Methods and products for transfecting cells
The present invention relates in part to nucleic acids encoding proteins, nucleic acids containing non-canonical nucleotides, therapeutics comprising nucleic acids, methods, kits, and devices for inducing cells to express proteins, methods, kits, and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, and therapeutics produced using these methods, kits, and devices. Methods for inducing cells to express proteins and for reprogramming and gene-editing cells using RNA are disclosed. Methods for producing cells from patient samples, cells produced using these methods, and therapeutics comprising cells produced using these methods are also disclosed.
1. A method for producing a gene-edited, reprogrammed cell, 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:
(i) at least one RNA molecule encoding one or more reprogramming factors, and
(ii) at least one RNA molecule encoding one or more gene-editing proteins;
wherein the transfecting results in the cell expressing the one or more reprogramming factors and the one or more gene editing proteins to result in a gene-edited, reprogrammed cell;
wherein step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the 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 1 , wherein the differentiated cell is a human skin cell.
4. The method of claim 1 , further comprising contacting the differentiated cell with at least one of poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, and laminin.
5. The method of claim 1 , wherein the medium is substantially free of immunosuppressants.
6. The method of claim 1 , wherein the one or more synthetic RNA molecules comprise at least one of a pseudouridine or a 5-methylcytidine residue.
7. The method of claim 1 , wherein the one or more synthetic RNA molecules further comprise one or more of a 5′-cap, a 5′-cap 1 structure, and a 3′-poly(A) tail.
8. The method of claim 1 , wherein the one or more reprogramming factor(s) are selected from Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, 1-Myc protein, Tert protein, Nanog protein, and Lin28 protein.
9. A method for producing a gene-edited, reprogrammed 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:
(i) at least one RNA molecule encoding one or more reprogramming factors, and
(ii) at least one RNA molecule encoding one or more gene-editing proteins;
wherein the transfecting results in the cell expressing the one or more reprogramming factors and the one or more gene editing proteins to result in a gene-edited, reprogrammed cell;
wherein step (c) is performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the cell.
10. The method of claim 9 , wherein the non-pluripotent cell is derived from a biopsy.
11. The method of claim 9 , wherein the non-pluripotent cell is a human skin cell.
12. The method of claim 9 , further comprising contacting the non-pluripotent cell with at least one of poly-L-lysine, poly-L-ornithine, RGD peptide, fibronectin, vitronectin, collagen, and laminin.
13. The method of claim 9 , wherein the medium is substantially free of immunosuppressants.
14. The method of claim 9 , wherein the one or more synthetic RNA molecules comprise at least one of a pseudouridine or a 5-methylcytidine residue.
15. The method of claim 9 , wherein the one or more synthetic RNA molecules further comprise one or more of a 5′-cap, a 5′-cap 1 structure, and a 3′-poly(A) tail.
16. The method of claim 9 , wherein the one or more reprogramming factor(s) are selected from Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, 1-Myc protein, Tert protein, Nanog protein, and Lin28 protein.