Composition and method of using miR-302 precursors as drugs for treating Alzheimer's diseases

• US 10,519,440 B2
• Filed: 02/19/2016
• Issued: 12/31/2019
• Est. Priority Date: 08/10/2012
• Status: Active Grant

• Current Assignee: Mello Biotechnology, Inc.
• Original Assignee: Mello Biotechnology, Inc.
• Inventors: Lin, Chih-Li, Li, Hsin-Hua, Lin, Shi-Lung, Lai, Te-Jen
• Primary Examiner(s): Wehbe, Anne Marie S
• Application Number:
  US15/048,964
• Publication Number:
  US 20170218362A1
• Time in Patent Office: 1,411 Days
• Field of Search: 435325, 4353201, 435368, 536 241
• CPC Class Codes: C12N 1/38 Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound C12N1/34 takes precedence
• 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 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/10 Cells modified by introduction of foreign genetic material

Abstract

This invention generally relates to a composition and method of using recombinant microRNAs (miRNA) and their hairpin-like precursors (pre-miRNA) as therapeutic drugs for treating Alzheimer'"'"'s diseases (AD). More specifically, the present invention relates to the use of man-made miRNA miR-302 precursors (pre-miR-302) for AD therapy in humans. These pre-miR-302 molecules can be mass produced in prokaryotes as a form of DNA expression-competent DNA vectors and/or hairpin-like RNAs. As prokaryotic cells do not transcribe or process hairpin-like RNAs, the present invention also teaches a method for expressing pre-miRNAs in prokaryotes, i.e. pro-miRNA, using a novel hairpin-like RNA transcription mechanism newly found in prokaryotes. Additionally, since miR-302 is a well-known embryonic stem cell (ESC)-specific factor in humans, our novel findings of this invention can be further used to advance the designs and development of novel regenerative medicine for treating many other ageing-related degenerative diseases, such as Parkinson'"'"'s diseases, osteoporosis, diabetes, and cancers.

Claims

1. A method of protecting living human brain neurons from Aβ

-induced neurotoxicity with hairpin-like RNA mimics of microRNA precursor (hairpin-like pre-miRNA mimics), comprising;

(a) treating and transfecting at least one living neuron with a vector in vitro, wherein the vector contains SEQ.ID.NO.2 and a eukaryotic promoter, and is capable of expressing at least one hairpin-like pre-miRNA mimic through the eukaryotic promoter; and

(b) expressing said at least one hairpin-like pre-miRNA mimic in the treated neurons.

2. The method as defined in claim 1, wherein said hairpin-like pre-miRNA mimics are miR-302 precursors (pre-miR-302) in a structural conformation selected from the group consisting of microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), their precursors and homologs, and a combination thereof.

3. The method as defined in claim 1, wherein said hairpin-like pre-miRNA mimics consist of SEQ.ID.NO3, SEQ.ID.NO4, SEQ.ID.NO5, and SEQ.ID.NO6.

4. The method as defined in claim 1, wherein said vector is pLenti-EF1alpha/CMV-RGFP/GFP-miR302.

5. The method as defined in claim 1, wherein the expression of said hairpin-like pre-miRNA mimics produces miR-302a, miR-302b, miR-302c and miR-302d in the treated neurons.

6. The method as defined in claim 1, further comprising a step of inducing Akt signaling activation.

7. The method as defined in claim 6, wherein said Akt signaling activation improves insulin resistance in the treated neurons.

8. The method as defined in claim 7, further comprising a step of suppressing p-307 IRS-1 serine phosphorylation, increasing IRS-1 tyrosine phosphorylation, or both.

9. The method as defined in claim 6, wherein said Akt signaling activation further stimulates Nanog expression to increase sensitivity of insulin signaling.

10. The method as defined in claim 1, further comprising a step of inducing Nrf2/HO-1 expression to reduce Aβ

-induced intracellular ROS accumulation and apoptosis.

11. The method as defined in claim 1, wherein said vector is formulated with glycylglycerin for being delivered into the treated neurons.

12. The method as defined in claim 1, wherein said vector is useful for developing drugs and therapies for treating Alzheimer'"'"'s diseases.

13. The method as defined in claim 1, wherein said hairpin-like pre-miRNA mimics are useful for developing drugs and therapies for treating Diabetes and Alzheimer'"'"'s diseases.

14. A method of inducing Akt signaling activation in living human brain neurons by silencing at least one gene, comprising:

(a) treating and transfecting at least one living neuron with a vector in vitro, wherein the vector contains SEQ.ID.NO.2 and a eukaryotic promoter, and is capable of expressing at least one hairpin-like pre-miRNA mimic through the eukaryotic promoter; and

(b) expressing said at least one hairpin-like pre-miRNA mimic in the treated neurons.

15. The method as defined in claim 14, wherein said at least one gene includes PTEN gene.