JP2015530988A - Production and use of new therapeutic anticancer drugs - Google Patents

Abstract

The present invention generally uses microribonucleic acid (microRNA, miRNA) and / or its small hairpin ribonucleic acid (shRNA) homolog / analog / derivative to design new anti-tumor and / or anti-cancer drugs, vaccines and treatments And a method. Specifically, the present invention relates to a microRNA precursor (pro-miRNA) composition produced using prokaryotic cells. When the composition is delivered to human cells, it is processed into mature miRNA effectors, causing specific silencing effects on genes that can be targeted by mir-302, and subsequently beneficial for tumor suppression and cancer treatment Has an effect. In nature, prokaryotic cells do not express or process eukaryotic miRNA precursors (pre-miRNAs). The present invention also discloses an inducible method of expressing pre-miRNAs, particularly mir-302 precursors, using a prokaryotic transcription system. Because mir-302 is a tumor suppressor for mankind, this new discovery facilitates development in the design and methods of new anticancer drugs, vaccines and / or treatments for various human tumors and cancers.

Description

Claimed Priority The present invention claims priority from a US provisional application filed Dec. 28, 2012, application number 61 / 761,890, entitled “Development of Generic Anticancer Drugs and Vaccines”. The present invention further claims the priority of the US application filed on June 2, 2010, with the application number 12 / 792,413, entitled “Development of Generic Anticancer Drugs and Vaccines”. The present invention further relates to an application number 13 / 572,263 filed on August 10, 2012, entitled “Inducible gene expression composition for using type II eukaryotic polymerase promoter-driven transcription in prokaryotic cells” Claiming priority of US applications that are "things and their applications". This application is based on US patent application 12 / 792,413, filed on June 2, 2010, entitled "Development of Generic Anticancer Drugs and Vaccines"; Is a continuation-in-part of U.S. patent application 13 / 572,263, "Inducible gene expression composition for using type II eukaryotic polymerase promoter-driven transcription and its application" . The entire contents of these applications are incorporated herein by reference.

FIELD OF THE INVENTION This invention relates generally to product design and related uses of novel DNA / RNA therapeutics and / or vaccines in the treatment of cancer. More particularly, the present invention relates to designs and methods using novel nucleic acid compositions, wherein the nucleic acid composition is processed into a small ribonucleic acid (small RNA) gene silencing effector after delivery to human cells, The cell cycle regulator and / or oncogene that is the target of 302 causes a silencing effect of a specific gene, and further has an effect of tumor suppression and / or cancer prevention against tumor / cancer cell growth, invasion and metastasis. Small ribonucleic acid gene silencing effectors are mir-302a, mir-302b, mir-302c, mir-302d, mir-302e, mir-302f, their precursors (pre-microribonucleic acid, pre-miRNAs), and those Tumor suppressor microRNA, TS, such as manually redesigned and / or modified homologues / derivatives such as small hairpin ribonucleic acid / small interfering ribonucleic acid (shRNA / siRNA), and combinations thereof -miRNA). The 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 / regeneration, to recover and / or regenerate damaged / aged tissues, or to treat degenerative diseases and prevent tumor / carcinogenesis. Contains active ingredients. Therefore, the inventor can understand that these stem cells are used as a means for identification and production of new drugs. From this, the new drugs obtained can be used in the development of pharmaceutical and / or therapeutic applications, such as the use of biological drugs, 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, cancer treatment and / or prevention, disease treatment, drug production, and combinations thereof.

