Use of microrna precursors as drugs for inducing cd34-positive adult stem cell expansion

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.

1 . A method for inducing CD34-positive cell expansion or regeneration using at least a microRNA or its precursor (pre-miRNA), comprising:
 (a) providing a cell substrate containing at least a CD34-positive cell,   (b) providing at least a microRNA or a pre-miRNA containing a shared seed sequence of SEQ.ID.NO:3, and   (c) contacting the microRNA or the pre-miRNA of (b) with the cell substrate containing said at least a CD34-positive cell of (a), so as to induce the expansion or regeneration of CD34-positive cell population; wherein the concentration of the microRNA or the pre-miRNA used for contacting is ranged from 50 to 500 micrograms per milliliter (50-500 μg/mL).   
     
     
         2 . The method as defined in  claim 1 , wherein the microRNA or the pre-miRNA is produced by eukaryotic promoter-driven RNA transcription in prokaryotic cells. 
     
     
         3 . The method as defined in  claim 2 , wherein said eukaryotic promoter-driven RNA transcription is induced by contacting a chemical agent containing 3-morpholinopropane-1-sulfonic acid (MOPS) with at least a transformed prokaryotic cell carrying at least an expression vector encoding a sequence of SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, or SEQ.ID.NO:9. 
     
     
         4 . The method as defined in  claim 3 , wherein said expression vector is a recombinant plasmid encoding the sequence of SEQ.ID.NO:5. 
     
     
         5 . The method as defined in  claim 3 , wherein said expression vector is pLenti-EF1alpha-RGFP-miR302 containing either a cytomegalovirus (CMV) or mammalian EF1alpha promoter, or both. 
     
     
         6 . The method as defined in  claim 2 , wherein said prokaryotic cells are  E. coli  competent cells. 
     
     
         7 . The method as defined in  claim 1 , wherein the pre-miRNA contains at least a hairpin-like sequence of SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, or SEQ.ID.NO:9. 
     
     
         8 . The method as defined in  claim 1 , wherein the pre-miRNA is a prokaryote-produced miR-302 precursor (pro-miR-302). 
     
     
         9 . The method as defined in  claim 8 , wherein said pro-miR-302 contains at least a sequence of miR-302a, miR-302b, miR-302c, or miR-302d. 
     
     
         10 . The method as defined in  claim 8 , wherein said pro-miR-302 is used as a part of drug ingredients for pharmaceutical and therapeutic applications. 
     
     
         11 . The method as defined in  claim 1 , wherein the pre-miRNA is used as a part of drug ingredients for pharmaceutical and therapeutic applications. 
     
     
         12 . The method as defined in  claim 11 , wherein the pre-miRNA is used to treat aging-related diseases. 
     
     
         13 . The method as defined in  claim 1 , wherein said induced CD34-positive cells are used as a part of treatment components in pharmaceutical or therapeutic applications. 
     
     
         14 . The method as defined in  claim 13 , wherein said induced CD34-positive cells are used to treat aging-related diseases. 
     
     
         15 . The method as defined in  claim 13 , wherein said induced CD34-positive cells reprogram the malignant properties of human cancer cells into a low-grade benign or normal-like state in vivo. 
     
     
         16 . The method as defined in  claim 13 , wherein said induced CD34-positive cells enhance scarless wound healing in vivo. 
     
     
         17 . The method as defined in  claim 1 , wherein said CD34-positive cells are adult stem cells and include skin, hair, muscle, blood (hematopoietic), mesenchymal, and neural stem cells, or a combination thereof.