WO2017095458A1 - Composition et méthode d'utilisation de précurseurs de mir-302 en tant que médicaments pour le traitement de la maladie d'alzheimer - Google Patents

Composition et méthode d'utilisation de précurseurs de mir-302 en tant que médicaments pour le traitement de la maladie d'alzheimer Download PDF

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WO2017095458A1
WO2017095458A1 PCT/US2016/018774 US2016018774W WO2017095458A1 WO 2017095458 A1 WO2017095458 A1 WO 2017095458A1 US 2016018774 W US2016018774 W US 2016018774W WO 2017095458 A1 WO2017095458 A1 WO 2017095458A1
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mir
hairpin
mirna
cells
rna
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Chih-Li Lin
Hsin-Hua Li
Shi-Lung Lin
Te-Jen LAI
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Chih-Li Lin
Hsin-Hua Li
Shi-Lung Lin
Lai Te-Jen
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors

Definitions

  • 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 drugs can be produced in prokaryotes as a form of expression-competent DNA vectors and/or hairpin-like structured RNAs.
  • MicroRNA (miRNA) miR-302 is the most abundant non-coding RNA species specifically found in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Our previous studies have shown that ectopic expression of miR-302 in mammalian somatic cells is able to reprogram the somatic cells to hESC-like iPSCs (as demonstrated in Lin et al., 2008, 2010 and 2011; EP 2198025; U.S. 12/149,725; U.S. 12/318,806; U.S. 12/792,413).
  • prokaryotic RNA promoters for expressing gene cDNA in prokaryotes and none of them were useful for expressing hairpin-like RNAs, such as miRNAs and shRNAs.
  • these miR-302-like shRNAs/siRNAs are transcribed from a recombinant miR-302 familial gene (SEQ.ID.NO.2, as shown in FIG.13A), of which the transcripts can be further processed into precursors (i.e. pre-miRNAs) of miR-302a (pro-miR-302a, SEQ.ID.NO.3), miR-302b (pro-miR-302b, SEQ.ID.NO.4), miR-302c (pro-miR-302c, SEQ.ID.NO.5), and (pro-miR-302d, SEQ.ID.NO.6), as shown in FIG.13B.
  • precursors i.e. pre-miRNAs
  • miR-302a pro-miR-302a, SEQ.ID.NO.3
  • miR-302b pro-miR-302b, SEQ.ID.NO.4
  • miR-302c pro-miR-302c,
  • ncRNAs non-coding RNAs
  • siRNAs short interfering RNAs
  • shRNAs small hairpin RNAs
  • ncRNAs preferably contain at least a sequence sharing 30% to 100% homology to a microRNA or a part of its precursor (pre-miRNA).
  • shRNAs/siRNAs can be manually designed to contain perfectly matched hairpin-stem regions, while native microRNA precursors (pre-miRNAs or pri-miRNAs) often contain mismatched base pairs.
  • the miR-302 molecules so obtained will remain as hairpin-like microRNA precursors (pre-miR-302s and pri-miR-302s), as shown in FIG. 6, which are useful for being isolated and delivered into eukaryotic cells for eliciting the desired function of miR-302.
  • both of the vector and miR-302 molecules can be simultaneously amplified in the transformed prokaryotic cells, such as E. coli.
  • the method for isolating the amplified pLenti-EFlalpha-RGFP-miR302 vector DNA and the transcribed miR-302 molecules are disclosed in Examples 5 and 6, respectively.
  • the result of induced hairpin-like miR-302/pre-miR-302 expression was confirmed by Northern blot analysis, as shown in FIG. 6.
  • the miR-302 expression was detected only in transformed cells treated with MOPS, glycerin and/or ethanol, but not in blank negative controls, indicating that in the absence of any chemical inducer no transcription activity can function through a gene promoter containing a hairpin-like structure in prokaryotic cells, as reported by McDowell et al (Science 1994).
  • the hairpin-like miR-302 expression shown in FIG. 6 is a specific result induced by the added transcription inducers of the present invention, not a random transcription leakage event.
  • the present invention provides a simple, cheap, fast and inducible composition and method for mass production of hairpin-like miR-302 molecules and/or their precursors/homologs in prokaryotes. Moreover, the isolation of miR-302 and/or its precursors from prokaryotic cells is relatively easy and cost-effective, as shown in FIG. 6 and Example 6 of the present invention.
