Methods and products for transfection

Patent metadata

Field	Value
Publication number	US-2016010060-A1
Application number	US-201514810123-A
Country	US
Kind code	A1
Filing date	Jul 27, 2015
Priority date	Dec 5, 2011
Publication date	Jan 14, 2016
Grant date	—

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.

First claim

1 .- 9 . (canceled)

10 . A method for reprogramming a cell to a less differentiated state, comprising:
(a) culturing a differentiated cell with a reprogramming medium, wherein the reprogramming medium is substantially free of feeders; (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, 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.

11 . The method of claim 10 , wherein the reprogramming medium is substantially free of immunosuppressants.

12 . The method of claim 10 , wherein the cell is a fibroblast.

13 . The method of claim 12 , wherein the cell is derived from a biopsy.

14 . The method of claim 13 , wherein the biopsy is a full-thickness dermal punch biopsy.

15 . The method of claim 10 , wherein the cell culture has a starting cell density of less than and 20,000 cells/cm 2 .

16 . The method of claim 10 , wherein the cell culture has a starting cell density of between 2,000 cells/cm 2 and 20,000 cells/cm 2 .

17 . The method of claim 10 , wherein the method further comprises a step of rinsing the cell after transfection.

18 . The method of claim 10 , wherein the reprogramming medium comprises a cell-adhesion molecule.

19 . The method of claim 18 , wherein the cell-adhesion molecule is selected from one or more fibronectin and vitronectin.

20 . The method of claim 19 , wherein the fibronectin and vitronectin are recombinant

21 . The method of claim 10 , 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.

22 . The method of claim 10 , wherein the one or more synthetic RNA molecules contains at least one member of the group: a pseudouridine residue and a 5-methylcytidine residue.

23 . The method of claim 10 , wherein the amount of one or more synthetic RNA molecules transfected in earlier transfections is between about 20 ng/cm 2 and 250 ng/cm 2 .

24 . The method of claim 10 , wherein the amount of one or more synthetic RNA molecules transfected in later transfections is between about 100 ng/cm 2 and 600 ng/cm 2 .

25 . The method of claim 10 , wherein the method comprises at least 5 transfections and the amount of one or more synthetic RNA molecules transfected is about 25 ng/cm 2 for the first transfection, about 50 ng/cm 2 for the second transfection, about 100 ng/cm 2 for the third transfection, about 200 ng/cm 2 for the fourth transfection, and about 400 ng/cm 2 for the fifth transfection.

Assignees

• Factor Bioscience Inc

Inventors

• Angel Matthew
• Rohde Christopher

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61P3/10

  for hyperglycaemia, e.g. antidiabetics · CPC title

• A61P37/04

  Immunostimulants · CPC title

• A61P7/06

  Antianaemics · CPC title

• A61P9/10

  for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis · CPC title