JP2020037599A - Production and utilization of novel therapeutic anti-cancer agents - Google Patents

Abstract

To provide compositions for reprogramming characteristics of malignant human cancer cells to the benign low-grade or normal somatic tissue cell-like conditions.SOLUTION: The composition comprises at least one micro-ribonucleic acid precursor (pre-miRNA) comprising a sequence of SEQ ID NO:3 at a concentration of 1 mg/mL, where the pre-miRNA is used in contact with a cellular sample containing at least one human cancer cell to induce reprogramming of the human cancer cell to benign low-grade or normal somatic tissue cell-like conditions, where the pre-miRNA comprising the sequence of SEQ ID NO:3 is produced by using RNA transcription driven by an eukaryotic promoter in a prokaryotic cell.SELECTED DRAWING: Figure 15

Description

Priority Claim The present invention claims the priority of a US provisional application filed December 28, 2012, filed with application number 61 / 761,890 and entitled "Development of Universal Anticancer Drugs and Vaccines." The present invention further claims the priority of a U.S. application filed June 2, 2010, filed 12 / 792,413, entitled "Development of Universal Anticancer Drugs and Vaccines." The present invention is further directed to an inducible gene expression composition for use of a type II eukaryotic polymerase promoter-driven transcription effect in prokaryotic cells, with application number 13 / 572,263, filed August 10, 2012. "Applications and their applications". This application is based on U.S. Patent Application 12 / 792,413, entitled `` Development of Universal Anticancer Drugs and Vaccines '' filed on June 2, 2010, and entitled `` Prokaryotic Cells US Patent Application No. 13 / 572,263, which is a continuation-in-part of US Pat. . The entire contents of these applications are incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates generally to product design and related uses of novel DNA / RNA therapeutics and / or vaccines in the treatment of cancer. More specifically, the present invention relates to designs and methods using the novel nucleic acid compositions, wherein the nucleic acid compositions are delivered to human cells, and then processed into a small ribonucleic acid (small RNA) gene silencing effector, and mir- It induces a silencing effect of a specific gene with 302 target cell cycle regulators and / or oncogenes, and further exerts tumor suppressive effects on tumor / cancer cell growth, invasion and metastasis, and / or a cancer prevention effect. Small ribonucleic acid gene silencing effectors include mir-302a, mir-302b, mir-302c, mir-302d, mir-302e, mir-302f, their precursors (pre-microribonucleic acid, pre-miRNAs), and Manually redesigned and / or modified homologues / derivatives such as small hairpin ribonucleic acid / small interfering ribonucleic acid (shRNA / siRNA), and tumor suppressor microribonucleic acid (TS) such as combinations thereof -miRNA). Such human cells of interest include normal somatic cells or tumor / cancer cells ex vivo and / or in vivo.

Background of the Invention Stem cells, like treasure chests, contain multiple types of stem cells used to induce stem cell growth / renewal, restore and / or regenerate damaged / aged tissue, or treat degenerative diseases or prevent tumor / carcinogenesis. Contains active ingredients. Therefore, the present inventor can understand that these stem cells can be used as a means for identifying or producing a new drug. From this, the new drugs obtained can be applied to pharmaceutical and / or therapeutic applications, such as biopharmaceutical applications, diagnostic devices and / or equipment, stem cell generation, stem cell research and / or treatment, tissue / organ recovery And / or regeneration, wound healing treatment, tumor suppression, treatment and / or prevention of cancer, disease treatment, drug production, and combinations thereof.

  Microribonucleic acid mir-302 is the most major microribonucleic acid (microRNA, miRNA) found in human embryonic stem cells (hES cells) and induced pluripotent stem (iPS cells). But many of its features are not yet clear. Previous studies have shown that ectopic overexpression of mir-302, i.e., higher than levels in human stem cells (hES cells), results in slower cell cycle rates (20-24 hours / cycle) and resting cell-like It has been shown that normal hES-like pluripotent stem cells having the cell morphology can reprogram human normal cells and cancer cells (Lin et al., 2008 and 2011 (Non-Patent Documents 1 and 2); US 12 / 792,413, EP2198025 (Patent Document 1)). Relative quiescence is one of the distinctive features of pluripotent stem cells (mirPS) generated by reprogramming with these mir-302s, while human stem cells (hES) and 3/4 IPS cells induced by a factor (i.e., Oct4-Sox2-Klf4-c-Myc or Oct4-Sox2-Nanog-Lin28) have a high proliferative capacity (12-15 hours / cycle) and a relentless tumorigenic tendency (Takahashi et al., 2006 (Non-patent document 3); Yu et al., 2007 (Non-patent document 4); Wernig et al., 2007 (Non-patent document 5)). Although most of the mechanisms supporting the anti-proliferative properties of mirPS cells have not been elucidated yet, they have discovered that two G1-checkpoint regulators, which are targets of mir-302, may be involved. These are cyclin-dependent kinase 2, CDK2, and cyclin D (Lin et al., 2010 (Non-Patent Document 6); US 12 / 792,413). Eukaryotic cell cycle progression is driven by the activation of cyclin-dependent kinases (CDKs), which are regulated by cyclin, a positive regulatory subunit, and negative regulatory subunits. It forms a functional complex with cyclin-dependent kinase inhibitors (CDK inhibitors, CKIs such as p14 / p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1 / Waf1, and p27Kip1), which are negative regulators. In mammalian cells, different cyclin-cyclin-dependent kinase complexes (cyclin-CDK complexes) are involved in regulating transitions at different times in the cell cycle, such as cyclin-D-CDK4 / 6, which is involved in G1 phase progression. , Cyclin-E-CDK2 involved in G1-S transition, cyclin-A-CDK2 involved in S-phase progression, and cyclinA / B-CDC2 (cyclin-A / B-CDK1) involved in M-phase entry is there. Thus, it can be understood that the antiproliferative function of mir-302 is a result of co-suppression of CDK2 and cyclin D during the G1-S phase transition period.

  However, studies on the mouse mir-291 / 294/295 family, analogs of human mir-302, have shown completely different functions from mir-302 in human cells. In mouse embryonic stem cells (mES), ectopic expression of mir-291 / 294/295 is expressed by p21Cip1 (or CDKN1A) and serine / threonine protein kinase Lats2 (Wang et al., 2008 (Non-Patent Document 7)) Directly promoted rapid cell growth and caused a high degree of tumorigenesis of the transfected cells. It is known that transgenic mice lacking the p21Cip1 / Waf1 gene showed normal growth when lacking control by the G1 checkpoint (Deng et al., 1995 (Non-Patent Document 8)). However, since Lats2 has functions such as recruiting λ-tubulin at the onset of mitosis and participating in spindle formation, its role has not been elucidated yet. The lack of Lats2 in mouse embryos also led to severe mitotic defects and was found to be fatal, indicating that silencing of Lats2 inhibits rather than promotes cell division. (Yabuta et al., 2007 (Non-Patent Document 9)). Thus, p21Cip1 silencing appears to be the major mechanism behind mir-291 / 294 / 295-induced tumorigenesis. However, when we screened the target site of mir-302 of the human p21Cip1 gene, all results were negative. Similar negative results indicate the two most famous online microribonucleic acid-target prediction programs, "TARGETSCAN" (http://www.targetscan.org/) and "PICTAR-VERT" (http: // pictar. mdc-berlin.de/). Thus, even though mir-302 and mir-291 / 294/295 are homologous analogs, their function in controlling tumorigenesis is substantially opposite. This indicated that homologous microribonucleic acids may not have the same function. The above findings also indicate that studies where mimics, such as small interfering ribonucleic acids, now replace natural microribonucleic acids, may not yield the same results as natural microribonucleic acids.

  The gene sequence encoding mir-302 is located at locus 4q25 on human chromosome 4, and is a conservative region that is often involved in longevity. Furthermore, mir-302 is encoded by the intron region of the La ribonucleoprotein domain family member 7 (LARP7) gene, and is expressed by the intron microribonucleic acid biosynthetic pathway (Barroso -delJesus, 2008 (Non Patent Literature 10); Ying and Lin, 2004 (Non Patent Literature 11); FIG. 13). Natural mir-302 contains four homologous sense family members (mir-302b, c, a, and d) and three distinct antisense members (mir-302b *, c *, and a *). Is transcribed, together with another miRNA, mir-367, into a polycistronic RNA cluster (Suh et al., 2004 (Non-Patent Document 12)). In US 12 / 792,413, our previous invention, the inventor has noted that mir-302 is not a miRNA, such as mir-92, mir-93, mir-367, mir-371, mir-372, mir-373, It was found that the expression of all mir-374 and mir-520 family members could be stimulated. Analysis using the online programs `` TARGETSCAN '' and `` PICTAR-VERT '' on the website `` Sanger miRBase :: Sequences '' (http://microrna.sanger.ac.uk/), furthermore, mir-302 and It was shown that miRNAs whose expression is stimulated share more than 400 target genes. This demonstrated that miRNAs whose expression is stimulated and mir-302 may play similar roles. These shared target genes are members of RAB / RAS-related oncogenes, ECT-related oncogenes, pleomorphic adenoma genes, E2F transcription factor, cyclin D-binding Myb-like transcription factor, HMG Box transcription factor, Sp3 transcription factor, transcription factor CP2-like protein, NFkB activating protein gene, cyclin-dependent kinase, MAPK / JNK-related kinase, SNF-related kinase, myosin light chain kinase, TNF-α-induction Protein gene, DAZ-related protein gene, LIM-related homeobox gene, DEAD / H box protein gene, forkhead box protein gene, BMP regulator, Rho / Rac guanine nucleotide exchange factor, IGF receptor (IGFR), endothelin Receptor, left / right determinant (Lefty), cyclin, p53-inducible nucleoprotein gene, RB-like 1, RB-binding protein gene, Max-binding protein Clast gene, c-MIR immune-recognizing cell regulator, Bcl-like apoptosis-promoting factor, cadherin, integrin s4 / s8, inhibin, ankyrin, SENP1, NUFIP2, FGF9 / 19, SMAD2, CXCR4, EIF2C, PCAF, MECP2, histone acetyl Including but not limited to transferase MYST3, nuclear RNP H3, and many nuclear receptors and factors. Most of these target genes are highly involved in embryonic development and cancer / tumor formation. In short, miR-302 further promotes its homologous miRNAs, such as miR-92, miR-93, miR-367, miR-371, miR-372, miR-373, miR-374 and miR-520 to function. Can be improved and / or maintained.

  MiRNA is a gene inhibitor in the cytoplasm, and its normal function is to bind to a specific target site with a high degree of complementarity and suppress the translation of transcripts (mRNAs) of that target gene And forms an RNA-induced silencing complex (RISC) to inhibit or degrade mRNAs. Therefore, the original function of the miRNA is determined by the rigor of the binding between the miRNA and the target gene. In contrast, our previous studies (Lin et al., 2010; US 12 / 792,413) provided an important interpretation of the mechanisms supporting mir-302 regulated tumor suppression. In human cells, multiple cell cycle regulators targeted by mir-302 silence, including the CDK2, cyclins D1 / D2, and BMI-1 genes, but, interestingly, do not include p21Cip1. As mentioned above, co-silencing of CDK2 and cyclin D in the cell cycle inhibits the transition from G1 to S phase and results in a relatively slow cell growth rate. In addition, silencing of BMI-1 stimulates the expression of two major tumor suppressors, p16Ink4a and p14ARF, which further reduces cell proliferation and suppresses tumor formation. In human cells, since the expression of p16Ink4a / p14ARF is increased and p21Cip1 is not affected, mir-302 is a pathway that co-suppresses cyclin-E-CDK2 and cyclin-D-CDK4 / 6, except for p16Ink4a-Rb and / or Alternatively, they may exert anti-tumorigenic functions through the p14 / 19ARF-p53 pathway.

  Based on the anti-tumorigenic properties of mir-302 described above, we can use mir-302 to treat human tumors and cancer. However, there may be four major problems with this approach: (1) As a gene silencing effector, mir-302 needs to function as its precursor. There is currently no effective ribonucleic acid synthesis technique to produce hairpin-like RNAs that are 73-75 nucleotides in length. To ensure the accuracy of the sequence, the maximum length of the synthesized RNA is about 45-55 nucleotides, but it is not enough to form the mir-302 precursor (pre-mir-302) . (2) We require mir-302 expression at concentrations higher than mir-302 levels in hES cells, thereby inducing its antitumor / cancer effect (Lin et al., 2010 (Non-Patent Document 6). )). However, there is currently no way to produce so many pre-mir-302s for drug production. Pre-mir-302 isolation from hES or iPS cells is expensive, of which bacterial-competent cells cannot produce pre-mir-302 with a high degree of secondary structure. (3) Synthetic mimics such as siRNA have never been successful in animal studies. Due to the short life cycle (3-5 days) of mimetics such as siRNA and their high toxicity, siRNA cannot be used in place of native mir-302 in in vivo conditions. In addition, there is a report that the use of a mimetic such as siRNA causes supersaturation of the microRNA pathway in cells, and also causes toxicity (Grimm et al., 2006 (Non-Patent Document 13)). (4) The full function of mir-302 depends on the mir-302 sense in its precursor and its mir-302 * antisense strand (eg, mir-302a *, mir-302b * and mir-302c *). Therefore, an economical and effective method for producing and / or isolating pre-mir-302 is a requirement from the development of anti-tumor / cancer therapy and its related drugs regulated by mir-302.

In short, there is a need for an effective and safe method of making, isolating and using mir-302 and its precursors and their related homologs / derivatives.
SUMMARY OF THE INVENTION The present invention is a design and method for using microRNA precursors (pre-miRNAs) as therapeutic agents and / or vaccines for treating cancer. Specifically, the present invention relates to a design and a method using the recombinant nucleic acid composition, wherein the recombinant nucleic acid composition is delivered to human cells, and then the small ribonucleic acid gene silencing effector (small RNA-based gene silencing) is used. effector) to cause a specific gene silencing effect on cell cycle regulators and / or oncogenes targeted by mir-302, resulting in effects such as tumor suppression and / or prevention of cancer, and thereby tumor / cancer It can be used to suppress cell growth, proliferation, invasion and metastasis. These small ribonucleic acid gene silencing effectors are tumor suppressor microribonucleic acids (TS-miRNA), such as mir-302a, mir-302b, mir-302c, mir-302d, mir-302e, mir-302f, their precursors. It is preferred to include bodies (pre-miRNAs) and their manually redesigned small hairpin ribonucleic acid (shRNA) -like homologs / derivatives, and combinations thereof. The design of small hairpin ribonucleic acid homologues / derivatives involves mismatches and perfect matches within individual single units or complex clusters in shRNA and small interfering ribonucleic acid (small interfering RNA, siRNA) structures homologous thereto. matched) nucleic acid composition. They can promote target specificity and reduce the amount of mir-302 required for delivery and treatment (copy number). The human cells include normal cells and / or tumor / cancer cells in vitro, ex vivo and / or in vivo.

  Natural microribonucleic acid (miRNA) is about 18-27 nucleotides (nucleotides, nt) in length, and its target messenger ribonucleic acid is complemented by microribonucleic acid (miRNA) and its target. (messemger RNA, mRNA) can be directly degraded, or the translation of the messenger ribonucleic acid (mRNA) as its target can be suppressed. The Mir-302 family (mir-302s) are microribonucleic acids (miRNAs) that have a high degree of homology and are conserved by many mammals. The Mir-302 family (mir-302s) contains four major family members, which are in 5 'to 3' order, mir-302b, mir-302c, mir-302a, mir-302d, and mir-302. It is transcribed into a non-coding RNA cluster containing 367 (Suh et al., 2004 (Non-Patent Document 12)). Recently, scientists have discovered the fifth and sixth mir-302 members, mir-302e and mir-302f, in addition to the original family cluster. Although mir-367 and mir-302s are co-expressed in natural contexts, their functions are quite different. In the present invention, based on the anti-tumorigenic function, we tend to express only mir-302s. In certain embodiments, we further provide 5′-GCTAAGCCAG GC-3 ′ (SEQ.ID.NO.1) and 5′-GCCTGGCTTA GC-3 ′ (SEQ.ID. Manually redesigned hairpin loops (NO.2) can be used to replace the original mir-302 precursor (pre-mir-302) hairpin loops. Normally, mir-302 is abundantly expressed only in non-mouse human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells, but not in other differentiated somatic cells (Tang Et al., 2007 (Non-Patent Document 14); Suh et al., 2004 (Non-Patent Document 12)). Since mir-302s are homologous small inhibitory RNAs that can silence target genes that are highly complementary to them, mir-302s is an errant and inactive form of oncogene activation in hES and iPS cells. May be related to the prevention of premature. Therefore, it is also used in the development of antitumor / cancer treatment drugs. In fact, hES cells before the morula stage (32-64 cell stage) usually have a relatively slow cell cycle rate, like the pluripotent stem (mirPS) cells induced by mir-302. (Lin et al., 2010 (Non-Patent Document 6)). This suggested that mir-302 plays a dual role in controlling normal stem cells and preventing tumor / cancer cell formation.

  All mir-302 members share a perfect match at the first 17 nucleotides of the 5 'terminal sequence, of which the seed motif is 5'-UAAGUGCUUC CAUGUUU-3' (SEQ.ID NO.3), and also has a sequence homology of 85% or more in a mature microribonucleic acid (miRNA) also having 23 nucleotides. Based on the current forecast results from the online calculation program "TARGETSCAN" (http://www.targetscan.org/) and "PICTAR-VERT" (http://pictar.mdc-berlin.de/), these members At the same time, they target nearly the same cellular genes, of which more than 607 human genes are included. Further, mir-302 may be somewhat similar in function to mir-92, mir-93, mir-200c, mir-367, mir-371, mir-372, mir-373, mir-374 and mir-302. The 520 family members also share many target genes. Many of these target genes are developmental signals and transcription factors involved in the initiation and / or establishment of lineage-specific cell differentiation during early embryonic development (Lin et al. , 2008 (Non-Patent Document 1)). And many of the target genes are also known oncogenes. Since many of these target developmental signals and differentiation-related transcription factors are oncogenes, mir-302 also has a tumor suppressor function, and normal hES cell growth progresses to tumor / cancer cell formation. Can be prevented.

  In one preferred embodiment, the present invention is a method of making and delivering a non-vector-based pre-mir-302 for use in treating human tumors / cancers. As mentioned above, the length of pre-mir-302 is about 73-75 nucleotides, but due to the limitation of 45-55 nucleotides, we cannot synthesize by conventional RNA synthesis techniques. Therefore, we need to adopt new methods to generate and increase the amount of pre-mir-302 to provide for drug production. Therefore, in the present invention, a mimetic of pre-mir-302 is prepared using a technique for producing a miRNA precursor (product is pro-miRNA) by a prokaryote. The technology is a derivative of US 13 / 572,263, which is a priority application. Pro-miRNA is a new hairpin-like pre-miRNA mimic produced from prokaryotic competent cells, such as the E. coli DH5α cell line. As is known to those skilled in the art, the transcription mechanisms of prokaryotes and eukaryotes are very different and incompatible with each other, so we need to modify the prokaryotic transcription system to be able to transcribe eukaryotic genes and pre-miRNAs. is there. The invention of US 13 / 572,263, which is the priority application of the present application, provides that a prokaryotic cell uses eukaryotic pol-2 and pol-2-like (i.e. It has already been disclosed that such hairpin-like RNAs (product pro-mir-302) can be transcribed. This transcription inducer not only makes the prokaryotic transcription system compatible with eukaryotic gene expression, but also stabilizes the secondary structure of the eukaryotic gene transcript in prokaryotic cells. The advantages of Pro-miRNA technology are, firstly, that due to the rapid and easy growth of bacterial competent cells, the production of a particular pre-miRNA or some (required) pre-miRNA meets the cost-benefit principle. Secondly, industrial scale production on the order of several grams to kilograms can be expected; and thirdly, since there is no need to culture and fuse tumors or mammalian cells, operation is simple and expensive. Absent; fourth, chemical reagent-induced transcription of the pol-2-like promoter ensures high sequence accuracy; and finally, lack of expression of Dicer and other miRNAs in prokaryotic cells Therefore, a pre-miRNA product with high purity can be obtained. The resulting pro-miRNA can be easily isolated from the bacterial extract and / or lysate (Example 20) and further purified (Example 21), and the extract and lysate Used in the development of pharmaceutical and therapeutic applications.

  We have already produced a number of pre-mir-302-like pro-miRNAs, pro-mir-302 (Examples 18-21) and used it to promote wound healing (Example 22). For therapeutic use, the isolated pro-mir-302 was prepared from a previously prepared ointment containing cocoa butter, cottonseed oil, olive oil, sodium pyruvate and white petrolatum. The concentration of pro-mir-302 in the ointment is 10 μg / mL. The skin was then cut open with a dissecting knife to form an open wound. Each ointment with or without pro-mir-302 was applied directly to the wound and covered the entire wound. This area was further sealed with a liquid bandage. As shown in FIG. 14, within two weeks, the rate of healing of the wound treated with pro-mir-302 was significantly improved, with results more than twice as fast as all of the other control groups. In the area healed by pro-mir-302, normal hair regeneration appears, and no scar remains (for example, the location indicated by the black arrow). In contrast, the other treatment groups leave a small scar and no hair growth. As is clear from these results, pro-mir-302 has a function of stimulating normal tissue recovery and regeneration.

  We also purified the pro-mir-302 and prepared it into a liquid drug and injected it to test the treatment of liver cancer / malignant liver tumor in vivo (Example 23). As shown in FIG. 15, after three injections, the drug containing pro-mir-302 reduced the volume of liver cancer cells / malignant liver tumors implanted in the body to close to 90%, i.e. compared to the untreated group. This reduced the average tumor size to 10% of the untreated group. In addition, the results of histological examination by H & E staining showed that the treatment results for this cancerous / malignant tumor were not recognized not only because of the antitumor forming properties of mir-302 (Lin et al., 2010). It was further shown that this was due to the newly discovered reprogramming feature. For example, as shown in FIG. 16, it was clearly shown that mir-302 can reprogram highly malignant human liver cancer transplants in the body to a relatively benign state that approximates normal liver tissue. The treated cancer cell transplant can further form normal liver structures such as typical hepatic lobules, central vein (CV) and portal triad (PT). Therefore, mir-302 not only can suppress the growth of tumor / cancer, but also contributes to the recovery and / or regeneration of normal tissues in the body environment, and achieves an advantageous effect for the treatment of cancer.

