PRODUCTION AND EXTRACTION OF MicroRNA PRECURSOR AS DRUG FOR CANCER THERAPY

• US 20140141470A1
• Filed: 12/27/2013
• Published: 05/22/2014
• Est. Priority Date: 08/10/2012
• Status: Active Grant

• Current Assignee: Mello Biotechnology, Inc., Wjwu & Lynn Institute For Stem Cell Research
• Original Assignee: David Ts Wu, Donald Chang, Shi-Lung Lin
• Inventors: LIN, Shi-Lung, WU, David TS, CHANG, Donald
• Granted Patent: US 9,399,773 B2
• Time in Patent Office: Days
• US Class Current: 435/91.32
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2330/50 Biochemical production, i.e. in a transformed host cell
• CPC Class Codes: C12N 2330/51 Specially adapted vectors

Abstract

This invention generally relates to a composition for developing novel anti-cancer drugs and/or vaccines and producing microRNA precursor (pre-miRNA) and/or its shRNA homologues/mimics/derivatives, and a method thereof. The present invention also relates to a use of a composition in producing novel prokaryote-produced microRNA precursor (pro-miRNA) capable of being delivered into human cells and processed by the cells into microRNA-like effectors to elicit specific silencing effects on certain targeted oncogenes, subsequently leading to a therapeutic result of tumor suppression and cancer therapy. Specifically, the method of the present invention includes inducing an expression of the pre-miRNA/pro-miRNAs, particularly human pre-miR-302, in prokaryotes through pol-2 or pol-2-like RNA promoter. Most importantly, the composition of the present invention is further a novel pre-miRNA-based drug that is capable of reprogramming the malignant properties of high-grade human liver cancers into a low-grade benign or even relatively normal stage—a mechanism called “Cancer Reversion”.

Claims

1. A composition for producing microRNA precursor (pre-miRNA) capable of reprogramming the malignant properties of human cancer cells into a low-grade benign or normal-like state and hence useful for developing anti-cancer drug for therapy, comprising:

(a) at least a chemical inducer agent, containing a molecular structure similar to 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol, glycerin, or a mixture thereof; and

(b) at least a line of transformed prokaryotic cells that carry at least a plasmid vector capable of expressing said pre-miRNA through a eukaryotic promoter;

wherein (a) and (b) are mixed together under a condition to induce the eukaryotic promoter-driven transcription of said pre-miRNA.

2. The composition as defined in claim 1, wherein said chemical inducer agent is a transcriptional inducer capable of stimulating eukaryotic promoter-driven RNA transcription in prokaryotes.

3. The composition as defined in claim 1, wherein said prokaryotic cells are in a bacterial culturing medium;

said chemical inducer agent is added into the bacterial culturing medium at a final volume to volume concentration of 0.001% to 4%.

4. The composition as defined in claim 3, wherein said bacterial culturing medium is Luria-Bertani (LB) broth.

5. The composition as defined in claim 1, wherein said prokaryotic cells are E. coli DH5alpha competent cells.

6. The composition as defined in claim 1, wherein said plasmid vector is a recombinant plasmid encoding a sequence of SEQ. ID. NO. 5.

7. The composition as defined in claim 1, wherein said plasmid vector is pLenti-EF1alpha-RGFP-miR302.

8. The composition as defined in claim 1, wherein said pre-miRNA contains at least a sequence homologous to SEQ. ID. NO. 6, SEQ. ID. NO. 7, SEQ. ID. NO. 8, or SEQ. ID. NO. 9.

9. The composition as defined in claim 1, wherein said pre-miRNA is a kind of prokaryote-produced microRNA precursor (pro-miRNA).

10. The composition as defined in claim 9, wherein said pro-miRNA contains at least a sequence of SEQ. ID. NO. 6, SEQ. ID. NO. 7, SEQ. ID. NO. 8, or SEQ. ID. NO. 9.

11. The composition as defined in claim 9, wherein said pro-miRNA is useful for pharmaceutical or therapeutic applications.