  • the present invention may also provide a composition and method for inducing iPSC derivation using isolated miR- 302 and/or pre-miR-302 molecules.
  • the methods for delivering miR- 302 and/or pre-miR-302 molecules into mammalian cells can be selected from the group of microinjection, lipid-/glycerin-/chemical-mediated infusion/perfusion, peptide-/sugar- /liposome-/chemical-mediated transfection, antigen-/antibody-/receptor-mediated endocytosis, transposon-/retrotransposon/caspase-mediated cell penetration, viral infection, gene gun penetration, electroporation, and a combination thereof.
  • the applications of isolated miR-302 and/or pre-miR-302 molecules may further include the induction and expansion of CD34-positive adult stem cells. As shown in FIGs.
  • MiR-302 Protects SK-N-MC Cells against ⁇ -induced Apoptosis.
  • DAPI staining To determine which kind of cell death induced by ⁇ , we further examined the nuclei fragmentation by DAPI staining. As shown in FIGs.
  • miR-302 plays a protective role in preventing ⁇ -induced cell apoptosis.
  • ⁇ -impaired insulin signaling may also lead to an increase of GSK3P activity as well as tau hyperphosphorylation, a relevant step in AD pathogenesis.
  • miR-302 may exert its protective effects mainly through activating and/or restoring the Akt/GSK3P signaling pathway.
  • Nrf2 a redox-sensitive transcription factor
  • HO-1 antioxidant-response elements
  • Nanog plays a protective role in ⁇ treatment
  • shRNA-mediated knockdown of Nanog in miR-302-transfected cells.
  • FIG. 17F downregulation of Nanog resulted in an increase of p-Ser307 IRS-1 expression as well as a decrease of both tyrosine phosphorylation and p-Ser 473-Akt/ p-Ser 9-GSK3P levels in miR-302-transfected cells after ⁇ treatment.
  • miR-302 may confer protection against ⁇ -induced neurotoxicity by downregulating PTEN to activate Akt and the downstream Nanog signaling.
  • Precursor messenger RNA primary RNA transcripts of a protein-coding gene, which are produced by eukaryotic type-II RNA polymerase ( ⁇ - ⁇ ) machineries in eukaryotes through an intracellular mechanism termed transcription.
  • a pre-mRNA sequence contains a 5 '-untranslated region (UTR), a 3'-UTR, exons and introns.
  • Intron a part or parts of a gene transcript sequence encoding non-protein-reading frames, such as in-frame intron, 5' -UTR and 3' -UTR.
  • RNA messenger RNA
  • mRNA messenger RNA
  • RNA splicing machineries e.g. spliceosomes
  • the peptides/proteins encoded by mRNAs include, but not limited, enzymes, growth factors, insulin, antibodies and their analogs/homologs as well as derivatives.
  • Nucleic Acid Template a double- stranded DNA molecule, double stranded RNA molecule, hybrid molecules such as DNA-RNA or RNA-DNA hybrid, or single-stranded DNA or RNA molecule.
  • Partial complementarity or complementation occurs when only some of the nucleic acid bases are matched according to the base pairing rules. Complete or total complementarity or complementation occurs when the bases are completely or perfectly matched between the nucleic acid strands.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as in detection methods that depend on binding between nucleic acids.
  • Percent complementarity or complementation refers to the number of mismatch bases over the total bases in one strand of the nucleic acid. Thus, a 50% complementation means that half of the bases were mismatched and half were matched. Two strands of nucleic acid can be complementary even though the two strands differ in the number of bases. In this situation, the complementation occurs between the portion of the longer strand corresponding to the bases on that strand that pair with the bases on the shorter strand.
  • Non-coding RNA an RNA transcript that cannot be used to synthesize peptides or proteins through intracellular translation machineries.
  • Non-coding RNA includes long and short regulatory RNA molecules such as microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) and double strand RNA (dsRNA). These regulatory RNA molecules usually function as gene silencers, interfering with expression of intracellular genes containing either completely or partially complementarity to the non-coding RNAs.
  • miRNA microRNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • dsRNA double strand RNA
  • MicroRNA single- stranded RNAs capable of binding to targeted gene transcripts that have partial complementarity to the miRNA.
  • MiRNA is usually about 17-27 oligonucleotides in length and is able to either directly degrade its intracellular mRNA target(s) or suppress the protein translation of its targeted mRNA, depending on the complementarity between the miRNA and its target mRNA.