  In another preferred embodiment, the present invention further provides a vector expressing mir-302 and treating tumor / cancer. The inventors have already designed and developed an inducible pTet-On-tTS-miR302s expression vector (FIG.1A), as disclosed in the priority application of the present application US 12 / 792,413, and further developed viral infection, Mir-302 is delivered into normal and / or human cancer cells by electroporation or liposome / polysomal transfection. The redesigned mir-302 structure contains four small non-coding RNA members, mir-302a, mir-302b, mir-302c, and mir-302d in the same cluster (mir-302 302s; FIG. 1B). Upon stimulation with Doxycycline (Dox), this mir-302s structure is driven and expressed by the cytomegalovirus (CMV) promoter, which is controlled by a tetracycline-responsive element (TRE). Following infection / transfection, expression of mir-302 occurs along the natural microribonucleic acid (miRNA) biosynthetic pathway. Among them, the mir-302s structure is co-transcribed with a reporter gene such as a red-shifted fluorescent protein (RGFP), and further spliceosomal components and / or in the cytoplasm. It is processed into a single mir-302 member by Dicers (FIG. 2A), which is an RNA endoribonuclease (RNase III) (Lin et al., 2003). The result of this measure was that in microribonucleic acid microarray analysis (Example 3), all of the sense (sense) mir-302 members were effectively expressed in transfected cells after stimulation with doxycycline (FIG.1C). . The steps for transducing human cells with the mir-302 expression vector are illustrated in FIG. 2B.

  The present inventors designed an intron microribonucleic acid expression system, that is, SpRNAi-RGFP, to transcribe the recombinant RGFP gene by mimicking the biosynthetic pathway of natural intronic microribonucleic acid (intronic miRNA) (FIG.2A). did. SpRNAi-RGFP contains an artificial / artificial splicable intron (splicing competent intron, SpRNAi), which is an intron microribonucleic acid (intronic miRNA) and / or small hairpin ribonucleic acid-like (shRNA-like) gene silencing Effectors can be generated (Lin et al., 2003 (Non-Patent Document 15); Lin et al., (2006) Methods Mol Biol. 342: 295-312). SpRNAi and RGFP are co-transcribed by type II RNA polymerase (Pol-II RNA polymerase) and are included in the messenger ribonucleic acid precursor / premessenger ribonucleic acid (pre-mRNA) of the SpRNAi-RGFP gene, and RGFP is RNA It is excised by splicing components (RNA splicing components). Then, the spliced SpRNAi is further processed into mature gene silencing effectors such as natural microribonucleic acids (native miRNA) and artificial small hairpin ribonucleic acids (shRNAs), so that specific ribonucleic acid interference effects on target genes ( Induces RNA interference, RNAi). At the same time, after intron splicing (intron splicing), the exons of the SpRNAi-RGFP gene transcript (transcript) are linked to form a mature messenger ribonucleic acid (mRNA), followed by miRNA / shRNA expression. Translated into an RGFP reporter protein used to identify Quantitatively, a one-fold concentration of RGFP corresponds to a four-fold concentration of mir-302 expression. Further, a functional protein exon, for example, a hES gene marker in somatic cell reprogramming (SCR) may be selectively used in place of RGFP to provide an additional gene function. Currently, more than 1000 types of natural microribonucleic acids of unknown function have been discovered in vertebrates, and more and more new microribonucleic acids have been identified one after another, so our intron microribonucleic acid expression system has been developed in vitro and in vivo. Can be an advantageous tool for measuring the above-mentioned microribonucleic acid function.

  SpRNAi intron has a 5 'splice site (5'-splice site), a branch point sequence (Branch-Point, BrP), a polypyrimidine tract (poly-pyrimidine tract), and a 3' splice site (3'-splice site). Including some common nucleotide components. It also includes a hairpin microribonucleic acid (miRNA) or a small hairpin ribonucleic acid (shRNA) precursor inserted between the 5 'splice site and the branch point sequence. This part of the intron usually forms a lariat structure during RNA splicing and processing. The 3 'end of SpRNAi contains various translational stop codon regions (T codon) in order to increase the accuracy of intronic RNA splicing and processing. This T codon, when expressed on messenger ribonucleic acid (mRNA) in the cytoplasm, generates an activation signal for the nonsense-mediated decay (NMD) system in the cell, and prevents cellular toxicity. Degrades any unstructured ribonucleic acid accumulated within. However, the highly structured small hairpin ribonucleic acid (shRNA) and microribonucleic acid precursor (pre-miRNA) are retained and further cleaved by Dicer, resulting in mature small interfering ribonucleic acid (siRNA) and microRNA, respectively. Form ribonucleic acid (miRNA). We manually incorporated SpRNAi into the DraII restriction site of the RGFP gene to express intronic miRNA / shRNA (Lin et al., 2006 and 2008 (Non-Patent Documents 16 and 1)) and used the recombinant SpRNAi-RGFP gene. Was formed. Cleavage of RGFP by the DraII restriction enzyme creates an AG-GN nucleotide cleavage site with three depressed nucleotides at each end, forming 5 ′ and 3 ′ splice sites after SpRNAi insertion, respectively. This intron insertion destroys the integrity of the RGFP protein, but this can be restored by intron splicing, so we use the red, red-shifted fluorescent protein (RGFP) that appears in transfected cells. The expression of mature miRNA / shRNA can be measured. The RGFP gene also has multiple exonic splicing enhancers (ESEs) to increase the accuracy and efficiency of RNA splicing.

  In particular, the SpRNAi intron has a 5'-GTAAGAGK-3 '(SEQ.ID.NO.4) or GU (A / G) AGU (SEQ.ID.NO.35) motif (i.e., 5'-GTAAGAGGAT-3 '(SEQ.ID.NO.36), 5'-GTAAGAGT-3' (SEQ.ID.NO.37), 5'-GTAGAGT-3 '(SEQ.ID.NO.38) and 5'-GTAAGT- 3 ′ (SEQ.ID.NO.39)), and the 3 ′ end of the SpRNAi intron is GWKSCYRCAG (SEQ.ID.NO.5). ) Or CT (A / G) A (C / T) NG (SEQ.ID.NO.40) motif (i.e., 5'-GATATCCTGCAG-3 '(SEQ.ID.NO.41), 5'-GGCTGCAG- It is a 3'-splicing site that is homologous to 3 '(SEQ. ID. NO. 42) and 5'-CCACAG-3' (SEQ. ID. NO. 43). In addition, the branch point sequence (branch point sequence) is located between the 5 'end and the 3' end splice site, 5'-TACTAAC-3 '(SEQ.ID.NO.44) and 5'-TACTTAT-3 Highly homologous to the 5'-TACTWAY-3 '(SEQ.ID.NO.6) motif, such as' (SEQ.ID.NO.45). The adenosine branch point sequence (adenosine) `` A '' nucleotides, in almost all spliceosomal introns (spliceosomal introns) 2'-5 'oligoadenylate synthase ((2'-5')-oligoadenylate Synthetases and spliceosomes can form part of a (2'-5 ')-linked lasso intron RNA. The poly-pyrimidine tract is located between the branch point sequence and the 3 ′ splice site, and has a high T (thymidylic acid) or high C (cytidylic acid) content. , 5 '-(TY) m (C /-) (T) nS (C /-)-3' (SEQ.ID.NO.7) or 5 '-(TC) nNCTAG (G /-)-3' (SEQ.ID.NO.8) is homologous to any of the motifs. Among them, the symbols “m” and “n” indicate a plurality of overlaps, ≧ 1, and most preferably, m is 1-3 and n is 7-12. The sign "-" indicates that there are nucleotides within the sequence that may be skipped. The sequence also includes several linker nucleotide sequences to link all of the synthesized intron components. Based on the guidelines for codes and formats indicating nucleotide and / or amino acid sequence data in 37 CFR 1.822, the code W is adenine (A) or thymine (T) / uracil (U), and the code K is guanine. (G) or thymine (T) / uracil (U), symbol S is cytosine (C) or guanine (G), symbol Y is cytosine (C) or thymine (T) / uracil (U), symbol R is adenine ( A) or guanine (G), and the symbol N indicates adenine (A), cytosine (C), guanine (G), or thymine (T) / uracil (U).

  In another possible embodiment, the present invention relates to a direct (exon-type) mir-302 miRNA / shRNA expression system, which does not undergo intracellular RNA splicing and / or NMD machinery, and Silencing effectors can be generated directly. However, a drawback of this method is that the expression of the mir-302-like gene silencing effector is not controlled by any intracellular surveillance system, such as nonsense-mediated decay (NMD), so that natural microribonucleic acid production is not possible. Cytotoxicity can be generated by supersaturation of the synthetic pathway (Grimm et al., 2006). The expression system used in this method includes a plasmid (plasmid), a viral vector (viral vector), a lentiviral vector (lentiviral vector), a transposon (transposon), a retrotransposon (retrotransposon), a jumping gene, and a protein encoding gene. It may be a linear or circular nucleic acid composition selected from (protein-coding gene), non-coding gene, artificially recombinant transgene, and combinations thereof. Mir-302-like gene silencing effectors, including microribonucleic acids, small hairpin ribonucleic acids, small interfering ribonucleic acids, and their precursors and homologues / derivatives, are capable of treating tissue-specific or non-specific ribonucleic acid (RNA) promoters. Expressed by control. RNA promoters are type II RNA polymerase (type-II RNA polymerase, Pol-II), viral polymerase (viral polymerase), type III RNA polymerase (type-III RNA polymerase, Pol-III), type I RNA polymerase (type-I RNA polymerase, Pol-I), and an RNA polymerase (TRE) promoter controlled by a tetracycline response element. The viral promoter is a Pol-II-like RNA promoter, and includes cytomegalovirus (cytomegalovirus, CMV), retrovirus long-terminal region (LTR), hepatitis B virus (HBV), adenovirus ( adenovirus, AMV) and adeno-associated virus (AAV), but are not limited thereto. As an example, the lentiviral LTR promoter can produce as many as 500,000 pre-messenger ribonucleic acid transcripts (copies) in a single cell. It is also possible to control the transcription rate of the gene silencing effector by inserting a drug-sensitive repressor (tTS) in front of the RNA polymerase promoter. Inhibitors are chemicals or G418, neomycin (neomycin), tetracycline (tetracycline), doxycycline (doxycycline), ampicillin (ampicillin), kanamycin (kanamycin), puromycin (puromycin), and antibiotics selected from such derivatives and the like Is suppressed by

  On the other hand, a plurality of transgenes and / or vectors expressing several intron gene silencing effectors may be used to achieve a gene silencing effect on the mir-302 target gene. Also, multiple gene silencing effectors can be generated by one intronic insert. For example, in ectopic expression of an anti-EGFP (anti-EGFP) intron containing microribonucleic acid precursors (pre-miRNA) in zebrafish, two different sized miRNAs, i.e., miR-EGFP (282/300) and miR-EGFP (280-302). This indicates that one SpRNAi insert can generate multiple gene silencing effectors (Lin et al. (2005) Gene 356: 32-38). In some instances, the intron gene silencing effector hybridizes with a target gene transcript (ie, messenger ribonucleic acid, mRNA) to form double-stranded small interfering ribonucleic acids (siRNAs), thereby producing a secondary ribonucleic acid interference effect. (RNAi). Since these gene silencing effectors are constantly produced by the transgene vector, the concern of rapid in vivo degradation of RNA is reduced. The advantage of this measure is that transfection or viral infection with a vector transgene can provide a reliable and long-term gene silencing effect.

Because the stem-loop structure of some natural pre-microribonucleic acids (pre-miRNAs) is excessively large and / or complicated and incompatible with microribonucleic acid expression systems / vectors, the present inventors have proposed that natural pre-microribonucleic acids In many cases, a manually redesigned methionyl-carrying ribonucleic acid loop (tRNA ^met loop, ie, 5 ′-(A / U) UCCAAGGGGG-3 ′ (SEQ. ID. NO. 46)) is used in place of the loop. The tRNA ^met loop transports manually redesigned microribonucleic acids (miRNAs) from the cell nucleus to the cytoplasm via Ran-GTP and Exportin-5 by a transport mechanism similar to natural microribonucleic acids Can be effectively executed (Lin et al., 2005 (Non-Patent Document 17)). An advantage of the present invention is that an artificially improved pair of preforms comprising 5'-GCTAAGCCAG GC-3 '(SEQ.ID.NO.1) and 5'-GCCTGGCTTA GC-3' (SEQ.ID.NO.2) The use of a microribonucleic acid loop improves the efficiency of manually redesigned microribonucleic acid (pre-miRNA) to the same nucleus export as natural pre-microribonucleic acid, and interferes with tRNA export. Can not. This improvement promotes the formation of the duplex of mir-302a-mir-302a * and the duplex of mir-302c-mir-302c *, and can improve the function and stability of the entire mir-302s. The design of these new pre-microribonucleic acid loops was completed by modifying the tRNA ^met loop and the short stem loop of mir-302b / mir-302a, including the short stem of mir-302b / mir-302a. The loop is highly expressed in human ES cells, but not in other differentiated tissue cells. Thus, the use of these recombinant / artificial / artificial hairpin loops within the mir-302 structure does not interfere with the natural miRNA pathway in the human body, thus reducing cytotoxicity and improving safety.

  The Mir-302 pre-microribonucleic acid family cluster is formed by hybridization and ligation / ligation of the synthesized mir-302 homologs, from the 5 'end to the 3' end, mir-302a, mir-302b, mir -302c and four parts of mir-302d pre-microribonucleic acids (pre-miRNAs, FIG. 1B). All these manually redesigned mir-302 miRNA / shRNA molecules have the same 17 nucleotides at the beginning of their 5 ′ terminal sequence [e.g., 5′-UAAGUGCUUC CAUGUUU-3 ′ (SEQ.ID.NO.3). )]. As a synthetic oligonucleotide used for the pre-microribonucleic acid cluster of Mir-302, mir-302a-sense: 5′-GTCACGCGTT CCCACCACTT AAACGTGGAT GTACTTGCTT TGAAACTAAA GAAGTAAGTG CTTCCATGTT TTGGTGATGG ATAGATCTCT C-3 ′ (SEQ.ID.NO.9), mir-302a-antisense: 5'-GAGAGATCTA TCCATCACCA AAACATGGAA GCACTTACTT CTTTAGTTTC AAAGCAAGTA CATCCACGTT TAAGTGGTGG GAACGCGTGA C-3 '(SEQ.ID.NO.10); GAGTCGCTCA TATGA-3 '(SEQ.ID.NO.11), mir-302b-antisense: 5'-TCATATGAGC GACTCCTACT AAAACATGGA AGCACTTACT TTCAAAGTCA CAGAAAGCAC TTCCATGTTA AAGTTGAAGG GAGCGAGAGA TCTAT-3' (SEQ.ID.NO.12), mir-302 302c-sense: 5'-CCATATGGCT ACCTTTGCTT TAACATGGAG GTACCTGCTG TGTGAAACAG AAGTAAGTGC TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3 '(SEQ.ID.NO.13); ' (SEQ.ID.NO.14), mir-302d-sense: 5'-CGTCTAGACA TAACACTCAA ACATGGAAGC ACTTAGCTAA GCCAGGCTAA GTGCTTCCAT GTTTGAGTGT TCGCGATCGC AT-3 '(SEQ.ID.NO.15), and mir-302d-antisense: 5 '-ATGCGATCGC GAACACTCAA ACATGGAAGC ACTTAGCCTG GCTTAGCTAA GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3' (SEQ.ID.NO.16) Alternatively, a manually redesigned small hairpin ribonucleic acid (shRNA) may be used. The manually redesigned small hairpin ribonucleic acid is synthetic miR-302s-sense: 5'-GCAGATCTCG AGGTACCGAC GCGTCCTCTT TACTTTAACA TGGAAATTAA GTGCTTCCAT GTTTGAGTGG TGTGGCGCGA TCGATATCTC TAGAGGATCC ACATC-3 '(SEQ.ID.NO.17) and mir-302s-antisense : 5′-GATGTGGATC CTCTAGAGAT ATCGATCGCG CCACACCACT CAAACATGGA AGCACTTAAT TTCCATGTTA AAGTAAAGAG GACGCGTCGG TACCTCGAGA TCTGC-3 ′ (SEQ.ID.NO.18) formed by hybridization, mir-302 pre-micro ribonucleic acid cluster (mir-302 pre-miRNA cluster) ) Instead of a simple intron insertion. Mir-302 small hairpin ribonucleic acid (mir-302 shRNA) has greater than 85% homology with all natural mir-302 members and targets the same human cellular genes. In designing the mir-302 homolog, thymine may be used instead of uracil, and vice versa.

  For intronic insertion of the pre-miRNA / shRNA of mir-302, on the side of the insertion site of the recombinant SpRNAi-RGFP transgene, there is a PvuI at the 5 'end and a MluI restriction / cloning site at the 3' end, respectively. Thus, the initial insert can be removed and replaced with various pre-miRNA / shRNA inserts (eg, mir-302 pre-miRNA / shRNA). These pre-miRNA / shRNA inserts have cohesive ends that match PvuI and MluI restriction sites. By altering intron inserts that target different gene transcripts, the intronic mir-302s (intronic mir-302s) expression system of the invention can be used in vitro, in vivo, ex vivo, and in vivo. Can be used as a powerful tool to induce target gene silencing. After intron insertion, this SpRNAi-RGFP transgene containing mir-302 at the intron insertion site is also in the restriction / cloning site of the pSingle-tTS-shRNA vector induced by doxycycline (i.e., the XhoI-HinDIII restriction / cloning site). Inserted to form a pTet-On-tTS-miR302s expression vector that can be expressed in cells (FIG. 1A).

  As a method of delivering a nucleic acid composition expressing pre-mir-302 (pro-mir-302) or mir-302 to human cells, liposome / polysome / chemical transfection (liposomal / polysomal / chemical transfection), DNA Recombination (DNA recombination), electroporation, gene gun penetration, transposon / retrotransposon insertion, jumping gene integration, micro-injection (micro- injection, viral infection, retroviral / lentiviral infection, and combinations thereof, or non-gene transfer methods or gene transfer methods. In order to prevent the risk of random transgene insertion and cell mutation, the present invention uses liposome or polysome transfection methods to create mir-302 expression vectors and / or mir-302-like gene silencing effectors (e.g., pre-mir -302 and / or pro-mir-302) are preferably delivered to target human cells (ie, tumor / cancer cells). After transduction, the expression level of mir-302s is determined by the expression rate of the TRE-controlled CMV promoter in the pTet-On-tTS-miR302s vector at the concentration of the mir-302-like gene silencing effector or at each concentration of doxycycline. . Thus, the present invention provides for in vitro, ex vivo, in vivo expression levels of mir-302 by providing a controllable mechanism by using known agents (i.e., doxycycline) or pre- / pro-mir-302 concentrations. Adjust This allows us to prevent any cytotoxicity due to RNA accumulation or supersaturation in the test cells. In addition, mir-302 expression controlled by the CMV promoter is always silenced by DNA methylation in human cells after an activation state of one month. The one-month activation mechanism is beneficial in the treatment of cancer because it can avoid long-term accumulation or supersaturation of RNA in the treated cells.

  In short, the present invention employs a novel design and strategy, and uses a vector-expressed or non-expressed mir-302-like gene silencing effector as a therapeutic agent for tumor suppression and cancer treatment. mir-302-like gene silencing effectors include mir-302a, mir-302b, mir-302c, mir-302d, their hairpin-like microribonucleic acid precursors (pre-miRNAs and / or pro-miRNAs), and manually. Includes redesigned small hairpin ribonucleic acid (shRNA) homologs / derivatives, as well as combinations thereof. In a preferred embodiment, the present invention is directed to mir-302-targeted cell cycle regulators and oncogenes that are processed into a sufficient amount of a mir-302-like gene silencing effector to be deliverable in the treated tumor / carcinoma cells. The present invention provides designs and methods for treating human tumors and cancers using recombinant nucleic acid compositions that suppress cancer and achieve beneficial effects in the treatment of tumors / cancers. This recombinant nucleic acid composition expresses a mir-302-like gene silencing effector by transcription driven by a persistent promoter (i.e., CMV) or a drug-inducible promoter (i.e., TRE-CMV) in the treated cells. Can be done. On the other hand, the recombinant nucleic acid composition is used for the production of pro-mir-302 driven by prokaryotic cells (product is pro-mir-302). The pro-mie-302 can be used as a mir-302-like gene silencing effector to treat human tumors / cancers. Preferably, the mir-302-like gene silencing effector is a recombinant mir-302 family cluster (mir-302s; hybrid of SEQ. ID. NOs. 9-16) or a manually redesigned mir-302 small hairpin ribonucleic acid homolog (shRNA, a hybrid of SEQ. ID. NOs. 17 and 18). The treated cell matrix can express a cellular gene targeted by mir-302 in vitro, ex vivo or in vivo. The present invention can suppress tumor / cancer cell formation by silencing mir-302 target cellular genes and reprogram highly malignant cancer cells to a relatively benign low-stage state You can also. This phenomenon is called cancer regression.

