# US 2025/0084409 A1 · Compositions and methods for capping rnas

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- Number: US 2025/0084409 A1
- Document ID: US20250084409A1
- Status: Published application
- Kind: A1
- Application number: 18/819,275
- Filed: 2024-11-25
- Published: 2025-03-13
- Earliest claimed: 2022-03-01
- Assignees: Verve Therapeutics, Inc.
- Applicants: Verve Therapeutics, Inc.
- Inventors: Christopher Cheng; Kallanthottathil Rajeev; Caroline Reiss; Kui Wang; Jay Leipheimer
- Web page: https://patentgrep.com/patent/US20250084409A1

## CPC

- C07H 21/02: with ribosyl as saccharide radical
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N 15/67: General methods for enhancing the expression
- C07K 2319/09: containing a nuclear localisation signal
- C12N 2310/315: Phosphorothioates
- C12N 2830/50: regulating RNA stability, not being an intron, e.g. poly A signal
- C12Y 305/04002: Adenine deaminase (3.5.4.2)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## Abstract

Provided herein are compositions and methods for preparation of 5′ end region-modified mRNAs. In particular, the instant disclosure relates to novel mRNA 5′ end region motifs and sequence initiators therefore together with assays that are capable of measuring the aspects of the functionality of those motifs and sequence initiators. Further provided herein are compositions and methods of treating conditions related to coronary disease.

## Claims

### Claim 1

An in vitro-transcribed (IVT) mRNA sequence initiator comprising a compound of Formula (I) or a salt or solvate thereof:
wherein
B1 is
each B2, B3, and Bn is independently a natural, a modified, or an unnatural nucleobase;
each Z1 and Z2 is independently hydrogen, fluorine, —OH, —SH, —CH3, —CH2CH3, —OCH3, —OCH2CH3, —SCH3, —NH2, NHCH3, or NHC(═O)CH3;
Z3 is —OCH3;
each Z4, and Zn is independently hydrogen, fluorine, —OH, —SH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3;
each Q1 and Q4 is independently —CH2—, —CH═CH—, —CH2O—, —CH2S—, —CH2CH2—, —CH2CF2—, —CH2NH2—, —CH2NH(CH3)—, or —CH2N(C(═O)CH3)—;
each Q2 and Q3 is independently —O—, —S—, —CH2—, —CF2—, —NH—, —N(CH3)—, or —N(C(═O)CH3)—;
X4 is —OH or —O−;
each X1, X2, X3, and Xn is independently —OH, —SH, —O−, —S−, —NH2, —NHCH3, —NH(C(═O)CH3), —CH3, —CH2CH3, —CH2CH2CH3, —CH(CH3)2, —OCH3, or —OCH2CH3;
Y4 is ═S;
each Y1, Y2, Y3 and Yn is independently ═O, ═S, ═NH, or ═NCH3;
each A, A1, and A2 is independently —O—, —S—, —CH2—, —NH—, —N(CH3)— or —N(C(═O)CH3)—; and
p=0, 1, 2, 3, 4, 5 or 6,
wherein the compound comprises a phosphorothioate, and where the phosphorothioate comprises a chiral phosphorous center.

### Claim at position 2

-4. (canceled)

### Claim 5

An in vitro-transcribed (IVT) mRNA sequence initiator comprising a compound of Formula (II) or a salt or solvate thereof:
wherein
B1 is
each B2, B3, and Bn is independently a natural, a modified, or an unnatural nucleobase;
each Z′ and Z″ is independently is hydrogen, fluorine, —OH, —SH, —CH3, —CH2CH3, —OCH3, —NH(CH3), —NH2, —NH(C(═O)CH3), or —SCH3;
Z′″ is hydrogen, fluorine, —CH3, —CH2CH3, —OCH3, or —OCH2CH3;
each Z1 and Z2 is independently hydrogen, fluorine, —OH, —SH, —CH3, —CH2CH3, —OCH3, —SCH3, —OCH2CH3, —NH2, NHCH3, or NHC(═O)CH3;
each Z3, Z4, and Zn is independently hydrogen, fluorine, —OH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3;
each Q1 and Q4 is independently —CH═CH—, —CH2—, —CH2O—, —CH2S—, —CH2CH2—, —CH2CF2—, —CH2NH2—, —CH2NH(CH3)—, or —CH2N(C(═O)CH3)—;
each Q2 and Q3 is independently —O—, —S—, —CH2—, —CF2—, —NH—, —N(CH3)—, or —N(C(═O)CH3)—;
each X1, X2, X3, X4, and Xn is independently —OH, —SH, —O−, —S−, —NH2, —NHCH3, —NH(C(═O)CH3), —CH3, —CH2CH3, —CH2CH2CH3, —CH(CH3)2, —OCH3 or —OCH2CH3;
each Y1, Y2, Y3, Y4, and Yn is independently ═O, ═S, ═NH, or ═NCH3;
each A, A1, and A2 is independently —O—, —S—, —CH2—, —NH—, —N(CH3)— or —N(C(═O)CH3)—; and
p=0, 1, 2, 3, 4, 5 or 6.