  • Natural miRNAs are found in almost all eukaryotes, functioning as a defense against viral infections and allowing regulation of gene expression during development of plants and animals.
  • Precursor MicroRNA hairpin-like single- stranded RNAs containing stem-arm and stem-loop regions for interacting with intracellular RNasein endoribonucleases to produce one or multiple microRNAs (miRNAs) capable of silencing a targeted gene or genes complementary to the microRNA sequence(s).
  • the stem- arm of a pre-miRNA can form either a perfectly (100%) or a partially (mis-matched) hybrid duplexes, while the stem- loop connects one end of the stem-arm duplex to form a circle or hairpin-loop conformation.
  • precursor of microRNA may also includes pri-miRNA.
  • Small interfering RNA small interfering RNA (siRNA): short double- stranded RNAs sized about 18-27 perfectly base-paired ribonucleotide duplexes and capable of degrading target gene transcripts with almost perfect complementarity.
  • the structure of a vector can be a linear or circular form of single- or double- stranded DNA selected form the group consisting of plasmid, viral vector, transposon, retrotransposon, DNA transgene, jumping gene, and a combination thereof.
  • Promoter a nucleic acid to which a polymerase molecule recognizes, perhaps binds to, and initiates RNA transcription.
  • a promoter can be a known polymerase binding site, an enhancer and the like, any sequence that can initiate synthesis of RNA transcripts by a desired polymerase.
  • Type-II RNA Polymerase (Pol-II or pol-2) Promoter: a RNA promoter that is recognized and used by eukaryotic type-II RNA polymerases (Pol-II or pol-2) which transcribe eukaryotic messenger RNAs (mRNAs) and/or microRNAs (miRNAs).
  • mRNAs eukaryotic messenger RNAs
  • miRNAs microRNAs
  • mammalian EF1 alpha promoter is a pol-2 promoter.
  • ⁇ - ⁇ -like Viral Promoter a viral RNA promoter capable of using the eukaryotic pol- 2 or equivalent transcription machinery for its gene expression.
  • cytomegaloviral (CMV) promoter and retroviral long terminal repeat (LTR) promoter are pol-2-like viral promoters.
  • Cistron a sequence of nucleotides in a DNA molecule coding for an amino acid residue sequence and including upstream and downstream DNA expression control elements.
  • Antibiotic Resistance Gene a gene capable of degrading antibiotics selected from the group consisted of penicillin G, streptomycin, ampicillin (Amp), neomycin, G418, kanamycin, erythromycin, paromycin, phophomycin, spectromycin, tetracycline (Tet), doxycycline (Dox), rifapicin, amphotericin B, gentamycin, chloramphenicol, cephalothin, tylosin, and a combination thereof.
  • Restriction/Cloning Site a DNA motif for restriction enzyme cleavage including but not limited to Aatll, Accl, Aflll/111, Agel, Apal/LI, Asel, Asp718I, BamHI, Bbel, BclI/II, Bglll, Bsml, Bspl20I, BspHI/LUl 11/1201, BsrI/BI/GI, BssHII/SI, BstBI/Ul/XI, CM, Csp6I, Dpnl, Dral/II, Eagl, Ecll36II, EcoRI/RII/47III/RV, Ehel, Fspl, Haelll, Hhal, HinPI, Hindlll, Hinfl, Hpal/II, KasI, Kpnl, Maell/III, Mfel, MM, MscI, Msel, Nael, Narl, Ncol, Ndel, NgoMI,
  • Gene Delivery a genetic engineering method selected from the group consisting of polysomal transfection, liposomal transfection, chemical transfection, electroporation, viral infection, DNA recombination, transposon insertion, jumping gene insertion, microinjection, gene-gun penetration, and a combination thereof.
  • Genetic Engineering a DNA recombination method selected from the group consisting of DNA restriction and ligation, homologous recombination, transgene
  • Cell Cycle Regulator a cellular gene involved in controlling cell division and proliferation rates, consisting but not limited of CDK2, CDK4, CDK6, cyclins, BMI-1, pl4/pl9Arf, pl5Ink4b, pl6Ink4a, pl8Ink4c, p21Cipl/Wafl, and p27Kipl, and a combination thereof.
  • Tumor Suppression a cellular anti-tumor and anti-cancer mechanism consisting but not limited of cell cycle attenuation, G0/G1 -checkpoint arrest, tumor suppression, anti- tumorigenecity, cancer cell apoptosis, and a combination thereof.
