# US 2019/0314527 A1 · In vivo production of proteins

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- Number: US 2019/0314527 A1
- Document ID: US20190314527A1
- Status: Published application
- Kind: A1
- Application number: 16/442,168
- Filed: 2019-06-14
- Published: 2019-10-17
- Earliest claimed: 2012-04-02
- Assignees: ModernaTX, Inc.
- Applicants: ModernaTX, Inc.
- Inventors: Antonin DE FOUGEROLLES; Jason P. SCHRUM; Justin GUILD
- Web page: https://patentgrep.com/patent/US20190314527A1

## CPC

- 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 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 9/1271: Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911})
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- A61K 9/5146: {obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides}
- A61K 9/5153: {Polyesters, e.g. poly(lactide-co-glycolide)}
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- C07H 21/02: with ribosyl as saccharide radical
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- C07K 14/505: Erythropoietin [EPO]
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- C07K 14/745: Blood coagulation or fibrinolysis factors
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
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- C12N 9/6435: {Plasmin (3.4.21.7), i.e. fibrinolysin}
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- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/85: for animal cells
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N 15/88: using microencapsulation, e.g. using {amphiphile} liposome vesicle
- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12P 21/005: {Glycopeptides, glycoproteins}
- C12Y 603/02019: Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
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- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- 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)
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The invention relates to compositions including polynucleotides encoding polypeptides which have been chemically modified by replacing the uridines with 1-methyl-pseudouridine to improve one or more of the stability and/or clearance in tissues, receptor uptake and/or kinetics, cellular access by the compositions, engagement with translational machinery, mRNA half-life, translation efficiency, immune evasion, protein production capacity, secretion efficiency, accessibility to circulation, protein half-life and/or modulation of a cell's status, function, and/or activity.

## Claims

### Claim 1

A method of expressing a protein in a mammalian subject, the method comprising administering a pharmaceutical composition comprising a plurality of lipid nanoparticles encapsulating a polynucleotide, wherein the lipid nanoparticle comprises a biodegradable cationic lipid, a neutral lipid, cholesterol, and a PEGylated lipid and the plurality of lipid nanoparticles has a mean particle size of between 80 nm to 160 nm, and
wherein the polynucleotide comprises:
(a) an open reading frame encoding the protein consisting of nucleotides selected from 1-methyl-pseudouridine, cytidine, adenosine, and guanosine;
(b) a 5′-UTR;
(c) at least one 5′ cap structure;
(d) a 3′-UTR; and
(e) a 3′ tailing sequence of linked nucleosides.

### Claim 2 (depends on 1)

The method of claim 1, wherein the biodegradable cationic lipid comprises an ester linkage.

### Claim 3 (depends on 1)

The method of claim 1, wherein the method comprises administering about 0.05 to about 0.5 mg/kg of polynucleotide.

### Claim 4 (depends on 1)

The method of claim 1, wherein the administration is intramuscular administration.

### Claim 5 (depends on 1)

The method of claim 1, wherein the administration is intravenous administration.

### Claim 6 (depends on 1)

The method of claim 1, wherein upon administration, expression of the protein is maximal at 8-24 hours.

### Claim 7 (depends on 1)

The method of claim 1, wherein the 3′-tailing sequence of linked nucleosides is selected from the group consisting of a poly-A tail and a polyA-G quartet.

### Claim 8 (depends on 7)

The method of claim 7, wherein the poly-A comprises approximately 160 nucleotides.

### Claim 9 (depends on 1)

The method of claim 1, wherein the at least one 5′ cap structure is selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

### Claim 10 (depends on 9)

The method of claim 9, wherein the at least one 5′-cap structure is cap0, cap1, or ARCA.

### Claim 11 (depends on 1)

The method of claim 1, wherein the plurality of lipid nanoparticles has a mean PDI of between 0.02 and 0.2.

### Claim 12 (depends on 1)

The method of claim 1, wherein the plurality of lipid nanoparticles has a mean lipid to polynucleotide ratio (wt/wt) of between 10 and 20.

### Claim 13 (depends on 1)

The method of claim 1, wherein the 3′-UTR comprises a miR binding site.

### Claim 14 (depends on 1)

The method of claim 1, wherein the 5′-UTR comprises a Kozak sequence.

### Claim 15 (depends on 1)

The method of claim 1, wherein the neutral lipid is a phospholipid.

### Claim 16 (depends on 1)

The method of claim 1, wherein the open reading frame is codon optimized to bias GC content.

### Claim 17 (depends on 1)

The method of claim 1, wherein the plurality of lipid nanoparticles comprise about 50 mol % biodegradable cationic lipid, about 38.5% cholesterol, about 10% neutral lipid and about 1.5% PEGylated lipid.

### Claim 18 (depends on 1)

The method of claim 1, wherein the polynucleotide includes at least two stop codons before the 3′ untranslated region (UTR).