# US 9,751,925 B2 · Alternative nucleic acid molecules containing reduced uracil content and uses thereof

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- Number: US 9,751,925 B2
- Document ID: US9751925B2
- Status: Granted
- Kind: B2
- Application number: 14/960,031
- Filed: 2015-12-04
- Published: 2016-08-18
- Granted: 2017-09-05
- Earliest claimed: 2014-11-10
- Assignees: ModernaTX, Inc.
- Applicants: ModernaTX, Inc.
- Inventors: Stephen G. Hoge; William Joseph Issa; Edward John Miracco; Jennifer Nelson; John Reynders; Matthew Stanton
- Web page: https://patentgrep.com/patent/US9751925B2

## CPC

- C07K 14/535: Granulocyte CSF; Granulocyte-macrophage CSF
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- C07K 14/505: Erythropoietin [EPO]
- C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12Y 113/12007: Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
- 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)
- 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)
- 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 alternative nucleosides, nucleotides, and nucleic acids, and methods of using them. In some aspects, the disclosure provides mRNA wherein the uracil content has been modified and which may be particularly effective for use in therapeutic compositions, because they may benefit from both high expression levels and limited induction of the innate immune response. In some aspects, the disclosure provides methods for the production of pharmaceutical compositions including mRNA without reverse phase chromatography.

## Claims

### Claim 1

An mRNA encoding a polypeptide, the mRNA comprising:
(i) a 5′-cap structure;
(ii) a 5′-UTR;
(iii) an open reading frame (ORF) encoding the polypeptide,
wherein at least 95% of uracils in the ORF are 5-methoxyuracils; and
wherein the uracil content in the ORF is between the theoretical minimum and 150% of the theoretical minimum,
(iv) a 3′-UTR; and
(v) a poly-A region;
wherein the level of expression in a mammalian cell of the encoded polypeptide from the mRNA is increased relative to a reference mRNA comprising a reference open reading frame (rORF) encoding the polypeptide,
wherein at least 95% of uracils in the rORF are 5-methoxyuracils, and wherein the uracil content in the rORF is from about 190% to about 200% of the theoretical minimum.

### Claim 2 (depends on 1)

The mRNA of claim 1, wherein the uracil content in the ORF is between the theoretical minimum and 125% of the theoretical minimum.

### Claim 3 (depends on 1)

The mRNA of claim 1, wherein the guanine content of the ORF is maximized for at least 50% of the codons, and wherein the ORF comprises at least one low frequency guanine maximized codon.

### Claim 4 (depends on 1)

The mRNA of claim 1, wherein the cytosine content of the ORF is maximized for at least 50% of the codons, and wherein the ORF comprises at least one low frequency cytosine maximized codon.

### Claim 5 (depends on 1)

The mRNA of claim 1, wherein the guanine and cytosine content of the ORF is maximized for at least 50% of the codons, and wherein the ORF comprises at least one low frequency guanine and cytosine maximized codon.

### Claim 6 (depends on 1)

The mRNA of claim 1, wherein the uracil content is less than 20% of the total nucleobase content in the ORF.

### Claim 7 (depends on 1)

The mRNA of claim 1, wherein the uracil content within any 20 nucleobase window within the ORF does not exceed 50%.

### Claim 8 (depends on 1)

The mRNA of claim 1, wherein expression of the polypeptide in HeLa cells transfected with the mRNA of claim 1 using Lipofectamine 2000, followed by an incubation for 18-22 hours at 37° C., is comparable to expression of said polypeptide from an mRNA comprising a modified reference open reading frame (mrORF) encoding said polypeptide, wherein 100% of the uracils in the mrORF are 1-methylpseudouracils.

### Claim 9 (depends on 1)

The mRNA of claim 1, wherein, upon intravenous administration of 0.05 mg/kg of the mRNA to a mouse, the mRNA expresses the encoded polypeptide, wherein the level of expression of the encoded polypeptide in the mouse is increased relative to a reference mRNA comprising a rORF encoding the polypeptide, wherein at least 95% of uracils in the rORF are 5-methoxyuracils, and wherein the uracil content in the rORF is about 190% of the theoretical minimum.

### Claim 10 (depends on 1)

The mRNA of claim 1, wherein the 5′-cap structure is cap0, cap1, or ARCA inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, or 2-azido-guanosine.

### Claim 11 (depends on 1)

The mRNA of claim 1, wherein the 3′-UTR is an alpha-globin 3′-UTR.

