and 0.2 U/ ⁇ L of the Ribolock ribonuclease inhibitor (Thermo Scientific). The reaction was performed for 0.5 h at 37°C.
  • the IVT were treated with alkaline phosphatase (FastAP) for 15 min. at 37°C.
  • the reaction mixture contained the FastAP buffer, 0.033 U/ ⁇ L FastAP and 0.5 U/ ⁇ L of the Ribolock ribonuclease inhibitor (Thermo Scientific).
  • the reaction mixture was purified using NucleoSpin® RNA Clean-Up (Macherey-Nagel) according to the instructions of the manufacturer. Transcript integrity was checked using non-denaturing 1% agarose gel and the concentration was determined using spectrophotometry. 2.2 Synthesis of short capped RNAs
  • the dsDNA template containing a sequence of the SP6 promotor or a sequence of the T7 promotor was prepared through hybridisation of the respective two complementary oligonucleotides.
  • the reaction was performed overnight at 37°C, in 20 pl containing: 200 x diluted dsDNA template, transcription buffer, 0.5 mM ATP/CTP/UTP, 0.125 mM GTP, 1.25 mM cap analogue with formula (II), 0.5 U/ ⁇ L of the Ribolock ribonuclease inhibitor and 1 U/ ⁇ L of the RNA SP6 polymerase (Thermo Fisher Scientific).
  • the dsDNA template has been removed by 30 minute digestion using DNase I (Thermo Fisher Scientific) at 37°C.
  • the obtained product was purified using a Oligo CleanUp & Concentration kit (Norgen Biotek), according to the instructions of the manufacturer.
  • the DNazyme was used (SEQ. 7).
  • the reaction mixture containing 30 moles of DNazyme per 400 ng RNA in 50 mM Tris pH 7.4 and 50 mM MgCl 2 was incubated for 1 h at 37°C.
  • the DNazyme was digested using DNase I (Thermo Fisher Scientific) for 30 minutes at 37°C, and the homogenous transcripts 24 or 25 nucleotides long and terminated with the respective cap analogue (no. 1 to 6) and (7, 8, 9, 12, 15), according to example 1, was purified using a Oligo CleanUp & Concentration kit (Norgen Biotek), according to the instructions of the manufacturer.
  • RNA with any desired length may be obtained using a similar method.
  • Oligonucleotides capped with compounds 1-6 and 7, 8, 9, 12, 15 were subjected to a 30-minute incubation with hNudt16 and separated into 15% polyacrylamide gel with added 7M urea, where C indicates a product with a cap which did not hydrolyse and D means an oligonucleotide without a cap.
  • Oligonucleotides capped with compounds 1-6 were checked in a reaction with hDcp2. The uncapped product at time “0” is a result of incomplete capping during the IVT reaction; Fig. 6 presents the hydrolysis level of a cap incorporated into mRNA using hNudt16.
  • the graphs presents the percentage share of mRNA which underwent cap hydrolysis during a 30-minute reaction with hNudt16.
  • the hydrolysis level was calculated on the basis of densitometric analysis, as a percentage loss of the capped band after enzyme addition.
  • the data presents average values ⁇ SD from at least 3 independent experiments.
  • mRNA Differently capped mRNA, according to example 2.1, were added to the reaction mixture of a cell-free translation system from rabbit reticulocyte lysate (RRL). The translation reaction was carried out for 60 minutes and its product, luciferase, was measured using luminometry, after adding a specific substrate (Table 1A, B). The study included three control mRNA capped with m 7 GpppG, m 73O GpppG (ARCA 3’)
  • the translation yield of a transcript containing an ARCA cap was 1.47 times (for the first studied group, Table 1A) and 1.51 times (for the second studied group, Table 1B) higher than in the case of a transcript containing m 7 GpppG, as was expected and according to previously published literature data [5],
  • the control mRNA capped with ApppG was translated at a very low level, which means that the conditions for monitoring the cap-dependent translation were selected correctly.
  • the mRNAs tested in group one three transcripts with analogues 1, 3, 5 can be identified, which showed the highest translational yield, more than three times higher than that obtained for m 7 GpppG-RNA (Table 1A).
  • the remaining three transcripts had a slightly lower translation yield, but it was still 2.37 to 2.88 times higher than the control m 7 GpppG-RNA (Table 1A).
  • mRNA with analogues 12, 13, 14 were characterised by the highest translation yield, achieving results ca. 3.2 - 3.7 times higher than m 7 GpppG-mRNA (Table 1B).
  • the mRNA with analogue 11 also achieved a very high translation level, ca. 2.9 times higher than m 7 GpppG- mRNA.