### Claim 6

An in vitro-transcribed (IVT) mRNA sequence initiator comprising a compound of Formula (III) or a salt or solvate thereof:
wherein
B1 is
B2 is a modified or unnatural nucleobase;
each B3, and Bn is independently a natural, a modified, or an unnatural nucleobase;
each Z1 and Z2 is independently hydrogen, fluorine, —OH, —SH, —CH3, —CH2CH3, —OCH3, —OCH2CH3, —SCH3, —NH2, NHCH3, or NHC(═O)CH3;
each Z3, Z4, and Zn is independently hydrogen, fluorine, —OH, —SH, —CH3, —CH2CH3, —OCH3, —NH2, —NHCH3, —NH(C(═O)CH3), —OCH2CH3, —OCH2OCH3, —OCH2CH2CH3, —OCH(CH3)2, —SCH3, or —OCH2CH2OCH3;
each Q1 and Q4 is independently —CH2—, —CH═CH—, —CH2O—, —CH2S—, —CH2CH2—, —CH2CF2—, —CH2NH2—, —CH2NH(CH3)—, or —CH2N(C(═O)CH3)—;
each Q2 and Q3 is independently —O—, —S—, —CH2—, —CF2—, —NH—, —N(CH3)—, or —N(C(═O)CH3)—;
each X1, X2, X3, X4, and Xn is independently —OH, —SH, —O−, —S−, —NH2, —NHCH3, —NH(C(═O)CH3), —CH3, —CH2CH3, —CH2CH2CH3, —CH(CH3)2, —OCH3, or —OCH2CH3;
each Y1, Y2, Y3, Y4, and Yn is independently ═O, ═S, ═NH, or ═NCH3;
each A, A1, and A2 is independently —O—, —S—, —CH2—, —NH—, —N(CH3)— or —N(C(═O)CH3)—; and
p=0, 1, 2, 3, 4, 5 or 6.

### Claim at position 5

-8. (canceled)

### Claim 9 (depends on 1)

The IVT mRNA sequence initiator of claim 1, wherein the initiator has a structure of Formula (I-c) or Formula (I-d):

### Claim 10

(canceled)

### Claim 11 (depends on 6)

The IVT mRNA sequence initiator of claim 6, wherein the initiator has a structure of Formula (III-a):

### Claim at position 9

-13. (canceled)

### Claim 14 (depends on 1)

The IVT mRNA sequence initiator of claim 1, wherein
each Z4 and Zn is independently —OH or —OCH3,
each Y1, Y2, Y3, and Yn is independently ═O or ═S,
each X1, X2, X3 and Xn is independently —O− or —S−.

### Claim at position 11

-17. (canceled)

### Claim 18 (depends on 6)

The IVT mRNA sequence initiator of claim 6, wherein B2 is

### Claim 19 (depends on 6)

The IVT mRNA sequence initiator of claim 6, wherein the compound is

### Claim 20 (depends on 5)

The IVT mRNA sequence initiator of claim 5, wherein the compound is

### Claim at position 15

-23. (canceled)

### Claim 24 (depends on 1)

The IVT mRNA sequence initiator of claim 1, wherein the compound is

### Claim at position 17

-31. (canceled)

### Claim 32 (depends on 1)

An mRNA sequence comprising the IVT mRNA sequence initiator of claim 1.

### Claim at position 19

-35. (canceled)

### Claim 36 (depends on 5)

An mRNA sequence comprising the IVT mRNA sequence initiator of claim 5.

### Claim 37 (depends on 6)

An mRNA sequence comprising the IVT mRNA sequence initiator of claim 6.

### Claim at position 22

-162. (canceled)

### Claim 163 (depends on 32)

A pharmaceutical composition comprising the mRNA sequence of claim 32 and one or more of pharmaceutically acceptable excipients.

### Claim at position 24

-169. (canceled)

### Claim 170 (depends on 163)

A method for reducing the risk of coronary disease in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition of claim 163.

### Claim 171 (depends on 9)

The IVT mRNA sequence initiator of claim 9, wherein
B2 is adenine and B3 is guanine;
each of Q1 and Q4 is —CH2O—;
each of Q2 and Q3 is —O—;
each of X1, X2, and X3 is —OH or —O−;
each of Y1, Y2, and Y3 is ═O; and
each of A1 and A is —O—.

### Claim 172 (depends on 32)

The mRNA sequence of claim 32, wherein the mRNA sequence comprises
(a) a 5′ untranslated region,
(b) a first region encoding a deaminase,
(c) a second region encoding a programmable nuclease,
(d) a third region encoding a nuclear localization sequence,
(e) a 3′ untranslated region, and
(f) a polyadenylic acid region.

