# US 11,027,025 B2 · Compositions comprising synthetic polynucleotides encoding CRISPR related proteins and synthetic sgRNAs and methods of use

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- Number: US 11,027,025 B2
- Document ID: US11027025B2
- Status: Granted
- Kind: B2
- Application number: 14/903,869
- Filed: 2014-07-11
- Published: 2016-12-22
- Granted: 2021-06-08
- Earliest claimed: 2013-07-11
- Assignees: ModernaTX, Inc.
- Applicants: ModernaTX, Inc.
- Inventors: Stephen G. Hoge; Eric Yi-Chun Huang; Tirtha Chakraborty
- Web page: https://patentgrep.com/patent/US11027025B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- 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}
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 9/16: acting on ester bonds (3.1)
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/907: {in mammalian cells}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C12N 2310/10: Type of nucleic acid
- C12N 2310/141: MicroRNAs, miRNAs
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/315: Phosphorothioates
- C12N 2310/346: having a combination of backbone and sugar modifications
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention relates to compositions comprising and methods of using synthetic polynucleotides, e.g., modified mRNA, encoding CRISPR related proteins including dCAS9 and synthetic sgRNAs targeting a gene of interest.

## Claims

### Claim 1

A composition comprising
(i) a plurality of lipid nanoparticles comprising a biodegradable cationic lipid, a non-cationic lipid, a sterol, and a PEG-lipid;
(ii) mRNA comprising:
(a) a first region of linked nucleosides consisting of nucleotides selected from 1-methyl-pseudouridine, cytidine, adenosine, and guanosine and encoding a protein comprising the sequence of SEQ ID NO: 7, 8, 9, 61, 62, 63, 64, or 65;
(b) a 5′-untranslated region (UTR); and
(c) a 3′-UTR comprising the sequence of SEQ ID NO: 81; and
(iii) sgRNA comprising:
(a) a first region of linked nucleosides complementary to either strand of a 5′ UTR of a gene of interest;
(b) a second region comprising a guide RNA scaffold sequence located at the 3′ terminus of the first region; and
(c) at least one phosphorothioate linkage and at least one nucleotide with a modification at the 2′-position of the sugar,
wherein the plurality of lipid nanoparticles encapsulate the mRNA and/or the sgRNA.

### Claim 2

A composition comprising
(i) a plurality of lipid nanoparticles comprising a biodegradable cationic lipid, a non-cationic lipid, a sterol, and a PEG-lipid;
(ii) mRNA comprising:
(a) a first region of linked nucleosides consisting of nucleotides selected from 1-methyl-pseudouridine, cytidine, adenosine, and guanosine and encoding a protein comprising the sequence of SEQ ID NO: 7, 8, 9, 61, 62, 63, 64, or 65;
(b) a 5′-UTR; and
a 3′-UTR comprising the sequence of SEQ ID NO: 82; and
(iii) sgRNA comprising:
(a) a first region of linked nucleosides complementary to either strand of a 5′ UTR of a gene of interest;
(b) a second region comprising a guide RNA scaffold sequence located at the 3′ terminus of the first region; and
(c) at least one phosphorothioate linkage and at least one nucleotide with a modification at the 2′-position of the sugar,
wherein the plurality of lipid nanoparticles encapsulate the mRNA and/or the sgRNA.

### Claim 3

A composition comprising
(i) a plurality of lipid nanoparticles comprising a biodegradable cationic lipid, a non-cationic lipid, a sterol, and a PEG-lipid;
(ii) mRNA comprising:
(a) a first region of linked nucleosides consisting of nucleotides selected from 1-methyl-pseudouridine, cytidine, adenosine, and guanosine and encoding a protein comprising the sequence of SEQ ID NO: 7, 8, 9, 61, 62, 63, 64, or 65;
(b) a 5′-UTR; and
(c) a 3′-UTR; and
(iii) sgRNA comprising:
(a) a first region of linked nucleosides complementary to either strand of a 5′ UTR of a gene of interest;
(b) a second region comprising a guide RNA scaffold sequence located at the 3′ terminus of the first region; and
(c) at least one phosphorothioate linkage and at least one nucleotide with a modification at the 2′-position of the sugar,
wherein the modification at the 2′-position of the sugar is a 2′-methoxy modification, and wherein the plurality of lipid nanoparticles encapsulate the mRNA and/or the sgRNA.