Method for adding cap structures to rna using immobilized enzymes

Abstract

The present invention relates to an immobilized capping enzyme, preferably an immobilized Vaccinia virus capping enzyme. Furthermore, the present invention relates to an immobilized cap-specific nucleoside 2′-O-methyltransferase, preferably an immobilized Vaccinia virus cap-specific nucleoside 2′-O-methyltransferase. Moreover, the present invention relates to a method for immobilizing said enzymes and to a method of using said enzymes for the addition of a 5′-cap structure to RNAs. Moreover, the present invention relates to an enzyme reactor for performing the capping reaction using said immobilized enzymes and the subsequent separation of the 5′-capped RNA product. In addition, the present invention relates to a kit comprising the capping enzyme and/or the cap-specific nucleoside 2′-O-methyltransferase.

1 . A capping enzyme being immobilized onto a solid support by covalent binding, entrapment, encapsulation or physical adsorption. 
     
     
         2 . The capping enzyme according to  claim 1 , being immobilized onto said solid support by covalent binding. 
     
     
         3 . The capping enzyme according to any one of  claims 1  and  2 , being immobilized by covalent binding to a thiol-activated solid support, haloacetyl functionalized solid support, pyridyl disulfide-functionalized solid support, maleimide-activated solid support, epoxy-activated solid support or a mixture thereof. 
     
     
         4 . The capping enzyme according to any one of the preceding claims, being immobilized via at least one thiol group, and/or amine group, and/or hydroxyl group. 
     
     
         5 . The capping enzyme according to any one of the preceding claims, being immobilized via a thiol group of at least one cysteine residue. 
     
     
         6 . The capping enzyme according to any one of  claims 2  to  5 , wherein the covalent binding is a disulfide bridge or a thioether bond. 
     
     
         7 . The capping enzyme according to any one of the preceding claims, wherein the solid support comprises a material selected from the group consisting of Sepharose™, thiopropyl-Sepharose™, Sephadex™, agarose, silica, magnetic beads, methacrylate beads and nanoparticles. 
     
     
         8 . The capping enzyme according to any one of the preceding claims, wherein the solid support is selected from the group consisting of activated thiol Sepharose™, thiopropyl-Sepharose™, thiol-activated Sephadex™, thiol-activated agarose, silica-based thiol-activated matrix, silica-based thiol-activated magnetic beads, pyridyl disulfide-functionalized nanoparticles, epoxy methacrylate beads, maleimide-activated agarose and mixtures thereof. 
     
     
         9 . The capping enzyme according to  claim 1 , being immobilized to an epoxy-activated solid support. 
     
     
         10 . The capping enzyme according to  claim 9 , being immobilized to an epoxy-activated solid support via a thiol group of at least one cysteine residue. 
     
     
         11 . The capping enzyme according to  claim 9  or  10 , wherein the epoxy-activated support is epoxy methacrylate beads. 
     
     
         12 . The capping enzyme according to any one of the preceding claims, wherein the capping enzyme has RNA triphosphatase (TPase), guanylyltransferase (GTase) and methyltransferase (MTase) activity independent of RNA polymerase II. 
     
     
         13 . The capping enzyme according to any one of the preceding claims, wherein the capping enzyme is a heterodimer of a catalytic and a regulatory polypeptide. 
     
     
         14 . The capping enzyme according to  claim 13 , wherein the capping enzyme is immobilized via the regulatory polypeptide. 
     
     
         15 . The capping enzyme according to any one of the preceding claims, wherein the capping enzyme is from Vaccina Virus. 
     
     
         16 . The capping enzyme according to any one of the preceding claims, wherein the wild-type capping enzyme is composed of:
 (i) a polypeptide comprising the amino acid sequence according to any one of SEQ ID NOs: 1 and 61-97 or a functional variant thereof having at least 80% sequence identity to the amino acid sequence according to any one of SEQ ID NOs: 1 and 61-97; and   (ii) a polypeptide comprising the amino acid sequence according to any one of SEQ ID NOs: 2 and 98-127 or a functional variant thereof having at least 80% sequence identity to the amino acid sequence according to any one of SEQ ID NOs: 2 and 98-127.   
     
