Production and extraction of MicroRNA precursor as drug for cancer therapy

• US 9,399,773 B2
• Filed: 12/27/2013
• Issued: 07/26/2016
• Est. Priority Date: 08/10/2012
• Status: Active Grant

• Current Assignee: Mello Biotechnology, Inc., Wjwu & Lynn Institute For Stem Cell Research
• Original Assignee: Mello Biotechnology, Inc.
• Inventors: Lin, Shi-Lung, Chang, Donald, Wu, David T S
• Primary Examiner(s): Schultz, Doug
• Application Number:
  US14/142,512
• Publication Number:
  US 20140141470A1
• Time in Patent Office: 942 Days
• Field of Search: None
• US Class Current: 1/1
• 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 homologs/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 method of producing microRNA precursor (pre-miRNA) containing a shared sequence SEQ ID NO:

3 capable of reprogramming the malignant properties of human cancer cells into a low-grade benign or normal-like state, comprising;

(a) providing at least a chemical inducer agent, containing a molecular structure similar to of 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 containing at least a eukaryotic pol-2 or pol-2-like RNA promoter and are capable of expressing said pre-miRNA containing the sequence SEQ ID NO;

3 through the eukaryotic promoter, wherein the expression of said pre-miRNA from said plasmid vector is inducible by the at least a chemical agent provided in (a), and wherein said transformed prokaryotic cells are E. coli competent cells and said eukaryotic promoter is cytomegalovirus (CMV) promoter; and

(c) mixing the at least a chemical agent from (a) and the at least a line of transformed prokaryotic cells from (b) together under a condition to induce the eukaryotic promoter-driven transcription of said pre-miRNA.

2. The method 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 method as defined in claim 1, wherein the step of providing at least a line of transformed prokaryotic cells further comprising providing said prokaryotic cells in a bacterial culturing medium;

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

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

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

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

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

8. The method as defined in claim 1, wherein said pre-miRNA contains at least a sequence of SEQ. ID. NO. 8.

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

C. with frequent agitation.

10. The method 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 vivo.

11. The method as defined in claim 1, wherein said human cancer cells are liver cancer cells.

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

13. The method as defined in claim 12, wherein said pharmaceutical or therapeutic applications includes anti-cancer drug development.