### Claim 12 (depends on 1)

The mRNA of claim 1, wherein the poly-A region is between 80 to 120 nucleotides in length.

### Claim 13 (depends on 1)

The mRNA of claim 1, wherein, upon contacting a BJ fibroblast cell or human keratinocyte cells, the mRNA induces a detectably lower level of IFN-β at 48 hours relative to an mRNA encoding the polypeptide and having unmodified uracils, and further having a uracil content above 150% of the theoretical minimum.

### Claim 14 (depends on 1)

The mRNA of claim 1, wherein, upon contacting the mammalian cell, the mRNA has a longer half-life, relative to an mRNA encoding the polypeptide and having unmodified uracils, and further having a uracil content above 150% of the theoretical minimum.

### Claim 15 (depends on 1)

The mRNA of claim 1, wherein the ORF further comprises at least one low-frequency codon.

### Claim 16 (depends on 1)

The mRNA of claim 1, wherein the 3′-UTR of the mRNA comprises at least one microRNA (miRNA) binding site.

### Claim 17 (depends on 16)

The mRNA of claim 16, wherein the miRNA is known to be expressed in liver cells or in immune cells.

### Claim 18 (depends on 17)

The mRNA of claim 17, wherein the miRNA is selected from miR-142-5p, miR-146-5p, and miR-146-3p.

### Claim 19 (depends on 17)

The mRNA of claim 17, wherein the miRNA is miR-142-3p.

### Claim 20 (depends on 16)

The mRNA of claim 16, wherein the miRNA is known to be expressed in liver cells, and is miR-122-5p or miR-122-3p.

### Claim 21

A method of producing a modified mRNA comprising an ORF encoding a polypeptide, the method comprising modifying an mRNA sequence to produce the modified mRNA by replacing at least one codon containing a uracil with a codon that encodes the same amino acid but contains a lower number of uracils,
wherein at least 95% of uracils in the ORF are 5-methoxyuracils, and
wherein the uracil content in the ORF is between the theoretical minimum and 150% of the theoretical minimum;
wherein the level of expression in a mammalian cell of the encoded polypeptide from the mRNA is increased relative to a reference mRNA comprising a reference open reading frame (rORF) encoding the polypeptide,
wherein at least 95% of uracils in the rORF are 5-methoxyuracils, and wherein the uracil content in the rORF is from about 190% to about 200% of the theoretical minimum.

### Claim 22 (depends on 21)

The method of claim 21, wherein the guanine content of the ORF is maximized for at least 50% of the codons, and wherein the ORF comprises at least one low frequency guanine maximized codon.

### Claim 23 (depends on 21)

The mRNA of claim 21, wherein the cytosine content of the ORF is maximized for at least 50% of the codons, and wherein the ORF comprises at least one low frequency cytosine maximized codon.

### Claim 24 (depends on 21)

The method of claim 21, wherein the guanine and cytosine content of the ORF is maximized for at least 50% of the codons, and wherein the ORF comprises at least one low frequency guanine and cytosine maximized codon.

### Claim 25 (depends on 21)

The method of claim 21, wherein the uracil content within any 20 nucleobase window within the ORF of the modified mRNA does not exceed 50%.

### Claim 26 (depends on 21)

The method of claim 21, wherein the uracil content is less than 20% of the total nucleobase content in the ORF.

### Claim 27

A composition comprising:
(a) a lipid nanoparticle comprising a cationic lipid; and
(2) an mRNA encoding a polypeptide, the mRNA comprising:
(i) a 5′-cap structure;
(ii) a 5′-UTR;
(iii) an ORF encoding the polypeptide
wherein at least 95% uracils in the ORF are 5-methoxyuracils; and
wherein the uracil content in the ORF is between the theoretical minimum and 150% of the theoretical minimum,
(iv) a 3′-UTR; and
(v) a poly-A region;
wherein the level of expression in a mammalian cell of the encoded polypeptide from the mRNA is increased relative to a reference mRNA comprising a reference open reading frame (rORF) encoding the polypeptide,
wherein at least 95% of uracils in the rORF are 5-methoxyuracils, and wherein the uracil content in the rORF is from about 190% to about 200% of the theoretical minimum.

### Claim 28 (depends on 27)

The composition of claim 27, wherein the 3′-UTR of the mRNA comprises at least one miRNA binding site.

### Claim 29 (depends on 28)

The composition of claim 28, wherein the miRNA binding site is a miR-143-3p binding site.