  • the other mRNA with the following analogues 7, 8, 9, 10, 15 showed higher translation levels in the range from ca. 2.5 to 2.7 times higher than m 7 GpppG-mRNA (Table 1B).
  • mRNA with modified caps and a sequence coding firefly luciferase was obtained as described in the example 2.1.2 in an in vitro transcription reaction and transfected into HEK293 in identical amounts.
  • the yield of translations of the modified mRNA was measured 6, 12, 24 and 48 hours after transfection as relative luciferase activity (Fig. 1A).
  • the relative, total luciferase expression has been defined as the surface area under the curve and normalised to a value obtained for mRNA with the m 7 GpppG cap (Fig.
  • Table 1 Translation properties of mRNA capped with newly synthesised cap analogues in the RRL system and the HEK293 cells.
  • the level of total luciferase expression in HEK293 cells was presented as the surface area under the curve normalised to the value obtained for mRNA capped with m 7 GpppG.
  • the data presents average values ⁇ SD from at least two independent experiments, each consisting of threereplicates.
  • cap analogues defined by formula (II) were determined.
  • the yield of ARCA-mRNA luciferase translation was measured in the presence of increasing concentrations of the given cap analogue.
  • Table 3 Hydrolysis of a cap incorporated into mRNA using human Nudt16.
  • the table presents the percentage of mRNA, which underwent cap hydrolysis during a 30-minute reaction with Nudt16, calculated on the basis of densitometric analysis as a percentage loss of the cap after enzyme addition.
  • the data represents average values ⁇ SD from at least 3 independent experiments.
  • the human Nudt16 enzyme displays specific, hydrolytic activity towards substrates including a nucleoside diphosphate connected to another X molecule. Previous studies of the Inventors proved that this enzyme preferentially hydrolyses GppG molecules. At higher concentrations, it can also hydrolyse GpppG substrates and GpppG connected to RNA chains, despite the fact that these are very unlikely to be its specific substrates. On the other hand, compounds with the same structure but methylated in the N7 position are very weakly hydrolysed or not hydrolysed at all by Nudt16 ([7], [8]).
  • RNA polymerases may initiate the transcription reaction in the presence of m 7 GpppG analogues and their derivatives both by an attack of the 3’-OH group of guanosine, as well as of m 7 G. This results in a mixture of transcripts with m 7 GpppG-RNA and Gpppm 7 G-RNA at the 5’ terminus. The latter product is inactive in terms of translation and significantly decreases the quantity of the obtained, heterologous protein from RNA preparation.
  • the problem of inverse incorporation of cap analogues was solved as a result of chemical modification of the 3’-O or 2’-O position of m 7 G.
  • the analogues containing such modifications are known as ARCA ( anti reverse cap analogue) and are incorporated only in the correct orientation.
  • RNA capped with m 7 GpppG is a mixture of two products m 7 GpppG-RNA and Gpppm 7 G- RNA, only one of which is hydrolysed by Nudt16. It was thus observed that only half of the material in this sample underwent hydrolysis. This means that the Nudt16 enzyme is a tool, which allows evaluation of the presence of unmethylated RNA in the prepared preparations. Indirect conclusions regarding the orientation of the cap analogue incorporation can also be drawn from the obtained data.
  • the decapping level for these compounds varied between 11.5 and 13.5%, while in the case of ARCA it was 6.35%.
  • Compounds 9 and 7 displayed a slightly slower progress of hydrolysis compared to m 7 GpppG.
  • the decapping level was 17.8%, 29.6% compared to m 7 GpppG with the value of 49.6%.
  • Tyr639 thus plays a specialised role in controlling the correct structure of the incorporated substrate ([12]). Both Tyr639 and the nearest amino acids are strictly conserved in the sequence of both polymerases T7 and SP6 (Tyr639 is marked with an arrow, and the nearby fragment with a black frame in the figure in the annex). It is thus unsurprising that dinucleotide analogues containing 2'-deoxy and 2'- O-methyldeoxyrybose on one side are incorporated into the RNA chain only on the other side of the unmodified nucleobase. The described results also indicate that modifications of the N 2 position have positive impact on the orientation of dinucleotide incorporation. In the case of some substituents, this modification is enough for the analogue to be incorporated in the correct orientation only, similar to ARCA.

