# US 9,376,669 B2 · Methods and products for expressing proteins in cells

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- Number: US 9,376,669 B2
- Document ID: US9376669B2
- Status: Granted
- Kind: B2
- Application number: 14/735,603
- Filed: 2015-06-10
- Published: 2015-10-01
- Granted: 2016-06-28
- Earliest claimed: 2012-11-01
- Assignees: Factor Bioscience Inc.
- Applicants: Factor Bioscience Inc.
- Inventors: Matthew Angel; Christopher Rohde
- Web page: https://patentgrep.com/patent/US9376669B2

## CPC

- C12N 15/907: {in mammalian cells}
- A61K 31/7115: Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
- A61K 35/14: Blood; Artificial blood (perfluorocarbons A61K31/02; umbilical cord blood A61K35/51; haemoglobin A61K38/42)
- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61P 1/02: Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 1/18: for pancreatic disorders, e.g. pancreatic enzymes
- A61P 5/00: Drugs for disorders of the endocrine system
- A61P 5/14: of the thyroid hormones, e.g. T3, T4
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 11/02: Nasal agents, e.g. decongestants
- A61P 11/04: for throat disorders
- A61P 13/02: of urine or of the urinary tract, e.g. urine acidifiers
- A61P 13/08: of the prostate
- A61P 13/10: of the bladder
- A61P 13/12: of the kidneys
- A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives
- A61P 17/00: Drugs for dermatological disorders
- A61P 19/00: Drugs for skeletal disorders
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 21/04: for myasthenia gravis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/14: for treating abnormal movements, e.g. chorea, dyskinesia
- A61P 25/16: Anti-Parkinson drugs
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 27/02: Ophthalmic agents
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C07K 14/195: from bacteria
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/1024: {In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair}
- C12N 15/86: Viral vectors
- C12Y 301/00: Hydrolases acting on ester bonds (3.1)
- C12Y 301/21004: Type II site-specific deoxyribonuclease (3.1.21.4)
- G01N 33/5014: {for testing toxicity}
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- 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
- 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)
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention relates in part to nucleic acids encoding proteins, therapeutics comprising nucleic acids encoding proteins, methods for inducing cells to express proteins using nucleic acids, methods, kits and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices. Methods and products for altering the DNA sequence of a cell are described, as are methods and products for inducing cells to express proteins using synthetic RNA molecules. Therapeutics comprising nucleic acids encoding gene-editing proteins are also described.

## Claims

### Claim 1

A composition comprising a nucleic acid encoding a gene-editing protein, the gene-editing protein comprising: (a) a DNA-binding domain and (b) a nuclease domain, wherein:
(a) the DNA-binding domain comprises a plurality of repeat sequences,
(i) at least one of the repeat sequences containing a region capable of binding to a binding site in a target DNA molecule, the binding site containing a defined sequence of between 1 and 5 bases in length, and
(ii) at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG (SEQ ID NO: 75) and is between 36 and 39 amino acids long, wherein:
“v” is E,
“w” is S,
“x” is N,
“y” is I, and
“z” is GGKQALETVQRLLPVLCQA (SEQ ID NO: 671);
(b) the nuclease domain comprises the catalytic domain of a protein selected from the group consisting of Fok1 and StsI; and
the gene-editing protein is capable of altering the target DNA sequence in a living cell.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the nuclease domain is capable of forming a dimer with another nuclease domain.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the composition is capable of generating a nick or double-strand break in the target DNA molecule.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the nucleic acid is a synthetic RNA molecule.

### Claim 5 (depends on 4)

The composition of claim 4, wherein the synthetic RNA molecule comprises one or more non-canonical nucleotides.

### Claim 6 (depends on 5)

The composition of claim 5, wherein the non-canonical nucleotide is 5-methylcytidine.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the nuclease domain comprises the catalytic domain of a protein comprising the amino acid sequence of SEQ ID NO: 53.