  • Targeted Cell a single or a plurality of human cells selected from the group consisting of a somatic cell, a tissue, a stem cell, a germ-line cell, a teratoma cell, a tumor cell, a cancer cell, and a combination thereof.
  • Cancerous Tissue a neoplastic tissue derived from the group consisting of skin cancer, prostate cancer, breast cancer, liver cancer, lung cancer, brain tumor/cancer, lymphoma, leukemia and a combination thereof.
  • Antibody a peptide or protein molecule having a pre- selected conserved domain structure coding for a receptor capable of binding a pre-selected ligand.
  • Prokaryote or Prokaryotic Cell an one-cell organism that lacks a distinct membrane- bound nucleus and has its genetic materials in the form of a continuous strand of DNA, such as bacteria.
  • Eukaryote or Eukaryotic Cell an one-cell or multiple-cell organism whose cells contain a nucleus and other structures (organelles) enclosed within membranes, such as yeast, plant and animal cells.
  • the present invention is a method of protecting human brain neurons from ⁇ -induced neurotoxicity in Alzheimer's diseases with hairpin-like RNA mimics of microRNA precursors (hairpin-like pre-miRNA mimics), comprising: (a) treating at least one neuron with a vector, wherein the vector contains SEQ.ID.NO.2 and is capable of expressing at least one hairpin-like pre-miRNA mimic through a eukaryotic promoter; and (b) inducing an expression of said at least one hairpin-like pre-miRNA mimic in the treated neurons with an administration of at least one transcription inducer.
  • RNA promoter The gene mediated by said eukaryotic RNA promoter is coded for either a non-coding or a protein-coding RNA transcript, or both, selected from the group consisted of microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), messenger RNA (mRNA), their precursors and homologs, and a combination thereof.
  • miRNA microRNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • mRNA messenger RNA
  • mRNA messenger RNA
  • FIGs. 1A and IB show the basic design of a eukaryotic promoter-driven hairpin RNA expression composition (A) and its related RNA processing and translation mechanisms (B).
  • Individual components of the eukaryotic promoter-driven hairpin RNA expression composition ⁇ i.e. the pLenti-EFlalpha-RGFP-miR302 plasmid vector which may carry both EFlalpha and CMV promoters) can be re-located in different places of the vector or even deleted for providing more compact and effective delivery into targeted cells.
  • FIG. 3 shows the results of different bacterial pellets after treated with about 0.1% (v/v) MOPS.
  • the E. coli bacteria were transformed by either pLVX-GFP-miR302+367 (green) or pLenti-EF lalpha-RGFP-miR302 (red) vector, respectively, before the MOPS treatment.
  • FIG. 4 shows the inducibility of different chemical inducers for stimulating EFlalpha and/or CMV promoter-driven gene expression in competent E. coli cells.
  • the top three most potent transcription inducers are MOPS, glycerin and ethanol.
  • the inducer concentrations used can be ranged from about 0.001% to about 10%, most preferably, from about 0.05 to about 4%.
  • FIG. 5 shows the Western blotting results of red RGFP protein expression induced by MOPS, glycerin, and ethanol, respectively.
  • Bacterial RuvB protein is used as a housekeeping standard to compare the levels of induced RGFP expression. Proteins and RNAs extracted from original E. coli cells without any vector transformation serve as negative controls.
  • FIG. 6 shows the Northern blotting results of miR-302 and its pre-miRNA/pri-miRNA cluster expression induced by MOPS, glycerin, and ethanol, respectively. RNAs extracted from original E. coli cells without any vector transformation serve as negative controls.
  • FIG. 7 shows iPS cell (iPSC) generation using miR-302 and pre-miR-302 isolated from bacterial extracts (BE), of which the miR-302/pre-miRNA expression has been confirmed by Northern blot analysis as shown in FIG. 6.
  • miR-302-reprogrammed iPS cells form sphere-like cell colonies and express strong ESC marker Oct4 proteins (labeled by Oct4-promoter-driven green fluorescent protein expression).
  • FIGs. 9 A and 9B show comparison of the healing results between untreated (9 A) and miR-302-treated (9B) wounds in vivo.
  • FIGs. 10A and 10B show the results of HPLC purification and analysis using a synthetic standard uDNA (made by Sigma-Genosys) and freshly extracted miR-302s/pre- miR-302s (or called pro-miR-302s) isolated from pLenti-EF lalpha-RGFP-miR302- transformed E. coli cells.