     
         17 . The capping enzyme according to any one of the preceding claims, wherein the wild-type capping enzyme is composed of:
 (i) a polypeptide comprising the amino acid sequence according to SEQ ID NO: 1 or a functional variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 1; and   (ii) a polypeptide comprising the amino acid sequence according to SEQ ID NO: 2 or a functional variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 2.   
     
     
         18 . The capping enzyme according to any one of the preceding claims, wherein the capping enzyme comprises at least one newly introduced cysteine residue compared to the wild-type capping enzyme. 
     
     
         19 . The capping enzyme according to  claim 18 , wherein the newly introduced cysteine residue is attached to the C terminus of the capping enzyme, preferably via a linker. 
     
     
         20 . The capping enzyme according to  claim 19 , wherein the linker is selected from the group consisting of SEQ ID NOs: 15-39. 
     
     
         21 . The capping enzyme according to any one of the preceding claims, wherein the capping enzyme comprises only one cysteine residue or is mutated to comprise only one cysteine residue. 
     
     
         22 . The capping enzyme according to  claim 21 , wherein the only one cysteine residue is a newly introduced cysteine residue. 
     
     
         23 . The capping enzyme according to  claim 22 , wherein the newly introduced cysteine residue is attached to the C terminus of the capping enzyme, preferably via a linker. 
     
     
         24 . The capping enzyme according to  claim 23 , wherein the linker is selected from the group consisting of SEQ ID NOs: 15-39. 
     
     
         25 . The capping enzyme according to any one of  claims 18  to  24 , wherein the capping enzyme comprises a polypeptide comprising the amino acid sequence according to any one of SEQ ID NOs: 4, 5 and 298-327 or a functional variant thereof having at least 80% sequence identity to the amino acid sequence according to any one of SEQ ID NOs: 4, 5 and 298-327. 
     
     
         26 . The capping enzyme according to  claim 25 , wherein the capping enzyme further comprises a polypeptide comprising the amino acid sequence according to any one of SEQ ID NOs: 1, 6, 7 and 261-297 or a functional variant thereof having at least 80% sequence identity to the amino acid sequence according to any one of SEQ ID NOs: 1, 6, 7 and 261-297. 
     
     
         27 . The capping enzyme according to any one of  claims 18  to  24 , wherein the capping enzyme comprises the amino acid sequence according to any one of SEQ ID NOs: 8, 9 and 10 or a functional variant thereof having at least 80% sequence identity to the amino acid sequence according to any of SEQ ID NOs: 8, 9 and 10. 
     
     
         28 . Method for producing the capping enzyme of any one of the preceding claims, comprising a step of a) contacting said capping enzyme with a solid support under conditions suitable for immobilizing the capping enzyme to the solid support by covalent binding. 
     
     
         29 . The method according to  claim 28 , wherein step a) comprises the formation of a disulfide bridge or thioether bond. 
     
     
         30 . The method according to  claim 28  or  29 , wherein step a) comprises the formation of a covalent bond between a cysteine residue of the capping enzyme and a thiol group, a haloacetyl group, a pyridyl disulfide, an epoxy group, or a maleimide group of the solid support. 
     
     
         31 . The method according to  claim 28 , wherein step a) comprises the formation of a covalent bond between a cysteine residue of the capping enzyme and an epoxy group of the solid support. 
     
     
         32 . The method according to  claim 31 , wherein the solid support is epoxy methacrylate beads. 
     
     
         33 . Use of a capping enzyme being immobilized onto a solid support for producing ribonucleic acid (RNA) molecules with 5′ cap0 structures. 
     
     
         34 . The use according to  claim 33 , wherein the capping enzyme is defined as in any one of  claims 1  to  27 . 
     