  The present invention has demonstrated the success of mir-302 mediated anti-tumor / cancer treatment in the following ways: First, safety against normal human cells. As shown in FIGS. 1E-1F and 3A-3B, transfection of mir-302 into normal human cells (mirPS-hHFC) slightly diminishes the cell cycle but does not cause apoptosis or cell death. Second, transfecting mir-302 into human cells allows the cells to be reprogrammed to a stem cell-like state, which is beneficial for treating damaged tissue (FIGS. 4A-4B and 5A-5D). Third, by transfecting mir-302 into human tumor / cancer cells (e.g., prostate cancer cells PC3, skin cancer cells Colo, breast cancer cells MCF7, liver cancer cells HepG2, and teratoma cells NTera-2), Tumor / cancer cell growth is strongly suppressed, causing> 98% cell death or apoptosis (FIGS. 6A-6D and 12A-12C). Fourth, mir-302 not only co-suppresses multiple cell cycle regulators such as cyclin-dependent kinase 2 (CDK2), cyclin D1 / D2 (cyclin D1 / D2) and BMI-1, but also Activation of suppressors, such as p16INK4a and p14 / p19Arf, can also suppress tumor formation (FIGS. 7B-7D). Finally, by delivering mir-302 to tumor / cancer cells in vivo,> 90% tumor / cancer cell growth can be inhibited (FIGS. 8A-8C and FIG. 15). Also, mir-302 does not cause cell senescence due to telomere shortening (FIGS. 9A-9C). Note that we have prepared a mir-302-like gene silencing effector and / or its expression vector and have successfully transfected into target tumor / cancer cells in vivo, The risk of mutation could be avoided (FIGS. 8A-8C and FIG. 15). These findings have provided strong evidence that mir-302-like gene silencing effectors can be used as therapeutics and / or vaccines for antitumor / cancer treatment. In addition, since mir-302 also has a function of recovering and regenerating damaged human tissue (Lin et al., 2008 and 2010 (Non-Patent Documents 1 and 6)), the present invention is intended for antitumor / cancer treatment Not only is it useful for applications of the present invention, but also used for tissue recovery and / or organ regeneration.

  Apparently, the drawings described below are by way of example only and do not limit the invention.

European Patent Application No. 2198025

Lin et al. (2008) .Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state.RNA 14, 2115-2124. Lin et al. (2011) .Regulation of somatic cell reprogramming through inducible mir-302 expression.Nucleic Acids Res. 39, 1054-1065. Takahashi et al. (2006) .Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell 126, 663-676. Yu et al. (2007) .Induced pluripotent stem cell lines derived from human somatic cells.Science 318, 1917-1920. Wernig et al. (2007) .In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.Nature 448, 318-324. Lin et al. (2010) .MicroRNA miR-302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of CDK2 and CDK4 / 6 cell cycle pathways.Cancer Res. 70, 9473-9482. Wang et al. (2008). Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat. Genet. 40, 1478-1483. Deng et al. (1995) .Mice lacking p21Cip1 / Waf undergo normal development, but are defective in G1 checkpoint control.Cell 82, 675-684. Yabuta et al. (2007) .Lats2 is an essential mitotic regulator required for the coordination of cell division.J Biol Chem. 282, 19259-19271. Barroso-deUesus et al. (2008) Embryonic stem cell-specific miR302-367 cluster: human gene structure and functional characterization of its core promoter.Mol Cell Biol. 28, 6609-6619. Ying SY and Lin SL. (2004) .Intron-derived microRNAs-Fine tuning of gene functions.Gene 342, 25-28. Suh et al. (2004) .Human embryonic stem cells express a unique set of microRNAs. Dev. Biol. 270, 488-498. Grimm et al. (2006) .Fatality in mice due to oversaturation of cellular microRNA / short hairpin RNA pathways.Nature 441, 537-541. Tang et al. (2007) .Maternal microRNAs are essential for mouse zygotic development.Genes Dev. 21, 644-648. Lin et al. (2003) .A novel RNA splicing-mediated gene silencing mechanism potential for genome evolution.Biochem Biophys Res Commun. 310, 754-760. Lin et al. (2006) Gene silencing in vitro and in vivo using intronic microRNAs.Methods Mol Biol. 342, 295-312. Lin et al. (2005) .Asymmetry of intronic pre-microRNA structures in functional RISC assembly.Gene 356, 32-38.

1A-1F show inducible mir-302 expression and the effect of proliferating normal human hair follicle cells (hHFC). FIG. 1A shows the structure of a pTet-On-tTs-miR302s vector inducible by doxycycline (Dox). Figure 1B shows the structure of the mir-302 family (mir-203s). FIG. 1C shows the result of microribonucleic acid microarray analysis of mir-302 induced and expressed after treatment with 10 μM doxycycline (Dox) (n = 3, p <0.01) and 6 hours later. FIG.1D shows the core-reprogrammed transcription factor Oct3 / 4-Sox2-Nanong and the protein product of the marker gene TRP1 in melanocytes (melanocytic), the dose-dependent effect of mir-302 on the expression pattern of cytokeratin (cytokeratin 16) (n = 5, p <0.01) were analyzed by Northern and Western blot analysis. FIG. 1E is a bar graph of the results of flow cytometry analysis showing the dose-dependent effects of mir-302 on changes in mitosis (M phase) and dormancy (G0 / G1 phase) of the mirPS-hHFC cell cluster. FIG. 1F shows the characteristics of DNA laddering of apoptotic DNA cleavage induced by mir-302 in each mirPS cell line after treatment with 10 μM doxycycline (Dox). 2A-2C show the biosynthesis of mir-302s and generation of mirPS cells. FIG. 2A shows the biosynthesis mechanism of intron mir-302. The Mir-302 family cluster is transcribed together with the gene encoding the red fluorescent protein (RGFP), and further isolated by the dicer of RNA endoribonuclease (RNaseIII), a spliceosomal complex and the cytoplasmic enzyme. Spliced to mir-302 member. Among them, the red fluorescent protein is used as an index of the mir-302 production amount. One-fold concentration of red fluorescent protein corresponds to four-fold concentration of mir-302. FIG. 2B shows the step of transfecting mir-302 by liposome / polysome / electroporation. Inducible mir-302 expression vector pTet-On-tTs-miR302s (FIG. Was introduced. 200,000 cultured hHFC cells from two human hair follicles (dermal papillae) were transfected with 10 μg pTet-On-tTs-miR302s per test. After being induced and expressed by doxycycline (Doxyxycline, Dox), the biosynthesis of mir-302 was based on the natural intronic microribonucleic acid (intronic miRNA) pathway. FIG. 2C shows the step of transfecting mir-302 by liposome / polysome / electroporation. Inducible mir-302 expression vector pTet-On-tTs-miR302s (FIG. Was introduced. 200,000 cultured hHFC cells from two human hair follicles (dermal papillae) were transfected with 10 μg pTet-On-tTs-miR302s per test. After being induced and expressed by doxycycline (Doxyxycline, Dox), the biosynthesis of mir-302 was based on the natural intronic microribonucleic acid (intronic miRNA) pathway. FIGS. 3A-3C show the changes that mir-302 expressed after being induced by doxycycline (Dox = 5 or 10 μg) made to mirPS-hHFC cell properties. FIG. 3A shows changes in cell morphology and cell cycle rate before and after inducing cell reprogramming by doxycycline. Flow cytometric analysis of each cell cycle stage and its corresponding DNA content is shown graphically above the cell morphology (n = 3, p <0.01). In the graph, the first (left) and second (right) peaks indicate the ratio of resting (G0 / G1) and mitotic (M) cells to all test cell clusters, respectively. Scale = 100 μm. FIG. 3B shows changes in cell morphology and cell cycle rate before and after inducing cell reprogramming by doxycycline. Flow cytometric analysis of each cell cycle stage and its corresponding DNA content is shown graphically above the cell morphology (n = 3, p <0.01). In the graph, the first (left) and second (right) peaks indicate the ratio of resting (G0 / G1) and mitotic (M) cells to all test cell clusters, respectively. Scale = 100 μm. FIG. 3C shows the time course of single mirPS-hHFC cells forming embryoid bodies (EB) after limiting dilution. The cell cycle was initially about 20-24 hours, but is presumed to have gradually accelerated after 72 hours. Scale = 100 μm. 4A-4B show analysis of pluripotency marker expression and in vivo pluripotency differentiation and assimilation. FIG.4A shows the expression pattern after the human embryonic stem cell (hES) marker gene is induced in mirPS cells by high concentration of mir-302, and the human embryonic stem cell (hES) marker gene is a human embryonic stem cell WA01- Results of comparison by Northern blot analysis with expression patterns in H1 (H1) and WA09-H9 (H9) (n = 5, p <0.01). The concentration of mir-302 in mirPS cells after 7.5 μM doxycycline (Dox) treatment was 30% higher than in H1 and H9 cells (> 30 times that of untreated hHFC), indicating that the major pluripotency marker, Oct3 / 4, began to induce co-expression of Sox2, Nanog, Lin28 and undifferentiated embryonic cell transcription factor 1 (UTF1). FIG. 4B shows that one week after transplantation of mirPS cells into immunodeficient SCID-beige mice, tissues differentiated by mirPS cells were assimilated into peripheral tissues near the injection site. White arrows indicate the direction of injection. The position of the intercalated disks is marked with a yellow triangle. Tissue cysts derived from mirPS-hHFC do not grow in organs / tissues other than the uterus and peritoneal cavity of mice. We further dissected one week after transplantation and examined tissue formation around the injection site. Mice were administered 10 μg doxycycline by tail vein injection the day before dissection. We show that RGFP-positive mirPS cells, including intestinal epithelial tissue formed after intraperitoneal injection, myocardium after cardiac injection, and skeletal muscle formed after dorsal flank injection, are transplanted into surrounding tissues at the transplant site. And differentiated into cells of the same type. Also, assimilated mirPS cells are similar to peripheral tissues such as intestinal epithelial MUC2, cardiac troponin T2 (troponin T type 2, cTnT), and skeletal muscle myosin heavy chain (myosin heavy chain, MHC). Tissue specific markers were expressed. FIGS. 5A-5D show that mirPS-hHFCs induced by doxycycline (Dox = 10 μM) obtained the properties of human embryonic-like stem cells (hES-like). FIG.5A shows the results of analyzing all gene expression patterns before and after somatic cell reprogramming (SCR) induced by mir-302 using a human genome gene chip (Human genome GeneChip U133 plus 2.0 array, Affymetrix) (n = 5 , P <0.01-0.05). Figure 5B shows that after cleavage with the restriction enzyme HpaII, the appearance and increase of relatively small DNA fragments was significantly greater than the genome-wide total CpG methylation in mirPS cells after treatment with 10 μM but not 5 μM doxycycline (Dox). Proven decrease. FIG. 5C is a detailed methylation map obtained by determining the sequences of the Oct3 / 4 and Nanog promoter regions by bisulfite DNA sequencing. Black and white circles represent methylated and unmethylated cytosine sites, respectively. FIG. 5D includes various tissues in which teratoma-like tissue cysts that have been pluripotently differentiated by mirPS-hHFCs are derived from three germ layers (embryonic germ layers). 6A-6E show an analysis of the in vitro tumorigenic potential of mirPS cells derived from various tumor / cancer cells after inducing and expressing mir-302 with 10 μM doxycycline (Dox). FIG. 6A shows changes in cell morphology and cell cycle rate before and after inducing mir-302 expression by doxycycline (Dox). Flow cytometric analysis of each cell cycle stage and its corresponding DNA content is shown graphically above the cell morphology (n = 3, p <0.01). FIG. 6B shows changes in cell morphology and cell cycle speed before and after inducing mir-302 expression by doxycycline (Dox). Flow cytometric analysis of each cell cycle stage and its corresponding DNA content is shown graphically above the cell morphology (n = 3, p <0.01). FIG.6C is a bar graph of flow cytometry analysis showing the dose-dependent effect of mir-302 on changes in mitosis (M phase) and dormancy (G0 / G1 phase) of mirPS cell clusters derived from various tumor / cancer cells. . FIG. 6D is a functional analysis (n = 4, p <0.05) of invasion in which suppression of tumors (and / or cancer cells) by Mir-302 appeared in the Matrigel chamber. FIG. 6E shows comparison results of the ratio of cells adhering to human bone marrow endothelial cell (hBMECs) monolayer before and after doxycycline-induced mir-302 expression (n = 4, p <0.05). 7A to 7D are luciferase 3'-terminal untranslated region reporter gene test (luciferase 3'-UTR reporter gene assay), target G1 checkpoint regulators (G1-checkpoint regulators) mir-302 induced genes Shows the silencing effect. FIG.7A shows that the structure of the luciferase 3′-terminal untranslated region reporter gene has two normal (T1 + T2), two mutant (M1 + M2), or normal and mutant There are mir-302 target sites in a mixture of (T1 + M2 or M1 + T2). The mutation site contains a mismatched TCC motif in place of the matching 3'-CTT end of the normal target site. FIG. 7B shows the effect of mir-302 expression induced by doxycycline (Dox) on luciferase expression (n = 5, p <0.01). Dox concentration = 5 or 10 μM. CCND1 and CCND2 indicate cyclin D1 and cyclin D2, respectively. Figures 7C and 7D show Western blot analysis of G1 checkpoint regulator, a major target of mir-302, at mir-302 induction at high (10 μM Dox) and low (5 μM Dox) concentrations. And comparison results of changes in human embryonic stem cells (hES) WA01-H1 (H1) and WA09-H9 (H9) (n = 4, p <0.01). FIGS.8A-8C show that tumor NTera2 cells were expressed by vector in mir-302s (NTera2 + mir-302s) or mir-302d * (NTera2 + mir-302d *) in which tumor NTera2 cells were expressed by vector. ) (n = 3, p <0.05). Mir-302s and mir-302d * in the transfected tumor cells NTera-2 were transcribed from the pCMV-miR302s and pCMV-miR302d * vectors, respectively. In FIG. 8A, morphological evaluation was performed on the average size of the tumor three weeks after the in-situ injection (post-is) was performed. All tumors were located at the original implantation site (points indicated by black arrows). No signs of cachexia or tumor metastasis were observed in any of the test mice. FIG.8B shows Northern and Western blot analyzes show that in vivo mir-302 is a G1 checkpoint regulator cyclin-dependent kinase 2 targeted by the core reprogrammed transcription factors Oct3 / 4-Sox2-Nanog and mir-302. 2 shows the effects on (CDK2), cyclins D1 / D2, cyclins D1 / D2, BMI-1, and expression patterns of p16Ink4a and p14Arf. FIG.8C shows immunohistochemical staining analysis that in vivo mir-302 is a core reprogrammed transcription factor Oct3 / 4-Sox2-Nanog and cyclin-dependent kinase 2 (CDK2 ), Cyclins D1 / D2, BMI-1, and the effect on expression patterns of p16Ink4a and p14Arf. 9A-9C show telomerase activity analysis of mirPS-hHFC and mirPS cell lines derived from various tumor / cancer cells in the expression of mir-302 induced by 10 μM doxycycline. FIG. 9A analyzed telomerase activity in the Telomere Repeat Amplification Protocol Test (TRAP) (n = 5, p <0.01). Telomerase activity is affected by RNase treatment (hHFC + RNase). FIG.9B demonstrated by Western blot analysis that expression of human telomerase reverse transcriptase (hTERT) in various mirPS cell lines was consistently increased, but decreased in AOF2 and HDAC2 expression (n = 5). , P <0.01). FIG. 9C shows telomerase activity measured by telomerase PCR ELISA test (OD470-OD680; n = 3, p <0.01). The analysis results of FIGS. 10A to 10D show the silencing effect induced by mir-302 on the targeted epigenetic gene. FIG.10A shows that the structure of the luciferase 3′-terminal untranslated region reporter gene has two normal (T1 + T2) or two mutated (M1 + M2), or normal and mutated 3′-terminal untranslated region. There are target sites of mixed (T1 + M2 or M1 + T2) mir-302. The mutation site contains a mismatched TCC motif instead of the matching 3 'terminal CTT of the normal target site. FIG. 10B shows the effect of mir-302 expressed by doxycycline (Dox) on luciferase expression (n = 5, p <0.01). Figures 10C and 10D show the western blot analysis of the epigenetic gene, which is a major target of mir-302 in inducing high (10 μM Dox) and low (5 μM Dox) mir-302. Shows the results of comparison of changes in expression in mirPS cells and human embryonic stem cells WA01-H1 (H1) and WA09-H9 (H9) (n = 4, p <0.01). FIG. 11 is a schematic diagram of mir-302-mediated somatic cell reprogramming (SCR) and a cell cycle regulation mechanism. Based on previous and current research, we have found two parallel events. First, mir-302 initiates reprogramming by potent silencing of several epigenetic regulators, AOF1 / 2, MECP1 / 2, and HDAC2, and de-encrypts whole genomic DNA. Inducing methylation further activates the cell marker genes of human embryonic stem cells required to induce SCR (grey marker). Second, co-suppression of the G1 checkpoint regulator cyclin-dependent kinase (CDK2), cyclins D1 / D2 (cyclins D1 / D2) and BMI-1, and activation of p16Ink4a and p14 / p19Arf reduce cell cycle decay. Inducing it suppresses whole cell activity to prepare for SCR (black marker). Resting of the dormant phase (G0 / G1) also prevents possible random growth and / or tumor-like transformation of reprogrammed pluripotent stem cells. In essence, the result of the synergistic effect of these two events is that a more accurate and safe reprogramming process can be simultaneously suppressed, as well as pre-mature and tumor formation. FIG. Shows the change in After transfection of mir-302, cells positive for mir-302 expression are hpESC + mir-302, PC3 + mir-302 and Colo + mir-302, respectively. FIG. 13 schematically shows the natural biosynthetic mechanism of intron miRNA miR-302. Mir-302 is a natural intron miRNA encoded by the intron region of La ribonucleoprotein domain family member 7 (LARP7). In the biosynthesis of mir-302, the primary miRNA precursor (pri-miRNA) is first transcribed by the type II RNA polymerase (Pol-II) along with the transcript of the LARP7 gene, and then the hairpin-like miRNA by cell spliceosomes. Modified into a precursor (pre-miRNA). Pre-miRNA is exported from the cell nucleus by exportin 5 (Xpo5), and further processed into mature miRNAs by Dicer-like ribonucleic acid endoribonuclease RNase III in the cytoplasm. Subsequently, the mature mir-302 molecule, together with the Argonaute protein (Ago 1-4), can bind to the RNA-induced silencing complex (RISC) and induce silencing of specific genes. FIG. 14 shows the results of an in vivo new drug preclinical test (pre-IND) of treating open skin wounds of mice using pro-mir-302. In the test, the upper blank group, that is, the treatment with the ointment alone, the ointment with a pro-mir-434 concentration of 10 μg / mL (negative control group), the ointment with the lower pro-mir-302 concentration of 10 μg / mL Including three different treatments. FIG. 15 shows the results of an in vivo new drug preclinical test (pre-IND) for treating human liver cancer cells xenografts in SCID nude mice using pro-mir-302 as an injection drug. After three treatments (once a week), the pro-mir-302 (pre-mir-302) drug reduced the tumor from 728 ± 328 mm ³ (untreated blank group, control group C) to 75 ± 15 mm ³ (treatment with pro-mir-302, group T), showing a reduction rate close to 90% of the mean tumor size. No significant therapeutic effect was found after treatment with a mimic such as a synthetic siRNA (siRNA-302) in the same step. Further histological examination (rightmost) showed normal liver lobule-like structures (circles with black arrows) only in xenografted liver cancer cells / tumors treated with pro-mir-302, This was not observed in other treatment groups or control groups. This result suggested that the reprogramming mechanism could restore malignant cancer cell properties to a relatively normal state (cancer reversion). FIG. 16 shows histological similarity between normal liver tissue and in vivo xenografted human liver cancer cells / tumors treated with pro-mir-302. After three treatments (once a week), the pro-mir-302 (pre-mir-302) drug is a relatively benign low grade (grade II) xenografted human liver cancer. (Lower) state. Like normal liver tissue (top), treated xenograft cancers (tumors) have central venous (CV) -like and portal triad (PT) -like structures (points indicated by black arrows) A typical hepatic lobule can be formed. In general, cancer cells are more acidic than normal hepatocytes, and the results of hematoxylin-eosin (H & E) staining indicate that the color of the cancer cells is relatively purple, and that the color of normal hepatocytes is comparable. Indicates that the target is red. FIG. 17 is a histopathological comparison of human liver cancer (tumor) xenografted with untreated siRNA treated with pro-mir-302 in SCID-beige nude mice and normal liver tissue Is shown. Untreated (top), xenografted human liver cancers (tumors) invasively invade normal tissues, such as muscle and blood vessels, forming large cell-cell and cancer-tissue fusion structures. Showed its high malignancy and metastasis. Treatment with mimics such as siRNA (siRNA-302) did not significantly reduce the malignancy of xenografted liver tumors (middle upper panel). The reason may be due to the short half-life of the siRNA molecule in vivo. In contrast, treatment with pro-mir-302 (= pre-mir-302) not only reprogrammed the xenografted cancer cells to a normal hepatocyte-like morphology (unfused), but also Successfully suppressed the invasion of normal tissue around (see middle and lower figures). Compared to normal liver tissue (bottom), pro-mir-302 treated malignant tumors have hepatic lobule-like structures, normal glandular-like sequences, and cell-cell and cancer-tissue junctions (black arrows) Showed very clear boundaries. This proved that the treated cancer (tumor) became relatively benign.

DETAILED DESCRIPTION OF THE INVENTION Certain specific embodiments of the present invention will be described with reference to the accompanying drawings, but these specific embodiments are merely examples, and a small number of examples may be used to represent the application of the principles of the present invention. It should be understood that only certain specific embodiments that are possible are given by way of example only. Those skilled in the art should consider various changes and modifications as falling within the spirit, scope and intention of the present invention, which is further defined in the appended claims.