  • the standard uDNA was designed to mimic a natural pre-miR- 302a as: 5'-CCACCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC UUCCAUGUUU UGGUGAUGG-3 ' (SEQ.ID.N0.3).
  • FIGs. 11A and 11B show the results of microRNA (miRNA) microarray analyses using small RNAs extracted from either blank E. coli competent cells or pLenti-EFlalpha- RGFP-miR302 (tfG P-mz7 «02)-transformed/transfected cells.
  • the extracted small RNAs were further purified by HPLC as shown in the green-labeled area of FIG. 10B.
  • FIG. 11A shows that RNAs from blank E.coli cells present almost no microRNA (green dots mean non- statistically significant whereas red dots indicate positive results). This is because prokaryotes lack several essential enzymes required for microRNA expression and processing, such as Pol-2, Drosha and RNase ⁇ Dicer.
  • FIG. 12 shows the lists of expressed microRNAs extracted from either blank E. coli cells (Group 1 as shown in FIG. 11 A) or pLenti-EFlalpha-RGFP-miR302- transformed/transfected cells (Group 2 as shown in FIG. 11B). Signals less than 500 are not statistically significant (as shown in green in FIGs. 11A and 11B), which may be caused by either low copy number expression or high background.
  • FIGs. 14A-14F show that treatments of miR-302 inhibit ⁇ -induced apoptosis in human SK-N-MC neuronal cells.
  • 14A Transfection of SK-N-MC cells with either the pLVX-Grn-miR302 vector (black bar, to form miR-302-overexpresed cells) or an empty vector (white bar, to serve as control cells), using a lipofectamine 2000 reagent. Positively transfected cells were detected by co-expression of a green fluorescent protein (AcGFP) under an inverted fluorescent microscope.
  • AcGFP green fluorescent protein
  • 14B RT-qPCR analyses of miR-302 expression using total RNA samples extracted from miR-302-transfected (black bar) or control (white bar) cells, respectively.
  • (16D) Cells of (16C) were further stained with JC-1 dye and observed under an inverted fluorescent microscope, showing that ⁇ treatment reduced the intensity of JC-1 green fluorescence in miR-302-transfected cells (n 3, p ⁇ 0.05), while further treatment of LY294002 (20 ⁇ ) prevented this effect.
  • FIGs. 17A-17F show that miR-302 targets PTEN and upregulates Nanog through Akt signaling.
  • (17B and 17C) Cells lysates were obtained from untreated control cells and miR-302-transfected cells, respectively, and further analyzed with western blotting for PTEN and Nanog, showing the downregulation of PTEN and upregulation of Nanog in miR-302- transfected cells (n 3, p ⁇ 0.05).
  • FIGs. 18A-18D show that Comparison of the expression levels of Naong and LARP7 mRNAs in vitro and in vivo after miR-302 treatments.
  • (18A) After 24-hour ⁇ treatment (2.5 ⁇ ), the expression of Nanog mRNA was markedly decreased in control cells in vitro (n 3, p ⁇ 0.05).
  • FIG. 19 shows a proposed scheme for the protective effects of miR-302 against ⁇ - induced neurotoxicity.
  • Upregulation of miR-302 can silence PTEN to activate Akt signaling, which subsequent (i) stimulates Nrf2/HO-l elevation and hence attenuates ⁇ - induced oxidative stress and apoptosis, and (ii) stimulates Nanog expression to suppress p- Ser307 IRS-1 expression, resulting in a significant increase of insulin/IRS-l/Akt signaling, so as to inhibit GSK3P -mediated tau hyperphosphorylation.
  • Table 1 shows data of AD patients and age-matched healthy individuals included in this trial study of miR-302 treatments for AD therapy.
  • the table presents gender, age, MMSE and CASI scores for AD patients and healthy individual controls, respectively.
  • Competent cells of E.coli DH5alpha strain were acquired from the z-competent E. coli transformation kit (Zymo Research, Irvine, CA) and transformed by mixing with about 1-10 ⁇ g of a desired plasmid vector such as pLVX-Grn-miR302 +367 and/or pLenti-EFlalpha- RGFP-miR302 vectors.
  • a desired plasmid vector such as pLVX-Grn-miR302 +367 and/or pLenti-EFlalpha- RGFP-miR302 vectors.
