# US 12,636,304 B2 · Metabolically stabilized double stranded mRNA

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- Number: US 12,636,304 B2
- Document ID: US12636304B2
- Status: Granted
- Kind: B2
- Application number: 17/235,535
- Filed: 2021-04-20
- Published: 2021-08-05
- Granted: 2026-05-26
- Earliest claimed: 2016-04-01
- Assignees: University of Iowa Research Foundation
- Applicants: University of Iowa Research Foundation
- Inventors: Kevin G. Rice; Samuel T. Crowley
- Web page: https://patentgrep.com/patent/US12636304B2

## CPC

- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 39/001191: {Melan-A/MART}
- A61K 39/12: Viral antigens
- C07H 21/02: with ribosyl as saccharide radical
- C12N 15/67: General methods for enhancing the expression
- A61K 2039/53: {DNA (RNA) vaccination}
- 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)
- 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)

## Abstract

Double stranded mRNA, e.g., produced in vitro, as well as method of making and using the ds mRNA, are provided. For example, the disclosure provides a method of expressing a prophylactic or therapeutic protein in mammalian cells in vivo, that includes introducing a composition comprising ds mRNA that encodes a protein to the mammalian cells in an amount effective to express the protein in vivo.

## Claims

### Claim 1

A method to induce an immune response in a mammal, comprising:
administering to the mammal a composition comprising a double-stranded (ds) mRNA encoding a protein, wherein the protein is an antigen, wherein the ds mRNA comprises a forward strand and a reverse strand, wherein the forward strand of the ds mRNA has a 5′ cap, a start codon, a poly A sequence and encodes the protein, wherein the reverse strand comprises a 5′ amino allyl modified uridine or cytidine, wherein the 5′ amino allyl modified uridine or cytidine is functionalized with an anhydride, or an N-hydroxysuccinamide ester, wherein the two strands of the ds mRNA are hydrogen bonded over at least 50% of the length of the strands, wherein the hydrogen bonded strands include at least a portion of the coding region for the protein;
wherein following administration of the composition to the mammal, the mammal expresses the protein, and wherein the protein induces an immune response in the mammal;
wherein the composition further comprises a carrier protein or a synthetic polymer.

### Claim 2 (depends on 1)

The method of claim 1, wherein the protein is a cancer antigen.

### Claim 3 (depends on 1)

The method of claim 1, wherein the composition is systemically administered to the mammal.

### Claim 4 (depends on 1)

The method of claim 1, wherein the composition further comprises a liposome or a nanoparticle.

### Claim 5 (depends on 1)

The method of claim 1, wherein at least one strand includes one or more non-natural nucleotides.

### Claim 6 (depends on 5)

The method of claim 5, wherein at least one of the non-natural nucleotides has a non-natural sugar or a non-natural nucleobase, or a combination thereof.

### Claim 7 (depends on 5)

The method of claim 5, wherein at least 5% of the nucleotides are non-natural nucleotides.

### Claim 8 (depends on 5)

The method of claim 5, wherein the non-natural nucleotide is a purine analog.

### Claim 9 (depends on 1)

The method of claim 1, wherein at least one strand includes at least one non-phosphodiester bond.

### Claim 10 (depends on 1)

The method of claim 1, wherein at least one strand includes 5-formyl cytidine or pseudouridine.

### Claim 11 (depends on 1)

The method of claim 1, wherein one of the strands is no more than 5 kb in length.

### Claim 12 (depends on 1)

The method of claim 1, wherein at least one strand has two or more different non-natural nucleotides.

### Claim 13 (depends on 1)

The method of claim 1, wherein the strands are not the same length.

### Claim 14 (depends on 1)

The method of claim 1, wherein the composition is administered intramuscularly (IM).

### Claim 15 (depends on 1)

The method of claim 1, wherein the protein is a viral antigen.

### Claim 16 (depends on 1)

The method of claim 1, wherein the protein is a bacterial antigen.