  The present invention provides novel nucleic acid compositions and methods of using recombinant mir-302-like gene silencing effectors to inhibit human tumor / cancer cell growth and tumor formation. Unlike conventional small hairpin ribonucleic acid (shRNA) designs, small hairpin ribonucleic acids according to the present invention include a mismatched stem-arm similar to the precursor of natural mir-302 (pre-mir-302). . The small hairpin ribonucleic acid according to the present invention is an improved pre-mir-302 stem loop, 5′-GCTAAGCCAG GC-3 ′ (SEQ.ID.NO.1) and 5′-GCCTGGCTTA GC-3 ′ (SEQ. .ID.NO.2), and the pre-mir-302 stem loop does not interfere with the transport of transport ribonucleic acid (tRNA) to the same nuclear export as natural microribonucleic acid precursors (pre-miRNAs). Transportation (nucleus export) efficiency can be provided. Without being limited to any particular theory, the anti-proliferative, anti-tumorigenic effects of the present invention can express newly discovered mir-302 family clusters (mir-302s) or small hairpin ribonucleic acids homologous to mir-302. Figure 4 shows the mir-302 mediated mechanism of gene silencing caused by transfecting a recombinant nucleic acid composition. All manually redesigned miRNA / shRNA molecules have a similar sequence 5'-UAAGUGCUUC CAUGUUU-3 '(SEQ.ID.NO.3) in the first 17 nucleotides of its 5' terminal sequence. . The steps for constructing a mir-302-like gene silencing effector and a nucleic acid composition expressing mir-302 are described in Examples 2 and 3. When designing a sequence homologous to mir-302, thymine (T) may be used instead of uracil (U).

  To position the role of mir-302 in the human cell cycle, we designed an inducible pTet-On-tTS-miR302s expression vector (FIG.1A, Example 2) in normal human cells and human cancer cells. Transfected. Mir-302s is a hES-specific microribonucleic acid (miRNA) family, and its family cluster consists of four small non-coding mir-302b, mir-302c, mir-302a, and mir-302d (mir-302s; FIG. 1B). (non-coding) RNA members are included (Suh et al., 2004). In the present invention, the expression of mir-302s is driven by a cytomegalovirus (CMV) promoter controlled by a tetracycline-response-element (TRE) under the stimulation of doxycycline (Dox). You. After transfection, mir-302 biosynthesis is based on the natural intron microribonucleic acid biosynthetic pathway, in which mir-302 is transcribed along with a reporter gene (RGFP) encoding a red fluorescent protein, and further spliced. Spliceosomal components and the cytoplasmic RNA endoribonuclease RNase III, Dicer, are spliced into a single mir-302 member (FIG. 2A) (Lin et al., 2008). Quantitatively, a 1-fold concentration of RGFP corresponds to a 4-fold concentration of mir-302. Microarray analysis of microribonucleic acids confirmed that under the stimulation of doxycycline (Dox), all mir-302 members except mir-302b * were effectively expressed in transfected cells (FIG. Example 3). The steps of transfecting cells with the pTet-On-tTS-miR302s expression vector are outlined in FIGS. 2B-2C.

  Also, nucleic acid compositions that express mir-302, such as pTet-On-tTS-miR302s, express the Kozak translation initiation common sequence (Kozak consensus translation initiation site) that improves translation efficiency in eukaryotic cells, expressing mir-302. A plurality of SV40 polyadenylation signals (SV40 polyadenylation signals) located downstream of the construct to be made (mir-302 construct), a pUC origin of replication for prokaryotic cell proliferation, a construct that expresses mir-302 (i.e., SpRNAi- RGFP) into the nucleic acid composition, at least two restriction sites, a selective SV40 origin of replication necessary for replication in mammalian cells expressing the SV40 T antigen, and an antibiotic resistance gene in a prokaryotic cell capable of replication. Includes an optional SV40 early promoter for expression. Expression of the antibiotic resistance gene is used as a selective marker to isolate positive cloning in which the transgene was expressed. These antibiotics, G418, neomycin (neomycin), puromycin (puromycin), penicillin G (penicillin G), ampicillin (ampicillin), kanamycin (kanamycin), streptomycin (streptomycin), erythromycin (erythromycin), spectromycin (spectromycin) ), Phophomycin, phophomycin, tetracycline, doxycycline, drifcycline, rifapicin, amphotericin B (amphotericin B), gentamicin (gentamycin), chloramphenicol, cephalothin (cephalothin), tyrosine (typhacine) tylosin), and combinations thereof.

Mir-302 attenuates the cell cycle of normal cells without apoptosis In our previous studies, in human melanoma cells Colo-829 and prostate cancer cells PC3, increased mir-302 expression was detected in these malignant cells. Can be reprogrammed to an hES-like pluripotent state (Lin et al., 2008). During the process of somatic cell reprogramming (SCR), mir-302 causes more than 98% (> 98%) of cancer cells to undergo apoptosis, while further increasing the growth rate of the remaining (<2%) reprogrammed cells. Significantly reduced. Although these properties are certainly beneficial in treating cancer, the function of mir-302 in normal human cells is not yet certain. To evaluate its effect, we introduced the inducible pTet-On-tTs-miR302s expression vector into normal human hair follicle cells (hHFCs). The reason for choosing hHFCs is that they are sufficient, reachable, and grow fast. We show that as the concentration of doxycycline (Dox) increases to 10 μM, the doxycycline concentration is higher than 7.5 μM (> 7.5 μM) (FIG.1D, Example 5), and that the core reprogramming factors ) Oct4-Sox2-Nanog was promoted simultaneously and proliferating cell clusters were reduced by 70%, i.e., from 37% ± 2% to 11% ± 2% (FIG.1E, M phase, Example 7) could be observed. In response, the dormant cell cluster increased by 41%, i.e. from 56% ± 3% of the original to 79% ± 5% (FIG.1E, G0 / G1, phase 7, Example 7). (MirPS cells; Lin et al., 2008) found in mir-302-reprogrammed pluripotent stem cells, resembling a potent antiproliferative effect. However, hHFC cells reprogrammed with mir-302 (mirPS-hHFC) do not show any signs of DNA laddering or cell death that can measure apoptosis (Figure 1F, Example 6), Normal cells were shown to be more tolerant of the antiproliferative effect of mir-302 compared to tumor / cancer cells. Tumor / cancer cells are considered to be extremely difficult to survive in such a dormant state due to their vigorous metabolism and rapid growth and expansion.

  Note that after treatment with doxycycline at concentrations higher than 7.5 μM, the morphology of mirPS-hHFC changed from spindle-shaped to dormant cell-like spheres (Figures 3A-3B, red RGFP-positive cells). . The mir-302 concentration of cells generated under the stimulation of 7.5 μM doxycycline is about 1.3 times the mir-302 concentration level in human embryonic stem cells H1 and H9 cells (FIG. 4A, Example 4). At this higher concentration level, mirPS-hHFCs strongly expressed Oct3 / 4, Sox2, Nanog, and other standard human embryonic stem cell (hES) markers (FIG. 4A). In microarray analysis of total gene expression, about half of the transcriptome changed from the hHFC somatic cell pattern to the hES-like expression pattern, and was higher than H1 / H9 cells and 93% (> 93%) It was further shown to have similarity (FIG. 5A, Example 8). Demethylation of total genomic DNA, the first sign of the onset of somatic cell reprogramming (SCR), can be clearly measured in these mirPS-hHFCs, and the pattern of demethylation is also in H1 / H9 cells. The same is true (FIGS. 5B and 5C, Example 9). In addition, individual mirPS-hHFC cells grow into a single embryoid body-like colony, and the cell division rate of 20-24 hours per cycle is also consistent with the antiproliferative effect of mir-302 (FIG. ). We show that these mirPS-hHFCs are pluripotent because they form teratoid cysts only in the uterus and peritoneal cavity of pseudopregnant immunodeficient SCID-beige mice. Special attention was given to having no tumoregenetic. These teratoid cysts include various tissues derived from three germ layer (embryonic germ layer) tissues: ectoderm, mesoderm, and endoderm (FIG. 5D, Example 10). When mirPS-hHFCs were xenotransplanted into normal male mice, mirPS-hHFCs were assimilated into surrounding tissues, and exhibited similar tissue markers. This also indicated that mir-302 could be used to heal damaged cells (FIG. 4B). Taken together, these findings suggest that mir-302 can reprogram somatic hHFCs into hES-like iPS cells (induced pluripotent stem cells). Transcription factors such as Oct3 / 4 and Sox2 are important for mir-302 expression (Marson et al., 2008; Card et al., 2008), and mir-302 replaces Oct3 / 4-Sox2 and replaces somatic cell reprogramming. (SCR) may be induced.

  What happens in parallel with Mir-302-induced SCR and cell cycle decay depends on the concentration of mir-302. We have found that when the concentration of mir-302 exceeds 1.3 times that in human embryonic stem cells H1 / H9, they occur almost simultaneously, so this particular concentration is the minimum that triggers the two events. The threshold was shown. In previous experiments, the event could not be triggered when a single mir-302 member, or a concentration of mir-302 corresponding to a relatively low concentration in H1 / H9 cells, was used. We have also demonstrated that concentrations of mir-302 induced by relatively low concentrations of 5 μM doxycycline cannot silence the target site or target G1 checkpoint modulator of the reporter gene. Compared to natural development, embryonic cells before the morula stage (32-64 cell stage) often show very slow cell cycle rates similar to those of mirPS cells. However, such a cell cycle regulatory effect was not found in blastocyst-derived hES cells. This suggests that relatively low concentrations of mir-302 in hES cytoplasm are not sufficient to silence the target G1 checkpoint regulator and oncogene. This may explain why hES from blastocysts has dramatic proliferative capacity and propensity to form tumors. Therefore, the present invention is also applicable to reducing the tumorigenicity of hES cells in stem cell therapy.

Mir-302 suppresses tumorigenic potential and induces apoptosis of various tumor / cancer cells
In view of mir-302's ability to cause apoptosis of cancer cells and cell cycle decay, we continued to explore the possibility of using mir-302 as a versatile human tumor / cancer cell therapeutic. Our previous studies have already shown potential in melanoma and prostate cancer cells (Lin et al., 2008), but current studies show breast cancer cells MCF7, liver cancer cells Hep G2, and embryonic The potential in teratoma cells NTera-2 was further tested. As shown in FIGS.6A-6B, the three tumor / cancer cells were transfected with the pTet-On-tTS-miR302s vector and all were reprogrammed into dormant mirPS cells under the stimulation of 10 μM doxycycline, and As a result, embryoid body-like colonies were formed. In all three tumor / cancer cells, mir-302 at this concentration level also caused significant apoptosis (> 95%) (FIG. 1F, Example 6). Furthermore, analysis of DNA content at each stage of the cell cycle by flow cytometry showed that mitotic cell clusters in all mirPS cells were significantly reduced (FIG. 6C, Example 7). Mitotic cell cluster (M phase) is reduced by 78% from 49% ± 3% to 11% ± 2% in mirPS-MCF7 cells, and from 46% ± 4% to 17% ± 2% in mirPS-HepG2 cells In mirPS-NTera2 cells, it was reduced by 62% from 50% ± 6% to 19% ± 4%. Conversely, dormant / dormant cell clusters (G0 / G1 phase), in mirPS-MCF7, mirPS-HepG2 and mirPS-NTera2 cells, from 41% ± 4% to 74% ± 5% in mirPS-MCF7 cells, The increase was 43% ± 3% to 71% ± 4% in mirPS-HepG2 cells and 40% ± 7% to 69% ± 8% in mirPS-NTera2 cells, by 80%, 65% and 72%, respectively. These results suggest that mir-302 could effectively reduce the fast cell cycle rate of these tumor / cancer cells and induce significant apoptosis.

  In vitro tumorigenecity assays, such as cell invasion tests (using a Matrigel chamber) and cell adhesion tests (cells adhere to a single cell layer of human bone marrow endothelial cells (hBMEC)), mir-302 Has anti-proliferative properties and two additional anti-tumorigenic effects. Cell invasion assays show that all three dormant mirPS-tumor / cancer cells lost their ability to migrate (down to <1%) but that the original tumor / cancer cells had higher nutrients. Forced entry into the replenished compartment area, occupying at least 9% ± 3% cell clusters in MCF7 cells, 16% ± 4% in Hep G2 cells, and 3% ± 2% in NTera-2 cells (FIG. 6D, Example 11). Cell adhesion assay (Cell adhesion assay) also consistently showed that these mirPS-tumor / cancer cells could not adhere to the hBMEC monolayer, but after the original MCF7 and Hep G2 cells were cultured for 50 minutes, A significant number of cell clusters (MCF7 7% ± 3%, Hep G2 20% ± 2%) rapidly translocated to the hBMEC monolayer (FIG. 6E, Example 12). Taken together, all the findings so far indicate that mir-302 is a human tumor suppressor, which can slow down the rapid growth of cells, induce apoptosis of tumor / cancer cells, invasion of tumor / cancer cells and It was once again strongly shown that metastasis could be suppressed. Most importantly, the novel function of this mir-302 is a variety of human tumors not limited to malignant skin cancer (Colo-829), prostate cancer (PC-3), breast cancer (MCF7), and liver cancer (HepG2) In view of the fact that teratoma (NTera-2) contains various different tissues, it can provide general-purpose treatment for various tumors.

Mir-302 mediated antiproliferative function is achieved by co-suppression on CDK2, cyclin-D1 / D2, and BMI-1
To confirm the actual interaction between mir-302 and its targeted G1 checkpoint regulator, we performed a luciferase 3'UTR region reporter test for luciferase. (FIG. 7A, Example 15). As a result, treatment with different concentrations of mir-302 resulted in cyclin-dependent kinase 2 (CDK2), cyclin D1 / D2 (cyclins-D1 / D2) and BMI-1 polycomb ring finger oncogene. Including the target G1 checkpoint modulators, they have significantly different inhibitory effects. In the presence of 10 μM doxycycline, mir-302 effectively binds to target sites on CDK2, cyclins D1 / D2, and BMI-1 transcripts, and increases expression of more than 80% (> 80%) of reporter luciferase. Silencing was successful (FIG. 7B, Example 15). The results of Western blot analysis confirming the inhibitory effect of Mir-302 on the true target gene in mirPS cells are consistent with the results from the 3'-terminal untranslated region reporter gene test for luciferase (Figure 7C, Example 5). . In contrast, the relatively low expression of mir-302 induced by 5 μM doxycycline is prominent either for the target site of the reporter gene or for a G1 checkpoint regulator other than cyclin D2. No significant silencing effect can be induced (FIGS. 7B and 7D). This demonstrates a dose-dependent manner of mir-302 expression and the ability of mir-302 to fine-tune cell cycle velocity based on the dose dependence. In the mammalian cell cycle, the G1-S phase transition is usually mediated by two compensatory cyclin-cyclin-dependent kinase complexes (cyclin-D-CDK4 / 6 and cyclin-E-CDK2). Controlled (Berthet et al., 2006). We show that high concentrations of mir-302 inhibit the two pathways that control the G1-S phase transition by inactivating the two complexes through co-suppression of CDK2 and cyclins D1 / D2, as well as being reprogrammed. Was found to reduce cell cycle velocity in mirPS cells. The limited expression of cyclin D3 in hHFCs and mirPS cells does not compensate for the loss of cyclins D1 / D2 in mirPS cells.

  We further detected that p16Ink4a and p14Arf expression increased slightly with silencing of BMI-1 (increase rates in hHFCs are 63% ± 17% and 57% ± 13%, respectively), but p21Cip1 Was not changed (FIG. 7C). BMI-1 deficiency, an oncogenic cancer stem cell marker, suppresses G1-S phase transition by increasing the activity of tumor suppressors such as p16Ink4a and p14Arf (Jacobs et al., 1999). In this situation, 16Ink4a directly inhibits the activity of cyclin-D-dependent CDK4 / 6 (cyclin-D-dependent CDK4 / 6) to phosphorylate retinoblastoma protein (Rb) To prevent Rb from releasing E2F necessary to enter the S phase, and perform E2F-dependent transcription (Parry et al., 1995; Quelle et al., 1995). P14Arf also allows p53-dependent transcription that plays a role in G1 arrest or apoptosis while preventing the binding of HDM2 to p53 (Kamijo et al., 1997). However, as currently known, embryonic stem cells are not regulated by cyclin-D-dependent CDKs (Burdon et al., 2002; Jirmanova et al., 2002; Stead et al.). Therefore, CDK2 is recognized as a major determinant for inducing G1-S transition in cell cycle regulation of hES cells. Therefore, CDK2 silencing is most likely to have the greatest effect on G1 arrest (G1-arrest) of mirPS cells, and co-suppression of cyclin-D and BMI-1 and co-activation of p16Ink4a and p14Arf Additionally suppresses cell growth induced by a tumorigenic signal-induced. Further, the loss of cyclin-D and the activation of p16Ink4a can be interpreted as the lack of cyclin-D-dependent CDK activity in embryonic stem cells.

  Therefore, the rigor of the interaction between the microribonucleic acid (miRNA) and the target gene can determine the intrinsic function of the microribonucleic acid. Due to different cell conditions, microribonucleic acids represent different preferences for gene targets. The present invention has discovered its important details and has disclosed for the first time that mir-302 has greatly different functions in human and mouse cells. mir-302 strongly targets CDK2, cyclins D1 / D2, and BMI-1 in human cells, but interestingly does not contain p21Cip1. Unlike mouse p21Cip1, human p21Cip1 does not contain any target sites for mir-302. This difference in gene targets has led to important schism in cell cycle regulation of both. In mouse embryonic stem cells (mES), mir-302 silences p21Cip1 and promotes tumor-like cell growth (Wang et al., 2008; Judson, 2009), but not in human mirPS cells. In this case, expression of p21Cip1 is maintained, resulting in relatively slow cell growth and relatively low tumorigenic potential. Also, mouse BMI-1 is not a target gene for mir-302 due to the lack of an appropriate target site. We show that in mirPS cells, silencing of human BMI-1 slightly stimulates p16Ink4a / p14ARF expression and attenuates cell proliferation, while mir-302 silences mouse BMI-1. Disclose that it cannot produce a similar effect in mouse cells. In mirPS cells, the expression of p16Ink4a / p14ARF is increased, and p21Cip1 is not affected. It should be understood that, apart from the suppressive pathway, it is most likely exerted through the p16Ink4a-Rb and / or p14 / 19ARF-p53 pathway. Significant differences in the preference of Mir-302 targeting human and mouse genes suggest that there is a fundamental difference in the cell cycle regulatory mechanisms of both.

Treatment of Mir-302 reduces tumor cell growth in vivo by more than 90% without altering stem cell pluripotency.We demonstrate that mir-302 has a tumor suppressor function and its normal versus tumor / cancer cells. Whether the mir-302 can be used as a drug to treat in vivo NTera2-derived teratomas in 8-week-old male athymic mice (BALB / c-nu / nu species) (Example 13). Tumor Tera-2 cell line (neoplastic Tera-2, NTera-2) is a pluripotent human embryonal teratocarcinoma cell line, and in vivo, various types of primitive somatic tissue ), Especially to primitive glandular tissue and primitive neural tissue (Andrews et al., 1984). Due to its pluripotency, NTera-2 derived teratomas can be a model for treating a variety of tumors in vivo. For drug administration, we injected pCMV-miR302s expression vector prepared with polyethylenimine (PEI) by in situ injection at the site as close as possible to the tumor. The pCMV-miR302s vector was controlled by a tetracycline response element (TRE-controlled), which consisted of changing the CMV promoter to a general CMV promoter (Example 2), but for DNA methylation, pCMV-miR302s human cells Expression lasts for about a month. After injection of the upper limit of 10 μg / mouse body weight (g) (maximum dose per injection) of the pCMV-miR302s vector, no signs of disease or cachexia were observed in the mice, so this method was used. Safety has been proven. Histological examination also showed that no tissue lesions were detected in brain, heart, lung, liver, kidney, and spleen.

In Examples of the present invention, when 5 treatments (treatment every 3 days) were performed with 2 μg / mouse weight (g) of pCMV-miR302s, there was a remarkable inhibitory effect on the growth of teratomas. I found that. As shown in FIG.8A (Example 13), after treatment with the pCMV-miR302s vector, the average size of NTera2-derived teratomas (11 ± 5 mm ³ , n = 6) was the untreated group (104 ± 23 mm ³ , Compared with n = 4), it decreased by more than 89% (> 89%). In contrast, administration of an equal volume of antisense-mir-302d (antisense-mir-302d) expression vector (pCMV-miR302d *) prepared with PEI reduced the size of the teratoma to 140% of its original size (250 ± 73 mm). ³ , n = 3). From the above results, the present inventors have found that NTera-2 cells express moderate mir-302 (FIG. 8B). Northern blot analysis also showed that in these differently treated teratoma cells, mir-302 expression was negatively correlated with tumor size (Figure 8B), i.e. It is suggested that regulating expression can effectively control teratoma growth in vivo. To demonstrate previous in vitro findings, we performed Western blot analysis to detect the G1 checkpoint modulator CDK2-cyclins-D1 / D2-BMI-1 in mir-302-treated teratomas. And the co-activation of the core reprogramming factor Oct3 / 4-Sox2-Nanog was confirmed (FIG. 8B). Analysis of these proteins in teratoma tissue by immunochemical (IHC) staining demonstrated similar results (FIG. 8C, Example 14). Most notably, we have found that mir-302 can suppress teratoma cell growth without affecting the natural pluripotency. This suggests that high concentrations of mir-302 play a role as both tumor suppressor and reprogramming factor. Based on the dual function of mir-302 and the consistent results obtained in vitro and in vivo (in vitro, in vivo), we conclude that the anti-proliferative mechanism of mir-302 discovered in vitro is It could be applied for suppression, so it was speculated that it has potential as a treatment for various tumors.

Mir-302 suppresses human epithelial primary culture cells (hpESC), human prostate cancer cells (PC3) and human melanoma cells Colo-829 (Colo) .We tested mir-302 in hpESC, PC3 and Colo cells tested. To silence the target gene by transfection, the pre-miRNA cluster (SEQ.ID.NOs. 9-16), which is the linked mir-302a-mir-302b-mir-302c-mir-302d, or manually Mimetic such as pre-mir-302 redesigned in (for example, 5'-UAAGUGCUUC CAUGUUU-3 '(SEQ.ID.NO.3) hairpin-like sequence containing (SEQ.ID.NO.3) containing a series of mir-302 configuration Designed and tested. The mature sequences of Mir-302a, mir-302b, mir-302c and mir-302d are respectively 5'-UAAGUGCUUC CAUGUUUUGG UGA-3 '(SEQ.ID.NO.71), 5'-UAAGUGCUUC CAUGUUUUAG UAG-3' ( SEQ.ID.NO.72), 5'-UAAGUGCUUC CAUGUUUCAG UGG-3 '(SEQ.ID.NO.73) and 5'-UAAGUGCUUC CAUGUUUGAG UGU-3' (SEQ.ID.NO.74). Note that these homologous mir-302-like gene silencing effectors are highly conserved (100% homology) in the first 17 nucleotides in the 5 'terminal region, i.e., the sequence 5'-UAAGUGCUUC CAUGUUU -3 '(SEQ.ID.NO.3). In these homologous sequences of mir-302, thymine (T) may be used instead of uracil (U).

