Nucleic acid constructs encoding reprogramming factors linked by self-cleaving peptides

• US 9,497,943 B2
• Filed: 06/15/2009
• Issued: 11/22/2016
• Est. Priority Date: 06/13/2008
• Status: Active Grant

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: Carey, Bryce Woodbury, Jaenisch, Rudolf
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US12/997,815
• Publication Number:
  US 20120028821A1
• Time in Patent Office: 2,717 Days
• US Class Current: 1/1
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: C07K 14/4705 stimulating, promoting or activating activity
• CPC Class Codes: C12N 15/79 Vectors or expression systems specially adapted for eukaryotic hosts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/605 Nanog
• CPC Class Codes: C12N 2501/606 c-Myc
• CPC Class Codes: C12N 2501/608 Lin28
• CPC Class Codes: C12N 2506/11 from blood or immune system cells
• CPC Class Codes: C12N 2506/115 from monocytes, from macrophages
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2740/15041 Use of virus, viral particle or viral elements as a vector
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2740/16043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2799/027 where the vector is derived from a retrovirus
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2840/203 : having an IRES
• CPC Class Codes: C12N 5/0606 : Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0696 : Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: G01N 33/5008 : for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Abstract

The disclosure relates to a method of reprogramming one or more somatic cells, e.g., partially differentiated or fully/terminally differentiated somatic cells, to a less differentiated state, e.g., a pluripotent or multipotent state. In further embodiments the invention also relates to reprogrammed somatic cells produced by methods of the invention, to chimeric animals comprising reprogrammed somatic cells of the invention, to uses of said cells, and to methods for identifying agents useful for reprogramming somatic cells.

Claims

1. A nucleic acid construct comprising at least three coding regions, a first coding region encoding a first reprogramming factor, a second coding region encoding a second reprogramming factor, and a third coding region that encodes a third reprogramming factor, wherein the first and second coding regions are linked by a nucleic acid that encodes a first self-cleaving peptide and the second and third coding regions are linked by a nucleic acid that encodes a second self-cleaving peptide so as to form a single open reading frame, wherein expression of the first, second, and third reprogramming factors is sufficient for reprogramming a mammalian somatic cell to pluripotency, wherein any two consecutive self-cleaving peptides are not the same, and

wherein the nucleic acid construct does not comprise Oct 3/4, Sox 2 and Klf4 ligated across the 2A sequence of foot-and-mouth disease virus in the order of Oct 3/4, Klf4 and Sox2;

and wherein the nucleic acid construct does not comprise c-Myc.

2. The nucleic acid construct of claim 1, further comprising a fourth coding region that encodes a fourth reprogramming factor.

3. The nucleic acid construct of claim 1, wherein:

the first or second self-cleaving peptide is a viral 2A peptide or the first and second self-cleaving peptide are a viral 2A peptide.

4. An expression cassette comprising:

the nucleic acid construct of claim 1 operably linked to a promoter, wherein the promoter drives transcription of a polycistronic message that encodes the reprogramming factors.

5. An expression vector comprising:

the expression cassette of claim 4.

6. An isolated mammalian cell comprising:

the expression cassette of claim 4.

7. The nucleic acid construct of claim 3, wherein the viral 2A peptide is an aphthovirus 2A peptide.

8. The nucleic acid construct of claim 3, wherein the viral 2A peptide is selected from the group consisting of:

a foot-and-mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a Thosea asigna virus (TaV) 2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a Theilovirus 2A peptide, and an encephalomyocarditis virus 2A peptide.

9. The nucleic acid construct of claim 1, wherein the coding regions encode a set of reprogramming factors selected from the group consisting of:

(i) Oct4, Klf4, and Sox2;

(ii) Oct4, Nanog, and Lin28;

(iii) Oct4, Nanog, and Sox2.

10. The nucleic acid construct of claim 1, wherein the coding regions encode a set of reprogramming factors selected from the group consisting of:

Oct4, Klf4, and Sox2.

11. The nucleic acid construct of claim 1, wherein:

(a) the reprogramming factors are selected from the group consisting of;

Oct4, Nanog, Sox2, Klf4, and Lin28, and c Myc;

or

(b) the construct does not encode one of the reprogramming factors selected from the group consisting of;

Oct4, Klf4, Sox2, Lin28, and Nanog.

12. The isolated mammalian cell of claim 6, wherein the cell expresses three reprogramming factors from the expression cassette.

13. The isolated mammalian cell of claim 6, wherein the cell expresses three reprogramming factors from the expression cassette, wherein the three reprogramming factors are not sufficient to reprogram mammalian fibroblasts with an efficiency of at least 0.05%.

14. The isolated mammalian cell of claim 6, wherein:

(a) the cell is selected from the group consisting of;

a somatic cell, a terminally differentiated somatic cell, and an iPS cell;

or

(b) the cell is a human cell;

or

(c) the cell further comprises a reporter gene integrated at a locus whose activation serves as a marker of reprogramming to pluripotency.

15. The isolated mammalian cell of claim 6, wherein the cell expresses three reprogramming factors from the expression cassette, wherein the three reprogramming factors are sufficient to reprogram mammalian fibroblasts with an efficiency of at least 0.05%.

16. The expression cassette of claim 4, further comprising one or more sites that mediate integration into the genome of a mammalian cell.

17. The expression vector of claim 5, wherein

(a) the vector is retroviral;

or

(b) the promoter is inducible.