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

a technology of precursors and microrna, which is applied in the direction of gene therapy, fermentation, biochemistry apparatus and processes, etc., can solve the problems of not being suitable for growing bacteria, achieve the effect of reducing the number of mitotic cells, enhancing scar-less wound healing, and effectively attenuating the fast cell cycle ra

Inactive Publication Date: 2017-11-30
LIN SHI LUNG +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]Based on the above knowledge, the present invention is a design and method for utilizing prokaryotic cells to produce human microRNA precursors (pre-miRNAs) and / or shRNAs as therapeutical drugs and / or vaccines for cancer therapy. More specifically, the present invention is a design and method of utilizing prokaryotic cells to produce a special kind of pre-miRNA-like agents, named pro-miRNA, that are capable of reprogramming the malignant properties of high-grade human cancer cells into a low-grade benign or even relatively normal-like state. Preferably, these pro-miRNAs are tumor suppressor microRNAs (TS-miRNA) similar to the precursors of miR-302a, b, c, d, e, and / or f (pre-miR-302s) and their natural familial cluster as well as their manually re-designed small hairpin RNA (shRNA) homologues / derivatives, and / or a combination thereof. The designs of pro-miRNA-like shRNA homologues / derivatives include imperfectly and perfectly matched hairpin compositions of the pro-miRNA and its homologous small interfering RNA (siRNA), which may be formed in a single unit or in a multiple unit cluster. These designs may improve the target specificity and / or reduce the copy number of pro-miR-302 required for effective delivery and therapy. The human cells suitable for such a drug treatment include normal, tumor, and cancerous cells in vitro, ex vivo and / or in vivo.
[0095]In principle, the present invention provides a novel composition design and its applicable strategy for inducing a quick adaptation of prokaryotes to use eukaryotic pol-2 and pol-2-like promoters for directly expressing certain desired microRNA precursors (pre-miRNA) without the need of using error-prone prokaryotic promoters or growing laborious and costly hybridomas or mammalian cells.

Problems solved by technology

For example, MOPS is frequently used as a buffering agent in bacterial cell lysis and hence is not suitable for growing bacteria.

Method used

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

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Experimental program
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Embodiment Construction

[0116]In the experimental disclosure which follows, the following abbreviations apply: M (molar); mM (millimolar); μm (micromolar); mol (moles); pmol (picomoles); gm (grams); mg (milligrams) μg (micrograms); ng (nanograms); L (liters); ml (milliliters); μl (microliters); ° C. (degrees Centigrade); RNA (ribonucleic acid); DNA (deoxyribonucleic acid); dNTP (deoxyribonucleotide triphosphate); PBS (phosphate buffered saline); NaCl (sodium chloride); HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid); HBS (HEPES buffered saline); SDS (sodium dodecyl sulfate); Tris-HCl (tris-hydroxymethylaminomethane-hydrochloride); ATCC (American Type Culture Collection, Rockville, Md.); hESC (human embryonic stem cells); and iPSC (induced pluripotent stem cells).

1. Bacterial Cell Culture and Chemical Treatments

[0117]E. coli DH5alpha competent cells were acquired as a part from the z-competent E. coli transformation kit (Zymo Research, Irvine, Calif.) and then transformed by mixing with 5 μg of a...

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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.

Description

PRIORITY[0001]This application is a Divisional of U.S. patent application Ser. No. 15 / 167,226 filed on May 27, 2016, which claims priority to the U.S. Provisional Application Ser. No. 62 / 262,280 filed on Dec. 2, 2015, which was entitled “miR-302 Attenuates Aβ-induced Neurotoxicity through Activation of Akt Signaling”. The present application also claims priority to the U.S. patent application Ser. No. 14 / 502,608 filed on Sep. 30, 2014, and Ser. No. 14 / 527,439 filed on Oct. 29, 2014, all of which are entitled “Inducible Gene Expression Composition for Using Eukaryotic Pol-2 Promoter-Driven Transcription in Prokaryotes and The Applications Thereof”. Also, the present application claims priority to the U.S. patent application Ser. No. 13 / 964,705 filed on Aug. 12, 2013, entitled “Production and Utilization of A Novel Anti-Cancer Drug in Therapy” and the U.S. patent application Ser. No. 14 / 142,512 filed on Dec. 27, 2013, entitled “Production and Extraction of MicroRNA Precursors as Drug ...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12N15/113C12P19/34C12N15/11C12N15/70
CPCC12N2310/14C12N2330/51C12N2330/50C12N15/113C12N15/111C12P19/34C12N2310/141C12N2320/30C12N15/70C12N15/1135
InventorLIN, SHI-LUNGCHANG, DONALDWU, DAVID TS
OwnerLIN SHI LUNG