# US 12,180,518 B2 · RNA polymerase variants for co-transcriptional capping

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- Number: US 12,180,518 B2
- Document ID: US12180518B2
- Status: Granted
- Kind: B2
- Application number: 17/816,696
- Filed: 2022-08-01
- Published: 2023-04-06
- Granted: 2024-12-31
- Earliest claimed: 2019-02-20
- Assignees: ModernaTX, Inc.
- Applicants: ModernaTX, Inc.
- Inventors: Athanasios Dousis; Kanchana Ravichandran; Amy E. Rabideau; Margaret Franklin; Kevin Smith; Michelle Lynn Hall
- Web page: https://patentgrep.com/patent/US12180518B2

## CPC

- C12N 9/1247: {DNA-directed RNA polymerase (2.7.7.6)}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Y 207/07006: DNA-directed RNA polymerase (2.7.7.6)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}
- C12Y: ENZYMES

## Abstract

The present disclosure provides RNA polymerase variants for high efficiency transcription.

## Claims

### Claim 1

A method comprising:
incubating a deoxyribonucleic acid (DNA) in an in vitro transcription reaction that comprises nucleoside triphosphates, buffer, and a ribonucleic acid (RNA) polymerase variant, wherein the RNA polymerase variant (i) comprises an amino acid substitution at position D351 relative to an RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1 and (ii) has at least 95% identity to the amino acid sequence of SEQ ID NO: 1, thereby producing a messenger RNA (mRNA).

### Claim 2 (depends on 1)

The method of claim 1, wherein the amino acid substitution at position D351 is selected from D351C, D351I, D351K, D351L, D351M, D351P, D351Q, D351R, D351S, D351T, D351V, and D351W.

### Claim 3 (depends on 2)

The method of claim 2, wherein the amino acid substitution at position D351 is D351V.

### Claim 4 (depends on 1)

The method of claim 1, wherein the RNA polymerase variant further comprises an amino acid substitution at position E350, relative to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

### Claim 5 (depends on 4)

The method of claim 4, wherein the RNA polymerase variant further comprises an amino acid substitution at position E350, and the amino acid substitution at position E350 is selected from E350W, E350A, E350K, and E350N.

### Claim 6 (depends on 5)

The method of claim 5, wherein the amino acid substitution at position E350 is E350W.

### Claim 7 (depends on 1)

The method of claim 1, wherein the RNA polymerase variant further comprises a C-terminal G and an amino acid substitution at positions G47 and E350, relative to a wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1.

### Claim 8 (depends on 7)

The method of claim 7, wherein the amino acid substitution at position G47 is G47A, the amino acid substitution at position E350 is E350W, and the amino acid substitution at position D351 is D351V.

### Claim 9 (depends on 1)

The method of claim 1, wherein the RNA polymerase variant has at least 98% identity to the amino acid sequence of SEQ ID NO: 1.

### Claim 10 (depends on 9)

The method of claim 9, wherein the RNA polymerase variant comprises the amino acid sequence of SEQ ID NO: 126.

### Claim 11 (depends on 1)

The method of claim 1, wherein the in vitro transcription reaction further comprises a cap analog.

### Claim 12 (depends on 11)

The method of claim 11, wherein the cap analog is a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap.

### Claim 13 (depends on 11)

The method of claim 11, wherein the DNA includes a 2′-deoxythymidine residue or 2′-deoxycytidine residue at template position +1.

### Claim 14 (depends on 11)

The method of claim 11, wherein the cap analog and nucleoside triphosphates are present in the reaction at equimolar concentrations.

### Claim 15 (depends on 11)

The method of claim 11, wherein the molar ratio of cap analog to nucleosides in the reaction is greater than 1:1.

### Claim 16 (depends on 11)

The method of claim 11, wherein the in vitro transcription reaction comprises a concentration of the cap analog that is at least 5-fold lower than a concentration of the cap analog required to produce an equivalent amount of mRNA using a control T7 RNA polymerase.

### Claim 17 (depends on 1)

The method of claim 1, wherein the buffer comprises dithiothreitol (DTT), magnesium ions, and tris.

### Claim 18 (depends on 1)

The method of claim 1, wherein the nucleoside triphosphates are naturally-occurring, synthetic, or modified.

### Claim 19 (depends on 1)

The method of claim 1, wherein greater than 50% of the mRNA produced is homogeneous at the 3′ end.

### Claim 20 (depends on 1)

The method of claim 1, wherein less than 5 ng of double-stranded RNA is produced per 25 μg of mRNA produced.