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Abstract

L'invention a pour objet une molécule d'ARN contenant un composé défini par la formule (II) à l'extrémité 5'. L'invention concerne également un procédé in vitro de synthèse de protéines ou de peptides dans des cultures cellulaires, ledit procédé impliquant la traduction d'une molécule d'ARN, l'utilisation d'une molécule d'ARN en médecine et l'utilisation de nouveaux analogues de coiffe dans le coiffage.
PCT/IB2023/053793 2022-04-13 2023-04-13 Molécule d'arn contenant des analogues de coiffe modifiés à l'extrémité 5', utilisation d'une molécule d'arn dans la synthèse in vitro de protéines ou de peptides, molécule d'arn destinée à être utilisée en médecine et utilisation d'analogues de coiffes modifiés pour le coiffage d'arn Ceased WO2023199261A1 (fr)

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EP23728425.2A EP4508060A1 (fr) 2022-04-13 2023-04-13 Molécule d'arn contenant des analogues de coiffe modifiés à l'extrémité 5', utilisation d'une molécule d'arn dans la synthèse in vitro de protéines ou de peptides, molécule d'arn destinée à être utilisée en médecine et utilisation d'analogues de coiffes modifiés pour le coiffage d'arn

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PL440921A PL440921A1 (pl) 2022-04-13 2022-04-13 Cząsteczka RNA zawierająca na końcu 5' zmodyfikowane analogi kapu, zastosowanie cząsteczki RNA w syntezie in vitro białka lub peptydu, cząsteczka RNA do stosowania w medycynie, zastosowanie modyfikowanych analogów kapu do kapowania RNA
PLPL440921 2022-04-13

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WO2023199261A1 true WO2023199261A1 (fr) 2023-10-19

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PCT/IB2023/053793 Ceased WO2023199261A1 (fr) 2022-04-13 2023-04-13 Molécule d'arn contenant des analogues de coiffe modifiés à l'extrémité 5', utilisation d'une molécule d'arn dans la synthèse in vitro de protéines ou de peptides, molécule d'arn destinée à être utilisée en médecine et utilisation d'analogues de coiffes modifiés pour le coiffage d'arn

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160032257A1 (en) * 2012-12-10 2016-02-04 Universität Hamburg Agent and method for modifying the 5' cap of rna

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160032257A1 (en) * 2012-12-10 2016-02-04 Universität Hamburg Agent and method for modifying the 5' cap of rna

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JOSEPHIN MARIE HOLSTEIN ET AL: "Enzymatic modification of 5'-capped RNA with a 4-vinylbenzyl group provides a platform for photoclick and inverse electron-demand Diels-Alder reaction", CHEMICAL SCIENCE, vol. 6, no. 2, 1 January 2015 (2015-01-01), United Kingdom, pages 1362 - 1369, XP055325281, ISSN: 2041-6520, DOI: 10.1039/C4SC03182B *
KAROLINA PIECYK ET AL: "Triazole-containing monophosphate mRNA cap analogs as effective translation inhibitors", RNA, vol. 20, no. 10, 22 August 2014 (2014-08-22), US, pages 1539 - 1547, XP055325195, ISSN: 1355-8382, DOI: 10.1261/rna.046193.114 *
PIECYK KAROLINA ET AL: "Isoxazole-containing 5' mRNA cap analogues as inhibitors of the translation initiation process", BIOORGANIC CHEMISTRY, vol. 96, 1 March 2020 (2020-03-01), US, pages 103583, XP093070354, ISSN: 0045-2068, Retrieved from the Internet <URL:https://pdf.sciencedirectassets.com/272552/1-s2.0-S0045206820X00022/1-s2.0-S004520681931819X/main.pdf?X-Amz-Security-Token=IQoJb3JpZ2luX2VjEG4aCXVzLWVhc3QtMSJGMEQCIDvMBlYJLuHeOWv8ZgbbbvV2vW9LNkz1f27cWz5ZlMzmAiAVJ8RyKj4xQUgy/jDBYbzBzxdbXInTuzy49bSX3LRcriqzBQgWEAUaDDA1OTAwMzU0Njg2NSIMR516X5Yg/9e2A8rlK> DOI: 10.1016/j.bioorg.2020.103583 *

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PL440921A1 (pl) 2023-10-16

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