Methods and means for enhancing rna production

Abstract

Further, the present invention relates to a bioreactor (1) for synthesizing RNA molecules of a given sequence, the bioreactor (1) having a reaction module (2) for carrying out in vitro RNA transcription reactions in a sequence-optimized reaction mix, a capture module (3) for temporarily capturing the transcribed RNA molecules, and a control module (4) for controlling the infeed of components of the sequence-optimized reaction mix into the reaction module (2), wherein the reaction module (2) comprises a filtration membrane (21) for separating nucleotides from the reaction mix, and the control of the infeed of components of the sequence-optimized reaction mix by the control module (4) is based on a measured concentration of separated nucleotides.

1 . A method for synthesizing an RNA molecule of a given sequence, comprising the following steps:
 a) determining the fraction (1) for each of the four nucleotides G, A, C and U in said RNA molecule, and   b) synthesizing said RNA molecule by in vitro transcription in a sequence-optimized reaction mix, wherein said sequence-optimized reaction mix comprises the four ribonucleoside triphosphates (NTPs) GTP, ATP, CTP and UTP, wherein the fraction (2) of each of the four ribonucleoside triphosphates in the sequence-optimized reaction mix corresponds to the fraction (1) of the respective nucleotide in said RNA molecule, a buffer, a DNA template, and an RNA polymerase.   
     
     
         2 . The method of  claim 1 , wherein step b) comprises the steps of
 b1) preparing a sequence-optimized ribonucleoside triphosphate (NTP) mix comprising the four ribonucleoside triphosphates (NTPs) GTP, ATP, CTP and UTP, wherein the fraction (2) of each of the four ribonucleoside triphosphates in the sequence-optimized ribonucleoside triphosphate (NTP) mix corresponds to the fraction (1) of the respective nucleotide in said RNA molecule, and   b2) synthesizing said RNA molecule by in vitro transcription in the sequence-optimized reaction mix comprising the NTP mix of step (b1), a buffer, a DNA template, and an RNA polymerase.   
     
     
         3 . The method of  claim 1  or  2 , wherein before the start of the in vitro transcription a start nucleotide is added to the sequence-optimized reaction mix which corresponds to the first nucleotide of said RNA molecule. 
     
     
         4 . The method of  claim 3 , wherein said start nucleotide is a nucleoside monophosphate, a nucleoside diphosphate, a nucleoside triphosphate or a di-nucleoside triphosphate. 
     
     
         5 . The method of  claim 3 , wherein said start nucleotide is a cap analog. 
     
     
         6 . The method of any of  claims 3  to  5 , wherein said start nucleotide is added in excess compared to the fraction of that nucleotide in said RNA molecule which is found at the first position of said RNA molecule. 
     
     
         7 . The method of any of  claims 1  to  6 , wherein for the nucleotides which do not correspond to the first nucleotide of the RNA molecule fraction (1) and fraction (2) differ by at most 10%. 
     
     
         8 . The method of any of  claims 1  to  7 , wherein a part or all of at least one ribonucleoside triphosphate is replaced by a modified nucleoside triphosphate. 
     
     
         9 . The method of  claim 8 , wherein said modified nucleoside triphosphate is selected from the group consisting of pseudouridine-5′-triphosphate, 1-methylpseudouridine-5 ‘-triphosphate, 2-thiouridine-5’-triphosphate, 4-thiouridine-5′-triphosphate and 5-methylcytidine-5′-triphosphate. 
     
     
         10 . The method of any of  claims 1  to  9 , wherein in the course of the in vitro transcription the sequence-optimized reaction mix is supplemented with the sequence-optimized ribonucleoside triphosphate (NTP) mix as defined in  claim 2  b1). 
     
     
         11 . The method of any of  claims 1  to  10 , wherein said RNA molecule is selected from the group consisting of non-coding and coding RNA molecules. 
     
     
         12 . The method of any of  claims 1  to  11 , wherein said RNA molecule is an mRNA. 
     
     
         13 . The method of any of  claims 1  to  12 , wherein said RNA molecule is longer than 100 nucleotides. 
     
     
         14 . The method of any of  claims 1  to  13 , wherein said synthesizing of an RNA molecule of a given sequence is performed as a large scale synthesis. 
     
     
         15 . The method of any of  claims 1  to  14 , wherein the NTP counter ion is tris(hydroxymethyl)-aminomethane (Tris). 
     
     
         16 . The method of any of  claims 1  to  15 , wherein the synthesizing of said RNA molecule by in vitro transcription is followed by separating and quantifying the unincorporated NTPs. 
     
