Method of de-differentiating and re-differentiating somatic cells using RNA
RNA prepared by in vitro transcription using a polymerase chain reaction (PCR)-generated template can be introduced into a cell to modulate cell activity. This method is useful in de-differentiating somatic cells to pluripotent, multipotent, or unipotent cells; re-differentiating stem cells into differentiated cells; or reprogramming of somatic cells to modulate cell activities such as metabolism. Cells can also be transfected with inhibitory RNAs, such as small interfering RNA (siRNA) or micro RNA (miRNA), or combinations thereof to induce reprogramming of somatic cells. For example, target cells are isolated from a donor, contacted with one or more RNA's causing the cells to be de-differentiated, re-differentiated, or reprogrammed in vitro, and administered to a patient in need thereof. The resulting cells are useful for treating one or more symptoms of a variety of diseases and disorders, for organ regeneration, and for restoration of the immune system.
1. A method of re-programming somatic cells comprising transiently transfecting the somatic cells ex vivo with one or more mRNAs in an effective amount to change the somatic cells to a less differentiated state,
wherein each of the one or more mRNAs are obtained by in vitro transcription of a linear double-stranded DNA template obtained by Polymerase Chain Reaction (PCR),
the DNA template comprising from the 5′ to 3′ direction:
an RNA polymerase promoter on the coding strand of the double-stranded DNA,
a 5′ untranslated region less than 3,000 nucleotides in length and effective for translation of the mRNAs into detectable polypeptides after transfection into the somatic cells,
an open reading frame that encodes the polypeptide, wherein the polypeptide is one that facilitates dedifferentiation of the cell selected from the group consisting of OCT4, SOX2, NANOG, LIN28, KLF4 and MYC,
a 3′ untranslated region effective for translation of the mRNAs into detectable polypeptides after transfection into a eukaryotic cell, and
a poly(A) stretch of 50-5,000 nucleotides on the coding strand of the double-stranded DNA,
wherein the promoter is heterologous to the open reading frame, and
wherein the DNA template is not contained within a DNA vector and terminates with the 3′ end of the poly(A) stretch.
2. The method of claim 1 wherein the somatic cells are transfected with at least two different mRNAs.
3. The method of claim 1 wherein the somatic cells are differentiated cells.
4. The method of claim 1 wherein at least one of the mRNAs is OCT4 mRNA.
5. The method of claim 1 wherein the one or more mRNAs comprise a combination of OCT4, SOX2, NANOG, and LIN28 mRNA.
6. The method of claim 5 wherein the one or more mRNAs comprise a combination of OCT4, SOX2, KLF4 and MYC mRNA.
7. The method of claim 1 further comprising the step of inducing the less differentiated cells to form differentiated cells.
8. The method of claim 7 wherein the differentiated cells are cells from a tissue selected from the group consisting of bone, connective tissue, organ tissue, vascular tissue, skin tissue and nervous tissue or hematopoietic cells.
9. The method of claim 1 further comprising contacting the somatic cells with one or more inhibitory RNAs that reduce expression of an mRNA encoding an allogeneic antigen expressed by the somatic cell.
10. The method of claim 1 wherein the somatic cells are lymphocytes which can be re-programmed into regulatory T cells.
11. The method of claim 6 further comprising growing the transfected cells on a feeder cell layer until small colonies form.
12. The method of claim 1 wherein the somatic cells are fibroblasts or keratinocytes.
13. The method of claim 11 wherein the one or more mRNAs are transiently transfected by multiple administrations staggered minutes, hours, days, or weeks apart.
14. The method of claim 13 wherein the one or more mRNAs comprise modified or artificial nucleotides, or nucleotide analogs.
15. The method of claim 1 further comprising inducing the less differentiated somatic cells to form differentiated cells of a cell type different from the starting somatic cells.