12. The composition as defined in claim 1, wherein said eukaryotic promoter is a pol-2 or pol-2-like RNA promoter.

13. The composition as defined in claim 12, wherein said pol-2 RNA promoter is the promoter of EF1 alpha.

14. The composition as defined in claim 12, wherein said pol-2-like RNA promoter is CMV promoter.

15. The composition as defined in claim 1, wherein said condition is LB broth at 37°

C. with frequent agitation.

16. The composition as defined in claim 1, wherein anti-cancer mechanisms of said anti-cancer drug include cancer reversion, in which the malignant properties of high-grade human cancers are reprogrammed into a low-grade benign or normal-like state in vitro, ex vivo or in vivo.

17. The composition as defined in claim 1, wherein said human cancers are liver cancers.

18. The composition as defined in claim 1, wherein said pre-miRNA is useful for pharmaceutical or therapeutic applications.

19. A method of producing microRNA precursor (pre-miRNA) capable of reprogramming the malignant properties of human cancer cells into a low-grade benign or normal-like state and hence useful for developing anti-cancer drug for therapy, comprising:

(a) providing at least a chemical inducer agent, containing a molecular structure similar to 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol, glycerin, or a mixture thereof;

(b) providing at least a line of transformed prokaryotic cells that carry at least a plasmid vector capable of expressing said pre-miRNA through a eukaryotic promoter; and

(c) mixing (a) and (b) together under a condition to induce the eukaryotic promoter-driven transcription of said pre-miRNA.

20. The method as defined in claim 19, wherein said chemical inducer agent is a transcriptional inducer capable of stimulating eukaryotic promoter-driven RNA transcription in prokaryotes.

21. The method as defined in claim 19, wherein the step of providing at least a line of transformed prokaryotic cells further comprises providing said prokaryotic cells in a bacterial culturing medium;

the step of mixing (a) and (b) further comprises adding said chemical inducer agent into the bacterial culturing medium at a final volume to volume concentration of 0.001% to 4%.

22. The method as defined in claim 21, wherein said bacterial culturing medium is Luria-Bertani (LB) broth.

23. The method as defined in claim 19, wherein said prokaryotic cells are E. coli DH5alpha competent cells.

24. The method as defined in claim 19, wherein said plasmid vector is a recombinant plasmid encoding a sequence of SEQ. ID. NO. 5.

25. The method as defined in claim 19, wherein said plasmid vector is pLenti-EF1alpha-RGFP-miR302.

26. The method as defined in claim 19, wherein said pre-miRNA contains at least a sequence homologous to SEQ. ID. NO. 6, SEQ. ID. NO. 7, SEQ. ID. NO. 8, or SEQ. ID. NO. 9.

27. The method as defined in claim 19, wherein said eukaryotic promoter is a pol-2 or pol-2-like RNA promoter.

28. The method as defined in claim 27, wherein said pol-2 RNA promoter is the promoter of EF1 alpha.

29. The method as defined in claim 27, wherein said pol-2-like RNA promoter is CMV promoter.

30. The method as defined in claim 19, wherein said condition is LB broth at 37°

C. with frequent agitation.

31. The method as defined in claim 19, wherein anti-cancer mechanisms of said anti-cancer drug include cancer reversion, in which the malignant properties of high-grade human cancers are reprogrammed into a low-grade benign or normal-like state in vitro, ex vivo or in vivo.

32. A use of a composition in producing microRNA precursor (pre-miRNA) capable of reprogramming the malignant properties of human cancer cells into a low-grade benign or normal-like state and useful for developing anti-cancer drug for therapy, wherein the composition comprises:

at least a line of transformed prokaryotic cells that carry at least a plasmid vector capable of expressing said pre-miRNA through a eukaryotic promoter.

33. The use as defined in claim 32, wherein said prokaryotic cells are mixed with at least a chemical inducer agent under a condition to induce the eukaryotic promoter-driven transcription of said pre-miRNA;

wherein said chemical inducer contains a molecular structure similar to 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol, glycerin, or a mixture thereof.

34. The use as defined in claim 33, wherein said condition is LB broth at 37°

C. with frequent agitation.