     
         35 . A method for producing capped ribonucleic acid (RNA) molecules, comprising a step of i) contacting a capping enzyme being immobilized onto a solid support with RNA molecules, a nucleotide and a methyl donor, preferably S-adenosylmethionine, under conditions suitable for forming a 5′-cap0 structure. 
     
     
         36 . The method according to  claim 35 , wherein the capping enzyme is defined as in any one of  claims 1  to  27 . 
     
     
         37 . The method according to any one of  claim 35  or  36 , wherein the RNA is messengerRNA (mRNA). 
     
     
         38 . The method according to any one of  claims 35  to  37 , further comprising a step of ii) converting the cap0 structure into a cap1 structure by contacting the RNA comprising a 5′-cap0 structure with a cap-specific nucleoside 2′-O-methyltransferase and a methyl donor. 
     
     
         39 . The method according to  claim 38 , wherein the cap-specific nucleoside 2′-O-methyltransferase is immobilized onto a solid support. 
     
     
         40 . The method according to  claim 39 , wherein the cap-specific nucleoside 2′-O-methyltransferase is immobilized onto said solid support by covalent binding. 
     
     
         41 . The method according to  claim 39  or  40 , wherein the cap-specific nucleoside 2′ methyltransferase is immobilized by covalent binding to a thiol-activated solid support, haloacetyl functionalized solid support, pyridyl disulfide-functionalized solid support, epoxy-activated solid support, maleimide-activated solid support or a mixture thereof. 
     
     
         42 . The method according to any one of  claims 39  to  41 , wherein the cap-specific nucleoside 2′-O-methyltransferase is immobilized via a thiol group of at least one cysteine residue. 
     
     
         43 . The method according to any one of  claims 40  to  42 , wherein the covalent binding is a disulfide bridge or a thioether bond. 
     
     
         44 . The method according to any one of  claims 39  to  43 , wherein the solid support is selected from the group consisting of Sepharose™, thiopropyl-Sepharose™, Sephadex™, agarose, silica, methacrylate beads, magnetic beads and nanoparticles. 
     
     
         45 . The method according to any one of  claims 39  to  44 , wherein the solid support is selected from the group consisting of activated thiol Sepharose™, thiopropyl-Sepharose™, thiol-activated Sephadex™, thiol-activated agarose, silica-based thiol-activated matrix, silica-based thiol-activated magnetic beads, pyridyl disulfide-functionalized nanoparticles, epoxy activated methacrylate beads, maleimide-activated agarose and mixtures thereof. 
     
     
         46 . The method according to  claim 39 , wherein the cap-specific nucleoside 2′-O-methyltransferase is immobilized to an epoxy-activated solid support. 
     
     
         47 . The method according to  claim 46 , wherein the cap-specific nucleoside 2′-O-methyltransferase is immobilized to an epoxy-activated solid support via a thiol group of at least one cysteine residue. 
     
     
         48 . The method according to  claim 46  or  47 , wherein the epoxy-activated support is epoxy methacrylate beads. 
     
     
         49 . The method according to any one of  claims 39  to  48 , wherein the wild-type cap-specific nucleoside 2′-O-methyltransferase comprises the amino acid sequence according to any one of SEQ ID NOs: 3 and 128-160 or a functional variant thereof having at least 80% sequence identity to the amino acid sequence according to any one of SEQ ID NOs: 3 and 128-160. 
     
     
         50 . The method according to any one of  claims 39  to  49 , wherein the cap-specific nucleoside 2′-O-methyltransferase comprises at least one newly introduced cysteine residue compared to the wild-type cap-specific nucleoside 2′-O-methyltransferase. 
     
     
         51 . The method according to  claim 50 , wherein the newly introduced cysteine residue is attached to the C terminus of the cap-specific nucleoside 2′-O-methyltransferase, preferably via a linker. 
     
     
         52 . The method according to  claim 51 , wherein the linker is selected from the group consisting of SEQ ID NOs: 15-39. 
     
