Generation of tumor-free embryonic stem-like pluripotent cells using inducible recombinant RNA agents

• US 20090203141A1
• Filed: 01/08/2009
• Published: 08/13/2009
• Est. Priority Date: 05/15/2003
• Status: Abandoned Application

• Current Assignee: David Ts Wu, Shi-Lung Lin
• Original Assignee: David Ts Wu, Shi-Lung Lin
• Inventors: Wu, David TS, Lin, Shi-Lung
• Application Number:
  US12/318,806
• Publication Number:
  US 20090203141A1
• Time in Patent Office: Days
• US Class Current: 435/456
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 2840/102 inhibiting translation
• CPC Class Codes: C12N 2840/44 being a specific part of the splice mechanism, e.g. donor, acceptor
• CPC Class Codes: C12N 2840/445 for trans-splicing, e.g. polypyrimidine tract, branch point splicing

Abstract

The present invention generally relates to a method for developing, generating and selecting tumor-free embryonic stem (ES)-like pluripotent cells using electroporation delivery of an inducible tumor suppressor mir-302 agent into mammalian cells. More particularly, the present invention relates to a method and composition for generating a Tet-On/Off recombinant transgene capable of expressing a manually re-designed mir-302 microRNA (miRNA)/shRNA agent under the control of doxycyclin (Dox) in human somatic/cancer cells and thus inducing certain specific gene silencing effects on the differentiation-associated genes and oncogenes of the cells, resulting in reprogramming the cells into an ES-like pluripotent state.

Claims

1. A method for reprogramming at least a mammalian cell into at least a pluripotent stem cell, said method comprising these steps of:

(a) Providing at least a cell substrate expressing a plurality of cellular genes targeted by mir-302;

(b) Providing at least a recombinant nucleic acid composition capable of being delivered, transcribed and processed into at least a gene silencing effector homologous to mir-302 in said cell substrate; and

(c) Treating said cell substrate with said recombinant nucleic acid composition under a condition that said cellular genes targeted by mir-302 are suppressed.

2. The method as defined in claim 1, wherein said recombinant nucleic acid composition includes a gene expression vector selected from the group consisting of plasmid, viral vector, retrotransposon, and a combination thereof.

3. The method as defined in claim 1, wherein said recombinant nucleic acid composition includes a drug-inducible gene expression vector.

4. The method as defined in claim 1, wherein said recombinant nucleic acid composition includes a Tet-On or Tet-Off gene expression vector.

5. The method as defined in claim 1, wherein the transcription of said recombinant nucleic acid composition is controlled by a tetracyclin derivative.

6. The method as defined in claim 5, wherein said tetracyclin derivative includes doxycyclin.

7. The method as defined in claim 1, wherein said recombinant nucleic acid composition further includes a recombinant intron.

8. The method as defined in claim 7, wherein said intron includes a 5′

-donor splice site, an intronic insert site, a branch point motif, a poly-pyrimidine tract, and a 3′

-acceptor splice site.

9. The method as defined in claim 8, wherein said intron is synthesized by a chemically synthesizing method.

10. The method as defined in claim 8, wherein said intron is ligated by a nucleotide recombination method.

11. The method as defined in claim 8, wherein said 5′

-donor splice site includes or is homologous to a SEQ.ID.NO.4 sequence.

12. The method as defined in claim 8, wherein said 5′

-donor splice site is homologous to 5′

-GTAAG-3′

.

13. The method as defined in claim 8, wherein said branch point motif includes or is homologous to a SEQ.ID.NO.6 sequence.

14. The method as defined in claim 8, wherein said branch point motif includes or is homologous to 5′

-TACTAAC-3′

.

15. The method as defined in claim 8, wherein said poly-pyrimidine tract includes or is homologous to a SEQ.ID.NO.7 or a SEQ.ID.NO.8 sequence.

16. The method as defined in claim 8, wherein said 3′

-acceptor splice site includes or is homologous to a SEQ.ID.NO.5 sequence.

