Generation of human embryonic stem-like cells using intronic RNA

• US 9,567,591 B2
• Filed: 05/07/2008
• Issued: 02/14/2017
• 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., University of Southern California
• Inventors: Lin, Shi-Lung, Ying, Shao-Yao, Wu, David Ts
• Primary Examiner(s): Schultz, Doug
• Application Number:
  US12/149,725
• Publication Number:
  US 20080293143A1
• Time in Patent Office: 3,205 Days
• Field of Search: None
• US Class Current: 1/1
• 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 method for inducing genomic DNA demethylation and hence activating Oct3/4 and Sox2 expression in mammalian cells, comprising the steps of:

(a) constructing a recombinant SpRNAi-RGFP nucleic acid composition that contains at least an intronic RNA gene silencing effector comprising SEQ. ID. NO. 39, SEQ. ID. NO. 40, SEQ. ID. NO. 41, SEQ. ID. NO. 42, SEQ. ID. NO. 43, SEQ. ID. NO. 44, SEQ. ID. NO. 45 and SEQ. ID. NO. 46, wherein said intronic RNA gene silencing effector is further processed into mir-302a, mir-302b, mir-302c and mir-302d in mammalian cells;

(b) introducing said recombinant SpRNAi-RGFP nucleic acid composition into a plurality of mammalian cells, wherein said plurality of mammalian cells generate a plurality of intronic RNA gene silencing effectors; and

(c) enabling the plurality of intronic RNA gene silencing effectors to reach a level that is sufficient to induce genomic DNA demethylation and hence activate Oct3/4 and Sox2 expression, which consequently results in reprogramming the cells into a pluripotent state.

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, further comprising a step of synthesizing nucleic acid components of said nucleic acid composition.

6. The method as defined in claim 1, wherein said recombinant SpRNAi-RGFP nucleic acid composition contains a recombinant cellular gene.

7. The method as defined in claim 1, wherein said recombinant SpRNAi-RGFP nucleic acid composition contains a marker protein gene.

8. The method as defined in claim 1, wherein said nucleic acid composition is constructed by a genetic engineering method.

9. The method as defined in claim 1, wherein said recombinant SpRNAi-RGFP nucleic acid composition is a nucleic acid sequence containing components of an intronic insert encoding an intronic RNA gene silencing effector, a branch point motif, a poly-pyrimidine tract, a donor splice site and an acceptor splice site.

10. The method as defined in claim 9, 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.

11. The method as defined in claim 9, wherein said intronic insert is a nucleic acid sequence containing either homology or complementarity, or both, to SEQ. ID. NO. 3.

12. The method as defined in claim 9, wherein the intronic insert is incorporated into said recombinant intron through at least a restriction/cloning site.

13. The method as defined in claim 9, 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.

14. The method as defined in claim 9, wherein said poly-pyrimidine tract is a high T or C content nucleic acid sequence containing or homologous to the SEQ. ID. NO. 7 sequence.

15. The method as defined in claim 9, wherein said donor splice site is a nucleic acid sequence either containing or homologous to the SEQ. ID. NO. 4 sequence.

16. The method as defined in claim 9, wherein said acceptor splice site is a nucleic acid sequence either containing or homologous to the SEQ. ID. NO. 5 sequence.

17. The method as defined in claim 1, wherein said recombinant SpRNAi-RGFP nucleic acid composition is a plasmid.

18. The method as defined in claim 1, wherein said recombinant SpRNAi-RGFP nucleic acid composition contains 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.

19. The method as defined in claim 18, wherein said restriction/cloning site is an oligonucleotide cleavage domain for an endonuclease.

20. The method as defined in claim 18, wherein said recombinant SpRNAi-RGFP nucleic acid composition 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.

21. The method as defined in claim 1, wherein said recombinant SpRNAi-RGFP nucleic acid composition is introduced into said mammalian cells by a gene delivery method.

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

23. The method as defined in claim 1, wherein the primary RNA transcript of said recombinant SpRNAi-RGFP nucleic acid composition contains a microRNA.

24. The method as defined in claim 1, wherein said intronic RNA gene silencing effector is released from said recombinant 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.

25. The method as defined in claim 1, wherein said intronic RNA gene silencing effector has an effect on intracellular posttranscriptional gene silencing, translational suppression, RNA interference, and/or nonsense-mediated decay mechanism.

26. The method as defined in claim 1, wherein said pluripotent cells express mir-302 microRNAs.

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

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

29. The method as defined in claim 1, wherein cells in said pluripotent state can differentiate into somatic cells.

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

31. The method as defined in claim 1, wherein said intronic RNA gene silencing effector shares some homologous target genes with mir-302 familial members.

32. The method as defined in claim 1, wherein cells in said pluripotent state can be selectively isolated using mir-302 microRNAs and Oct3/4 as markers.