  Microribonucleic acid mir-302 is the most important microribonucleic acid (microRNA, miRNA) discovered in human embryonic stem (hES cell) and induced pluripotent stem (iPS cell) But many of its features are not yet clear. In previous studies, ectopic overexpression of mir-302, i.e. higher than the level in human stem cells (hES cells), has a slow cell cycle rate (20-24 hours / cycle) and dormant cell-like It has been shown that human normal cells and cancer cells can be reprogrammed into normal hES-like pluripotent stem cells having the cell morphology (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 distinct features of these mir-302 reprogrammed pluripotent stem cells (mirPS), whereas human stem cells (hES) and 3/4 in previous reports IPS cells induced by various factors (i.e., Oct4-Sox2-Klf4-c-Myc or Oct4-Sox2-Nanog-Lin28) are highly proliferative (12-15 hours / cycle) and have an unrelenting tendency to form tumors (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 that support the anti-proliferative properties of mirPS cells have not yet been elucidated, we have discovered that two G1-checkpoint regulators that 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). The progression of the eukaryotic cell cycle is driven by the activation of cyclin-dependent kinases (CDKs), which are cyclins that are positive regulatory subunits and negative regulatory subunits. It forms a functional complex with cyclin-dependent kinase inhibitors (CDK inhibitors, CKI, 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 the regulation of transitions at different times in the cell cycle, for example, cyclin-D-CDK4 / 6, which is involved in G1 progression. Cyclin-E-CDK2 involved in G1-S phase transition, cyclin-A-CDK2 involved in S phase progression, and cyclinA / B-CDC2 (cyclin-A / B-CDK1) involved in M phase entry is there. Thereby, it can be understood that the anti-proliferative function of mir-302 is the 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, which are analogs of human mir-302, showed results that were completely different from mir-302 in human cells. In mouse embryonic stem cells (mES), ectopic expression of mir-291 / 294/295 is p21Cip1 (or CDKN1A) and serine / threonine protein kinase Lats2 (Wang et al., 2008 (non-patent document 7)) Direct silencing promoted rapid cell growth and caused a high degree of tumor formation in the transfected cells. It is known that transgenic mice deficient in the p21Cip1 / Waf1 gene showed normal growth when lacking control by G1 checkpoint (Deng et al., 1995 (Non-patent Document 8)). However, since Lats2 has functions such as recruiting λ-tubulin at the start of mitosis and participating in the formation of spindles, its role has not yet been elucidated. Lats2 deficiency in mouse embryos also caused severe mitotic defects and was found to be fatal, indicating that Lats2 silencing inhibits rather than promotes cell division (Yabuta et al., 2007 (Non-Patent Document 9)). Thus, p21Cip1 silencing appears to be a major mechanism behind tumor formation induced by mir-291 / 294/295. However, all of the results we screened for the mir-302 target site of the human p21Cip1 gene were negative. Similar negative results are the two most famous online microribonucleic acid-target prediction programs, `` TARGETSCAN '' (http://www.targetscan.org/) and `` PICTAR-VERT '' (http: // pictar. It was also shown by mdc-berlin.de/). Thus, even though mir-302 and mir-291 / 294/295 are homologous analogs, their functions in controlling tumorigenesis are substantially reversed. This indicated that the homologous microribonucleic acid may not have the same function. The above findings also indicate that studies that currently replace mimics such as small interfering ribonucleic acids may not yield the same results as natural microribonucleic acids.

  The gene sequence encoding mir-302 is located at the locus 4q25 of human chromosome 4, and is a conservative region frequently associated with longevity. Furthermore, mir-302 is encoded by the intron region of the La ribonucleoprotein domain family member 7 (LARP7) gene and expressed by the biosynthetic pathway of intron microribonucleic acid (Barroso -delJesus, 2008 (Non-Patent Document 10); Ying and Lin, 2004 (Non-Patent Document 11); FIG. 13). Natural mir-302 contains 4 homologous sense family members (mir-302b, c, a and d) and 3 distinct antisense members (mir-302b *, c * and a *) Is transcribed into a polycistronic RNA cluster together with another miRNA, mir-367 (Suh et al., 2004 (Non-patent Document 12)). In our past invention, US 12 / 792,413, the inventor found that mir-302 is another miRNA, such as mir-92, mir-93, mir-367, mir-371, mir-372, mir-373, It was found that the expression of the entire mir-374 and mir-520 family members can be stimulated. An analysis using the online programs “TARGETSCAN” and “PICTAR-VERT” on the website “Sanger miRBase :: Sequences” (http://microrna.sanger.ac.uk/) These miRNAs whose expression was stimulated were shown to share more than 400 target genes. This demonstrated that miRNA and mir-302, whose expression is stimulated, may play a similar role. These shared target genes are members of RAB / RAS-related oncogenes, ECT-related oncogenes, pleomorphic adenoma genes, E2F transcription factors, cyclin D binding Myb-like transcription factors, 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 nuclear protein gene, RB-like 1, RB-binding protein gene, Max-binding protein Protein gene, c-MIR immune recognition cell regulator, Bcl-like pro-apoptotic 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 functions by homologous miRNAs such as miR-92, miR-93, miR-367, miR-371, miR-372, miR-373, miR-374 and miR-520. 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 by a high degree of complementarity and to suppress translation of transcripts (mRNAs) of the target gene And forms RNA-induced silencing complexes (RISCs) to inhibit or degrade mRNAs. Therefore, the original function of the miRNA is determined by the stringency of the binding between the miRNA and the target gene. In contrast, our previous study (Lin et al., 2010 (Non-Patent Document 6); US 12 / 792,413) provided an important interpretation of the mechanism supporting tumor suppression regulated by mir-302. In human cells, multiple cell cycle regulators targeted by mir-302 silence by including the CDK2, cyclins D1 / D2, and BMI-1 genes, but interestingly, they do not contain p21Cip1. As described above, co-silencing between CDK2 and cyclin D in the cell cycle inhibits the transition from G1 phase to S phase and results in a relatively slow cell growth rate. In addition, silencing BMI-1 stimulates the expression of two major tumor suppressors, p16Ink4a and p14ARF, thereby further reducing cell proliferation and suppressing tumor formation. In human cells, the expression of p16Ink4a / p14ARF is increased and p21Cip1 is not affected. Alternatively, it may exert an anti-tumorigenic function through the p14 / 19ARF-p53 pathway.