17. The method as defined in claim 8, wherein said 3′

-acceptor splice site is homologous to 5′

-CTGCAG-3′

.

18. The method as defined in claim 8, wherein said intronic insert site includes said gene silencing effector homologous to mir-302.

19. The method as defined in claim 1, wherein said recombinant nucleic acid composition further includes a plurality of exons, said exons are selected from the group consisting of fluorescent protein marker genes, luciferase genes, lac-Z reporter genes, embryonic stem cell marker genes, viral genes, bacterial genes, cellular marker genes, jumping genes, transposons and a combination thereof.

20. The method as defined in claim 1, wherein said gene silencing effector is homologous to either a SEQ.ID.NO. 1 or a SEQ.ID.NO.2 sequence.

21. The method as defined in claim 1, wherein said gene silencing effector is homology or complementarity, or both, to a SEQ.ID.NO.3 sequence.

22. The method as defined in claim 1, wherein said gene silencing effector is a recombinant hairpin-like RNA including a sequence homologous to a SEQ.ID.NO.9 sequence.

23. The method as defined in claim 1, wherein said gene silencing effector is a recombinant nucleic acid sequence formed by the hybrid of a SEQ.ID.NO.27 and a SEQ.ID.NO.28 sequence.

24. The method as defined in claim 1, wherein said gene silencing effector includes a recombinant RNA homologous to the group including a SEQ.ID.NO.9, a SEQ.ID.NO.10, a SEQ.ID.NO.11, a SEQ.ID.NO.12, a SEQ.ID.NO.13 sequence, and a combination thereof.

25. The method as defined in claim 1, wherein said gene silencing effector is formed by ligation linkage of a hybrids of a SEQ.ID.NO.29, a SEQ.ID.NO.30, a SEQ.ID.NO.31, a SEQ.ID.NO.32, a SEQ.ID.NO.33, a SEQ.ID.NO.34, a SEQ.ID.NO.35, a SEQ.ID.NO.36 sequence, and a combination thereof.

26. The method as defined in claim 1, wherein said recombinant nucleic acid composition is selected from the group consisting of a tetracycline responsive element, a viral or a type-II RNA polymerase (Pol-II) promoter, or both, a Kozak consensus translation initiation site, polyadenylation signals, a plurality of restriction/cloning sites and a combination thereof.

27. The method as defined in claim 1, wherein said recombinant nucleic acid composition is selected from the group consisting of a pUC origin of replication, a SV40 early promoter for expressing at least an antibiotic resistance gene in replication-competent prokaryotic cells, an optional SV40 origin for replication in mammalian cells and a combination thereof.

28. The method as defined in claim 1, wherein said recombinant nucleic acid composition is introduced into said mammalian cell by a gene delivery method selected from the group consisting of liposomal transfection, chemical transfection, transgenic DNA recombination, viral infection, transposon insertion, jumping gene insertion, micro-injection, electroporation, gene-gun penetration, and a combination thereof.

29. The method as defined in claim 1, wherein said mammalian cell is a human cell.

30. The method as defined in claim 1, wherein said mammalian cell is a normal somatic cell.

31. The method as defined in claim 1, wherein said mammalian cell is a diseased somatic cell.

32. The method as defined in claim 1, wherein said mammalian cell is a tumor or cancerous cell.

33. The method as defined in claim 1, wherein said mammalian cell is a human hair follicle cell.

34. The method as defined in claim 1, wherein said mammalian cell is a human skin cell.

35. The method as defined in claim 1, wherein said pluripotent stem cell expresses embryonic stem cell marker Oct3/4.

36. The method as defined in claim 1, wherein said pluripotent stem cell is cultured under a feeder-free cell cultural condition.

