Methods and products for expressing proteins in cells

• US 10,724,053 B2
• Filed: 10/17/2019
• Issued: 07/28/2020
• Est. Priority Date: 11/01/2012
• Status: Active Grant

• Current Assignee: Factor Bioscience Inc
• Original Assignee: Factor Bioscience Inc
• Inventors: Angel, Matthew, Rohde, Christopher
• Primary Examiner(s): Nguyen, Quang
• Application Number:
  US16/655,744
• Publication Number:
  US 20200032297A1
• Time in Patent Office: 285 Days
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 31/7115 Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
• CPC Class Codes: A61K 35/14 Blood; Artificial blood perfluorocarbons A61K31/02; umbilical cord blood A61K35/51; haemoglobin A61K38/42
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61P 1/02 Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/02 Nasal agents, e.g. decongestants
• CPC Class Codes: A61P 11/04 for throat disorders
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 13/08 of the prostate
• CPC Class Codes: A61P 13/10 of the bladder
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 : for myasthenia gravis
• CPC Class Codes: A61P 25/00 : Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 : for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 : Anti-Parkinson drugs
• CPC Class Codes: 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
• CPC Class Codes: A61P 27/02 : Ophthalmic agents
• CPC Class Codes: A61P 31/00 : Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 5/00 : Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 5/14 : of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/195 : from bacteria
• CPC Class Codes: C07K 2319/80 : containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: 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
• CPC Class Codes: C12N 15/86 : Viral vectors
• CPC Class Codes: C12N 15/907 : in mammalian cells
• CPC Class Codes: C12N 2800/80 : Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/00 : Hydrolases acting on ester bonds (3.1)
• CPC Class Codes: C12Y 301/21004 : Type II site-specific deoxyribonuclease (3.1.21.4)
• CPC Class Codes: G01N 33/5014 : for testing toxicity

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

1. An in vitro method for inserting a nucleic-acid sequence into a safe-harbor location of a genome of a cell comprising transfecting in vitro a cell comprising a safe-harbor location with (i) a first nucleic acid comprising a nucleic-acid sequence for insertion and (ii) a second nucleic acid encoding a gene-editing protein targeting the safe-harbor location, 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 and 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 Q, D or E,

“

w”

is S or N,

“

x”

is N or H,

“

y”

is D, A, H, N, K, or G, and

“

z”

is GGKQALETVQRLLPVLCQD (SEQ ID NO;

     670) or GGKQALETVQRLLPVLCQA (SEQ ID NO;

     671); and

(b) the nuclease domain comprises a catalytic domain of a nuclease,

to result in insertion of the nucleic-acid sequence into the safe-harbor location, wherein the cell is a human cell and the safe-harbor location is the AAVS1 locus or wherein the cell is a rodent cell and the safe-harbor location is the Rosa26 locus.

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

3. The method of claim 1, wherein the gene-editing protein is capable of generating a nick or double-strand break in a target DNA molecule.

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

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

6. The method of claim 5, wherein the non-canonical nucleotide is selected from the group consisting of pseudouridine, 5-methylpseudouridine, 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, N4-methylcytidine, N4-acetylcytidine, and 7-deazaguanosine.

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

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7 or SEQ ID NO;

53.

8. The method of claim 1, wherein the nuclease is selected from the group consisting of FokI and StsI.

9. The method of claim 1, wherein at least one of the repeat sequences contains 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.