Generation of human embryonc stem-like cells using intronic RNA

• US 20080293143A1
• Filed: 05/07/2008
• Published: 11/27/2008
• Est. Priority Date: 05/15/2003
• Status: Active Grant

• Current Assignee: Mello Biotechnology, Inc., Shi-Lung Lin, University of Southern California
• Original Assignee: Mello Biotechnology, Inc.
• Inventors: Ying, Shao-Yao, Lin, Shi-Lung, Wu, David Ts
• Granted Patent: US 9,567,591 B2
• 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

Abstract

This invention generally relates to a method for developing, generating and selecting human embryonic stem (hES)-like pluripotent cells using transgenic expression of intronic microRNA-like RNA agents. More particularly, the present invention relates to a method and composition for generating a non-naturally occurring intron and its intronic components capable of being processed into mir-302-like RNA molecules in mammalian cells and thus inducing certain specific gene silencing effects on differentiation-related and fate-determinant genes of the cells, resulting in reprogramming the cells into a pluripotent embryonic stem (ES)-cell-like state. The ES-like cells so obtained are strongly express hES cell markers, such as Oct3/4, SSEA-3 and SSEA-4, and can be guided into various tissue cell types by treating certain hormones and/or growth factors under a feeder-free cell culture condition in vitro, which may be used for transplantation and gene therapies. Therefore, the present invention offers a simple, effective and safe gene manipulation approach for not only reprogramming somatic cells into ES-like pluripotent cells but also facilitating the maintenance of pluripotent and renewal properties of ES cells under a feeder-free cell culture condition, preventing the tedious retroviral insertion of four large transcription factor genes into one single cell as used in the previous iPS methods.

Claims

1. A transgenic method for inducing intronic mir-302-mediated gene silencing effects in mammalian cells comprises the steps of:

(a) Constructing a recombinant nucleic acid composition that contains at least an intron encoding a mir-302-like gene-silencing effector flanked with exons, wherein said intron can be cleaved out of the exons for inducing mir-302-mediated gene silencing;

(b) Cloning said recombinant nucleic acid composition into an expression-competent vector; and

(c) Introducing said expression-competent vector into a plurality of mammalian cells, wherein said cells generate a plurality of primary RNA transcripts of said recombinant nucleic acid composition, and wherein the cells splice said intron out of the primary RNA transcripts so as to provide a mir-302-mediated gene silencing effect on genes containing complementarity to the sequence of the mir-302-like gene-silencing effector, which consequently results in reprogramming the cells into a plurality of stem cell-like pluripotent cells.

2. The method as defined in claim 1, wherein said mammalian cells are human cells.

3. The method as defined in claim 1, wherein said mammalian cells are somatic cells.

4. The method as defined in claim 1, wherein said mammalian cells are cancerous cells.

5. The method as defined in claim 1, wherein the mir-302-like gene-silencing effector of said intron is a synthetic DNA sequence.

6. The method as defined in claim 1, further comprising the step of synthesizing the nucleic acid components of said intron or exon sequences, or both.

7. The method as defined in claim 1, wherein said recombinant nucleic acid composition is a recombinant cellular gene.

8. The method as defined in claim 1, wherein said recombinant nucleic acid composition is a recombinant gene derived from GFP genes, viral genes, mammalian genes, jumping genes, transposons, and a combination thereof.

9. The method as defined in claim 1, wherein said intron is constructed by a genetic engineering method selected from the group consisting of DNA restriction and ligation, homologous recombination, transgene incorporation, transposon insertion, jumping gene integration, retroviral infection, and a combination thereof.

10. The method as defined in claim 1, wherein said intron is a nucleic acid sequence containing components of an intronic insert encoding a mir-302-like gene-silencing effector, a branch point motif, a poly-pyrimidine tract, a donor splice site and an acceptor splice site.