  In these experiments, we redesigned the pre-miRNA cluster of mir-302a-mir-302b-mir-302c-mir-302d in hpESC and PC3 cells and redesigned the mir-302 homologue in PC3 cells (SEQ.ID .NO.75). After transfection of these mir-302s, all cell lines change their cell morphology (bottom) from a spindle or amoeboid shape to a more rounded shape, which may cause the cells to lose cell migration ability, and It suggests that it has a very slow cell replication rate, similar to stem cell growth (FIGS. 12A-12C). Flow cytometry analysis showing DNA content (y-axis) at different cell cycle stages (x-axis) (upper panel) showed a mitotic cell population reduction of more than 67% and slow cell growth of mir-302 transfected cells Although rates were confirmed, the number of cell populations is indicated by the DNA content at each cell cycle stage. The first (left) and second (right) peaks indicate the level of cell population numbers in the dormant G0 / G1 and mitotic M phases, respectively, in the entire test cell population. After mir-302 transfection, the number of mitotic cell populations decreased from 36.1% to 10.9% for hpESC cells, from 38.4% to 12.6% for PC3 cells, and from 36.5% to 11.5% for Colo cells, but remained empty. Groups transfected with vectors or vectors expressing pre-miRNA, including mir-gfp, have no significant changes in either cell morphology or cell proliferation after transfection. Pre-miRNAs, including mir-gfp, are used to target firefly EGFP genes that have no homology to human and mouse genes. Based on these findings, we show that transgene expression of the mir-302 homolog can transform primary human cultured cells and cancer cells, such as stem cell (ES) morphology and replication rate, Similar to the changes observed in previous iPS cell studies (Okita et al., (2007) Nature 448: 313-317; Wernig et al., (2007) Nature 448: 318-324).

The progression of cancer has been described as irreversible due to a cumulative mutation in the gene by which Mir-302 reprograms highly malignant human liver cancer xenografted in vivo to a low-stage benign state. However, the present invention discloses a novel function of the precursor of the microribonucleic acid mir-302 (pre-mir-302), i.e., reprogramming a highly malignant cancer / tumor to a low stage to a benign state, It may also belong to a rare natural healing process called spontaneous cancer regression. Spontaneous cancer regression occurs very rarely in 1 in 100,000 cancer patients. The present inventor has discovered that treatment with pre-mir-302 can increase the incidence of rare cancer regression to about 90%. As shown in FIG. 15, mimics such as pre-mir-302 (pro-mir-302) were used as therapeutic agents for malignant human liver tumors xenografted in SCID-beige nude mice, pro-mir -302 drug successfully reduced malignant tumor from 728 ± 328 mm ³ (untreated blank group, control group C) to 75 ± 15 mm ³ (pro-mir-302 treatment, group T) And showed a reduction rate close to 90% of the average tumor size. In comparison, treatment with mimetics such as other synthetic siRNAs (siRNA-302) did not show any approximate therapeutic effect. Further histological examination (rightmost) showed that normal liver lobule-like structures (circles with black arrows) were observed only in the group of xenograft malignancies treated with pro-mir-302. No treatment was observed in the control group or control group, demonstrating that the occurrence of the reprogramming mechanism restored the malignant cancer cell properties to a relatively normal state (cancer reversion, cancer reversion) . This novel reprogramming mechanism may be related to the function of mir-302's tumor suppressor. This mechanism silences multiple human oncogenes simultaneously, specifically targeting, and eliminates / prevents activation of tumor signaling pathways. However, this reprogramming mechanism may differ from the previously reported reprogramming function of mir-302 on somatic cells, since the appearance of pluripotent Oct4-positive stem cells has not been confirmed (Lin et al., 2008 and 2011). ).

  In a more detailed histological study, pro-mir-302 drug reprograms high-grade (xenograft IV) xenografted human liver cancer cells / tumors to a relatively benign, low-grade (lower than grade II) state He further disclosed that he was successful. As shown in FIG. 16, the treated xenografted cancer (tumor) is a typical hepatic lobule with central vein (CV) -like and portal triad (PT) -like structures (indicated by black arrows) And highly similar to normal liver histology (top). In the SCID-beige nude mouse body, untreated, treated with siRNA, compared with xenografted human liver cancer (tumor) treated with pro-mir-302 and normal liver tissue (FIG. 17), Untreated (top), xenografted human liver cancers (tumors) invasively invade surrounding normal tissues, such as muscle and blood vessels, creating large cell-cell and cancer-tissue fusion structures. It was found to have formed, indicating its advanced malignancy and metastasis. Treatment with mimics such as siRNA (siRNA-302) did not significantly reduce the malignancy of xenografted liver cancer cells / tumors (middle upper panel). The reason may be due to the short half-life of the siRNA molecule in vivo. In contrast, treatment with pro-mir-302 (= pre-mir-302) not only reprogrammed xenografted cancer cells into a normal hepatocyte-like morphology (unfused), but also cancer (cells). Successfully suppressed the invasion of normal tissue around (see middle and lower figures). Compared to normal liver tissue (bottom), pro-mir-302 treated malignant tumors have hepatic lobule-like structures, normal glandular-like sequences, and cell-cell and cancer-tissue junctions (black arrows) Showed very clear boundaries. This proved that the treated cancer (tumor) became relatively benign. Next, six or more consecutive treatments with the drug, including pro-mir-302, completely eliminated xenografted cancer cells / tumors in all six samples.

Mir-302 does not shorten telomeres, but activates cell senescence by activating p16Ink4a / p14ARF.Human induced pluripotent stem cells (iPS) accelerate senescence and limit expansion. (Banito et al., 2009; Feng et al., 2010). Normal mature cells undergo a finite number of divisions and ultimately reach a quiescence state called replicative senescence. Replicative senescence is a normal defense mechanism against the formation of tumor / cancer cells, since cells that have escaped replicative senescence often become immortal cells, such as tumor / cancer cells. In the present invention, we have discovered that mir-302 can trigger p16Ink4a / p14ART-related cell cycle regulation by directly silencing BMI-1. In other studies, BMI-1 activates the transcription of human telomerase reverse transcriptase (hTERT), which enhances telomerase activity to avoid replication senescence and increase cell life. It was further pointed out (Dimri et al., 2002). This indicates that overexpression of mir-302 causes hTERT-associated senescence of mirPS cells. To elucidate this, we performed a telomere repeat amplification protocol (TRAP) (Example 16) to measure telomerase activity. As shown in FIG. 9A, all mirPS cells treated with 10 μM doxycycline surprisingly resembled their native tumor / cancer cells and human stem cell H1 / H9 cells and showed intense telomerase activity. Western blot analysis also showed that hTERT expression was increased, but not decreased, in these mirPS cells (FIG. 9B). Telomerase PCR ELISA tests demonstrated that the relative activity of telomerase was increased (FIG. 9C). We were also able to further detect silencing of lysine-specific histone demethylase (also called lysine-specific histone demethylase AOF2, or KDM1 / LSD1) and histone deacetylase (histone deacetylase HDAC2) in mirPS cells (FIG. ). Previous studies have indicated that AOF2 is required for repression of hTERT transcription and that deficiency of both AOF2 and HDAC2 causes overexpression of hTERT (Won et al., 2002; Zhu et al., 2008). We also found in the present invention that AOF2 and HDAC2 were strong targets of mir-302 and were both silenced in mirPS cells (FIG. 10). Thus, in mirPS cells, mir-302 does not actually induce hTERT-associated senescence, but enhances telomerase activity in mirPS cells. However, the effect of increased hTERT activity was neutralized by suppression of BMI-1 and activation of p16Ink4a / p14ARF caused by mir-302, reaching a balance that prevented tumor / cancer cell formation in mir-302 cells. I have.

  In short, in the present invention, mir-302 having a novel tumor suppressor function is used as a therapeutic drug for cancer. We found that mir-302-mediated cell cycle regulation involves a highly coordinated mechanism between co-suppression of G1 checkpoint regulators and activation of CDK inhibitors. These events need to happen simultaneously to prevent any mistakes during G1-S progression. G0 / G1 quiescence during the cell cycle is crucial for the initiation of somatic cell reprogramming (SCR). In the dormant state, the somatic genome is heavily demethylated and more than 91% of the cell transcriptome is reprogrammed into a hES-like gene expression pattern. By elucidating the interaction between mir-302 and its target gene, we were able to recognize the complex mechanisms of cell cycle regulation associated with mir-302 during SCR (FIG. 11). In our previous studies, mir-302 silenced its targeted epigenetic regulators, while activating co-expression of Oct3 / 4-Sox2-Nanog, and subsequently reprogramming these Transcription factors have been shown to cause SCR (Lin et al., 2008). The present invention further proactively demonstrated that mir-302 attenuates cell division by simultaneously silencing CDK2, cyclins D1 / D2, and BMI-1 during SCR. Moderate control of cell cycle speed has biological significance in preventing the oncogenic ability of oncogenes that are well activated during SCR. In this regard, mir-302 silences CDK2 and cyclins D1 / D2 and inhibits G1-S transition. At the same time, suppression of BMI-1 further increases the activity of p16Ink4a and p19Arf tumor suppressors. Through the coordinated regulation of these cell cycle pathways, mir-302 can initiate the progression of somatic cell reprogramming (SCR) without exacerbating cell tumorigenesis.

  The invention has at least five frontal innovations. First, mir-302-like gene silencing effectors replace human cells with hES-like by replacing all four core reprogramming transcription factors Oct-Sox2-Klf-c-Myc and Oct4-Sox2-Nanog-Lin28. The stem cells can be reprogrammed. These reprogrammed cells are useful for stem cell therapy. Second, since the mir-302-like gene silencing effector has a small size (including about 23 ribonucleotides), the expression vector of such a small mir-302-like gene silencing effector is structurally By being more compact, transfection efficiency in vivo can be improved. Third, intracellular nonsense-mediated decay (NMD) systems and inducible expression systems can prevent cytotoxicity associated with RNA. Fourth, mir-302-mediated apoptosis occurs only in tumor / cancer cells and not in normal human cells. Finally, the present invention uses polysomal, liposomal, and electroporation transfection instead of retroviral / lentiviral infection to transform nucleic acid compositions expressing mir-302 into tumor / cancer. Delivery to cells ensures safety and use of mir-302-like gene silencing effectors in in vitro and in vivo treatments. Taken together, these innovations show the potential to treat tumors / cancers by using mir-302-like gene silencing effectors and compositions expressing the mir-302-like gene silencing effectors, A new design has been provided that can be developed into a therapeutic drug and / or vaccine.

A. Definitions To better understand the present invention, some terms are defined as follows.
Nucleotide: A unimolecular deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) containing a sugar component (pentose), a phosphate group, and a nitrogenous heterocyclic base. The base is linked to the sugar component by a glycosidic carbon (1 ′ carbon of the pentose), and the combination of the base and the sugar is a nucleoside. The nucleoside in which at least one phosphate group is bonded at the 3′-end or the 5′-end of the pentose is a nucleotide. That is, DNA and RNA are composed of different nucleotide units, that is, deoxyribonucleotide and ribonucleotide units, respectively.

  Oligonucleotide: A molecule containing two or more, preferably three or more, usually ten or more deoxyribonucleotides (DNAs) or ribonucleotides (RNAs). Oligonucleotides longer than 13 nucleotide monomers in length are also referred to as polynucleotides. The exact length will depend on many factors, as well as on the optimal function or use of the oligonucleotide. Oligonucleotides may be produced by any method, including chemical synthesis, DNA replication, RNA transcription, reverse transcription, or a combination thereof.

  Nucleotide Analog: a purine or pyrimidine nucleotide, which in structure is A (adenine), T (thymine), G (guanine), C (cytosine) or U (uracil) nucleotide Are different but sufficiently similar that they can replace normal nucleotides in a nucleic acid molecule.

  Nucleic Acid Composition: a mixture of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a mixture of deoxyribonucleic acid and ribonucleic acid (DNA / RNA) in the form of a single-stranded or double-stranded molecular structure Refers to such an oligonucleotide or polynucleotide.

  Gene: A nucleic acid composition, the nucleotide sequence of which encodes RNA and / or polypeptide (protein). The gene may be either RNA or DNA. Genes are non-coding RNAs, such as small hairpin RNA (shRNA), microRNA (miRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), It may encode molecular RNA (snRNA) and their precursors and derivatives. On the other hand, genes may also encode protein-coding RNAs essential for the synthesis of proteins / peptides, such as messenger RNA (mRNA) and its precursors and derivatives. In some examples, the protein-encoding RNA may include at least one miRNA or shRNA sequence.

  Primary ribonucleic acid transcript (Primary RNA Transcript): An RNA sequence transcribed by a gene without any RNA treatment or modification. Primary ribonucleic acid transcripts include mRNA, heteronuclear RNA (hnRNA), rRNA, tRNA, snoRNA, snRNA pre-microRNA, viral RNA, and their precursors and derivatives.

  Premessenger ribonucleic acid (Precursor messenger RNA, pre-mRNA): In eukaryotic cells, a gene encoding a protein is produced by a type II eukaryotic RNA polymerase (Pol-II) by an intracellular mechanism called transcription. Primary RNA transcript. The premessenger ribonucleic acid sequence includes a 5'-untranslated region (5'-UTR), a 3'-untranslated region (3'-untranslated region, 3-UTR), an exon (exon) and Introns are included.

  Intron: A portion or portions of a gene transcript sequence encoding a non-protein reading frame. For example, an in-frame intron, a 5'-terminal untranslated region (5'-UTR), and a 3'-terminal untranslated region (3'-UTR).

  Exon: A portion or portions of a gene transcript sequence that encodes a protein reading frame (cDNA). Said cDNAs are, for example, cellular genes, growth factors, insulin, antibodies and their analogs / homologs and derivatives.

  Messenger RNA (mRNA): Combined from pre-messenger ribonucleic acid exons. Premessenger ribonucleic acid is formed after removal of introns by intracellular RNA splicing machines (spliceosomes), and functions as protein-encoding RNA in peptide / protein synthesis. The peptides / proteins encoded by the mRNA include, but are not limited to, enzymes, growth factors, insulin, antibodies, and analogs / homologues and derivatives thereof.

Complementary deoxyribonucleic acid (cDNA): A single- or double-stranded deoxyribonucleic acid (DNA) having a sequence complementary to an mRNA sequence and containing no intron sequence.
Sense nucleic acid (Sense): A nucleic acid molecule identical in sequence and composition to mRNA. The sign “+”, “s” or “sense” indicates the sense nucleic acid structural form.

  Antisense nucleic acid: A nucleic acid molecule that is complementary to an individual mRNA molecule. The symbol "-" indicates this antisense nucleic acid structural form, or "a" or "antisense" is prefixed to DNA or RNA, such as "aDNA" or "aRNA".

  Base Pair (bp): In a double-stranded DNA molecule, adenine (adenine, A) and thymine (thymine, T) or cytosine (cytosine, C) and guanine (guanine, G) match (partnership) is there. In RNA, uracil (uracil, U) is used instead of thymine (thymine, T). In RNA, uracil may match guanine. Generally, the matches are linked via hydrogen bonding. As an example, the sense nucleic acid sequence "5'-A-T-C-G-U-3 '" is complementary to its antisense nucleic acid sequence "5'-A-C-G-A-T-3'" or "5'-G-C-G-A-T-3 '".

  5'-end: The end lacking one nucleotide at the 5 'end position of a contiguous nucleotide. In the contiguous nucleotides, the 5 'hydroxyl group of one nucleotide is linked to the 3' hydroxyl group of the next nucleotide by a phosphodiester bond. Other groups may be present at the terminus, such as one or more phosphate groups.

  3'-end: The end that lacks one nucleotide at the 3 'end position of a contiguous nucleotide. In the contiguous nucleotides, the 5 'hydroxyl group of one nucleotide is linked to the 3' hydroxyl group of the next nucleotide by a phosphodiester bond. At the end, other groups may be present, usually a hydroxyl group.

  Template: A nucleic acid molecule that can be copied by a nucleic acid polymerase. Depending on the different polymerases, the template may be single-stranded, double-stranded, or partially double-stranded. The synthesized copy is complementary to this template, at least one strand of the double-stranded template, or a partial double-stranded template. Both RNA and DNA are synthesized in the direction from the 5 'end to the 3' end. Since the two strands of a nucleic acid duplex are always aligned, the 5 'end of these duplexes is located at the opposite end of the duplex (if necessary, the The same applies to the 3 'end).

Nucleic Acid Template: a double-stranded DNA molecule, a double-stranded RNA molecule, a hybrid molecule (eg, a DNA-RNA or RNA-DNA hybrid), or a single-stranded DNA or RNA molecule.
Conserved: When a nucleotide sequence hybridizes non-randomly to one that is exactly complementary to a preselected sequence (the reference sequence), both sequences are conserved.

  Homologous or Homology: Refers to homology between a polynucleotide and a gene or messenger RNA sequence. A nucleic acid sequence may be partially or completely homologous to a particular gene or messenger RNA sequence. By way of example, homology can be expressed as a percentage of the total number of nucleotides occupied by the same nucleotide.

  Complementary (or complementarity or complementation): Two polynucleotides (ie, mRNA and cDNA sequences) whose bases have been matched according to the base pair (bp) rule. For example, the sequence "5'-A-G-T-3 '" is complementary to the sequences "5'-A-C-T-3'" and "5'-A-C-U-3 '", respectively. Complementation may occur between two DNA strands, between one DNA strand and one RNA strand, or between two RNA strands. The complement may be “partial” or “complete” or “whole”. If only some of the nucleobases are matched according to the base pairing rules, there is partial complementarity or complementation. When bases match completely or completely between these nucleic acid chains, complete or total complementarity or complementation is obtained. The degree of complementation between nucleic acid strands greatly affects the efficiency and strength of hybridization between nucleic acid strands. This is very important for amplification reactions, as well as detection methods that rely on binding between nucleic acids. Percent complementarity or complementation is the ratio of the number of mismatched bases to the total number of bases in a single strand of the nucleic acid. Thus, a 50% complementarity means that half of the bases are mismatched and half are matched. The two strands of a nucleic acid can be complementary even if the number of bases of the two strands is different. In this case, complementation occurs between some of the longer strands that are paired with the bases of the shorter strand.

Complementary base: A normally matching nucleotide when DNA or RNA has a double-stranded structure.
Complementary Nucleotide Sequence: The nucleotide sequence in the DNA or RNA of a single-stranded molecule is sufficiently complementary to the nucleotide sequence of the other single-stranded molecule, and is unique between the two strands by hydrogen bonding Hybridization.

  Hybridization (Hybridization and Hybridization): The formation of a duplex between nucleotide sequences that are sufficiently complementary to form a complex by base pairing. When a primer (or splice template) "hybridizes" with a target (template), the complex (or hybrids) formed by hybridization is sufficiently stable to initiate DNA synthesis by DNA polymerase. The priming function required for this is provided. There is a specific interaction between two complementary polynucleotides that is competitively inhibited (ie, non-random).

  Posttranscriptional Gene Silencing: The effect of target gene knockout or knockdown on mRNA degradation or translational repression, generally exogenous / viral DNA or RNA transgene or small repression Triggered by any of the sex RNAs.

  Ribonucleic acid interference (RNAi): A post-transcriptional gene silencing mechanism in eukaryotic cells, which is a small suppression such as microribonucleic acid (miRNA), small hairpin ribonucleic acid (shRNA) and small interfering ribonucleic acid (siRNA) It can be triggered by sex RNA molecules. These small RNA molecules generally function as gene silencers and interfere with the expression of genes that are completely or partially complementary to these small RNAs in cells.

  Non-coding RNA: Non-coding RNA is an RNA transcript that cannot be used for the synthesis of peptides or proteins by the mechanism of intracellular translation. Non-coding RNAs include long and short regulatory RNA molecules such as miRNAs, small hairpin RNAs, small interfering RNAs and double-stranded RNAs. These regulatory RNA molecules generally function as gene silencers, interfering with the expression of a gene in a cell that is completely or partially complementary to a non-coding RNA.

  Microribonucleic acid (MicroRNA, miRNA): A single-stranded RNA that can bind to a target gene transcript partially complementary to the microribonucleic acid (miRNA). MiRNAs are usually about 17-27 oligonucleotides in length and, depending on the degree of complementation between the miRNA and its target mRNA, directly degrade mRNA targets in cells or inhibit protein translation of that target mRNA can do. Natural miRNAs are found in almost all eukaryotic cells, and their role is like a defense against viral infection, as well as being able to regulate gene expression during animal and plant development. In principle, one miRNA targets multiple target RNAs to achieve its full function, or promotes complete gene silencing by targeting the same gene targeted by multiple miRNAs.

  Microribonucleic acid precursor (MicroRNA Precursor, Pre-miRNA): One hairpin like stem-arm and stem-loop region that interacts with Dicer, an intracellular RNA endoribonuclease RNase III Producing one or more mature microribonucleic acids (miRNAs) and silencing target genes of these microribonucleic acids or genes complementary to these microribonucleic acid sequences. Can be. The stem-arm region of the pre-microribonucleic acid (pre-miRNA) forms a completely (100%) or partially (mismatched) hybridized duplex, and the stem-loop region is linked to one end of the stem-arm duplex, The RNA-induced silencing complex (RISC) is combined with the Argonaute protein (AGO) by forming a circular or hairpin loop-like circular structure.