37. The method as defined in claim 1, wherein said pluripotent stem cell differentiates into a germ line-like cell.

38. The method as defined in claim 1, wherein said pluripotent stem cell differentiates into a spermatogonia-like cell.

39. The method as defined in claim 1, wherein said pluripotent stem cell differentiates into a normal somatic cell.

40. The method as defined in claim 1, wherein said pluripotent stem cell differentiates into a fibroblast.

41. The method as defined in claim 1, wherein said pluripotent stem cell differentiates into a chondrocyte.

42. The method as defined in claim 1, wherein said pluripotent stem cell is selectively isolated by mir-302 microRNAs or Oct3/4 as markers.

43. A recombinant nucleic acid composition for inducing mir-302-mediated gene silencing effects comprising:

At least an intron encoding a gene silencing effector flanked with exons, wherein said intron is cleaved out of said exons for inducing mir-302-mediated gene silencing effects, and said exons are linked to form a coding sequence with desired function.

50. The recombinant nucleic acid composition as defined in claim 50, wherein said poly-pyrimidine tract includes or is homologous to the SEQ.ID.NO.7 or SEQ.ID.NO.8 sequence.

44. The recombinant nucleic acid composition as defined in claim 49, wherein said intron comprises:

(a) An intronic insert encoding a gene silencing effector homologous to mir-302;

(b) 5′

-donor and acceptor splice sites;

(c) A branch point motif; and

(d) At least a poly-pyrimidine tract.

45. The recombinant nucleic acid composition as defined in claim 50, wherein said intronic insert is a hairpin-like nucleic acid sequence including a stem-loop structure homologous to either SEQ.ID.NO.1 or SEQ.ID.NO.2.

46. The recombinant nucleic acid composition as defined in claim 50, wherein said intronic insert is homology or complementarity, or both, to SEQ.ID.NO.3.

47. The recombinant nucleic acid composition as defined in claim 50, wherein said intronic insert includes a nucleic acid sequence selected from the group consisting of SEQ.ID.NO.9, SEQ.ID.NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12, SEQ.ID.NO. 13 and a combination thereof.

48. The recombinant nucleic acid composition as defined in claim 50, wherein said branch point motif includes or is homologous to the SEQ.ID.NO.6 sequence.

49. The recombinant nucleic acid composition as defined in claim 50, wherein said branch point motif includes or is homologous to 5′

-TACTAAC-3′

.

51. The recombinant nucleic acid composition as defined in claim 50, wherein said 5′

-donor splice site includes or is homologous to the SEQ.ID.NO.4 sequence.

52. The recombinant nucleic acid composition as defined in claim 50, wherein said 5′

-donor splice site includes or is homologous to 5′

-GTAAG-3′

.

53. The recombinant nucleic acid composition as defined in claim 50, wherein said 3′

-acceptor splice site includes or is homologous to the SEQ.ID.NO.5 sequence.

54. The recombinant nucleic acid composition as defined in claim 50, wherein said 3′

-acceptor splice site includes or is homologous to 5′

-CTGCAG-3′

.

55. The recombinant nucleic acid composition as defined in claim 49, wherein said gene-silencing effector is either homology or complementarity, or both, to SEQ.ID.NO.3.

56. The recombinant nucleic acid composition as defined in claim 49, wherein said gene-silencing effector includes SEQ.ID.NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12, and/or SEQ.ID.NO.13.

57. The recombinant nucleic acid composition as defined in claim 49, wherein said gene silencing effector is formed by ligation linkage of the hybrids of SEQ.ID.NO.29, SEQ.ID.NO.30, SEQ.ID.NO.31, SEQ.ID.NO.32, SEQ.ID.NO.33, SEQ.ID.NO.34, SEQ.ID.NO.35, SEQ.ID.NO.36 and a combination thereof.

58. The recombinant nucleic acid composition as defined in claim 49, wherein said gene-silencing effector includes SEQ.ID.NO.9.

59. The recombinant nucleic acid composition as defined in claim 49, wherein said gene silencing effector is formed by the hybrids of SEQ.ID.NO.27 and SEQ.ID.NO.28.