11. The method as defined in claim 10, wherein said intronic insert is a hairpin-like nucleic acid sequence containing a stem-loop structure homologous to either SEQ.ID.NO.1 or SEQ.ID.NO.2.

12. The method as defined in claim 10, wherein said intronic insert is a nucleic acid sequence containing either homology or complementarity, or both, to SEQ.ID.NO.3.

13. The method as defined in claim 10, wherein said intronic insert is a hairpin-like precursor microRNA (pre-miRNA) sequence containing 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, and SEQ.ID.NO.13.

14. The method as defined in claim 10, wherein the intronic insert is incorporated into said intron through at least a restriction/cloning site selected from the group consisting of AatII, AccI, AflII/III, AgeI, ApaI/LI, AseI, Asp 718I, BamHI, BbeI, BclI/II, BglII, BsmI, Bsp120I, BspHI/LU1II/120I, BsrI/BI/GI, BssHII/SI, BstBI/U1/XI, ClaI, Csp6I, DpnI, DraI/II, EagI, Ecl136II, EcoRI/RII/47III, EheI, FspI, HaeIII, HhaI, HinPI, HindIII, HinfI, HpaI/II, KasI, KpnI, MaeII/III, MfeI, MluI, MscI, MseI, NaeI, NarI, NcoI, NdeI, NgoMI, NotI, NruI, NsiI, PmlI, Ppu101, PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI, XmaI cleavage site, and a combination thereof.

15. The method as defined in claim 10, wherein said branch point is an adenosine (A) nucleotide located within a nucleic acid sequence containing or homologous to the SEQ.ID.NO.6 sequence.

16. The method as defined in claim 10, wherein said branch point is an adenosine (A) nucleotide located within a nucleic acid sequence containing at least an oligonucleotide motif homologous to 5′

-TACTAAC-3′

.

17. The method as defined in claim 10, wherein said poly-pyrimidine tract is a high T or C content nucleic acid sequence containing or homologous to the SEQ.ID.NO.7 or SEQ.ID.NO.8 sequence.

18. The method as defined in claim 10, wherein said donor splice site is a nucleic acid sequence either containing or homologous to the SEQ.ID.NO.4 sequence.

19. The method as defined in claim 10, wherein said donor splice site is a nucleic acid sequence containing or homologous to 5′

-GTAAG-3′

.

20. The method as defined in claim 10, wherein said acceptor splice site is a nucleic acid sequence either containing or homologous to the SEQ.ID.NO.5 sequence.

21. The method as defined in claim 10, wherein said acceptor splice site is a nucleic acid sequence containing or homologous to 5′

-CTGCAG-3′

.

22. The method as defined in claim 1, wherein said mir-302-like gene-silencing effector is a nucleic acid sequence containing either homology or complementarity, or both, to SEQ.ID.NO.3.

23. The method as defined in claim 1, wherein said mir-302-like gene-silencing effector is a nucleic acid sequence containing SEQ.ID.NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12, and/or SEQ.ID.NO.13.

24. The method as defined in claim 1, wherein said mir-302-like gene-silencing effector is a nucleic acid sequence containing SEQ.ID.NO.9.

25. The method as defined in claim 1, wherein said expression-competent vector is selected from the group consisting of DNA transgene, plasmid, transposon, retrotransposon, jumping gene, viral vector, and a combination thereof.

26. The method as defined in claim 1, wherein said vector contains comprises a viral or type-II RNA polymerase (Pol-II) promoter or both, a Kozak consensus translation initiation site, polyadenylation signals and a plurality of restriction/cloning sites.