  • Non-transformed bacterial cells were normally grown in Luria- Bertani (LB) broth supplemented with 10 mM MgS0 4 and 0.2 mM glucose at 37°C with frequent agitation at 170 rpm, whereas the transformed bacterial cells were cultivated under the same condition with further addition of 100 ⁇ g/mL ampicillin.
  • hpESCs human epidermal skin cells
  • hpESCs human epidermal skin cells
  • the mixture was added into a 100-mm cell culture dish containing 50%-60% confluency of hpESCs or the cancer/tumor cells, respectively.
  • the medium was replaced by fresh EpiLife medium with HKGS supplements or the conditioned medium suggested by ATCC 12 to 18 hours later. This transfection procedure could be repeated 3 to 4 times every three-four days to increase transfection efficiency.
  • mirPSCs were grown and passaged in knockout DMEM/F-12 medium (Invitrogen, CA) supplemented with 20% knockout serum, 1% MEM nonessential amino acids, 100 ⁇ ⁇ -mercaptoethanol, 1 mM GlutaMax, 1 mM sodium pyruvate, 10 ng/mL bFGF, 10 ng/mL FGF-4, 5 ng/mL LIF, 100 IU/ml penicillin/ 100 ⁇ g/mL streptomycin, 0.1 ⁇ A83-01, and 0.1 ⁇ valproic acid (Stemgent, San Diego, CA), at 37°C under 5% C0 2 .
  • knockout DMEM/F-12 medium Invitrogen, CA
  • MEM nonessential amino acids 100 ⁇ ⁇ -mercaptoethanol
  • 1 mM GlutaMax 1 mM sodium pyruvate
  • 10 ng/mL bFGF 10 ng/mL FGF-4
  • 5 ng/mL LIF 100
  • human neuroblastoma SK-N-MC cells were obtained from the American Type Culture Collection (ATCC, Bethesda, MD, USA). Cells were maintained in Minimal Eagle's medium (MEM, Gibco), supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, and 2 mM L-glutamine at 37 °C, 5% C02.
  • MEM Minimal Eagle's medium
  • a pLVX-Grn-miR-302 vector was applied to transfect the SK-N-MC cells, using a lipofectamine 2000 reagent (Invitrogen) following the manufacturer's instructions, so as to form miR-302-transfected cells.
  • the miR-302-transfected cells were identified by the presence of a co-expressed AcGFP green fluorescent protein.
  • shRNA-Nanog another shRNA gene silencer vector directed against human Nanog mRNAs, called shRNA-Nanog, was obtained from Academia Sinica in Taiwan. In some experiments, we further transfected the shRNA-Nanog vector into the miR-302-transfected cells with the lipofectamine 2000 reagent.
  • Proteins are resolved by SDS-polyacrylamide gel electrophoresis (PAGE), electroblotted onto a nitrocellulose membrane and incubated in Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) for 2 hours at room temperature. Then, a primary antibody is applied to the reagent and incubated the mixture at 4°C.
  • PAGE SDS-polyacrylamide gel electrophoresis
  • Primary antibodies include Oct3/4 (Santa Cruz Biotechnology, Santa Cruz, CA), Sox2 (Santa Cruz), Nanog (Santa Cruz), CDK2 (Santa Cruz), cyclin Dl (Santa Cruz), cyclin D2 (Abeam), BMI-1 (Santa Cruz), keratin 16 (Abeam), ⁇ -actin (Chemicon, Temecula, CA), RuvB (Santa Cruz) and RGFP (Clontech). After overnight, the membrane is rinsed three times with TBS-T and then exposed to goat anti- mouse IgG conjugated secondary antibody to Alexa Fluor 680 reactive dye (1:2,000; Invitrogen-Molecular Probes), for 1 hour at the room temperature. After three additional TBS-T rinses, fluorescent scanning of the immunoblot and image analysis are conducted using Li-Cor Odyssey Infrared Imager and Odyssey Software v.10 (Li-Cor).
  • RNAs (10 ⁇ g) are isolated with a mz ' rVanaTM miRNA isolation kit (Ambion, Austin, TX), fractionated by either 15% TBE-urea polyacrylamide gel or 3.5% low melting point agarose gel electrophoresis, and electroblotted onto a nylon membrane.
  • Detection of miR-302 and/or pre-miR-302 is performed with a [LNA]-DNA probe (5'-[TCACTGAAAC] ATGGAAGCAC TTA-3') (SEQ.ID.NO. l) probe.
