Method of producing rna from circular dna and corresponding template dna

Abstract

The present invention is concerned with a method of producing a target RNA using a circular DNA, wherein said method does not comprise a step of linearizing said circular DNA. The present invention further relates to a circular DNA comprising an RNA polymerase promoter sequence, followed by a sequence encoding a target RNA, followed by a sequence encoding a self-cleaving ribozyme, followed by an RNA polymerase terminator sequence element, wherein the latter element may comprise several RNA polymerase terminator sequences. Multimeric DNA obtained by rolling circle amplification of said circular DNA is also within the scope of the present invention.

1 . A method of producing a linear target RNA comprising the steps of:
 a) providing a circular DNA as template DNA comprising the following sequence elements from 5′ to 3′:
 i. an RNA polymerase promoter sequence operably linked to 
 ii. a sequence encoding said target RNA operably linked to 
 iii. a sequence encoding a self-cleaving ribozyme, wherein said self-cleaving ribozyme cleaves close to or at its 5′ end, operably linked to 
 iv. an RNA polymerase terminator sequence element; and 
   b) in vitro transcription of said template DNA to obtain said target RNA;   c) purifying said target RNA by at least one purification step in order to obtain purified target RNA;   
       wherein said method does not comprise a step of linearizing said circular DNA provided in step a). 
     
     
         2 . The method according to  claim 1 , wherein said circular DNA provided in step a) comprises an RNA polymerase terminator sequence element iv which comprises at least one Class II termination sequence and fails to comprise a Class I termination sequence. 
     
     
         3 . The method according to  claim 1  or  2 , wherein said circular DNA provided in step a) comprises an RNA polymerase terminator sequence element iv which comprises at least one VSV terminator sequence and/or at least one 1R11 variant rrnB T1 downstream terminator sequence. 
     
     
         4 . The method according to  claim 1  or  2 , wherein said circular DNA provided in step a) comprises an RNA polymerase terminator sequence element iv which comprises at least one VSV terminator sequence. 
     
     
         5 . The method according to any one of  claims 1  to  4 , wherein the in vitro transcription in step b) is carried out in the presence of naturally occurring nucleotides and at least one modified nucleotide, wherein said at least one modified nucleotide at least partially replaces at least one naturally occurring nucleotide. 
     
     
         6 . The method according to any one of  claims 1  to  5 , wherein the in vitro transcription in step b) is carried out in the presence of a cap analog. 
     
     
         7 . The method according to any one of  claims 1  to  6 , wherein said target RNA is purified in step c) by at least one first and at least one second purification step. 
     
     
         8 . The method according to  claim 7 , wherein the at least one first purification step comprises a precipitation step and the at least one second purification step comprises a chromatographic step. 
     
     
         9 . The method according to  claim 8 , wherein the at least one first purification step comprises an alcohol precipitation step or a LiCl precipitation step and the at least one second purification step comprises a chromatographic step selected from the group consisting of HPLC, anion exchange chromatography, affinity chromatography, hydroxyapatite chromatography and core bead chromatography. 
     
     
         10 . The method according to  claim 7 , wherein the at least one first purification step comprises a tangential flow filtration step and the at least one second purification step comprises a chromatographic step. 
     
     
         11 . The method according to  claim 10 , wherein the at least one first purification step comprises a diafiltration step using tangential flow filtration and/or a concentration step using tangential flow filtration and the at least one second purification step comprises a chromatographic step selected from the group consisting of HPLC, anion exchange chromatography, affinity chromatography, hydroxyapatite chromatography and core bead chromatography. 
     
     
         12 . The method according to any one of the preceding claims, wherein said circular DNA provided in step a) is amplified by rolling circle amplification prior to the in vitro transcription in step b). 
     
     
         13 . The method according to any one of  claims 1  to  11 , wherein said method does not comprise a step of amplifying said circular DNA and said circular DNA is provided in step a) at a concentration ranging from about 0.075 g/L to about 0.3 g/L, preferably from about 0.1 g/L to about 0.2 g/L. 
     
