Methods and products for reprogramming cells to a less differentiated state

• US 8,497,124 B2
• Filed: 05/07/2012
• Issued: 07/30/2013
• 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:
  US13/465,490
• Publication Number:
  US 20130029418A1
• Time in Patent Office: 449 Days
• Field of Search: 435/377, 435/375, 435/325
• US Class Current: 435/377
• 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 containing albumin, wherein the albumin is treated with an ion-exchange resin or charcoal;

b. transfecting the cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules includes 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 to result in the cell being reprogrammed to a less differentiated state.

2. The method of claim 1, wherein the albumin was treated with sodium octanoate.

3. The method of claim 2, wherein the albumin was brought to a temperature of at least 40°

C.

4. 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.

5. The method of claim 1, wherein the one or more synthetic RNA molecules includes at least one RNA molecule encoding Oct4 protein, at least one RNA molecule encoding Sox2 protein, at least one RNA molecule encoding Klf4 protein, and at least one RNA molecule encoding c-Myc protein.

6. The method of claim 1, further comprising contacting the cell with at least one member of the group:

poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, and laminin.

7. 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.

8. The method of claim 1, wherein the cell is a skin cell.

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

a. culturing a differentiated cell with a reprogramming medium containing albumin, wherein the albumin is treated with sodium octanoate;

brought to a temperature of at least 40°

C.; and

treated with an ion-exchange resin or charcoal;

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

c. repeating step (b) at least twice during 5 consecutive days to result in the cell being reprogrammed to a less differentiated state.

10. The method of claim 9, 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.

11. The method of claim 9, wherein one or more synthetic RNA molecules includes at least one RNA molecule encoding Oct4 protein, at least one RNA molecule encoding Sox2 protein, at least one RNA molecule encoding Klf4 protein, and at least one RNA molecule encoding c-Myc protein.

12. The method of claim 9, further comprising contacting the cell with at least one member of the group:

poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, and laminin.

13. The method of claim 9, 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 9, wherein the cell is a skin cell.