     
         17 . The method of any of  claims 1  to  16 , wherein the synthesizing of said RNA molecule by in vitro transcription is carried out in a bioreactor (1). 
     
     
         18 . The method of any of  claim 17 , wherein said bioreactor (1) comprises a DNA template immobilized on a solid support. 
     
     
         19 . The method of any of  claims 17  to  18 , wherein said bioreactor (1) comprises a filtration membrane (21) for separating nucleotides from the sequence-optimized reaction mix. 
     
     
         20 . The method of  claim 19 , wherein said filtration membrane (21) is selected from the group of regenerated cellulose, modified cellulose, poly sulfone (PSU), polyacrylonitrile (PAN), polymethylmethacrylate (PMMA), polyvinyl alcohol (PVA) and polyarylethersulfone (PAES). 
     
     
         21 . The method of any of  claims 19  to  20 , wherein said filtration membrane (21) has a molecular weight cut-off in a range of approximately 10 to 50 kDa. 
     
     
         22 . The method of any of  claims 17  to  21 , wherein said bioreactor (1) comprises a sensor unit (41) for the real-time measurement of the nucleotide concentration during the reaction. 
     
     
         23 . The method of  claim 22 , wherein said sensor unit (41) measures the nucleotide concentration by photometric analysis. 
     
     
         24 . The method of any of  claims 17  to  23 , wherein said bioreactor comprises a control module (4) which controls the addition of the sequence-optimized ribonucleoside triphosphate (NTP) mix as defined in  claim 2  b1). 
     
     
         25 . The method of any of  claims 17  to  24 , wherein said bioreactor (1) comprises an actuator (43) which adds the sequence-optimized ribonucleoside triphosphate (NTP) mix as defined in  claim 2  b1). 
     
     
         26 . The method of any of  claims 17  to  25 , wherein said bioreactor (1) comprises a resin to capture the RNA molecules and to separate the RNA molecules from the other components of the transcription reaction mix. 
     
     
         27 . The method of any of  claims 17  to  26 , wherein said bioreactor (1) operates in a semi-batch mode or in a continuous mode. 
     
     
         28 . The method of any of  claims 17  to  27 , wherein said bioreactor (1) comprises at least one ion-selective electrode, 
     
     
         29 . The method of  claim 28 , wherein the at least one ion-selective electrode is used for measuring the concentration of one or more types of ions in a liquid comprised in at least one compartment of the bioreactor (1). 
     
     
         30 . The method of  claim 29 , wherein the ion is selected from the group consisting of H + , Na + , K + , Mg 2+ , Ca 2+ , Cl −  and PO 4   3− . 
     
     
         31 . An RNA molecule obtainable by the method according to any one of  claims 1  to  30 . 
     
     
         32 . Use of a sequence-optimized ribonucleoside triphosphate (NTP) mix optimized for an RNA molecule of a given sequence for the synthesis of said RNA molecule. 
     
     
         33 . The use of  claim 32 , wherein the sequence-optimized NTP mix has been optimized by a method comprising the steps of:
 a) determining the fraction (1) of each of the four nucleotides G, A, C and U in said RNA molecule, and   b) preparing the sequence-optimized ribonucleoside triphosphate (NTP) mix comprising the four ribonucleoside triphosphates GTP, ATP, CTP and UTP, wherein the fraction (2) of each of the four ribonucleoside triphosphates in the sequence-optimized ribonucleoside triphosphate (NTP) mix corresponds to the fraction (1) of the respective nucleotide in said RNA molecule.   
     
     
         34 . The use of any of  claims 32  to  33 , wherein a part or all of at least one ribonucleoside triphosphate is replaced by a modified nucleoside triphosphate. 
     
     
         35 . The use of any of  claims 32  to  34 , wherein said modified nucleoside triphosphate is selected from the group consisting of pseudouridine-5′-triphosphate, 1-methylpseudouridine-5′-triphosphate, 2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate and 5-methylcytidine-5′-triphosphate. 
     
     
         36 . A sequence-optimized ribonucleoside triphosphate (NTP) mix for the synthesis of an RNA molecule of a given sequence comprising the four nucleoside triphosphates GTP, ATP, CTP and UTP, wherein the fraction (2) of each of the four nucleoside triphosphates in the sequence-optimized ribonucleoside triphosphate (NTP) mix corresponds to the fraction (1) of the respective nucleotide in said RNA molecule. 
     
