Kit for making induced pluripotent stem cells using modified RNAs
AssigneeChildren's Medical Center Corporation
InventorsDerrick Rossi, Luigi Warren
Life of this patent
Estimating the term…
Abstract
Described herein are synthetic, modified RNAs for changing the phenotype of a cell, such as expressing a polypeptide or altering the developmental potential. Accordingly, provided herein are compositions, methods, and kits comprising synthetic, modified RNAs for changing the phenotype of a cell or cells. These methods, compositions, and kits comprising synthetic, modified RNAs can be used either to express a desired protein in a cell or tissue, or to change the differentiated phenotype of a cell to that of another, desired cell type.
Claims16
A kit for making induced pluripotent stem (iPS) cells from a fibroblast cell comprising the following components:
- (a) a synthetic mRNA encoding OCT4, wherein each cytosine of the synthetic mRNA encoding OCT4 is replaced with 5-methylcytosine and each uracil is replaced with pseudouracil;
- (b) a synthetic mRNA encoding SOX2, wherein each cytosine of the synthetic mRNA encoding SOX2 is replaced with 5-methylcytosine and each uracil is replaced with pseudouracil;
- (c) a synthetic mRNA encoding KLF4, wherein each cytosine of the synthetic mRNA encoding KLF4 is replaced with 5-methylcytosine and each uracil is replaced with pseudouracil;
- and
- (d) a synthetic mRNA encoding c-MYC, wherein each cytosine of the synthetic mRNA encoding c-MYC is replaced with 5-methylcytosine and each uracil is replaced with pseudouracil.
A method for making induced pluripotent stem (iPS) cells from a fibroblast cell comprising:
- a) subjecting the fibroblast cell in culture medium to a daily transfection with a transfection mixture comprising
- (i) a synthetic mRNA mixture comprising a synthetic mRNA encoding OCT4;
- a synthetic mRNA encoding SOX2;
- a synthetic mRNA encoding KLF4;
- and a synthetic mRNA encoding c-MYC, wherein the synthetic mRNA encoding OCT4 is provided in molar excess of at least three times the concentration of the remaining three synthetic mRNAs, and wherein each cytosine of each of the synthetic mRNAs is replaced with 5-methylcytosine, and each uracil of each of the synthetic mRNAs is replaced with pseudouracil;
- and
- (ii) a synthetic mRNA encoding a short half-life nuclear green fluorescent protein (GFP);
- b) providing culture conditions comprising 5% O2;
- c) removing the transfection mixture four hours after each daily transfection, from the cell in culture medium;
- d) culturing the cells in a culture medium not comprising the transfection mixture of step (a) for about three days to allow expansion of the cells;
- e) picking cells from the expanded cells of step (d), thereby generating iPS cells to generate iPS cell lines.
Dependencies are inferred from the claim text.