  Based on the antitumor characteristics of mir-302 described above, we can use mir-302 as a therapeutic agent for human tumors and cancers. However, there may be four major problems with this attempt: (1) As a gene silencing effector, mir-302 needs to function as its precursor. There is currently no effective ribonucleic acid synthesis technique that produces RNA that is 73-75 nucleotides in length like a hairpin. To ensure the accuracy of its sequence, the maximum length of the synthesized RNA is about 45-55 nucleotides, but that is not enough to form the mir-302 precursor (pre-mir-302) . (2) We need mir-302 expression at a higher concentration than mir-302 levels in hES cells, thereby inducing its anti-tumor / carcinogenic effect (Lin et al., 2010 (Non-Patent Document 6 )). However, there is currently no method for producing so much pre-mir-302 for drug production. The isolation of pre-mir-302 from hES or iPS cells is expensive, of which pre-mir-302 with a high degree of secondary structure cannot be produced by bacterial competent cells. (3) Synthesized miRNAs 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 its high toxicity, siRNA cannot be used in place of natural mir-302 in in vivo conditions. In addition, there is a report that the use of mimics such as siRNA causes supersaturation of the intracellular microRNA pathway and 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 related drugs regulated by mir-302.

In short, there is a need for an effective and safe method for producing, isolating and using mir-302 and its precursors, and related homologues / derivatives.
SUMMARY OF THE INVENTION The present invention is a design and method for using microRNA precursors (pre-miRNAs) as therapeutics and / or vaccines for the treatment of cancer. Specifically, the present invention relates to a design and method using a recombinant nucleic acid composition, and after the recombinant nucleic acid composition is delivered to a human cell, a small ribonucleic acid gene silencing effector (small RNA-based gene silencing). effector), causing specific gene silencing effects on cell cycle regulators and / or oncogenes that are targets of mir-302, resulting in tumor suppression and / or cancer prevention, etc. It can be used to inhibit cell growth, proliferation, invasion and metastasis. These small ribonucleic acid gene silencing effectors are tumor suppressor microribonucleic acid (TS-miRNA) such as mir-302a, mir-302b, mir-302c, mir-302d, mir-302e, mir-302f, their precursors The body (pre-miRNAs), and their manually redesigned small hairpin ribonucleic acid (shRNA) -like homologues / derivatives, and combinations thereof are preferred. Small hairpin ribonucleic acid homologues / derivatives are designed for mismatched and perfect matches within individual single units or complex clusters in shRNA and its homologous small interfering RNA (siRNA) structures. matched) nucleic acid composition. They can promote target specificity and reduce the quantity (copy number) of mir-302 required for delivery and therapy. Said human cells include in vitro, ex vivo and / or in vivo normal cells and / or tumor / cancer cells.

  Natural microribonucleic acid (miRNA) is about 18-27 nucleotides (nts) in length, and the target messenger ribonucleic acid by complementation between microribonucleic acid (miRNA) and its target (messemger RNA, mRNA) can be directly degraded, or translation of the target messenger ribonucleic acid (mRNA) can be suppressed. The Mir-302 family (mir-302s) are microribonucleic acids (miRNAs) that have a high degree of homology and are conserved in 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- It is transcribed into a non-coding RNA cluster containing 367 (Suh et al., 2004 (Non-Patent Document 12)). Recently, in addition to the original family cluster, scientists have discovered the fifth and sixth mir-302 members, mir-302e and mir-302f. Although mir-367 and mir-302s are co-expressed in nature, their functions are very different from each other. In the present invention, based on the function of anti-tumor formation, we tend to express only mir-302s. In certain embodiments, we further have 5′-GCTAAGCCAG GC-3 ′ (SEQ.ID.NO.1) and 5′-GCCTGGCTTA GC-3 ′ (SEQ.ID. Manual redesigned hairpin loops such as NO.2) can be used to replace the original mir-302 precursor hairpin loop. 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 2007 (Non-Patent Document 14); Suh et al. 2004 (Non-Patent Document 12)). mir-302s are homologous small inhibitory RNAs that can silence target genes that are highly complementary to them, so mir-302s is an oncogenic errant in hES and iPS cells and May be related to prevention of premature. Therefore it is also used in the development of anti-tumor / cancer treatment drugs. In fact, hES cells before the morula stage (32-64 cell stage) usually have a relatively slow cell cycle rate, like 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 regulating normal stem cells and preventing tumor / cancer cell formation.