  Prokaryote-produced MicroRNA Precursor (Pro-miRNA): A small hairpin-like ribonucleic acid similar to natural ribonucleic acid precursor (pre-miRNA), but artificially produced in miRNA expression plasmid of prokaryotic competent cells. Introduce and transfer. By way of example, pro-miRNA is structurally similar to pre-mir-302, but is transcribed from the pLVX-Grn-miR302 + 367 or pLenti-EF1alpha-RGFP-miR302 vector of the competent E. coli DH5α cell line (implemented Example 18). Since prokaryotic cells do not normally express highly secondary structures like eukaryotic pre-miRNAs, the production of pro-miRNAs in prokaryotic cells usually involves chemical stimulation to stabilize the production of RNA secondary structures. Required (Lin, UA 13 / 572,263).

  Small interfering ribonucleic acid (Small interfering RNA, siRNA): A short double-stranded ribonucleic acid, a ribonucleotide duplex having about 18 to 27 perfect base pairs, which is almost completely complementary thereto Can degrade target gene transcripts.

  Small hairpin or short hairpin ribonucleic acid (small hairpin or short hairpin RNA, shRNA): a single-stranded ribonucleic acid comprising a pair of partially or completely matched stem arm nucleotide sequences, wherein the stem arm sequence is Separated by looped oligonucleotides, forming a hairpin-like structure. Many natural microribonucleic acids (miRNAs) are derived from hairpin-like ribonucleic acid precursors, immediately pre-microribonucleic acids (pre-miRNAs).

  Vector: A recombinant nucleic acid composition that can move or colonize in different genetic environments, for example, recombinant DNA (recombinant DNA, rDNA). Generally, other nucleic acid molecules will be operatively linked thereto. The vector is capable of autonomous replication in a cell, of which the vector and its ligated fragment are also replicated. Preferred vectors are episomes, ie, nucleic acid molecules that are extrachromosomally replicable. Preferred vectors are self-replicating and expressible nucleic acids. A vector capable of inducing the expression of a gene encoding one or more polypeptides and / or non-coding (non-coding) RNA is herein referred to as an "expression vector" or an "expression-competent vector". vector) ". Particularly important vectors allow the cloning of cDNA from mRNAs using reverse transcriptase. The components of the vector are a viral promoter, a type II (Type-II) RNA polymerase (Pol-II or pol-2) promoter or both, a Kozak translation initiation common sequence (Kozak consensus translation initiation site), a polyadenylation signal (polyadenylation). signals), multiple restriction / cloning sites, pUC origin of replication, SV40 early promoter to express at least one antibiotic resistance gene in replication competent prokaryotic cells (SV40 early promoter), a selective SV40 origin of replication for replication in mammalian cells (SV40 origin), and / or a tetracycline response element. The structure of the vector may be linear or circular of single-stranded or double-stranded DNA, and is selected from plasmids, viral vectors, transposons, retrotransposons, DNA transgenes, jumping genes and combinations thereof.

  Promoter: A nucleic acid that is recognized by or bound to a polymerase molecule and represses RNA transcription. In the present invention, a promoter may be a known polymerase binding site, an enhancer or analog thereof, or a sequence that initiates the synthesis of an RNA transcript by the required polymerase.

  Eukaryotic Promoter: A nucleic acid motif sequence required for gene transcription, which is a type II eukaryotic RNA polymerase (type II RNA polymerase (pol-2)), pol-2 equivalent and / or pol -2 Recognized by a compatible viral polymerase.

  Type-II RNA polymerase promoter (Type-II RNA polymerase (Pol-II or pol-2) promoter): An RNA promoter recognized by and bound to a type II eukaryotic RNA polymerase (Pol-II or pol-2). To transcribe eukaryotic messenger ribonucleic acid (mRNA) and / or microribonucleic acid (miRNA). By way of example, the pol-2 promoter may be, but is not limited to, a mammalian RNA promoter or a cytomegalovirus (CMV) promoter.

  Type-II RNA polymerase (Pol-II or pol-2) Equivalent: Eukaryotic transcription machinery, compatible with mammalian type II RNA polymerase (Pol-II or pol-2) and Pol-II Selected from the group consisting of viral RNA polymerase.

  Type II RNA polymerase compatible virus promoter (Pol-II (pol-2) Compatible Viral Promoter): a viral RNA promoter capable of gene expression using eukaryotic pol-2 or eukaryotic transcription machinery. By way of example, the pol-2 compatible viral promoter may be, but is not limited to, a cytomegalovirus (CMV) promoter or a lentiviral LTR (retroviral long terminal repeat) promoter.

Cistron: A nucleotide sequence in a DNA molecule that encodes an amino acid residue sequence and contains upstream and downstream DNA expression control elements.
Intron Excision: a cellular mechanism responsible for RNA processing, maturation, and degradation, including RNA splicing, exosome difestion, nonsense-mediated decay (NMD), and Including these combinations.

  Ribonucleic acid processing (RNA Processing): A cellular mechanism related to RNA maturation, modification, and degradation, including RNA splicing, exosome digestion, nonsense-mediated decay (NMD), and RNA editing ( RNA editing), RNA processing and combinations thereof.

Donor Splice Site: Includes SEQ.ID.NO.4 sequence, sequence homologous to SEQ.ID.NO.4, or 5'-GTAAG-3 '(SEQ.ID.NO.47) sequence It is a nucleic acid sequence.
Acceptor Splice Site: Includes SEQ.ID.NO.5 sequence, sequence homologous to SEQ.ID.NO.5, or 5'-CTGCAG-3 '(SEQ.ID.NO.48) sequence It is a nucleic acid sequence.

  Branch Point: Adenosine in a nucleic acid sequence, wherein the nucleic acid sequence is a SEQ.ID.NO.6 sequence, a sequence homologous to SEQ.ID.NO.6, or 5'-TACTAAC-3 '( SEQ.ID.NO.44) sequence.

  Poly-Pyrimidine Tract: a nucleic acid sequence containing a high percentage of thymidylic acid and cytidylic acid, wherein the nucleic acid sequence is SEQ.ID.NO.7, SEQ.ID.NO.8 or homologous thereto Including sequences.

Targeted Cell: One or more human cells selected from somatic cells, tissues, stem cells, germ cells, teratoma cells, tumor cells, cancer cells, and combinations thereof.
Cancerous Tissue: A tumor tissue selected from skin cancer, prostate cancer, breast cancer, liver cancer, lung cancer, brain tumor / cancer, lymph cancer, leukemia, and combinations thereof.

  Expression-Competent Vector: a linear or circular single-stranded or double-stranded DNA, such as a plasmid, a viral vector, a transposon, a retrotransposon, a DNA recombination gene, a jumping gene, and any of these. Selected from a combination.

  Antibiotic Resistance Gene: A gene expressed to have the ability to degrade an antibiotic, wherein the antibiotic is penicillin G, streptomycin, ampicillin (Amp), neomycin, G418, kanamycin, erythromycin, paromycin. Fofomycin, spectromycin, tetracycline (Tet), doxycycline (Dox), rifapicin, amphotericin B, gentamicin, chloramphenicol, cephalothin, tyrosine and combinations thereof.

  Restriction / Cloning Site: Restriction / Cloning Site: DNA motifs that are cleavage sites for restriction enzymes, and these restriction / cloning sites are AatII, AccI, AflII / III, AgeI, ApaI / LI, AseI. , Asp718I, BamHI, BbeI, BclI / II, BglII, BsmI, Bsp120I, BspHI / LU11I / 120I, BsrI / BI / GI, BssHII / SI, BstBI / U1 / XI, ClaI, Csp6I, DpnI, DraI / II, EagI , Ecl136II, EcoRI / RII / 47III / RV, EheI, FspI, HaeIII, HhaI, HinPI, HindIII, HinfI, HpaI / II, KasI, KpnI, MaeII / III, MfeI, MluI, MscI, MseI, NaeI, NaeI, NcoI , NdeI, NgoMI, NotI, NruI, NsiI, PmlI, Ppu10I, PstI, PvuI / II, RsaI, SacI / II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoIxma But not limited thereto.

  Gene Delivery: Genetic engineering methods, including polysomal transfection, liposomal transfection, chemical transfection, electroporation, viral infection, DNA recombination, transposon insertion, jumping gene insertion. , Microinjection, gene gun penetration, and combinations thereof.

  Genetic Engineering: A method of DNA recombination, selected from DNA restriction and ligation reactions, homologous recombination, transgene integration, transposon insertion, jumping gene insertion, retroviral infection, and combinations thereof. .

  Cell Cycle Regulator: A cell gene involved in controlling the rate of cell division and cell proliferation, and includes cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), and cyclin-dependent Sex kinase 6 (CDK6), cyclins, BMI-1, p14 / p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1 / Waf1, p27Kip1, and combinations thereof, but are not limited thereto.

  Tumor Suppression Effect: Mechanism and response of cell anti-tumor and / or anti-cancer, including cell cycle decay, cell cycle arrest, suppression of tumor cell growth, cell tumorigenesis Inhibition of tumor / cancer cell transformation, induction of tumor / cancer apoptosis, induction of recovery to normal cells, reprogramming of highly malignant cancer cells to a relatively benign low-stage state (tumor regression) ) And combinations thereof, but are not limited thereto.

  Cancer Therapy Effect: A cell response and / or cellular mechanism by drug treatment, which suppresses the expression of oncogenes, suppresses the growth of cancer cells, suppresses invasion and / or migration of cancer cells, and cancer Inhibits cell metastasis, induces cell death of cancer cells, avoids tumor / cancer development, avoids cancer recurrence, suppresses cancer progression, recovers damaged tissue cells, relatively benign and relatively benign of advanced malignant tumors This includes, but is not limited to, reprogramming to a low-stage state (cancer regression / remission) and combinations thereof.

  Gene Silencing Effect: Cell response after suppression of gene function, suppression of oncogene expression, suppression of cell proliferation, cell cycle arrest, G0 / G1 checkpoint arrest, cell cycle decay , Tumor suppression, antitumor formation, cancer regression, cancer avoidance, cancer cell apoptosis, cell recovery / regeneration, cell reprogramming, reprogramming diseased cells to a relative normal state (spontaneous treatment) and these Including, but not limited to, combinations.

  Transcription Inducer: A chemical agent capable of inducing and / or enhancing transcription of a eukaryotic gene against a pol-2 or pol-2-like promoter in prokaryotic cells. By way of example, the transcriptional derivative may have a chemical structure similar to, but not including, 3- (N-Morpholino) propanesulfonic acid (MOPS), ethanol, glycerin, or mixtures thereof. Not exclusively.

Antibody: A peptide or protein molecule having a preselected conserved domain structure, which can be a receptor that binds a preselected ligand.
Pharmaceutical and / or therapeutic applications: biomedical uses, devices and / or equipment, including diagnosis, stem cell generation, stem cell research and / or therapeutic development, tissue / organ recovery and / or regeneration, wound repair It is used for healing treatment, tumor suppression, cancer treatment and / or prevention, disease treatment, drug production, and combinations thereof.

B. Compositions and Methods Compositions and methods for inducing full tumor suppression and / or therapeutic effect on cancer, wherein nucleic acid compositions are used. The nucleic acid composition is delivered and treated with a mir-302-like gene silencing effector in a human cell matrix to suppress cell cycle regulators and / or oncogenes that are targeted by mir-302 in cells, resulting in tumors. / Suppress and / or avoid cancer cell growth. Wherein the mir-302-like gene silencing effector comprises the sequence SEQ.ID.NO.3, and the human cell matrix may be normal, or may comprise at least one tumor and / or cancer cell. .

  Preferably, the present invention uses a novel design and strategy to deliver a mir-302-like gene silencing effector that is induced or continuously expressed in transfected cells. mir-302-like gene silencing effectors are mir-302a, mir-302b, mir-302c, mir-302d, their hairpin-like microribonucleic acid precursors (pre-miRNAs), and manually redesigned Mimetics such as small hairpins (shRNAs), pro-miRNAs and / or small interfering RNAs (siRNAs) and homologs / derivatives thereof, and combinations thereof. Transfection of the mir-302-like gene silencing effector is achieved by vector or non-vector gene transfer systems. Among them, this transmission method is polysomal transfection, liposomal transfection, chemical transfection, electroporation, viral infection, DNA recombination, transposon insertion. (transposon insertion), jumping gene insertion, microinjection, gene gun penetration and combinations thereof. Due to the mode of vector transfer, expression of the mir-302-like gene silencing effector is driven by a sustained expression promoter (ie, CMV) or a drug-inducible (ie, TRE-CMV) promoter. The drug-inducible recombinant nucleic acid composition can be a recombinant mir-302 family cluster (mir-302s; hybrid of SEQ ID NOs. 9-16) or a manually redesigned mi-302 shRNA homolog (i.e., It is preferably a Tet-On vector containing a recombinant transgene into which a hybrid of SEQ. ID. NOs. 17 and 18 has been inserted. The cell matrix expresses the target gene of mir-302 in vitro, ex vivo or in vivo. By silencing cell cycle regulators and oncogenes targeted by this mir-302, the present invention suppresses the tumorigenic potential of cells and reprograms treated cells into non-tumor / cancer cells. Can be.

The following examples illustrate certain preferred specific embodiments and aspects of the invention by way of example, but do not limit the scope of the invention.
In the experimental files disclosed below, the following abbreviations apply: M (molar, molar), mM (millimolar, millimolar), μm (micromolar, micromolar), mol (mole, moles), pmol (picomole, picomole), gm (gram, grams), mg (milligram, milligrams), μg (micrograms, micrograms), ng (nanograms, nanograms), L (liters, liters), ml (milliliters, milliliters), μl (microliters, microliters), ° C (degrees Celsius, degrees Centigrade), cDNA (copy Or complementary DNA, copy or complementary DNA), DNA (deoxyribonucleic acid, deoxyribonucleic acid), ssDNA (single-stranded DNA, single stranded DNA), dsDNA (double-stranded DNA, double-stranded DNA), dNTP (deoxyribonucleoside- Triphosphate, deoxyribonucleotide triphosphate), RNA (ribonucleic acid, ribonucleic acid), PBS (phosphate buffered saline, phosphate buffered saline), NaCl (sodium chloride, sodium chloride), HEPES (N-2-hydroxyethylpiperazine- N-2-ethanesulfonic acid, N-2-hydroxyethylpi perazine-N-2-ethanesulfonic acid), HBS (HEPES buffered saline, HEPES buffered saline), SDS (sodium dodecyl sulfate, sodium dodecyl sulfate), Tris-HCl (tris-hydroxymethylaminomethane-hydrochloride, tris-hydroxymethylaminomethane -hydrochloride), ATCC (American Type Culture Collection, Rockville, MD), hES (human embryonic stem cells), iPS (induced pluripotent stem cells), And SCR (somatic cell reprogramming).

Example 1
Cell culture and transfection Human cancer cell lines NTera-2, HepG2, MCF7, PC3 and Colo829 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and human hair follicle cells (hHFCs) were obtained from at least two hairs. Obtained by digesting hair dermal papillae with 4 mg / ml collagenase, of which collagenase digestion works for 45 minutes in fresh RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS) I let it. Melanocytes (melanocytes) is a _{37 ° C, 5% CO 2} , human melanocyte growth supplement -2 (HMGS-2, Invitrogen, Carlsbad, CA) is added and 254 culture does not contain antibiotics (Medium 254). When cells have grown to 70% -80% confluency, cells are isolated by exposure to trypsin-EDTA solution for 1 minute, and phenol red-free DMEM culture (phenol red-free DMEM medium, Invitrogen), and the isolated cells were diluted 1:10 before subculturing in fresh 254 medium containing HMGS-2 supplement. For the step of gene transfer by electroporation, a mixture of the pTet-On-tTS-miR302s vector (10 μg) and the pTet-On-Adv-Neo (-) vector (50 μg) was added to a low osmotic buffer solution. In addition to the isolated cells (20,000-50,000) in (200 μl, Eppendorf, Westbury, NY), electroporation was also performed using an electroporator at 300-400 V for 150 μsec and the corresponding vectors were used. Delivered into these cells. Cells after electroporation are first phenol red-free, 20% serum replacement (Knockout serum), 1% MEM non-essential amino acids, 10 ng / ml basic fibroblast growth factor (bFGF), 1 mM GlutaMax, and 1 mM pyruvate. The cells were cultured in a DMEM culture solution containing sodium (Invitrogen) at 37 ° C. and 5% CO ₂ for 24 hours. Subsequently, G418 at 850 μg / ml and tetracycline (Dox) at a concentration higher than 3.75 μg / ml were added and continued for 3-5 days until the cells expressed intense red fluorescence (RGFP), as replaced daily. . Next, individual red fluorescent cells (mirPS) were monitored on a TE200 inverted microscope system (Nikon, Melville, NY) and collected individually in 96-well plates with a MO-188NE 3D micromanipulation system (Nikon). Under doxycycline (Dox) deficiency conditions, 20% Knockout serum, 1% MEM non-essential amino acids, 100 μM β-mercaptoethanol, 1 mM GlutaMax, 1 mM sodium pyruvate, 10 ng / ml basic fibroblasts DMEM / F- containing growth factor (bFGF), 100 IU / ml penicillin / 100 μg / ml streptomycin / 250 μg / ml G418, 0.1 μM A83-01, and 0.1 μM valproic acid (Stemgent, San Diego, CA) MirPS cells were cultured in 12 media under the conditions of 37 ° C. and 5% CO ₂ . On the other hand, mirPS cells were cultured under the same feeder-free culture conditions in the presence of doxycycline (Dox, 3.75 to 5 μg / ml, Sigma-Aldrich, St. Louis, MO) and further 0.05%. μM GSK inhibitor SB216763 (Stemgent) was added. Addition of GSK inhibitors promoted mirPS proliferation, but showed a slight tendency to induce neuronal differentiation. In terms of nerve cell induction, mirPS cells were cultured under the above feeder-free culture conditions containing 0.05 μM SB216763 and no doxycycline (Dox).

Example 2
Construction of recombinant vector expressing mir-302s
The generation of the Mir-302 family cluster (mir-302s) is as previously reported (Lin et al., 2008). The Mir-302s cluster contains four parts: pre-miRNAs of mir-302a, mir-302b, mir-302c, and mir-302d. Synthetic oligonucleotides (Sigma-Genosys, St. Louis, MO) used to construct the mir-302s cluster are listed below. In constructing the expression vector, we mixed an equal amount (1: 1) of mir-302s with the pre-made SpRNAi-RGFP recombinant gene (Lin et al., 2006 and 2008) and used the MluI / PvuI restriction enzyme. The mixture was digested at 37 ° C for 4 hours. Next, the digested mixture was purified with a gel extraction kit (Qiagen, CA), collected in 30 μl of double-distilled water (ddH ₂ O), and incubated at 8 ° C using T4 DNA ligase (T4 ligase). Working for 16 hours, the mixture was ligated. In this step, a recombinant SpRNAi-RGFP gene expressing mir-302 is formed, and further cleaved by XhoI / HindIII restriction enzymes, and then induced by doxycycline, a pSingle-tTS-shRNA vector (Clontech, Palo Alto, CA) Insertion into it formed an inducible pTet-On-tTS-miR302s expression vector. Subsequently, we further modified the pTet-On-tTS-miR302s vector, i.e., using the TRE-CMV promoter isolated from the pTRE-Tight plasmid (Clontech), the pTet-On-tTS-miR302s vector native U6. The promoter was replaced. In order to generate a non-inducible pCMV-miR302s persistent expression vector, we cut the modified pTet-On-tTS-miR302s vector with EcoRI restriction enzyme, and then upstream of the tTS-TRE by gel electrophoresis. By removing the sequence (1.5 kb) and obtaining a new vector cut from the gel, a DNA ligation step was performed to complete the construction of a non-inducible pCMV-miR302s vector.

  As a synthetic oligonucleotide used for DNA recombination of the mir-302 family preribonucleic acid (pre-miRNA) cluster, mir-302a-sense: 5′-GTCACGCGTT CCCACCACTT AAACGTGGAT GTACTTGCTT TGAAACTAAA GAAGTAAGTG CTTCCATGTT TTGGTGATGG ATAGATCTCT C -3 '(SEQ.ID.NO.9); mir-302a-antisense: 5'-GAGAGATCTA TCCATCACCA AAACATGGAA GCACTTACTT CTTTAGTTTC AAAGCAAGTA CATCCACGTT TAAGTGGTGG GAACGCGTGA C-3' (SEQ.ID.NO.10); mir-302b- Sense: 5'-ATAGATCTCT CGCTCCCTTC AACTTTAACA TGGAAGTGCT TTCTGTGACT TTGAAAGTAA GTGCTTCCAT GTTTTAGTAG GAGTCGCTCA TATGA-3 '(SEQ.ID.NO.11); '(SEQ.ID.NO.12); mir-302c-sense: 5'-CCATATGGCT ACCTTTGCTT TAACATGGAG GTACCTGCTG TGTGAAACAG AAGTAAGTGC TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3' (SEQ.ID.NO.13); mir-302c-antisense: 5 '-ATGTCTAGAC GCCTCCACTG AAACATGGAA GCACTTACTT CTGTTTCACA CA GCAGGTAC CTCCATGTTA AAGCAAAGGT AGCCATATGG-3 '(SEQ.ID.NO.14); mir-302d-sense: 5'-CGTCTAGACA TAACACTCAA ACATGGAAGC ACTTAGCTAA GCCAGGCTAA GTGCTTCCAT GTTTGAGTGT TCGCGATCGC AT-3' (SEQ.ID.NO.15); and mir -302d-antisense: 5'-ATGCGATCGC GAACACTCAA ACATGGAAGC ACTTAGCCTG GCTTAGCTAA GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3 '(SEQ.ID.NO.16). In addition, instead of the mir-302 pre-micro ribonucleic acid (pre-miRNA) cluster, synthesized miR-302s-sense: 5′-GCAGATCTCG AGGTACCGAC GCGTCCTCTT TACTTTAACA TGGAAATTAA GTGCTTCCAT GTTTGAGTGG TGTGGCGCGA TCGATATCTC TAGAGGATCC ACATC-3 ′ (SEQ. ID.NO.17) and mir-302s-antisense: 5'-GATGTGGATC CTCTAGAGAT ATCGATCGCG CCACACCACT CAAACATGGA AGCACTTAAT TTCCATGTTA AAGTAAAGAG GACGCGTCGG TACCTCGAGA TCTGC-3 '(SEQ.ID.NO.18) Simple intron insertion was performed by using the designed shRNA. In designing the mir-302 homolog, thymine (T) may be used instead of uracil (U), and vice versa. All of these synthetic sequences were extracted and purified by polyacrylamide gel electrophoresis (PAGE) prior to ligation.