     
         53 . The method according to any one of  claims 39  to  52 , wherein the cap-specific nucleoside 2′-O-methyltransferase comprises only one cysteine residue or is mutated to comprise only one cysteine residue. 
     
     
         54 . The method according to  claim 53 , wherein the only one cysteine residue is a newly introduced cysteine residue. 
     
     
         55 . The method according to  claim 54 , wherein the newly introduced cysteine residue is attached to the C terminus of the cap-specific nucleoside 2′-O-methyltransferase, preferably via a linker. 
     
     
         56 . The method according to any one of  claims 50  to  55 , wherein the cap-specific nucleoside 2′-O-methyltransferase comprises the amino acid sequence according to any one of SEQ ID NOs: 11, 12 and 328-360 or a functional variant thereof having at least 80% sequence identity to the amino acid sequence according to any one of SEQ ID NOs: 11, 12 and 328-360. 
     
     
         57 . The method according to any one of  claims 35  to  56 , further comprising a step of ii) isolating the capped RNA molecules by filtration or chromatography. 
     
     
         58 . The method according to  claim 57 , wherein the filtration comprises ultrafiltration and/or diafiltration. 
     
     
         59 . The method according to any one of  claims 57  and  58 , further comprising a step of iii) formulating the capped RNA for administration to a human subject. 
     
     
         60 . An enzyme reactor comprising the capping enzyme according to any one of  claims 1  to  27 . 
     
     
         61 . The enzyme reactor according to  claim 60 , further comprising a cap-specific nucleoside 2′-O-methyltransferase. 
     
     
         62 . The enzyme reactor according to  claim 61 , wherein the cap-specific nucleoside 2′-O-methyltransferase is immobilized. 
     
     
         63 . The enzyme reactor according to  claim 62 , wherein the cap-specific nucleoside 2′-O-methyltransferase is defined as in any one of  claims 40  to  56 . 
     
     
         64 . The enzyme reactor according to  claim 62  or  63 , wherein the enzyme reactor is divided into 2 modules, one module comprising the immobilized capping enzyme and one module comprising the immobilized cap-specific nucleoside 2′-O-methyltransferase. 
     
     
         65 . Use of the enzyme reactor according to  claim 60  in a method according to any one of  claims 35  to  37  and  57  to  59 . 
     
     
         66 . Use of the enzyme reactor according to any one of  claims 61  to  64  in a method according to any one of  claims 38  to  59 . 
     
     
         67 . A capping enzyme comprising an amino acid sequence selected from the group consisting of:
 a) the amino acid sequence according to any one of SEQ ID NOs: 4, 5, 198-227 and 298-327 or a functional variant thereof having at least 80% sequence identity to the amino acid sequence according to any one of SEQ ID NOs: 4, 5, 198-227 and 298-327;   b) the amino acid sequence according to any one of SEQ ID NOs: 6, 7, 161-197 and 261-297 or a functional variant thereof having at least 80% sequence identity to the amino acid sequence according to any of SEQ ID NOs: 6, 7, 161-197 and 261-297; and   c) the amino acid sequence according to any one of SEQ ID NOs: 8, 9 and 10 or a functional variant thereof having at least 80% sequence identity to the amino acid sequence according to any one of SEQ ID NOs: 8, 9 and 10.   
     
     
         68 . A kit comprising a capping enzyme being immobilized onto a solid support, and/or a cap-specific nucleoside 2′-O-methyltransferase being immobilized onto a solid support, a reaction buffer, a methyl donor (SAM) and nucleoside triphosphates. 
     
     
         69 . The kit according to  claim 68 , further comprising one or more of a nucleotide mixture (optionally comprising modified nucleotides), an RNA polymerase, and an RNA in vitro transcription buffer. 
     
     
         70 . The kit according to  claim 68  or  69 , wherein the capping enzyme has a sequence as defined in  claim 67 .