Methods and products for transfection

• US 10,443,045 B2
• Filed: 04/03/2019
• Issued: 10/15/2019
• 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:
  US16/374,482
• Publication Number:
  US 20190225944A1
• Time in Patent Office: 195 Days
• 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 15/85 for animal cells
• 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 differentiated cell to a less differentiated state, comprising:

(a) providing a differentiated cell;

(b) culturing the differentiated cell; and

(c) transfecting the differentiated 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;

wherein the transfecting results in the cell expressing the one or more reprogramming factors; and

wherein step (c) is performed at least twice and 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 and occurs in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

2. The method of claim 1, wherein the differentiated cell is derived from a biopsy.

3. The method of claim 2, wherein the differentiated cell is derived from a dermal punch biopsy sample.

4. The method of claim 1, wherein the differentiated cell is from a human subject.

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

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 contain at least one of a pseudouridine or a 5-methylcytidine residue.

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

9. A method for reprogramming a non-pluripotent cell, comprising:

(a) providing a non-pluripotent cell;

(b) culturing the non-pluripotent cell; and

(c) transfecting the non-pluripotent 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;

wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed; and

wherein step (c) is performed at least twice and 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 non-pluripotent cell being reprogrammed and occurs in the presence of a medium containing ingredients that support reprogramming of the non-pluripotent cell.

10. The method of claim 9, wherein the non-pluripotent cell is derived from a biopsy.

11. The method of claim 10, wherein the non-pluripotent cell is derived from a dermal punch biopsy sample.

12. The method of claim 9, wherein the non-pluripotent cell is from a human subject.

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

14. 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.

15. The method of claim 9, wherein the one or more synthetic RNA molecules contain at least one of a pseudouridine or a 5-methylcytidine residue.

16. The method of claim 9, wherein the medium is substantially free of immunosuppressants.