Methods and products for transfecting cells

• US 10,662,410 B1
• Filed: 01/30/2020
• Issued: 05/26/2020
• Est. Priority Date: 12/05/2011
• Status: Active Grant

• Current Assignee: Factor Bioscience Inc
• Original Assignee: Factor Bioscience Inc
• Inventors: Angel, Matthew, Rohde, Christopher
• Primary Examiner(s): Noble, Marcia S
• Application Number:
  US16/776,765
• Publication Number:
  US 20200157504A1
• Time in Patent Office: 117 Days
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• 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 25/02 for peripheral neuropathies
• 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 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C08G 77/08 characterised by the catalysts used
• CPC Class Codes: C08K 5/5399 Phosphorus bound to nitrogen
• CPC Class Codes: C08L 83/04 : Polysiloxanes
• CPC Class Codes: C12N 15/87 : Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/907 : in mammalian cells
• CPC Class Codes: C12N 2500/25 : Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/40 : Nucleotides, nucleosides, bases cyclic nucleotides C12N2501/01, anti-neoplasic drugs C12N2501/06
• CPC Class Codes: C12N 2500/44 : Thiols, e.g. mercaptoethanol
• CPC Class Codes: C12N 2501/115 : Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/155 : Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/165 : Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/2303 : Interleukin-3 (IL-3)
• CPC Class Codes: C12N 2501/26 : Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2501/91 : Heparin
• CPC Class Codes: C12N 2501/998 : Proteins not provided for elsewhere
• CPC Class Codes: C12N 2506/09 : from epidermal cells, from skin cells, from oral mucosa cells
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 2800/80 : Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 5/0647 : Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0657 : Cardiomyocytes; Heart cells
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: C12N 9/16 : acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12P 21/00 : Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12Y 301/21 : Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
• CPC Class Codes: H01L 31/048 : Encapsulation of modules
• CPC Class Codes: Y02E 10/50 : Photovoltaic [PV] energy
• CPC Class Codes: Y02E 10/52 : PV systems with concentrators

Abstract

The present invention relates in part to nucleic acids encoding proteins, nucleic acids containing non-canonical nucleotides, therapeutics comprising nucleic acids, methods, kits, and devices for inducing cells to express proteins, methods, kits, and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices. Methods for inducing cells to express proteins and for reprogramming and gene-editing cells using RNA are disclosed. Methods for producing cells from patient samples, cells produced using these methods, and therapeutics comprising cells produced using these methods are also disclosed.

Claims

1. A method for producing a gene-edited cell, comprising:

(a) providing a cell comprising a target DNA sequence;

(b) culturing the cell; and

(c) transfecting the cell with a plurality of synthetic RNA molecules, wherein the synthetic RNA molecules include;

i. a first synthetic RNA molecule encoding a first fusion protein comprising a DNA-binding domain and a catalytic domain of a nuclease; and

ii. a second synthetic RNA molecule encoding a second fusion protein comprising a DNA-binding domain and a catalytic domain of a nuclease;

wherein;

the first fusion protein and the second fusion protein are independently a transcription activator-like effector nuclease (TALEN);

the transfecting results in the cell expressing the first fusion protein and the second fusion protein to result in a double-stranded break in the target DNA sequence; and

the first synthetic RNA molecule and the second synthetic RNA molecule are independently synthesized by in vitro transcription from a DNA template.

2. The method of claim 1, wherein the DNA template encodes a plurality of monomer repeats wherein each monomer repeat comprises a repeat variable domain (RVD), wherein the RVDs are selected to target a sequence within the target DNA sequence.

3. The method of claim 1, wherein the cell is a human cell.

4. The method of claim 1, further comprising contacting the cell with at least one of poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, and laminin.

5. The method of claim 1, further comprising contacting the cell with a medium.

6. The method of claim 5, wherein the medium is substantially free of immunosuppressants.

7. The method of claim 1, wherein the first synthetic RNA molecule, the second synthetic RNA molecule, or both the first synthetic RNA molecule and the second synthetic RNA molecule comprise at least one of a pseudouridine or a 5-methylcytidine residue.

8. The method of claim 1, wherein the first synthetic RNA molecule, the second synthetic RNA molecule, or both the first synthetic RNA molecule and the second synthetic RNA molecule further comprise one or more of a 5′

-cap, a 5′

-cap 1 structure, and a 3′

-poly(A) tail.

12. The method of claim 7, further comprising contacting the cell with at least one of poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, and laminin.

9. A method for producing a gene-edited cell comprising an inserted DNA sequence, comprising:

(a) providing a cell comprising a target DNA sequence;

(b) culturing the cell;

(c) transfecting the cell with a plurality of synthetic RNA molecules, wherein the synthetic RNA molecules include;

i. a first synthetic RNA molecule encoding a first fusion protein comprising a DNA-binding domain and a catalytic domain of a nuclease; and

ii. a second synthetic RNA molecule encoding a second fusion protein comprising a DNA-binding domain and a catalytic domain of a nuclease;

wherein the first fusion protein and the second fusion protein are independently a transcription activator-like effector nuclease (TALEN), the transfecting results in the cell expressing the first fusion protein and the second fusion protein to result in a double-stranded break in the target DNA sequence; and

wherein the first synthetic RNA molecule and the second synthetic RNA molecule are independently synthesized by in vitro transcription from a DNA template; and

(d) transfecting the cell with a DNA repair template comprising a sequence for insertion and one or more regions of homology to the DNA of the cell, wherein the one or more regions of homology comprise regions upstream and/or downstream of the double-stranded break, to result in insertion of the sequence in the region of the double-stranded break.

10. The method of claim 9, wherein the DNA template encodes a plurality of monomer repeats, wherein each monomer repeat comprises a repeat variable domain (RVD), and wherein the RVDs are selected to target a sequence within the target DNA sequence.

11. The method of claim 9, wherein the cell is a human cell.

13. The method of claim 9, further comprising contacting the cell with a medium.

14. The method of claim 13, wherein the medium is substantially free of immunosuppressants.

15. The method of claim 9, wherein the first synthetic RNA molecule, the second synthetic RNA molecule, or both the first synthetic RNA molecule and the second synthetic RNA molecule comprise at least one of a pseudouridine or a 5-methylcytidine residue.

16. The method of claim 9, wherein the first synthetic RNA molecule, the second synthetic RNA molecule, or both the first synthetic RNA molecule and the second synthetic RNA molecule further comprise one or more of a 5′

-cap, a 5′

-cap 1 structure, and a 3′

-poly(A) tail.