  Recombined mir-302 family pre-microribonucleic acid cluster (mir-302s) contains mir-302a-sense and mir-302a-antisense, mir-302b-sense and mir-302b-antisense, mir-302c It was formed by linking four mir-302a-d hybrids, including -sense and mir-302c-antisense, and mir-302d-sense and mir-302d-antisense. These mir-302a, mir-302b, mir-302c, and mir-302d hybrids were digested with PvuI / XhoI, XhoI / NheI, NheI / XbaI and XbaI / MluI restriction enzymes, extracted with gel, and then filtered. Collected together in a column (Qiagen, CA) in 35 μl of sterile double distilled water. The hybrid mixture was ligated with T4 DNA ligase (Roche, 20U) to form clusters. Finally, a pre-microribonucleic acid cluster of the mir-302 family was inserted into the SpRNAi-RGFP recombinant gene linearized by PvuI / MluI restriction enzymes. Further, after hybridizing SEQ.ID.NO.17 and SEQ.ID.NO.18, the hybrid was cut using PvuI / MluI restriction enzyme, and the cut hybrid was linearized with PvuI / MluI. Mir-302 shRNA was formed by insertion into the resulting SpRNAi-RGFP.

  In order to propagate the pTet-On-tTS-miR302s and CMV-miR302s vectors, a strain of Escherichia coli DH5a was cultured in an LB medium containing ampicillin (Sigma Chemical, St. Louis, MO) at a concentration of 100 μg / ml. Then, using an endotoxin-free maxi-prep plasmid extraction kit (Endo-Free Maxi-Prep Plasmid Extraction Kit, Qiagen, CA), the pTet-On-tTS-miR302s and CMV-miR302s vectors after growth were isolated and purified. .

Example 3
In microarray analysis confluency of 70% of the micro-ribonucleic acid (miRNA), mirVana ^TM micro ribonucleic acid isolation kit ^{(mirVana TM miRNA isolation kit, Ambion} ) using a small RNAs were isolated from each cell culture. To evaluate the purity and content of isolated small RNAs by 1% formaldehyde-agar gel electrophoresis and spectrometer measurement (Bio-Rad), immediately freeze on dry ice, and perform microarray analysis of microribonucleic acid Was sent to LC Sciences (San Diego, CA). Each microarray chip was hybridized with a single sample labeled with Cy3 or Cy5, respectively, or with a pair of samples labeled with Cy3 and Cy5. Next, background subtraction and normalization were performed. In duplicate sample analysis, p-values were calculated and transcripts expressed with a difference greater than three-fold were listed. The results are shown in FIG. 1C.

Example 4
Northern blot analysis
mirVana ^TM micro ribonucleic acid isolation kit (Ambion, Austin, TX) was isolated total RNAs (10 [mu] g) in the該全RNAs by 15% TBE-urea polyacrylamide gels or 3.5% low melting point agarose gel electrophoresis After fractionation, the fractionated total RNAs were electroblotted on a nylon membrane. Subsequently, mir-302 was detected with [LNA] -DNA probe (5 '-[TCACTGAAAC] ATGGAAGCAC TTA-3') (SEQ. ID. NO. 19). Probes for detecting other genes were further synthesized, and their sequences are shown in Table 1. All probes were purified by high performance liquid chromatography (HPLC) and in the presence of [ ³² P] -dATP (> 3000 Ci / mM, Amersham International, Arlington Heights, Ill.) In the presence of terminal transferase, 20 units). The hybridization reaction is a mixture of 50% freshly deionized formamide (pH 7.0), 5x Denhardt's solution, 0.5% SDS, 4x SSPE, and 250 mg / mL denatured salmon sperm DNA fragment. (18 hours, 42 ° C). Next, the membrane was washed twice consecutively with 2 × SSC, 0.1% SDS (15 minutes, 25 ° C.), washed once with 0.2 × SSC, 0.1% SDS (45 minutes, 37 ° C.), Then, autoradiography was performed. The results are shown in FIGS. 1D, 4A and 8B.

Example 5
Western blot analysis According to the manufacturer's suggestion, cells were treated with CelLytic-M lysis / extraction reagent (Sigma) using CelLytic-M lysis / extraction reagent supplemented with protease inhibitor, leupeptin, TLCK, TAME and PMSF. (1,000,000) were dissolved. Next, the lysate was centrifuged at 12,000 rpm at 4 ° C. for 20 minutes to obtain a supernatant after centrifugation. The protein concentration was measured using an improved SOFTmax protein measurement package software using an E-max microplate reader (microplate reader, Molecular Devices, CA). Under reducing (reducing, +50 mM DTT) and non-reducing (non-reducing, no DTT) conditions, 30 μg of each cell lysate was added to the SDS-PAGE sample buffer, and the mixture was boiled for another 3 minutes. Was loaded on a 6% -8% polyacrylamide gel. Next, after analyzing the protein by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was electroblotted on a nitrocellulose membrane, and an Odyssey blocking reagent (Li-Cor The membrane was incubated for 2 hours at room temperature in Biosciences, Lincoln, NB). Then, the primary antibody was added to the reagent, and the mixture was cultured at 4 ° C. Primary antibodies used were Oct3 / 4 (Santa Cruz Biotechnology, Santa Cruz, CA), Sox2 (Santa Cruz), Nanog (Santa Cruz), Lin28 (Abcam Inc., Cambridge, MA), UTF1 (Abcam), Klf4 ( Santa Cruz), TRP1 (Santa Cruz), keratin 16 (Abcam), CDK2 (Santa Cruz), cyclin D1 (Santa Cruz), cyclin D2 (Abcam), BMI-1 (Santa Cruz), AOF2 (Sigma), HDAC2 (Sigma) Abcam), hTERT (Santa Cruz), s-actin (Chemicon, Temecula, CA), and RGFP (Clontech). After overnight, the membrane was rinsed three times with TBS-T and exposed to goat anti-mouse IgG (goat anti-mouse IgG) for 1 hour at room temperature. The goat anti-mouse IgG was combined with Alexa Fluor 680 reactive dye (1: 2,000, Invitrogen-Molecular Probes) to form a secondary antibody. After rinsing three more times with TBS-T, fluorescence scanning and image analysis of immunoblots were performed using a Li-Cor Odyssey Infrared Imager and Odyssey software V.10 (Li-Cor). went. The results are shown in FIGS. 1D, 4B, 7C to 7D, 8B and 9B.

Example 6
DNA ladder test for apoptotic cells Isolate and isolate genomic DNA from approximately 2,000,000 cells using the DNA ladder kit for apoptotic cells (Apoptotic DNA Ladder Kit, Roche Biochemicals, Indianapolis, Iowa) as suggested by the manufacturer. 2 μg of the DNA thus obtained was evaluated by 2% agar gel electrophoresis. The result is shown in FIG. 1F.

Example 7
Flow cytometric analysis of DNA content After completing the necessary experiments, cells were hydrolyzed with trypsin, centrifuged into granules, and further suspended in pre-cooled PBS containing 1 ml 70% methanol. Cells were fixed at -20 ° C for 1 hour. Then, these cells were centrifuged into granules and washed once with 1 ml of PBS. The cells were again centrifuged into a granule and incubated at 37 ° C for 30 minutes in 1 ml of a PBS solution containing 1 mg / ml propidium iodide (PI), 0.5 μg / ml ribonuclease (RNase). Resuspended. Thereafter, about 15,000 cells were analyzed by BD FACSCalibur flow cytometry (San Jose, CA). A pulse width vs. pulse area diagram was drawn and cell doublets were excluded by gating single cells. The collected data was analyzed with the "Watson Pragmatic" algorithm using the package software Flowjo. The results are shown in FIGS. 3A to 3B and FIGS. 6A to 6C.

Example 8
Genome-wide microarray analysis Using a human genome GeneChip U133 plus 2.0 array (Affymetrix, Santa Clara, CA), changes in the expression pattern of more than 47,000 human genes in test cells were detected. In accordance with proposal of the manufacturer, mirVana ^TM micro ribonucleic acid isolation kit ^{(mirVana TM miRNA Isolation Kit, Ambion} ) using the total RNA was isolated from each test sample. The purity and content of the isolated RNA were evaluated by 1% formaldehyde-agar gel electrophoresis and spectrometer measurements (Bio-Rad). These sample signals were normalized by the overall average difference between perfect and mismatched probes. Then, using Affymetrix Microarray Suite 5.0 version, Expression Console ^™ 1.1.1 version (Affymetrix) and Genesprings (Silicon Genetics) software, changes in gene expression patterns of the entire genome were analyzed. If the change in gene expression was greater than one-fold, it was considered a positive difference gene. In the gene clustering test (gene clustering), a plug-in program Genetrix (Epicenter Software) was used in combination with Affymetrix software. The signal of the sample was normalized by the average of the control group housekeeping genes in each microarray. The result is shown in FIG. 5A.

Example 9
DNA demethylation test
Genomic DNA in about 2,000,000 cells was isolated using a DNA isolation kit (Roche). 1 μg of the isolated DNA was taken and subjected to bisulfite treatment (CpGenome DNA modification kit, Chemicon, Temecula, CA) according to the manufacturer's proposal. On the other hand, 2 μg of the isolated DNAs was taken and digested with CCGG cleavage restriction enzyme HpaII, and then demethylation of the whole genome was determined by 1% agar gel electrophoresis (FIG. 5B). Treatment of the DNA with bisulfite converted all unmethylated cytosine to uracil, while maintaining methylated cytosine as cytosine. In bisulfite DNA sequencing analysis, we used the polymerase chain reaction (PCR) to amplify the Oct3 / 4 and Nanog promoter regions. Primers used for amplification of the Oct3 / 4 promoter region were 5′-GAGGCTGGAG CAGAAGGATT GCTTTGG-3 ′ (SEQ.ID.NO.20) and 5′-CCCTCCTGAC CCATCACCTC CACCACC-3 ′ (SEQ.ID.NO.21 )including. Primers used for amplification of the Nanog promoter region are 5'-TGGTTAGGTT GGTTTTAAAT TTTTG-3 '(SEQ.ID.NO.22) and 5'-AACCCACCCT TATAAATTCT CAATTA-3' (SEQ.ID.NO.23 )including. First, mix bisulfite-modified DNAs (50 ng) with these primers (total 100 pmole) in 1x PCR buffer, heat to 94 ° C, keep for 2 minutes, and immediately cool on ice did. Thereafter, 25 cycles of PCR were performed using a high sensitivity PCR kit (Expand High Fidelity PCR kit, Roche) at 94 ° C for 1 minute and at 70 ° C for 3 minutes. After completion of the amplification reaction, the DNA product having the correct length was further fractionated by 3% agar gel electrophoresis, and then the DNA product was purified using a gel extraction kit (Qiagen) and subjected to DNA sequencing. Was. The detailed pattern of DNA methylation sites was obtained by comparing unchanged cytosine in bisulfite-modified DNA sequences with unchanged cytosine in bisulfite-unmodified DNA sequences. . The result is shown in FIG. 5C.

Example 10
Transplantation and Teratoma Formation Approximately 5 to 10 mirPS cell-derived embryoid bodies (4 to 8 cell stages) are suspended in 50 μl of a 2: 1 mixture of DMEM culture medium and Matrigel, and then MirPS cell-derived embryoid bodies were transplanted into the uterus of 6-week-old female pseudopregnant immunodeficient SCID-beige mice. A method for making pseudopregnant mice is to inject 1 IU of human menopausal gonadotrophin (HMG) into the peritoneum for 2 days, and then add another human chorionic gonadotrophin (hCG). Injected for days. The cells and the mice are not treated with doxycycline (Dox) before or after transplantation. During the implantation period, a single mouse was anesthetized with a 2.5% tribromoethanol (Avertin) solution in a volume of 0.4 ml. After transplantation or after said transplant cell population is grown to 100 mm ³ larger size was monitored for 4 weeks heterologous transplant population (Xenografted masses) from 3. After disconnecting the該嚢tumor / teratoma, to calculate the volume using equations (length × width ²⁾ / 2, it was carried out further counted, weighed and further histological analysis. Teratoma-like tissue cysts can usually be observed about 2.5 weeks after transplantation. The result is shown in FIG. 5D.

Example 11
Cell penetration test First, use 200 μg / ml Matrigel alone, or use Matrigel supplemented with a phenol red-free DMEM culture solution containing 20% FBS and 1% L-glutamine (L-Glutamine) to insert the chamber. (chamber inserts, hole diameter: 12 μm, Chemicon), and dried under a sterile environment until the next day. Cells were collected, rinsed, and resuspended to a final cell density of 100,000 cells / ml using phenol red-free DMEM culture. A 500 μl aliquot of the cell suspension was dispensed into a top chamber and a chemotactic gradient was created by adding 1.5 ml DMEM conditioned medium to the bottom chamber. After culturing at 37 ° C overnight for 16 hours, the invasion state was measured. First, wipe the top chamber with absorbent cotton, and fix the invading cells located on the lower side of the membrane using 100% methanol for 10 minutes, air-dry, stain with cresyl violet (cresyl violet) for 20 minutes, and then use water. Washed gently. After drying, the cresyl violet stain on the membrane was washed with a washing solution containing 1: 1 100% ethanol and 0.2 M sodium citrate (NaCitrate) for 20 minutes, and a wavelength of 570 was collected with a Precision Microplate Reader (Molecular Dynamics). The absorbance at nm was read. The method of indicating the cell invasion rate was the percentage of the absorbance of the test sample to the absorbance obtained when the membrane layer of the chamber insert was not dried (total number of cells). The result is shown in FIG. 6D.

Example 12
Cell adhesion test The cell adhesion test was performed as described in a previous report (Lin et al., 2007). Human bone marrow endothelial cells (hBMECs) are seeded at a density of 100,000 cells / ml in a 96-well plate, and an adhesion medium [RPMI 1640 / 0.1% BSA / 20 mM HEPES (pH 7.4)] before the test. I was rinsed. The test cells are isolated using trypsin (used for tumor / cancer cells) or collagenase (used for mirPS cells), rinsed with sterile saline, and then 10 μM fura-4 acetoxymethyl ester. The cells were resuspended at a density of 1,000,000 cells / ml in PBS containing a fluorescent probe (acetoxymethyl ester, fluorescent probe, Sigma) for 1 hour in a dark environment at 37 ° C. Subsequently, the cells were centrifuged, and the cells were rinsed with a serum-free culture medium containing 1% (v / v) probenecid (probenecid, 100 mM) .The cells were then placed in an adherent culture medium at 37 ° C in a dark environment. The cells were cultured for 20 minutes to activate the fluorescent probe in the cells. Thereafter, the 100,000 cells (300 μl of cell suspension / well) were added to a confluent hBMEC endothelial cell monolayer and cultured at 37 ° C. for 50 minutes. Non-adhered cells were washed away by rinsing twice with 250 μl of adhesion medium. Fluorescence was read at 37 ° C at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a fluorescent plate reader (Molecular Dynamics). The result is shown in FIG. 6E.

Example 13
In Vivo Tumor Growth TestsWe injected NTera-2 cells (2,000,000 cells in Matrigel-PBS with a total volume of 100 μl) into 8-week-old male mice (BALB / c-nu / nu species) in the flank ( That is, xenotransplantation was performed on the hind limb. The tumors were monitored weekly and one week after NTera-2 xenograft, the pCMV-miR302s or pCMV-miR302d * vectors were introduced by in situ injection. Five treatments (every 3 days) were performed with 2 μg (per 1 g of mouse body weight) of pCMV-miR302s or pCMV-miR302d * vector (total 10 μg) prepared with polyethyleneimine (PEI). In vivo-jetPEI delivery reagent (Polyplus-transfection Inc., New York, NY) was used according to the manufacturer's use suggestions. Sampling began 3 weeks after injection or after non-vector-treated tumors grew to about 100 mm ³ in average size. All major organs such as blood, brain, heart, lung, liver, kidney and spleen, and xenograft tumors were removed for histological evaluation of tumors and immunoreactivity cytotoxicity studies. The tumor formation was monitored by palpation, and the tumor volume was calculated by the formula (length × width ² ) / 2. Tumors were also counted, dissected, and weighed, and histologically examined by hematoxylin-eosin staining (H & E) and immunostaining tests. Histological examination did not detect any detectable histological lesions in the brain, heart, lung, liver, kidney and spleen. The results are shown in FIGS.

Example 14
Immunostaining test Tissue samples were fixed overnight at 4 ° C with 4% paraformaldehyde. Before embedding the sample in paraffin wax, it was washed with 1 × PBS, methanol, isopropanol and tetrahydronaphthalene in this order. The embedded samples were then cut with a microtome to a thickness of 7-10 μm and fixed on clean TESPA-coated glass slides. The glass slide was then dewaxed with xylene, fixed under a cover glass with mounting media (Richard Allan Scientific, Kalamazoo, MI), and hematoxylin and eosin (eosin). ) (H & E, Sigma) and observed for morphology. Immunohistochemistry (IHC) staining kit was purchased from Imgenex (San Diego, CA). Antibody dilution and immunostaining steps were performed according to the manufacturer's suggestions. The primary antibodies used were Oct3 / 4 (Santa Cruz), Sox2 (Santa Cruz), Nanog (Santa Cruz), CDK2 (Santa Cruz), cyclin D1 (Santa Cruz), cyclin D2 (Abcam), BMI-1 ( Santa Cruz) and RGFP (Clontech). Biotin-conjugated goat anti-rabbit or biotin-conjugated horse anti-mouse antibody (Chemicon, Temecula, CA) was used as the secondary antibody. Horseradish peroxidase (Streptavidin-HRP) with streptavidin as a tertiary antibody was added. After the slide glass was washed three times with PBT, the bound antibody was detected with a DAB substrate. Positive results were observed with a 100 × microscope equipped with full-field scanning, and quantitative analysis was performed using a Metamorph image processing program (Nikon 80i microscopic quantitative analysis system) at a magnification of 200 ×. The results are shown in FIG. 8C.

Example 15
Luciferase 3'-terminal untranslated region reporter gene test The luciferase test was performed using a modified pMir-Report miRNA expression reporter vector system (pMir-Report miRNA Expression Reporter Vector System, Ambion). The target site of Mir-302 (normal and / or mutated) was inserted into the cloning site of the 3 'untranslated region (3'-UTR) of the pMir-Report Luciferase Reporter reporter vector. The two synthesized target sites were separated by 12 -CAGT-overlapping sequences. Another pMir-Reportβ-gal Control vector was used as a control vector without reporter. 50,000 mirPS cells were transfected with 200 ng of reporter vector using FuGene HD reagent (Roche) with or without doxycycline (Dox) according to the manufacturer's suggestion. Cell lysates were collected 48 hours after transfection. The level of luciferase degradation is normalized (normalized) and expressed as relative luciferase activity (relative luciferase activity, RLA), and its calculation method is based on the luciferase activity level of mirPS cells treated with doxycycline (Dox-on). Dividing by the luciferase activity level of mirPS cells not treated with doxycycline (Dox-off). Further, cells expressing mir-434 were generated by introducing pTet-On-tTS-miR434-5p into hHFCs by electroporation, and were used as a negative control group. The results are shown in FIGS. 7B and 10B.

Example 16
Telomere repeat amplification protocol test According to the manufacturer's proposal, about 1,000,000 cells were treated with a cell lysis / extraction reagent (CelLytic-M lysis / extraction reagent, Sigma) supplemented with protease inhibitors, leupeptin, TLCK, TAME and PMSF. Dissolved. The lysate was centrifuged at 4 ° C. for 20 minutes at a rotation speed of 12,000 rpm, and the lysate (supernatant) after centrifugation was collected. Subsequently, the protein concentration was measured with an E-max microplate reader (Molecular Devices, CA) using the improved SOFTmax protein measurement package software. Infrared fluorescent dye (infrared Alexa Fluor 680 dye, TS Primer, Sigma-Genosys) labeled oligonucleotide 5'-AATCCGTCGAGCAGAGTT-3 '(SEQ.ID.NO.24) and 5'-GTGTAACCCTAACCCTAACCC-3' (CX primer , 30 μM) (SEQ. ID. NO. 25) was used to detect polymerase chain reaction (PCR) products. Telomerase inhibitors were added directly to the main mixture. For all test cell lines, optimal results were achieved using 50 ng of protein per reaction. After incubating for 30 minutes at 30 ° C, place the sample in a polymerase chain reactor, heat to 94 ° C, hold for 2 minutes, then synthesize denaturation at 94 ° C for 30 seconds, 57 ° C The synthesis was performed for 35 cycles of 30 seconds of PCR. Finally, a single post-synthesis step was performed at 57 ° C. for 30 seconds. Next, the PCR products were fractionated by 6% non-denaturing polyacrylamide gel (Nondenaturing polyacrylamide gel) electrophoresis, and a Li-Cor Odyssey infrared imager (Li-Cor Odyssey Infrared Imager) and Odyssey software V.10 ( The images were detected and analyzed using Li-Cor). The result is shown in FIG. 9A.

Example 17
Statistical analysis In immunostaining, Western blot and Northern blot analysis, any change in signal intensity greater than 75% was considered a positive result, and after these results were analyzed, the mean ± standard deviation (mean ± SE) It was represented by Statistical analysis of the data was calculated by one-way ANOVA. When the main effect was significant, a group significantly different from the control group was determined by Dunnett's post-hoc test. A two-tailed student ttest was used when comparing and comparing two treatment groups. In experiments involving more than two treatment groups, post-hoc multiple range tests were performed after ANOVA. A probability p <0.05 was considered statistically significant. All p's were determined from two-tailed tests.