  • the probe has been purified by high- performance liquid chromatography (HPLC) and tail-labeled with terminal transferase (20 units) for 20 min in the presence of [ 32 P]-dATP (> 3000 Ci/mM, Amersham International, Arlington Heights,IL).
  • Competent E. coli DH5alpha cells treated with plasmid transformation are cultivated overnight in LB broth supplemented with 10 mM MgS0 4 and 0.2 mM glucose at 37°C with frequent agitation at 170 rpm.
  • 0.5 to 2 ml of MOPS, glycerin, and/or ethanol is added into every 1 litter of LB broth for the above bacterial cultivation and amplification.
  • All amplified plasmid DNAs and expressed mRNAs/microRNAs are isolated together using a HiSpeed plasmid purification kit (Qiagen, Valencia, CA), following the manufacturer's protocol but with a minor modification that RNase A is not added into the PI buffer.
  • the final extracted products containing both plasmids and mRNAs/microRNAs are dissolved in DEPC-treated ddH 2 0 and stored at -80°C before use.
  • RNase A is added into the PI buffer and the extraction procedure is performed following the manufacturer's protocol.
  • RGFP mRNA can be used to identify the transfected cells, while pre-miR-302s are used to reprogram somatic cells to ESC-like iPS cells.
  • the purified pre-miR-302s can also be added into stem cell culture medium to facilitate and maintain the reprogramming process.
  • Embedding, sectioning and immunostaining tissue samples are performed as reported (Lin et al., RNA 2008).
  • Primary antibodies include Oct4 (Santa Cruz), Sox2 (Santa Cruz), Nanog (Santa Cruz), and RGFP (Clontech).
  • Fluorescent dye-labeled goat anti-rabbit or horse anti-mouse antibody is used as the secondary antibody (Invitrogen-Molecular Probes). Positive results are examined and analyzed at lOOx or 200x magnification under a fluorescent 80i microscopic quantitation system with a Metamorph imaging program (Nikon). 8.
  • Genomic DNAs are isolated from about two million cells using a DNA isolation kit (Roche, Indianapolis, IA) and 1 ⁇ g of the isolated DNAs are further treated with bisulfite (CpGenome DNA modification kit, Chemicon, Temecula, CA), according to the manufacturers' suggestions.
  • the treatment with bisulfite converts all unmethylated cytosine to uracil, while methylated cytosine remains as cytosine.
  • bisulfite DNA sequencing analyses we amplify the promoter regions of Oct4 and Nanog with PCR.
  • Primers include 5 ' -GAGGCTGGAG CAGAAGGATT GCTTTGG-3'(SEQ.ID.N0.2) and 5'-CCCTCCTGAC CCATCACCTC CACCACC-3'(SEQ.ID.N0.3) for Oct4, and 5 ' -TGGTTAGGTT GGTTTTAAAT TTTTG-3' (SEQ.ID.NO.4) and 5'-AACCCACCCT TATAAATTCT CAATTA-3'(SEQ.ID.N0.5) for Nanog.
  • the bisulfite-modified DNAs 50 ng
  • the bisulfite-modified DNAs (50 ng) are first mixed with the primers (total 100 pmole) in lx PCR buffer, heated to 94°C for 2 min, and immediately cooled on ice.
  • PCR 25 cycles of PCR are performed as follows: 94°C for 1 min and 70°C for 3 min, using an Expand High Fidelity PCR kit (Roche).
  • the amplified DNA product with a correct size is further fractionized by 3% agarose gel electrophoresis, purified with a gel extraction filter (Qiagen), and then used in DNA sequencing.
  • a detailed profile of the DNA methylation sites is then generated by comparing the unchanged cytosine in the converted DNA sequence to the unconverted one.
  • MMP was investigated using a vital mitochondrial cationic dye JC-1, which accumulates in mitochondria in a potential-dependent matter.
  • JC-1 a vital mitochondrial cationic dye
  • MMP was quantified by fluorescent intensity using Image J software (NIH, Bethesda, MD). Then, the normalized fluorescence intensity levels from control cells were set as 100% for comparing the relative expression levels of the fluorescent intensities in tested groups.
  • AD patients 7
  • age-matched healthy individuals 6
  • Age- matched healthy individuals were recruited by local advertisement at the Aging Research Unit, Chung Shan Medical University, Taichung, Taiwan.
