Use of microRNA precursors as drugs for inducing CD34-positive adult stem cell expansion

• US 9,879,263 B2
• Filed: 05/27/2016
• Issued: 01/30/2018
• Est. Priority Date: 08/12/2011
• Status: Active Grant

• Current Assignee: Mello Biotechnology, Inc., Wjwu & Lynn Institute For Stem Cell Research
• Original Assignee: Mello Biotechnology, Inc., Wjwu & Lynn Institute For Stem Cell Research
• Inventors: Lin, Shi-Lung, Chang, Donald, Wu, David T S
• Primary Examiner(s): SCHULTZ, JAMES
• Application Number:
  US15/167,226
• Publication Number:
  US 20160264974A1
• Time in Patent Office: 613 Days
• Field of Search: None
• 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 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2330/50 Biochemical production, i.e. in a transformed host cell
• CPC Class Codes: C12N 2330/51 Specially adapted vectors
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

This invention generally relates to a composition and its production method useful for developing drugs/vaccines and/or therapies against a variety of degenerative diseases in humans. Particularly, the present invention teaches the essential steps of production and purification processes necessary for producing small hairpin-like RNA (shRNA) compositions, such as microRNA precursors (pre-miRNA) and short interfering RNAs (siRNA), which are useful for treating human ageing related diseases, such as, but not limited, Alzheimer'"'"'s diseases, Parkinson'"'"'s diseases, osteoporosis, diabetes, and cancers. The novelty of the present invention is to create an artificially enhanced adaptation environment for prokaryotic cells to adopt eukaryotic pol-2 and/or pol-2-like promoters for transcribing desired ncRNAs and/or their precursors without going through error-prone prokaryotic promoters, so as to improve the productive efficiency and reading fidelity of the shRNA transcription in the prokaryotic cells. The resulting shRNAs, preferably pre-miRNAs and siRNAs, are useful for developing therapeutic drugs against human degenerative diseases, particularly through a mechanism to induce CD34-positive stem cell expansion and/or regeneration.

Claims

1. A method of producing microRNA precursors (pre-miRNAs) containing a sequence of SEQ.ID.NO.3 and capable of inducing CD34-positive adult stem cell expansion or regeneration in a human, comprising:

(a) providing at least a chemical inducer agent, containing a molecular structure 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 of SEQ.ID.NO.3 through the eukaryotic pol-2 or pol-2-like RNA promoter, wherein the expression of said pre-miRNA from said plasmid vector is inducible by the at least a chemical inducer agent provided in (a), and wherein said transformed prokaryotic cells are E. coli competent cells and said eukaryotic pol-2 or pol-2-like RNA promoter is cytomegalovirus (CMV) or mammalian EF1alpha promoter; and

;

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

wherein said pre-miRNA induces said CD34-positive adult stem cell expansion or regeneration by contacting with at least a CD34-positive adult stem cell in a cell substrate, wherein a concentration of said pre-miRNA used for contacting ranges from 50 to 500 micrograms per milliliter.

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 said chemical inducer agent is added into a bacterial culturing medium at a final volume to volume concentration of about 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 transformed 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 the sequence of SEQ.ID.NO.5.

7. The method as defined in claim 6, wherein said plasmid vector is pLenti-EF1alpha-RGFP-miR302.

8. The method as defined in claim 1, wherein said pre-miRNAs contain 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 method as defined in claim 1, wherein said pre-miRNA is a prokaryote-produced microRNA precursor (pro-miRNA).

10. The method 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 method as defined in claim 1, wherein said pre-miRNA is useful for pharmaceutical or therapeutic applications by contacting said pre-miRNA with the cell substrate containing said at least a CD34-positive adult stem cell.

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

C. with frequent agitation.

13. The method as defined in claim 1, wherein said CD34-positive adult stem cell expansion or regeneration induced by contacting said pre-miRNA with the cell substrate containing said at least a CD34-positive adult stem cell is useful for pharmaceutical or therapeutic applications.

14. The method as defined in claim 13, wherein said CD34-positive adult stem cell expansion or regeneration induced by contacting said pre-miRNA with the cell substrate is useful for reprogramming the malignant properties of human cancer cells into a low-grade benign or normal-like state in vivo.

15. The method as defined in claim 13, wherein said CD34-positive adult stem cell expansion or regeneration induced by contacting said pre-miRNA with the cell substrate is useful for enhancing scarless wound healing in vivo.