27. The method as defined in claim 26, wherein said restriction/cloning site is an oligonucleotide cleavage domain for an endonuclease selected from the group consisting of AatII, AccI, AflII/III, AgeI, ApaI/LI, AseI, Asp 718I, BamHI, BbeI, BclI/II, BglII, BsmI, Bsp120I, BspHI/LU11I/120I, BsrI/BI/GI, BssHII/SI, BstBI/U1/XI, ClaI, Csp6I, DpnI, DraI/II, EagI, Ecl136II, EcoRI/II/47III, EheI, FspI, HaeIII, HhaI, HinPI, HindIII, HinfI, HpaI/II, KasI, KpnI, MaeII/III, MfeI, MluI, MscI, MseI, NaeI, NarI, NcoI, NdeI, NgoMI, NotI, NruI, NsiI, PmlI, Ppu10I, PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI, XmaI restriction enzyme, and a combination thereof.

28. The method as defined in claim 26, wherein said vector further contains a pUC origin of replication, a SV40 early promoter for expressing at least an antibiotic resistance gene in replication-competent prokaryotic cells and an optional SV40 origin for replication in mammalian cells.

29. The method as defined in claim 28, wherein said antibiotic resistance gene is selected from the group consisted of penicillin G, ampicillin, neomycin, paromycin, kanamycin, streptomycin, erythromycin, spectromycin, phophomycin, tetracycline, rifapicin, amphotericin B, gentamycin, chloramphenicol, cephalothin, tylosin, G418, and a combination thereof.

30. The method as defined in claim 1, wherein said vector is introduced into said mammalian cells 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.

31. The method as defined in claim 1, wherein the primary RNA transcript of said recombinant nucleic acid composition is generated by transcription machinery selected from the group consisting of type-II (Pol-II), type-III (Pol-I), type-I (Pol-I), and viral RNA polymerase transcription machineries.

32. The method as defined in claim 1, wherein the primary RNA transcript of said recombinant nucleic acid composition is an ribonucleotide sequence selected from the group consisting of mRNA, hnRNA, rRNA, tRNA, snoRNA, snRNA, pre-microRNA, viral RNA and their RNA precursors as well as derivatives.

33. The method as defined in claim 1, wherein said mir-302-like gene-silencing effector is released from said intron by an intron excision mechanism selected from the group consisting of RNA splicing, exosome digestion, nonsense-mediated decay (NMD) processing, and a combination thereof.

34. The method as defined in claim 1, wherein said mir-302-mediated gene silencing effect is caused by an intracellular posttranscriptional gene silencing, translational suppression, RNA interference, and/or nonsense-mediated decay mechanism.

35. The method as defined in claim 1, wherein said stem cell-like pluripotent cells express mir-302 microRNAs.

36. The method as defined in claim 1, wherein said stem cell-like pluripotent cells express embryonic stem cell markers Oct3/4, SSEA-3, and SSEA-4.

37. The method as defined in claim 1, wherein said stem cell-like pluripotent cells can be cultured under a feeder-free cell culture condition.

38. The method as defined in claim 1, wherein said stem cell-like pluripotent cells can be cultured under DMEM with 10% charcoal-stripped FBS.

39. The method as defined in claim 1, wherein said stem cell-like pluripotent cells can differentiate into germ line-like cells.

40. The method as defined in claim 1, wherein said stem cell-like pluripotent cells can differentiate into spermatogonia-like cells.

41. The method as defined in claim 1, wherein said stem cell-like pluripotent cells can differentiate into fibroblast-like cells.

42. The method as defined in claim 1, wherein said stem cell-like pluripotent cells can differentiate into chondrocyte-like cells.

43. The method as defined in claim 1, wherein said stem cell-like pluripotent cells can form embryoid body-like colonies.

44. The method as defined in claim 1, wherein said mir-302-like gene-silencing effector shares some homologous target genes with mir-93, mir-367, mir-371, mir-372, mir-373, and mir-520 familial members.

45. The method as defined in claim 1, wherein said stem cell-like pluripotent cells can be selectively isolated using mir-302 microRNAs as markers.

46. A recombinant nucleic acid composition for inducing intronic mir-302-mediated gene silencing effects comprises:

at least an intron encoding a mir-302-like gene silencing effector flanked with exons, wherein said intron can be cleaved out of said exons for inducing mir-302-mediated gene silencing effects, and said exons can be linked to form a gene with desired function.

