Methods of reprogramming cells to a less differentiated state

• US 9,399,761 B2
• Filed: 07/27/2015
• Issued: 07/26/2016
• 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:
  US14/810,123
• Publication Number:
  US 20160010060A1
• Time in Patent Office: 365 Days
• US Class Current: 1/1
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/02 for peripheral neuropathies
• 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 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: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/33 other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/84 from mammals
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2501/608 : Lin28
• 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 2506/45 : from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 5/0018 : Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• CPC Class Codes: C12N 5/0625 : Epidermal cells, skin cells; Cells of the oral mucosa
• CPC Class Codes: C12N 5/0647 : Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0656 : Adult fibroblasts
• CPC Class Codes: C12N 5/0657 : Cardiomyocytes; Heart cells
• CPC Class Codes: C12N 5/0676 : Pancreatic cells
• CPC Class Codes: C12N 5/0695 : Stem cells; Progenitor cells; Precursor cells
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS

Abstract

The present invention relates in part to methods for producing tissue-specific cells from patient samples, and to tissue-specific cells produced using these methods. Methods for reprogramming cells using RNA are disclosed. Therapeutics comprising cells produced using these methods are also disclosed.

Claims

1. A method for reprogramming a cell to a less differentiated state, comprising:

(a) culturing a differentiated cell with a reprogramming medium;

(b) transfecting the cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and

(c) repeating step (b) at least twice during 5 consecutive days, wherein the amount of one or more synthetic RNA molecules transfected in one or more later transfections is greater than the amount transfected in one or more earlier transfections, to result in the cell being reprogrammed to a less differentiated state,

wherein steps (a)-(c) are performed without using feeder cells and occur in the presence of a feeder cell conditioned medium.

2. The method of claim 1, wherein the reprogramming medium is substantially free of immunosuppressants.

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

4. The method of claim 3, wherein the cell is derived from a biopsy.

5. The method of claim 4, wherein the biopsy is a full-thickness dermal punch biopsy.

6. The method of claim 1, wherein the cell culture has a starting cell density of less than and 20,000 cells/cm².

7. The method of claim 1, wherein the cell culture has a starting cell density of between 2,000 cells/cm² and 20,000 cells/cm².

8. The method of claim 1, wherein the method further comprises a step of rinsing the cell after transfection.

9. The method of claim 1, wherein the reprogramming medium comprises a cell-adhesion molecule.

10. The method of claim 9, wherein the cell-adhesion molecule is selected from one or more fibronectin and vitronectin.

11. The method of claim 10, wherein the cell-adhesion molecule is recombinant.

12. The method of claim 1, wherein the one or more synthetic RNA molecules includes at least one RNA molecule encoding at least one member of the group:

Oct4 protein, Sox2 protein, Klf4 protein, and c-Myc protein.

13. The method of claim 1, wherein the one or more synthetic RNA molecules contains at least one member of the group:

a pseudouridine residue and a 5-methylcytidine residue.

14. The method of claim 1, wherein the amount of one or more synthetic RNA molecules transfected in earlier transfections is between about 20 ng/cm² and 250 ng/cm².

15. The method of claim 1, wherein the amount of one or more synthetic RNA molecules transfected in later transfections is between about 100 ng/cm² and 600 ng/cm².

16. The method of claim 1, wherein the method comprises at least 5 transfections and the amount of one or more synthetic RNA molecules transfected is about 25 ng/cm² for the first transfection, about 50 ng/cm² for the second transfection, about 100 ng/cm² for the third transfection, about 200 ng/cm² for the fourth transfection, and about 400 ng/cm² for the fifth transfection.