Generation of human iPS cells by a synthetic self-replicative RNA

Patent metadata

Field	Value
Publication number	US-9862930-B2
Application number	US-201314402924-A
Country	US
Kind code	B2
Filing date	May 21, 2013
Priority date	May 21, 2012
Publication date	Jan 9, 2018
Grant date	Jan 9, 2018

Abstract

The disclosure provides methods and compositions useful for obtaining induced stem cells, methods of making and use thereof.

First claim

What is claimed is:

1. A RNA replicon comprising:
a plurality of non-structural replicase domains from an alphavirus and at least four heterologous polynucleotide sequences that encode reprogramming factors (RFs) for inducing the generation of pluripotent stem cells when expressed in a somatic cell:
wherein the RNA replicon comprises from 5′ to 3′: polynucleotide sequences encoding the plurality of non-structural replicase domain sequences obtained from an alphavirus; a promoter; RF 1 ; a coding sequence for a first self-cleaving peptide; RF 2 ; a coding sequence for a second self-cleaving peptide; RF 3 ; an IRES; RF 4 ; an optional IRES or an optional promoter; an optional sequence encoding an optional selectable marker; an alphavirus 3′ UTR and polyA tail;

wherein RF 1-4 are heterologous polynucleotide sequences which encode reprogramming factors that induce de-differentiation of a somatic cell to a pluripotent cell; and
wherein RF 1-4 are polynucleotides encoding RFs selected from the group consisting of Oct-3, Oct-4, Klf, Sox-2, c-Myc, n-Myc, L-Myc, Nanog, and Glis1.

2. The RNA replicon of claim 1 , wherein the polynucleotide sequences encoding the plurality of non-structural replicase domain sequences are obtained from an alphavirus selected from the group consisting of Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Equine Encephalitis virus (WEE), Sindbis virus, Semliki Forest virus, Middelburg virus, Chikungunya virus, O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus and Buggy Creek virus.

3. The RNA replicon of claim 1 , wherein the polynucleotide encoding the Klf polypeptide encodes a Klf polypeptide having a sequence selected from:
(a) having at least 95% sequence identity to the sequence of SEQ ID NO:8 and having KLF4 activity; or
(b) having the sequence as set forth in SEQ ID NO:8.

4. The RNA replicon of claim 1 , wherein the polynucleotide encoding the Klf polypeptide comprises the sequence as set forth in SEQ ID NO:7, wherein the thymidine residues are replaced with uracil resides.

5. The RNA replicon of claim 1 , wherein the polynucleotide encoding the Sox-2 polypeptide encodes a Sox-2 polypeptide having a sequence selected from:
(a) having at least 95% sequence identity to the sequence of SEQ ID NO:6 and having Sox-2 activity, or
(b) having the sequence as set forth in SEQ ID NO:6.

6. The RNA replicon of claim 1 , wherein the polynucleotide encoding the Sox-2 polypeptide comprises the sequence as set forth in SEQ ID NO:5, wherein the thymidine residues are replaced with uracil resides.

7. The RNA replicon of claim 1 , wherein the polynucleotide encoding the Oct-4 polypeptide encodes a Oct-4 polypeptide having a sequence selected from:
(a) having at least 95% sequence identity to the sequence of SEQ ID NO:4 and having Oct-4 activity; or
(b) having the sequence as set forth in SEQ ID NO:4.

8. The RNA replicon of claim 1 , wherein the polynucleotide encoding the Oct-4 polypeptide comprises the sequence as set forth in SEQ ID NO:3, wherein the thymidine residues are replaced with uracil resides.

9. The RNA replicon of claim 1 , wherein the polynucleotide encoding the c-Myc polypeptide encodes a c-Myc polypeptide having a sequence selected from:
(a) having at least 95% sequence identity to the sequence of SEQ ID NO:10 and having c-Myc activity; or
(b) having the sequence as set forth in SEQ ID NO:10.

10. The RNA replicon of claim 1 , wherein the polynucleotide encoding the c-Myc polypeptide comprises the sequence as set forth in SEQ ID NO:9, wherein the thymidine residues are replaced with uracil resides.

11. The RNA replicon of claim 1 , wherein the polynucleotide encoding the Glis1 polypeptide encodes a Glis1 polypeptide having a sequence selected from:
(a) having at least 95% sequence identity to the sequence of SEQ ID NO:34 and having Glis1 activity; or
(b) having the sequence as set forth in SEQ ID NO:34.

12. The RNA replicon of claim 1 , wherein the polynucleotide encoding the Glis1 polypeptide comprises the sequence as set forth in SEQ ID NO:33, wherein the thymidine residues are replaced with uracil resides.

13. The RNA replicon of claim 1 , wherein the polynucleotide encoding the Nanog polypeptide encodes a Nanog polypeptide having a sequence selected from:
(a) having at least 95% sequence identity to the sequence of SEQ ID NO:2 and having Nanog activity; or
(b) having the sequence as set forth in SEQ ID NO:2.

14. The RNA replicon of claim 1 , wherein the polynucleotide encoding the Nanog polypeptide comprises the sequence as set forth in SEQ ID NO:1, wherein the thymidine residues are replaced with uracil resides.

15. The RNA replicon of claim 1 , wherein the replicon comprises from 5′ to 3′:
VEE polynucleotide sequences encoding the plurality of nonstructural replicase domain sequences; a promoter; RF 1 ; a coding sequence for a first self-cleaving peptide; RF 2 ; a coding sequence for a second self-cleaving peptide; RF 3 ; an IRES; RF 4 ; an optional IRES or an optional promoter; an optional sequence encoding an optional selectable marker; a VEE 3′ UTR and polyA tail;
wherein RF 1-4 are selected from the group consisting of Oct-4, Klf4, Sox-2, c-Myc, Nanog, and Glis1.

16. The RNA replicon of claim 1 , wherein the replicon comprises a sequence that is 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:29, 30, 31, or 32 from about position 1 to about position 7561 wherein “T” of the sequence is substituted with “U”.

17. The RNA replicon of claim 16 , wherein the replicon comprises a sequence that is 100% identical to the sequence of SEQ ID NO:29, 30, 31, or 32, wherein the thymidine residues are replaced with uracil resides.

18. The RNA replicon of claim 1 , wherein the promoter located 3′ to the polynucleotide sequences encoding the plurality of non-structural replicase domains and 5′ to RF 1 , is a 26S internal promoter.

19. The RNA replicon of claim 1 , wherein the coding sequence for the first self-cleaving peptide is a coding sequence for a T2A or E2A self-cleaving peptide.

20. The RNA replicon of claim 1 , wherein the coding sequence for the second self-cleaving peptide is a coding sequence for a T2A or E2A self-cleaving peptide.

21. The RNA replicon of claim 15 , wherein RF 1 is Oct4, RF 2 is cMyc, RF 3 is Klf4, and RF 4 is Sox2.

22. The RNA replicon of claim 15 , wherein RF 1 is Oct4, RF 2 is Klf4, RF 3 is Sox2, and RF 4 is cMyc.

23. The RNA replicon of claim 15 , wherein RF 1 is Oct4, RF 2 is Klf4, RF 3 is Sox2, and RF 4 is Glis1.

Assignees

• Univ California

Inventors

• Dowdy Steven F
• Yoshioka Naohisa

Classifications

• C12N2840/206

  having multiple IRES · CPC title

• C12N5/0696 Primary

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title

• C12N2501/604

  Klf-4 · CPC title

• C12N2501/60

  Transcription factors · CPC title

• C12N9/127

  RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase · CPC title