     
         14 . A circular DNA comprising the following sequence elements from 5′ to 3′:
 a) an RNA polymerase promoter sequence operably linked to 
 b) a sequence encoding a target RNA operably linked to 
 c) a sequence encoding a self-cleaving ribozyme, wherein said self-cleaving ribozyme cleaves close to or at its 5′ end and fails to catalyze a self-circularization of the resulting RNA, operably linked to 
 d) an RNA polymerase terminator sequence element. 
 
     
     
         15 . The circular DNA according to  claim 14 , wherein said RNA polymerase promoter sequence is a T7 RNA polymerase promoter sequence. 
     
     
         16 . The circular DNA according to  claim 14  or  15 , wherein said target RNA is selected from the group consisting of mRNA, viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA, CRISPR RNA, ribozymes, aptamers, riboswitches, immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA). 
     
     
         17 . The circular DNA according to any one of  claims 14  to  16 , wherein said self-cleaving ribozyme encoded in sequence element c) is selected from the group consisting of a hepatitis delta virus (HDV) ribozyme, a hammerhead ribozyme and a hairpin ribozyme. 
     
     
         18 . The circular DNA according to any one of  claims 14  to  17 , wherein said RNA polymerase terminator sequence element comprises at least one Class II termination sequence and fails to comprise a Class I termination sequence. 
     
     
         19 . The circular DNA according to any one of  claims 14  to  18 , wherein said RNA polymerase terminator sequence element comprises at least one VSV terminator sequence and/or at least one 1R11 variant rrnB T1 downstream terminator sequence. 
     
     
         20 . The circular DNA according to any one of  claims 14  to  18 , wherein said RNA polymerase terminator sequence element comprises at least one VSV terminator sequence. 
     
     
         21 . The circular DNA according to any one of  claims 14  to  18 , wherein said RNA polymerase terminator sequence element comprises at least two Class II termination sequences, wherein said at least two Class II termination sequences are optionally separated by a spacer sequence. 
     
     
         22 . The circular DNA according to  claim 21 , wherein said RNA polymerase terminator sequence element comprises at least three Class II termination sequences, wherein said at least three Class II termination sequences are optionally separated by a spacer sequence. 
     
     
         23 . The circular DNA according to  claim 21  or  22 , wherein said RNA polymerase terminator sequence element comprises at least four Class II termination sequences, wherein said at least four Class II termination sequences are optionally separated by a spacer sequence. 
     
     
         24 . The circular DNA according to any one of  claims 18  and  21  to  23 , wherein said Class II termination sequences are independently selected from the group consisting of the VSV terminator sequence, the PTH terminator sequence, the rrnB T1 downstream terminator sequence, the rrnC terminator sequence, the concatemer junction sequence of the replicating T7 DNA, and a variant of any of the foregoing. 
     
     
         25 . The circular DNA according to any one of  claims 18  and  21  to  24 , wherein said Class II termination sequences are identical. 
     
     
         26 . The circular DNA according to  claim 25 , wherein said Class II termination sequences are VSV terminator sequences or 1R11 variant rrnB T1 downstream terminator sequences. 
     
     
         27 . A multimeric DNA obtained by rolling circle amplification of said circular DNA according to any one of  claims 14  to  26 . 
     
     
         28 . Use of a circular DNA according to any one of  claims 14  to  26  or a multimeric DNA according to  claim 27  in a method according to any one of  claims 1  to  13 . 
     
     
         29 . Use of a method according to any one of  claims 1  to  13  to produce linear mRNA as target RNA, wherein said mRNA provides at least the expression capability of mRNA produced by a method not resulting in a 2′3′ cyclic phosphate at the 3′ end of the mRNA. 
     
     
         30 . The use according to  claim 29 , wherein said method not resulting in a 2′3′ cyclic phosphate at the 3′ end of the mRNA is a method comprising a step of in vitro transcription using linearized DNA. 
     
     
         31 . The use according to  claim 29 , wherein said method not resulting in a 2′3′ cyclic phosphate at the 3′ end of the mRNA is a method not comprising a template DNA comprising a sequence encoding a self-cleaving ribozyme.