# US 2024/0189417 A1 · Substitution of nucleotide bases in self-amplifying messenger ribonucleic acids

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

- Number: US 2024/0189417 A1
- Document ID: US20240189417A1
- Status: Published application
- Kind: A1
- Application number: 18/425,426
- Filed: 2024-01-29
- Published: 2024-06-13
- Earliest claimed: 2021-09-03
- Assignees: Glaxosmithkline Biologicals SA
- Applicants: GlaxoSmithKline Biologicals SA
- Inventors: Giulietta MARUGGI; Qiongman KONG; Varnika ROY; Dong YU; Meng ZHANG
- Web page: https://patentgrep.com/patent/US20240189417A1

## CPC

- A61K 39/215: Coronaviridae, e.g. avian infectious bronchitis virus
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- A61K 31/7105: Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
- A61K 31/7115: Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
- A61K 39/0002: {Fungal antigens, e.g. Trichophyton, Aspergillus, Candida}
- A61K 39/0011: {Cancer antigens}
- A61K 39/0013: {Therapeutic immunisation against small organic molecules, e.g. cocaine, nicotine}
- A61K 39/02: Bacterial antigens
- A61K 39/12: Viral antigens
- A61P 31/14: for RNA viruses
- A61P 37/04: Immunostimulants
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- A61K 48/0066: {Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid}
- A61K 2039/53: {DNA (RNA) vaccination}
- C12N 2770/20034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)}

## Abstract

Provided herein are RNA that: are collectively self-amplifying in an intracellular environment, comprise N1-methyl-pseudouridines and uridines, and have a mole percentage or mole proportion of the N1-methylpseudouridines to the total of the uridines and the Ni-methylpseudouridines or a mole ration of the N1-methylpseudouridines to the uridines.

## Claims

### Claim 1

A self-amplifying messenger (SAM) ribonucleic acid (RNA) comprising N1-methylpseudouridines, uridines, a first RNA segment, and a second RNA segment; the first RNA segment comprising a heterologous nucleic acid; the second RNA segment encoding one or more proteins capable of replicating the SAM RNA in an intracellular environment; the SAM RNA having a first mole percentage of the N1-methylpseudouridines to the total of the N1-methylpseudouridines and the uridines from 15% to 75%.

### Claim 2 (depends on 1)

The SAM RNA of claim 1, the first mole percentage being to 70%.

### Claim 3 (depends on 1)

The SAM RNA of claim 1, the first mole percentage being to 65%.

### Claim 4 (depends on 1)

The SAM RNA of claim 1, the first mole percentage being to 60%.

### Claim 5 (depends on 1)

The SAM RNA of claim 1, the first mole percentage being to 55%.

### Claim 6 (depends on 1)

The SAM RNA of claim 1, the first mole percentage being to 50%.

### Claim 7 (depends on 4)

The SAM RNA of claim 4, the first mole percentage being from 20%.

### Claim 8 (depends on 4)

The SAM RNA of claim 4, the first mole percentage being from 25%.

### Claim 9 (depends on 1)

The SAM RNA claim 1, the one or more proteins capable of replicating the SAM RNA in an intracellular environment comprising an alphavirus non-structural protein-1 (nsP1), an alphavirus nsP2, an alphavirus nsP3, and an alphavirus nsP4.

### Claim 10 (depends on 1)

The SAM RNA claim 1, the heterologous nucleic acid encoding a heterologous protein.

### Claim 11 (depends on 10)

The SAM RNA of claim 10, the heterologous protein comprising an immunogen, an antibody, or an immunotherapeutic molecule.

### Claim 12 (depends on 10)

The SAM RNA of claim 10, the heterologous protein comprising an immunogen or an antibody against the immunogen.

### Claim 13 (depends on 1)

The SAM RNA of claim 1 further comprising a poly-adenosine monophosphate (poly(A)) tail.

### Claim 14 (depends on 13)

The SAM RNA of claim 13 further comprising a 5′ untranslated region (5′ UTR), the 5′ UTR being 5′ of the first RNA segment and the second RNA segment.

### Claim 15 (depends on 14)

The SAM RNA of claim 14 further comprising a 3′ untranslated region (3′ UTR), the 3′ UTR being 3′ of the first RNA segment and the second RNA segment, and being 5′ of the poly(A) tail.

### Claim 16 (depends on 15)

The SAM RNA of claim 15 further comprising a 5′ cap.

### Claim 17 (depends on 16)

The SAM RNA of claim 16, the 5′ cap being a cap-0, a cap-1, or a cap-2.

### Claim 18 (depends on 16)

The SAM RNA of claim 16, the 5′ cap being a cap-1.

### Claim 19 (depends on 16)

The SAM RNA of claim 16, the 5′ cap being a cap-0.

### Claim 20 (depends on 1)

A composition comprising the SAM RNA of claim 1 and a pharmaceutically acceptable delivery vehicle.