Example 18
Cell transformation and induction of pre-miRNA expression
Competent Escherichia coli DH5α cell line (competent E. coli DH5α) obtained from z-competent E. coli transformation kit (z-competent E. coli transformation kit, Zymo Research, Irvine, CA) and pLVX-Grn-miR302 + 367 Alternatively, transformation was performed by mixing with a selected plasmid vector such as pLenti-EF1α-RGFP-miR302. Untransformed bacterial cells were frequently shaken at 170 rpm while growing normally in LB (Luria-Bertani) medium at 37 ° C. supplemented with 10 mM magnesium sulfate (MgSO ₄ ) and 0.2 mM glucose. The transformed bacterial cells were further cultured in LB medium supplemented with 100 μg / ml ampicillin. On the other hand, in an LB medium supplemented with 10 mM magnesium sulfate and 0.2 mM glucose and supplemented with 100 μg / ml ampicillin, 0.5 to 2 ml of MOPS was added per liter for chemical induction. In addition, the transformed bacterial cells were used as a negative control group and cultured in the LB medium to which the above-mentioned ampicillin was added but no chemical derivative was added.

Example 19
Human cell culture and intracellular transmission of Mir-302Human primary epidermal skin cells (hpESCs) were prepared in fresh RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS). in mg / ml collagenase I, is obtained by at least 2 mm ³ were treated for 35 minutes from volume isolated at 37 ° C.. The isolated keratinocytes are supplemented with human keratinocyte growth supplements (HKGS, Invitrogen, Carlsbad, CA) at 37 ° C and 5% carbon dioxide and contain antibiotics. Cultured in no EpiLife serum-free cell culture. When the cells have grown to 50% to 60% confluency, the cells are exposed for 1 minute in a trypsin / EDTA solution and phenol red-free DMEM medium (Invitrogen). Rinse once and the isolated cells were diluted 1:10 and subcultured in fresh EpiLife broth containing HKGS supplements. The human cancer / tumor cell lines Colo-829, PC3, MCF7, HepG2 and Tera-2 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured under the conditions suggested by the manufacturer. For microRNA transfection, 15 μg of isolated miR-302 and / or its precursor were dissolved in EpiLife broth mixed with 50 μl liposome / polysome DNA transfection agent (X-tremeGENE HP DNA transfection reagent). After culturing for 10 minutes, the mixture was added to hpESCs or cancer / tumor cells that reached 50% to 60% confluency, respectively, in a 100 mm cell culture dish. After 12-18 hours, the original culture was replaced with fresh EpiLife culture containing HKGS supplement or the culture proposed by ATCC. The transfection step may be repeated 3-4 times every 3-4 days to improve transfection efficiency. After cell morphology changes into a ball, 20% Knockout serum, 1% MEM non-essential amino acids, 100 μM β-mercaptoethanol, 1 mM GlutaMax, 1 mM sodium pyruvate, 10 ng / ml basic Fibroblast growth factor (bFGF), 10 ng / ml FGF-4, 5 ng / ml LIF, 100 IU / ml penicillin / 100 μg / ml streptomycin, 0.1 μM A83-01, and 0.1 μM valproic acid (Stemgent, Sangen These cells (mirPSCs) were cultured and subcultured in a knockout DMEM / F-12 culture medium supplemented with Diego, CA) at 37 ° C. and 5% CO ₂ .

Example 20
Plasmid DNA / total RNA / microRNA extractionCompetent Escherichia coli DH5α cells transformed with the plasmid (Example 18) were cultured overnight at 37 ° C. in LB medium supplemented with 10 mM magnesium sulfate and 0.2 mM glucose. Shake frequently at rpm. In addition to bacterial culture and amplification, the addition of an additional 0.5-2 ml MOPS per liter of the medium induced production of ribonucleic acids and / or proteins driven by eukaryotic promoters. Using a plasmid purification kit (HiSpeed plasmid purification kit, Qiagen, Valencia, CA), all the amplified plasmid DNAs and the expressed mRNA / microRNAs precursor (pre-miRNAs) were isolated together. Among them, the isolation step followed the one suggested by the manufacturer but was slightly adjusted, that is, the ribonuclease RNase A was not added to the P1 buffer. Final extraction products, including both plasmids and mRNAs / pre-miRNAs, were dissolved in DEPC-treated double distilled water (ddH ₂ O) and stored at -80 ° C until used. If it is desired to purify only the amplified plasmid vector, the ribonuclease RNase A should be added to the P1 buffer according to the steps suggested by the manufacturer.

Example 21
Purification of Mir-302 and its precursor Further, the entire RNA isolated by the method in Example 20 was purified using the mirVana ^™ miRNA isolation kit (Ambion, Austin, TX) according to the manufacturer's proposal. The final product was dissolved in double distilled water (ddH ₂ O) treated with DEPC and stored at -80 ° C until used. Bacterial RNAs degrade very quickly in nature (several hours), while eukaryotic poly-A RNAs (mRNAs) and hairpin-like microRNA precursors (pre-miRNA / pro-miRNA) are relatively stable at 4 ° C. Because (the half-life can reach 3-4 days), we can take advantage of this difference to obtain pure mRNAs and / or pre-miRNAs / pro-miRNAs for later application. Was. As an example, RGFP mRNA can be used to determine cell transfection, and pre-miR-302s / pro-miRNAs can be used to reprogram somatic cells into embryonic stem cell-like (hES-like) iPS cells, or Used to treat tumor / cancer cells. Purified pre-miR-302s / pro-mir-302s can also be used to reprogram highly malignant cancer cells to a relatively benign, low-stage state. This is a beneficial result for treating cancer.

Example 22
In vivo wound healing and tissue recovery / regeneration studies
Pro-miR-302s and related plasmid vectors were amplified and extracted by the methods described in Examples 18 and 20, and further purified by the methods described in Examples 20 and 21. Next, the isolated pro-miR-302s was prepared with an ointment base containing previously prepared cocoa butter, cottonseed oil, olive oil, sodium pyruvate, and white petrolatum. The concentration of this pro-mir-302 in the prepared ointment base was about 10 μg / mL. The skin was cut open with a dissecting scalpel to form an open wound on the skin, approximately 0.5 x 0.5 square centimeters. Ointment (about 0.3 mL) was applied directly to the wound and covered the entire wound. Subsequently, the area treated with a further liquid bandage was sealed.

Example 23
In Vivo Therapeutic Study of Liver Cancer We have xenografted malignant human liver tumors into immunodeficient SCID-beige mice and constructed an effective animal model to study liver cancer metastasis and treatment. To construct this model, we mixed 5 million human liver cancer cells (HepG2) with 100 μL matrix gel and implanted this mixture subcutaneously in the hind flank of the mouse hind limb. Therefore, there are approximately equal amounts of cancer cell transplants in the hind legs on both sides of the mouse. About 2 weeks after transplantation, the malignant tumor was observed. Among them, the average size of the tumor was about 15.6 ± 8 mm ³ (ie, the initial size of the tumor before treatment). In each mouse, we selected the side with the larger tumor as the experimental group and the other side with the smaller tumor as the control group. One side of the same mouse was treated with a blank formulation (negative control group) and the other side was treated with a preparation containing pro-mir-302, resulting in minimal inter-individual differences.

  To deliver pro-mir-302 in vivo to the targeted cancer area, we employed the methods and products of a specialized pharmaceutical company, Latitude (San Diego, CA), wrapped pro-mir-302 in liposomes, Granules of 160-200 nm in diameter were formed. Tests show that these granules containing pro-mir-302 were almost 100% stable after 2 weeks at room temperature and 1 month at 4 ° C, whereas other synthetic The mimetic such as siRNA (siRNA-302) was rapidly degraded by more than 50% in 3 to 5 days, indicating that pro-miRNA was more stable than siRNA as a therapeutic agent. To perform the toxicity test, we additionally injected pro-mir-302 (1 mg / mL), prepared to a maximum volume of 300 μL, into the tail vein (n = 8) of each mouse. Over a period of more than 6 months, we observed no measurable side effects in all of the test mice. In general, unmodified nucleic acids are relatively insensitive to immunity and can be readily metabolized from tissue cells, making them a safe tool for in vivo therapeutics.

To test efficacy, we injected 200 μL of pro-mir-302 prepared on one side of the mouse subcutaneously, injected 200 μL of blank prescription reagent on the other side, and once a week in the same manner. Three consecutive injections were made. The drug and reagent were administered to the surrounding area where the tumor was located, and were absorbed into the tumor and surrounding tissue within 18 hours. Samples were collected one week after the third injection. Heart, liver, kidney and transplanted malignant tumors were excised for further histological evaluation. The tumor formation was monitored by palpation, and the tumor volume was calculated by the formula (length × width ² ) / 2. Tumors were also counted, dissected, and weighed, and histologically examined by hematoxylin-eosin staining (H & E) and immunostaining tests. Histological examination did not detect any detectable histological lesions in the heart, liver, and kidney. The results are shown in FIGS.

REFERENCES The following references are incorporated herein by reference in their entirety.
1. Lin et al. (2008). Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state.RNA 14, 2115-2124.
2. Lin et al. (2010) .MicroRNA miR-302 inhibits the tumorigenecity of human pluripotent stem cells by coordinate suppression of CDK2 and CDK4 / 6 cell cycle pathways. Cancer Res. 70, 9473-9482.
3. Lin et al. (2011). Regulation of somatic cell reprogramming through inducible mir-302 expression. Nucleic Acids Res. 39, 1054-1065.
4. Takahashi et al. (2006) .Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell 126, 663-676.
5. Yu et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920.
6. Wernig et al. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318-324.
7. Wang et al. (2008). Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat. Genet. 40, 1478-1483.
8. Deng et al. (1995) .Mice lacking p21Cip1 / Waf undergo normal development, but are defective in G1 checkpoint control.Cell 82, 675-684.
9. Yabuta et al. (2007). Lats2 is an essential mitotic regulator required for the coordination of cell division. J Biol Chem. 282, 19259-19271.
10. Judson et al. (2009). Embryonic stem cell-specific microRNAs promote induced pluripotency. Nat Biotechnol. 27, 459-461.
11. Barroso-deUesus et al. (2008) Embryonic stem cell-specific miR302-367 cluster: human gene structure and functional characterization of its core promoter.Mol Cell Biol. 28, 6609-6619.
12. Ying SY and Lin SL. (2004). Intron-derived microRNAs-Fine tuning of gene functions. Gene 342, 25-28.
13. Suh et al. (2004) .Human embryonic stem cells express a unique set of microRNAs. Dev. Biol. 270, 488-498.
14. Tang et al. (2007). Maternal microRNAs are essential for mouse zygotic development. Genes Dev. 21, 644-648.
15. Lin et al. (2003) .A novel RNA splicing-mediated gene silencing mechanism potential for genome evolution.Biochem Biophys Res Commun. 310, 754-760.
16. Lin et al. (2006) Gene silencing in vitro and in vivo using intronic microRNAs.Methods Mol Biol. 342, 295-312.
17. Grimm et al. (2006) .Fatality in mice due to oversaturation of cellular microRNA / short hairpin RNA pathways.Nature 441, 537-541.
18. Lin et al. (2005). Asymmetry of intronic pre-microRNA structures in functional RISC assembly. Gene 356, 32-38.
19. Marson et al. (2008). Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells.Cell 134, 521-533.
20. Card et al. (2008). Oct4 / Sox2-regulated miR-302 targets cyclin D1 in human embryonic stem cells. Mol. Cell Biol. 28, 6426-6438.
21. Jacobs et al. (1999) .The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature 397, 164-168.
22. Parry et al. (1995) .Lack of cyclin D-Cdk complexes in Rb-negative cells correlated with high levels of p16INK4 / MTS1 tumor suppressor gene product.EMBO J. 14, 503-511.
23. Quelle et al. (1995) .Alternative reading frames of the NK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest.Cell 83, 993-1000.
24. Kamijo et al. (1997) .Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF.Cell 91, 649-659.
25. Burdon et al. (2002). Signaling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol. 12, 432-438.
26. Jirmanova et al. (2002). Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1 / S transition in mouse embryonic stem cells.Oncogene 21, 5515-5528.
27. Stead et al. (2002) .Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A / E and E2F activities.Oncogene 21, 8320-8333.
28. Andrews et al. (1984) .Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro. Lab Invest. 50, 147-162.
29. Banito et al. (2009). Senescence impairs successful reprogramming to pluripotent stem cells. Gene Dev. 23, 2134-2139.
30. Feng et al. (2010). Hemangioblastic derivatives from human induced pluripotent stem cells exhibit limited expansion and early senescence. Stem Cell in press.
31. Dimri et al. (2002). The Bmi-1 oncogene induces telomerase activity and immortalizes human mammary epithelial cells. Cancer Res. 62, 4736-4745.
32. Won et al. (2002) .Sp1 and Sp3 recruit histone deacetylase to repress transcription of human telomerase reverse transcriptase (hTERT) promoter in normal human somstic cells. J Biol Chem. 277, 38230-38238.
33. Zhu et al. (2008) .Lysine-specific demethylase 1 (LSD1) is required for the transcriptional repression of the telomerase reverse transcriptase (hTERT) gene.PloS One 3, e1446.
34. EU Pat. No. EP2198025 to Lin.
35. US Pat. No. 5,843,780, 6,200,806, 7,029,913, and 7,220,584 to Thomson.
36. US Pat. No. 6,090,622, 6,245,566, and 6,331,406 to Gearhart.
37. US Pat. No. 6,875,607 to Reubinoff.
38. US Pat. No. 7,250,255 to Shinya Yamanaka.
The examples and embodiments described herein are for illustrative purposes only, and various modifications and alterations therefrom will occur to those skilled in the art, and are not intended to limit the scope of the appended claims. It is understood that the spirit and scope of the invention described in the scope should be included. All publications and patent documents cited herein are hereby incorporated by reference in their entirety for various purposes.

[Appendix 1]
By reprogramming a malignant human cancer to a benign or normal state at a relatively low stage, a composition used for the development of a therapeutic anticancer drug,
(A) 3-morpholinopropane-1-sulfonic acid (3-morpholinopropane-1-sulfonic acid, MOPS), ethanol, glycerin, or at least one chemical derivative agent having a molecular structure of a mixture thereof,
(b) at least one transformed prokaryotic cell line having at least one expression vector that expresses the microribonucleic acid precursor with a eukaryotic promoter, and under certain conditions (a) and (b) A composition for producing a microribonucleic acid precursor (pre-miRNA), which is mixed and induces transcription of the microribonucleic acid precursor driven by the eukaryotic promoter.
[Appendix 2]
The composition according to claim 1, wherein the chemical inducer agent is a transcription inducer that stimulates transcription of ribonucleic acid driven by the eukaryotic promoter in prokaryotic cells.
[Appendix 3]
The prokaryotic cells are present in a bacterial medium, the chemical inducer agent is added to the bacterial medium, and has a final concentration of 0.05% (v / v) to 0.2% (v / v) in the bacterial medium. The composition according to Supplementary Note 1.
[Appendix 4]
4. The composition according to claim 3, wherein the bacterial medium is an LB medium.
[Appendix 5]
The composition according to claim 1, wherein the prokaryotic cells are competent cells of E. coli DH5α.
[Appendix 6]
3. The composition according to claim 1, wherein said expression vector is a recombinant plasmid encoding the sequence shown in SEQ. ID. NO.
[Appendix 7]
2. The composition according to claim 1, wherein the expression vector is pLenti-EF1α-RGFP-miR302.
[Appendix 8]
The composition according to claim 1, wherein the microribonucleic acid precursor contains at least the sequence shown in SEQ. ID. NO.
[Appendix 9]
2. The composition according to claim 1, wherein the microribonucleic acid is a microribonucleic acid (pro-miRNA) produced by a prokaryotic cell.
[Appendix 10]
The microribonucleic acid produced by the prokaryotic cell is at least SEQ.ID.NO.9, SEQ.ID.NO.11, SEQ.ID.NO.13, SEQ.ID.NO.15 or SEQ.ID.NO. 18. The composition according to claim 9, comprising the sequence shown in 17.
[Appendix 11]
2. The composition according to claim 1, wherein said microribonucleic acid is used for pharmaceutical or therapeutic applications.
[Supplementary Note 12]
The composition according to claim 1, wherein the eukaryotic promoter is a type II ribonucleic acid polymerase (pol-2) promoter or a pol-2-like promoter.
[Appendix 13]
13. The composition according to appendix 12, wherein the pol-2 promoter is an EF1α promoter.
[Appendix 14]
13. The composition according to appendix 12, wherein the pol-2-like promoter is a CMV promoter.
[Appendix 15]
The composition according to claim 1, wherein the condition is an LB medium that is frequently shaken at 37 ° C.
[Appendix 16]
The composition of claim 1, wherein the anticancer mechanism of the anticancer drug comprises reversing the cancer to reprogram malignant and advanced human cancers to a low stage or normal state in vitro, ex vivo, or in vivo.
[Appendix 17]
2. The composition according to claim 1, wherein said human cancer comprises a tumor or a cancer cell.
[Appendix 18]
By reprogramming malignant human cancer at a low stage benign or normal state, a method for producing microribonucleic acid precursors used for the development of therapeutic anticancer drugs,
(a) providing at least one chemical derivative agent having a molecular structure of 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol, glycerin, or a mixture thereof; When,
(b) providing at least one transformed prokaryotic cell line having at least one expression vector that expresses the microribonucleic acid precursor with a eukaryotic promoter,
(c) mixing (a) and (b) under certain conditions to induce transcription of the microribonucleic acid precursor driven by the eukaryotic promoter, comprising: method of producing miRNA).
[Appendix 19]
19. The method according to claim 18, wherein said chemical inducer agent is a transcription inducer that stimulates transcription of ribonucleic acid driven by said eukaryotic promoter in prokaryotic cells.
[Appendix 20]
The step of providing the prokaryotic cell further comprises the step of providing the prokaryotic cell in a bacterial medium, wherein the step of mixing (a) and (b) comprises adding the chemical derivative to the bacterial medium at 0.05% (v 19. The method according to claim 18, further comprising the step of adding to a final concentration of between 0.2 / v) and 0.2% (v / v).
[Appendix 21]
21. The method according to supplementary note 20, wherein the bacterial medium is an LB medium.
[Supplementary Note 22]
19. The method according to claim 18, wherein the prokaryotic cell is a competent cell of E. coli DH5α.
[Appendix 23]
19. The method according to claim 18, wherein said expression vector is a recombinant plasmid encoding the sequence shown in SEQ. ID. NO.
[Appendix 24]
19. The method according to supplementary note 18, wherein the expression vector is pLenti-EF1α-RGFP-miR302.
[Appendix 25]
19. The method according to claim 18, wherein the microribonucleic acid precursor contains at least the sequence shown in SEQ. ID. NO.
[Supplementary Note 26]
19. The method according to claim 18, wherein the microribonucleic acid is a microribonucleic acid (pro-miRNA) produced by a prokaryotic cell.
[Appendix 27]
The microribonucleic acid produced by the prokaryotic cell is at least SEQ.ID.NO.9, SEQ.ID.NO.11, SEQ.ID.NO.13, SEQ.ID.NO.15 or SEQ.ID.NO. 27. The method according to supplementary note 26, comprising the sequence shown in 17.
[Appendix 28]
19. The method according to Appendix 18, wherein said microribonucleic acid is used for pharmaceutical or therapeutic applications.
[Appendix 29]
19. The method according to claim 18, wherein the eukaryotic promoter is a type II ribonucleic acid polymerase (pol-2) promoter or a pol-2-like promoter.
[Appendix 30]
28. The method according to supplementary note 27, wherein the pol-2 promoter is an EF1α promoter.
[Appendix 31]
28. The method according to Appendix 27, wherein said pol-2-like promoter is a CMV promoter.
[Supplementary Note 32]
19. The method according to supplementary note 18, wherein the condition is an LB medium that is frequently shaken at 37 ° C.
[Appendix 33]
19. The method of claim 18, wherein the anti-cancer mechanism of the anti-cancer drug comprises reversing the cancer to reprogram malignant and advanced human cancer to a low stage or normal state in vitro, ex vivo or in vivo.
[Appendix 34]
19. The method according to claim 18, wherein said human cancer comprises a tumor or a cancer cell.

Claims (9)

  A composition for reprogramming the properties of a malignant human cancer cell to a cell-like state of low-grade benign or normal somatic tissue, said composition comprising the sequence of SEQ.ID.NO.3. At least one microribonucleic acid precursor (pre-miRNA) at a concentration of 1 mg / mL or less, wherein said pre-miRNA is capable of transforming a human into a low-stage benign or normal somatic tissue cell-like state. The pre-miRNA used in contact with a cell specimen containing at least one human cancer cell to induce reprogramming of the cancer cell, wherein the pre-miRNA comprising the sequence of SEQ. ID. NO. A composition produced by using the transcription of an RNA driven by a eukaryotic promoter in E. coli.   2. The composition of claim 1, wherein said prokaryotic cells are cultured in Luria-Bertani (LB) medium.   The composition according to claim 1, wherein the prokaryotic cells are competent cells of E. coli DH5α.   3. The composition of claim 1, wherein the pre-miRNA comprising the sequence of SEQ. ID. NO. 3 is transcribed from a recombinant plasmid encoding a mir-302 family cluster.   The composition according to claim 1, wherein the pre-miRNA comprising the sequence of SEQ.ID.NO.3 comprises at least the sequence of mir-302a, mir-302b, mir-302c or mir-302d.   2. The composition of claim 1, wherein reprogramming the properties of the malignant human cancer cells to a low-grade benign or normal somatic cell-like state is an anti-cancer mechanism called cancer reversal.   2. The composition of claim 1, wherein said human cancer cells are liver cancer cells.   The composition according to claim 1, wherein the pre-miRNA is used for pharmaceutical or therapeutic use.   9. The composition of claim 8, wherein said pharmaceutical or therapeutic use comprises the development of an anti-cancer drug.