  • PBMCs peripheral blood mononucleated cells
  • RNAs were extracted from patients' PBMCs and cells, respectively, using a Qiagen RNeasy Kit (Qiagen) and further quantified spectrophotometrically.
  • RT-qPCR was carried out using 1 ⁇ g of total RNAs and following the protocols of an ABI High-Capacity cDNA Archive Kit (ABI). Then, we diluted the resulting cDNA into ten folds and used only 5 ⁇ of the diluted cDNA in each of triplicate qPCRs run on a Applied Biosystems 7300 Real Time PCR System with Maxima SYBR Green qPCR Master Mix (2X), ROX solution provided (Thermo), according to the manufacturer's instructions.
  • Levels of relative mRNA or miRNA expression were acquired with the SDS software version 1.2.3 (Sequence Detection Systems 1.2.3-7300 Real Time PCR System, Applied Biosystems) and then further normalized with the level of housekeeping GAPDH expression in the same sample.
  • the normalized mRNA levels from control cells or normal healthy individuals were set as 100% for comparing the relative expression levels of the mRNA expression in tested groups.
  • the measured values of mRNA expression were first normalized with the expression level of housekeeping GAPDH, and then compared to the normalized mRNA levels from control cells or normal healthy individuals, of which the control mRNA levels were set as 100% for comparing the relative expression levels of the mRNA in tested groups.
  • the normalized fluorescence intensity levels from control cells were set as 100% for comparing the relative expression levels of the fluorescent intensities in tested groups.
  • Statistical significance of differences between compared groups was determined by one-way analysis of variance (ANOVA) following Dunnett's post-hoc test for multiple comparisons with a SPSS statistical software (SPSS, Inc., Chicago, IL, USA) as well as the two-tailed Student's t-test.
  • Nrf2 as a master redox switch in turning on the cellular signaling involved in the induction of cytoprotective genes by some chemopreventive phytochemicals. Planta Med. 74, 1526-1539. Kwon SH, Ma SX, Hwang JY, Lee SY, and Jang CG. (2015) Involvement of the Nrf2/HO- 1 signaling pathway in sulfuretin-induced protection against amyloid beta neurotoxicity. Neuroscience 304, 14-28. Alva JA, Lee GE, Escobar EE, and Pyle AD. (2011) Phosphatase and tensin homolog regulates the pluripotent state and lineage fate choice in human embryonic stem cells. Stem Cells 29, 1952-1962. Kuijk EW, van Mil A, Brinkhof B, Penning LC, Colenbrander B, and Roelen BA. (2010) PTEN and TRP53 independently suppress Nanog expression in

Abstract

La présente invention concerne, de manière générale, une composition et une méthode d'utilisation de micro-ARN (miARN) de recombinaison et leurs précurseurs (pré-miARN) à structure en épingle à cheveux en tant que médicaments thérapeutiques pour le traitement de la maladie d'Alzheimer (MA). Plus particulièrement, la présente invention concerne l'utilisation de précurseurs de miR-302 (pré-miR-302) de miARN synthétique pour une thérapie MA chez l'homme. Ces molécules pré-miR-302 peuvent être produites en masse dans des procaryotes comme une forme de vecteurs d'ADN à compétence d'expression d'ADN et/ou d'ARN à structure en épingle à cheveux. Étant donné que les cellules procaryotes ne transcrivent ou ne traitent pas l'ARN à structure en épingle à cheveux, la présente invention porte également sur une méthode permettant d'exprimer le pré-miARN dans des procaryotes, à savoir le pro-miARN, à l'aide d'un nouveau mécanisme de transcription d'ARN à structure en épingle à cheveux récemment découvert dans des procaryotes. De plus, étant donné que le miR-302 constitue un facteur spécifique bien connu de cellules souches embryonnaires (ESC) chez l'homme, les nouveaux résultats de cette invention peuvent en outre être utilisés pour faire avancer la conception et la mise au point d'un nouveau médicament régénérateur pour le traitement de nombreuses autres maladies dégénératives liées au vieillissement, telles que la maladie de Parkinson, l'ostéoporose, le diabète, et le cancer.
PCT/US2016/018774 2015-12-02 2016-02-19 Composition et méthode d'utilisation de précurseurs de mir-302 en tant que médicaments pour le traitement de la maladie d'alzheimer WO2017095458A1 (fr)

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