47. The recombinant nucleic acid composition as defined in claim 46, wherein said intron comprises:

(a) an intronic insert encoding a mir-302-like gene silencing effector;

(b) donor and acceptor splice sites;

(c) a branch point motif; and

(d) at least a poly-pyrimidine tract.

48. The recombinant nucleic acid composition as defined in claim 47, wherein said intronic insert is a hairpin-like nucleic acid sequence containing a stem-loop structure homologous to either SEQ.ID.NO.1 or SEQ.ID.NO.2.

49. The recombinant nucleic acid composition as defined in claim 47, wherein said intronic insert is a nucleic acid sequence containing either homology or complementarity, or both, to SEQ.ID.NO.3.

50. The recombinant nucleic acid composition as defined in claim 47, wherein said intronic insert is a hairpin-like precursor microRNA (pre-miRNA) sequence containing 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, and SEQ.ID.NO.13.

51. The recombinant nucleic acid composition as defined in claim 47, wherein said intronic insert is incorporated into said intron through at least a restriction/cloning site selected from the group consisting of AatII, AccI, AflII/III, AgeI, ApaI/LI, AseI, Asp718I, BamHI, BbeI, BclI/II, BglII, BsmI, Bsp120I, BspHI/LU1II/120I, BsrI/BI/GI, BssHII/SI, BstBI/UI/XI, ClaI, Csp6I, DpnI, DraI/II, EagI, Ecl136II, EcoRI/RII/47III, EheI, FspI, HaeIII, HhaI, HinPI, HindIII, HinfI, HpaI/II, KasI, KpnI, MaeII/III, MfeI, MluI, MscI, MseI, NaeI, NarI, NcoI, NdeI, NgoMI, NotI, NruI, NsiI, PmlI, Ppu101, PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI, XmaI cleavage site, and a combination thereof.

52. The recombinant nucleic acid composition as defined in claim 47, wherein said branch point is an adenosine (A) nucleotide located within a nucleic acid sequence containing or homologous to the SEQ.ID.NO.6 sequence.

53. The recombinant nucleic acid composition as defined in claim 47, wherein said branch point is an adenosine (A) nucleotide located within a nucleic acid sequence containing at least an oligonucleotide motif homologous to 5′

-TACTAAC-3′

.

54. The recombinant nucleic acid composition as defined in claim 47, wherein said poly-pyrimidine tract is a high T or C content nucleic acid sequence containing or homologous to the SEQ.ID.NO.7 or SEQ.ID.NO.8 sequence.

55. The recombinant nucleic acid composition as defined in claim 47, wherein said donor splice site is a nucleic acid sequence either containing or homologous to the SEQ.ID.NO.4 sequence.

56. The recombinant nucleic acid composition as defined in claim 47, wherein said donor splice site is a nucleic acid sequence containing or homologous to 5′

-GTAAG-3′

.

57. The recombinant nucleic acid composition as defined in claim 47, wherein said acceptor splice site is a nucleic acid sequence either containing or homologous to the SEQ.ID.NO.5 sequence.

58. The recombinant nucleic acid composition as defined in claim 47, wherein said acceptor splice site is a nucleic acid sequence containing or homologous to 5′

-CTGCAG-3′

.

59. The recombinant nucleic acid composition as defined in claim 46, wherein said mir-302-like gene-silencing effector is a nucleic acid sequence containing either homology or complementarity, or both, to SEQ.ID.NO.3.

60. The recombinant nucleic acid composition as defined in claim 46, wherein said mir-302-like gene-silencing effector is a nucleic acid sequence containing SEQ.ID.NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12, and/or SEQ.ID.NO.13.

61. The recombinant nucleic acid composition as defined in claim 46, wherein said mir-302-like gene-silencing effector is a nucleic acid sequence containing SEQ.ID.NO.9.