# US 10,501,768 B2 · Method of producing RNA from circular DNA and corresponding template DNA

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,501,768 B2
- Document ID: US10501768B2
- Status: Granted
- Kind: B2
- Application number: 15/743,988
- Filed: 2016-07-13
- Published: 2018-07-19
- Granted: 2019-12-10
- Earliest claimed: 2015-07-13
- Assignees: CureVac AG
- Applicants: CureVac AG
- Inventors: Fabian Johannes Eber; Stefanie Sewing; Wenke Wagner
- Web page: https://patentgrep.com/patent/US10501768B2

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12Q 1/6865: Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## 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.

## Claims

### Claim 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 transcribing 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),
wherein said RNA polymerase terminator sequence element comprises at least two Class II termination sequences.

### Claim 2 (depends on 1)

The method of claim 1, wherein the RNA polymerase terminator sequence element comprises at least two Class II termination sequences and does not comprise a Class I termination sequence.

### Claim 3 (depends on 1)

The method of claim 1, wherein the RNA polymerase terminator sequence element comprises at least one VSV terminator sequence or at least one 1R11 variant rrnB T1 downstream terminator sequence.

### Claim 4 (depends on 1)

The method of claim 1, wherein the in vitro transcription 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.

### Claim 5 (depends on 1)

The method of claim 1, wherein the in vitro transcription is carried out in the presence of a cap analog.

### Claim 6 (depends on 1)

The method of claim 1, wherein said target RNA is purified by at least one first and at least one second purification step.

### Claim 7 (depends on 6)

The method of claim 6, wherein the at least one first purification step comprises a precipitation step, and the at least one second purification step comprises a chromatographic step.

### Claim 8 (depends on 7)

The method of claim 7, 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.

### Claim 9 (depends on 6)

The method of claim 6, 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.

### Claim 10 (depends on 9)

The method of claim 9, wherein the at least one first purification step comprises a diafiltration step using tangential flow filtration 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.

### Claim 11 (depends on 1)

The method of claim 1, wherein said circular DNA provided in step a) is amplified by rolling circle amplification prior to the in vitro transcription in step b).

### Claim 12 (depends on 1)

The method of claim 1, 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.

### Claim 13 (depends on 1)

The method of claim 1, wherein said RNA polymerase promoter sequence is a T7 RNA polymerase promoter sequence.

### Claim 14 (depends on 1)

The method of claim 1, 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).

### Claim 15 (depends on 1)

The method of claim 1, 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.

### Claim 16 (depends on 1)

The method of claim 1, wherein said Class II termination sequences are 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.

### Claim 17 (depends on 1)

The method of claim 1, wherein said Class II termination sequences are identical.

### Claim 18 (depends on 17)

The method of claim 17, wherein said Class II termination sequences are VSV terminator sequences or 1R11 variant rrnB TI downstream terminator sequences.

### Claim 19 (depends on 1)

The method of claim 1, wherein the purified target RNA does not comprise a self-cleaving ribozyme sequence.