### Claim 173 (depends on 172)

The mRNA sequence of claim 172, wherein
the deaminase is an adenine base editor, and
the programmable nuclease is a Cas9 protein.

### Claim 174 (depends on 32)

The mRNA sequence of claim 32, wherein the mRNA sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3 or 4.

### Claim 175 (depends on 1)

A method of producing an mRNA sequence using an IVT reaction comprising
(a) mixing a DNA template, a polymerase enzyme, the IVT mRNA sequence initiator of claim 1, and nucleoside triphosphates (NTPs) at a specified molar ratio of the IVT mRNA sequence initiator to the NTPs to generate a mixture;
(b) incubating the mixture at a specified temperature and duration; and
(c) harvesting and purifying the mRNA sequence from the mixture,
wherein said molar ratio of the IVT mRNA sequence initiator to the NTPs is about 1:5, about 1:2.5, about 1:1.67, about 1:1.25, or about 1:1;
wherein said method is capable of producing a yield of
(i) at least 80%, or
(ii) at least 3 mg of mRNA per mL of the IVT reaction,
with a capping efficiency of at least 80%; and
wherein said NTPs is GTP, ATP, CTP, UTP, or a modified NTP, or a combination thereof.

### Claim 176 (depends on 36)

A pharmaceutical composition comprising the mRNA sequence of claim 36 and one or more of pharmaceutically acceptable excipients.

### Claim 177 (depends on 176)

A method for reducing the risk of coronary disease in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition of claim 176.

### Claim 178 (depends on 36)

The mRNA sequence of claim 36, wherein the mRNA sequence comprises
(a) a 5′ untranslated region,
(b) a first region encoding a deaminase,
(c) a second region encoding a programmable nuclease,
(d) a third region encoding a nuclear localization sequence,
(e) a 3′ untranslated region, and
(f) a polyadenylic acid region.

### Claim 179 (depends on 178)

The mRNA sequence of claim 178, wherein
the deaminase is an adenine base editor, and
the programmable nuclease is a Cas9 protein.

### Claim 180 (depends on 36)

The mRNA sequence of claim 36, wherein the mRNA sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3 or 4.

### Claim 181 (depends on 5)

A method of producing an mRNA sequence using an IVT reaction comprising
(a) mixing a DNA template, a polymerase enzyme, the IVT mRNA sequence initiator of claim 5, and nucleoside triphosphates (NTPs) at a specified molar ratio of the IVT mRNA sequence initiator to the NTPs to generate a mixture;
(b) incubating the mixture at a specified temperature and duration; and
(c) harvesting and purifying the mRNA sequence from the mixture,
wherein said molar ratio of the IVT mRNA sequence initiator to the NTPs is about 1:5, about 1:2.5, about 1:1.67, about 1:1.25, or about 1:1;
wherein said method is capable of producing a yield of
(i) at least 80%, or
(ii) at least 3 mg of mRNA per mL of the IVT reaction,
with a capping efficiency of at least 80%; and
wherein said NTPs is GTP, ATP, CTP, UTP, or a modified NTP, or a combination thereof.

### Claim 182 (depends on 37)

A pharmaceutical composition comprising the mRNA sequence of claim 37 and one or more of pharmaceutically acceptable excipients.

### Claim 183 (depends on 182)

A method for reducing the risk of coronary disease in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition of claim 182.

### Claim 184 (depends on 37)

The mRNA sequence of claim 37, wherein the mRNA sequence comprises
(a) a 5′ untranslated region,
(b) a first region encoding a deaminase,
(c) a second region encoding a programmable nuclease,
(d) a third region encoding a nuclear localization sequence,
(e) a 3′ untranslated region, and
(f) a polyadenylic acid region.

### Claim 185 (depends on 184)

The mRNA sequence of claim 184, wherein
the deaminase is an adenine base editor, and
the programmable nuclease is a Cas9 protein.

### Claim 186 (depends on 37)

The mRNA sequence of claim 37, wherein the mRNA sequence comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 3 or 4.

### Claim 187 (depends on 6)

A method of producing an mRNA sequence using an IVT reaction comprising
(a) mixing a DNA template, a polymerase enzyme, the IVT mRNA sequence initiator of claim 6, and nucleoside triphosphates (NTPs) at a specified molar ratio of the IVT mRNA sequence initiator to the NTPs to generate a mixture;
(b) incubating the mixture at a specified temperature and duration; and
(c) harvesting and purifying the mRNA sequence from the mixture,
wherein said molar ratio of the IVT mRNA sequence initiator to the NTPs is about 1:5, about 1:2.5, about 1:1.67, about 1:1.25, or about 1:1;
wherein said method is capable of producing a yield of
(i) at least 80%, or
(ii) at least 3 mg of mRNA per mL of the IVT reaction,
with a capping efficiency of at least 80%; and
wherein said NTPs is GTP, ATP, CTP, UTP, or a modified NTP, or a combination thereof.