     
         37 . A kit comprising a sequence-optimized ribonucleoside triphosphate (NTP) mix optimized for an RNA molecule of a given sequence as defined in  claim 32  or the components thereof. 
     
     
         38 . A bioreactor (1) for synthesizing RNA molecules of a given sequence, comprising:
 i) a reaction module (2) for carrying out in vitro transcription reactions in a sequence-optimized reaction mix as defined in  claim 1  (b);   ii) a capture module (3) for temporarily capturing the transcribed RNA molecules; and   iii) a control module (4) for controlling the infeed of components of the sequence-optimized reaction mix into the reaction module (2), wherein
 the reaction module (2) comprises a filtration membrane (21) for separating nucleotides from the sequence-optimized reaction mix, and 
 wherein 
 the control of the infeed of components of the sequence-optimized reaction mix by the control module (4) is based on a measured concentration of separated nucleotides. 
   
     
     
         39 . The bioreactor (1) of  claim 38 , wherein the filtration membrane (21) is an ultrafiltration membrane (21) for separation of high molecular weight components from low molecular weight components, preferably wherein said filtration membrane (21) has a molecular weight cut-off in a range from 10 to 100 kDa, 10 to 75 kDa, 10 to 50 kDa, 10 to 25 kDA or 10 to 15 kDa, further preferably the filtration membrane has a molecular weight cut-off value in a range of approximately 10 to 50 kDa. 
     
     
         40 . The bioreactor (1) of  claim 38  or  39 , wherein the filtration membrane (21) is selected from the group of regenerated cellulose, modified cellulose, polysulfone (PSU), polyacrylonitrile (PAN), polymethylmethacrylate (PMMA), polyvinyl alcohol (PVA) and polyarylethersulfone (PAES). 
     
     
         41 . The bioreactor (1) of any one of  claims 38  to  40 , wherein said reaction module (2) comprises a DNA template immobilized on a solid support as basis for the RNA transcription reaction. 
     
     
         42 . The bioreactor (1) of any one of  claims 38  to  41 , wherein said capture module (3) comprises a resin to capture the transcribed RNA molecules and to separate the transcribed RNA molecules from other soluble components of the transcription reaction mix. 
     
     
         43 . The bioreactor (1) of any one of  claims 38  to  42 , wherein said capture module (3) comprises means (31) for purifying the captured transcribed RNA molecules. 
     
     
         44 . The bioreactor (1) of any one of  claims 38  to  43 , wherein said capture module (3) comprises means (31) for eluting the captured transcribed RNA molecules, preferably by means of an elution buffer. 
     
     
         45 . The bioreactor (1) of any one of  claims 38  to  44 , wherein the bioreactor (1) further comprises a reflux module (5) for returning the residual filtrated reaction mix to the reaction module (2) from the capture module (3) after capturing the transcribed RNA molecules, preferably wherein the means (51) for returning the residual filtrated reaction mix is a pump (51). 
     
     
         46 . The bioreactor (1) of  claim 45 , wherein the reflux module (5) comprises immobilized enzymes or resin to capture disruptive components. 
     
     
         47 . The bioreactor (1) of any one of  claims 38  to  46 , wherein said control module (4) comprises a sensor unit (41) for the real-time measurement of the concentration of separated nucleotides during the reaction. 
     
     
         48 . The bioreactor (1) of  claim 47 , wherein said sensor unit (41) measures, as a transcription reaction parameter, the concentration of separated nucleotides by photometric analysis. 
     
     
         49 . The bioreactor (1) of  claim 48 , wherein said sensor unit (41) measures further transcription reaction parameters in the filtrated reaction mix, preferably wherein the further transcription reaction parameters are pH-value and/or salinity. 
     
     
         50 . The bioreactor (1) of any one of  claims 38  to  49 , wherein said control module (4) controls an addition of the sequence-optimized ribonucleoside triphosphate (NTP) mix of  claim 32  to the sequence-optimized reaction mix, preferably wherein said bioreactor (1) comprises an actuator (43) for addition of the sequence-optimized ribonucleoside triphosphate (NTP) mix to the sequence-optimized reaction mix. 
     
     
         51 . The bioreactor (1) of any one of  claims 38  to  49 , wherein said bioreactor (1) operates in a semi-batch mode or in a continuous mode 
     
     
         52 . The bioreactor (1) of any one of  claims 38  to  49 , wherein said bioreactor (1) is adapted to carry out the method of any one of  claims 1  to  30 .