### Claim 21 (depends on 20)

The composition of claim 20, the pharmaceutically acceptable delivery vehicle comprising a lipid nanoparticle (LNP).

### Claim 22 (depends on 21)

The composition of claim 21, the LNP encapsulating the SAM RNA.

### Claim 23 (depends on 12)

A method of eliciting an immune response in a subject to the immunogen, the method comprising administering to the subject an effective amount of the SAM RNA of claim 12.

### Claim 24 (depends on 23)

The method of claim 23, the immune response being a protective immune response.

### Claim 25 (depends on 23)

The method of claim 23, the immune response being a therapeutic immune response.

### Claim 26 (depends on 23)

The method of claim 23, the immunogen comprising a venom, a poison, an allergen, a cancer antigen, a bacterial antigen, a viral antigen, a fungal antigen, a parasite antigen, or a fragment thereof.

### Claim 27 (depends on 23)

The method of claim 23, the heterologous protein comprising the antibody against the immunogen.

### Claim 28 (depends on 23)

The method of claim 23, the heterologous protein comprising the immunogen.

### Claim 29 (depends on 23)

The method of claim 23, the subject being human.

### Claim 30 (depends on 1)

A method of manufacturing the SAM RNA of claim 1, the method comprising admixing an RNA polymerase, the N1-methylpseudouridines, the uridines, and a template nucleic acid comprising a sequence of the SAM RNA, thereby obtaining an admixture; the admixture having a second mole percentage of the N1-methylpseudouridines to the total of the N1-methylpseudouridines and the uridines; the second mole percentage being the same as the first mole percentage; the admixing being under conditions wherein the RNA polymerase produces the SAM RNA from the template nucleic acid.

### Claim 31 (depends on 16)

A method of manufacturing the SAM RNA of claim 16, the method comprising:
a first admixing of an RNA polymerase, the N1-methylpseudouridines, the uridines, and a template nucleic acid comprising a sequence of the SAM RNA, thereby obtaining an admixture; the admixture having a second mole percentage of the N1-methylpseudouridines to the total of the N1-methylpseudouridines and the uridines; the second mole percentage being the same as the first mole percentage; the first admixing being under conditions wherein the RNA polymerase produces the SAM RNA from the template nucleic acid, thereby obtaining an uncapped SAM RNA; and
a second admixing of the uncapped SAM RNA, a messenger RNA guanylyltransferase, guanosine triphosphate, a (guaninine-N7-)-methyltransferase, and S-adenosyl-L-methionine, the second admixing being under conditions that cause a 5′ to 5′ triphosphate cap linkage, the second admixing optionally further comprising a 2′-O-methyltransferase, and optionally the second admixing being under conditions that form cap-1 or cap-2.

### Claim 32 (depends on 30)

The method of claim 30, the RNA polymerase being a T7 RNA polymerase.

### Claim 33 (depends on 20)

A method of eliciting an immune response in a subject to an immunogen, the method comprising administering to the subject an effective amount of the composition of claim 20, and the heterologous nucleic acid encoding the immunogen or an antibody against the immunogen.

### Claim 34 (depends on 22)

A method of eliciting an immune response in a subject to an immunogen, the method comprising administering to the subject an effective amount of the composition of claim 22, and the heterologous nucleic acid encoding the immunogen or an antibody against the immunogen.

### Claim 35

A plurality of auto-amplifying messenger (AAM) RNAs comprising N1-methylpseudouridines, uridines, a first RNA, and one or more second RNA; the first RNA comprising a heterologous nucleic acid; the one or more second RNA encoding one or more proteins capable of replicating the AAM RNA in an intracellular environment; the plurality of AAM RNAs having a first mole percentage of the N1-methylpseudouridines to the total of the N1-methypseudouridines and uridines from 15% to 75%.

### Claim 36 (depends on 35)

A composition comprising the AAM RNA of claim 35 and a pharmaceutically acceptable delivery vehicle.

### Claim 37 (depends on 36)

The composition of claim 36, the pharmaceutically acceptable delivery vehicle comprising a LNP.

### Claim 38 (depends on 35)

A method of eliciting an immune response in a subject to an immunogen, the method comprising administering to the subject an effective amount of the plurality of AAM RNAs of claim 35; the heterologous nucleic acid encoding an immunogen or an antibody against the immunogen.

### Claim 39 (depends on 35)

A method of manufacturing the plurality of AAM RNAs of claim 35, the method comprising one or more admixing of an RNA polymerase, the N1-methylpseudouridines, the uridines, and one or more template nucleic acids, thereby obtaining an admixture wherein the one or more template nucleic acids comprise a sequence of the plurality of AAM RNAs; the one or more admixture having a second mole percentage of the N1-methylpseudouridines to the total of the N1-methylpseudouridines and the uridines; the second mole percentage being the same as the first mole percentage; the one or more admixing being under conditions wherein the RNA polymerase produces the plurality of AAM RNAs from the one or more template nucleic acids.