  All mir-302 members share the exact sequence at the first 17 nucleotides of the 5 'terminal sequence, of which the seed motif 5'-UAAGUGCUUC CAUGUUU-3' (SEQ.ID) NO.3) and also has a sequence homology of 85% or more in mature microribonucleic acid (miRNA) having 23 nucleotides as well. Based on the current prediction results from the online computation program "TARGETSCAN" (http://www.targetscan.org/) and "PICTAR-VERT" (http://pictar.mdc-berlin.de/), these members At the same time, it targets almost the same cellular genes, including over 607 human genes. In addition, mir-302 may be somewhat functionally similar to mir-92, mir-93, mir-200c, mir-367, mir-371, mir-372, mir-373, mir-374 and mir- 520 family members also share a number of 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 embryogenesis (Lin et al. , 2008 (Non-Patent Document 1)). Many of the target genes are also known oncogenes. Since many of these targeted developmental signals and differentiation-related transcription factors are oncogenes, mir-302 also has a tumor suppressor function, and the growth of normal hES cells proceeds to the formation of tumor / cancer cells. Can be prevented.

  In certain preferred embodiments, the present invention is a method for the production and delivery of non-vector-based pre-mir-302 for use in the treatment of human tumors / cancers. As described above, the length of pre-mir-302 is about 73 to 75 nucleotides, but due to the limitation of 45 to 55 nucleotides in length, we cannot synthesize it by conventional RNA synthesis techniques. Therefore, we need to increase the amount of pre-mir-302 by adopting a new method to be used for drug production. Therefore, in the present invention, a miRNA precursor (product is pro-miRNA) is produced using a prokaryote, and a mimic of pre-mir-302 is prepared. The technology is a derivative invention of US 13 / 572,263, 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 between prokaryotic and eukaryotic are very different and incompatible with each other, so we need to modify the prokaryotic transcription system to transcribe eukaryotic genes and pre-miRNAs. is there. In the invention of US 13 / 572,263, the priority application of the present application, some chemical induction causes prokaryotic cells to use eukaryotic pol-2 and pol-2 like (ie CMV) promoters, and a large amount of pre-mir-302. It has already been disclosed that such hairpin-like RNAs (product is 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 advantage of Pro-miRNA technology is that, firstly, because of the fast and easy growth of bacterial competent cells, the production of a specific pre-miRNA or some (necessary) pre-miRNA meets the cost-benefit principle. Secondly, we can expect mass production on the scale of several grams to kilograms. Third, it is not necessary to culture and fuse tumors or mammalian cells, so it is easy and expensive to operate. Fourth, transcription of the pol-2-like promoter induced by chemical reagents ensures high sequence accuracy; and finally, lack of expression of Dicer and other miRNAs in prokaryotes Therefore, a highly pure pre-miRNA product is obtained. The resulting pro-miRNA can be easily isolated from bacterial extracts and / or lysates (Example 20) and further purified (Example 21). 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 them to promote wound healing (Example 22). For use in therapy, isolated pro-mir-302 was prepared from a pre-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 cut open with a scalpel 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 region was further sealed with a liquid bandage. As shown in FIG. 14, within 2 weeks, the healing rate of the wound treated with pro-mir-302 was significantly improved, and more than twice as fast as all other control groups. In the region healed with pro-mir-302, normal hair regeneration appears, and no scar remains (for example, a portion indicated by a black arrow). In contrast, in the other treatment groups, a slight scar remains and there is no hair growth. As is clear from this result, pro-mir-302 has a function of stimulating normal tissue recovery and regeneration.

  We also purified the pro-mir-302 into a liquid drug and injected it to test the treatment of liver / malignant liver tumors in vivo (Example 23). As shown in Figure 15, after 3 injections, drugs containing pro-mir-302 reduced the volume close to 90% of liver cancer cells / malignant liver tumors implanted in the body, 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 with H & E staining indicate that the treatment result for this cancer / malignant tumor is not only due to the antitumor formation property of mir-302 (Lin et al., 2010), but has not been previously recognized. It was further shown that it depends on the newly discovered reprogramming function. 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 approximating normal liver tissue. Treated cancer cell transplants can also form normal hepatic fistula structures, such as typical hepatic lobule, central vein (CV) and portal trivial (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, reaching an advantageous effect for cancer treatment.

  In another preferred embodiment, the present invention further provides a vector that expresses mir-302 and treats tumors / cancers. As disclosed in US 12 / 792,413, the priority application of the present application, the inventor has already designed and developed an inducible pTet-On-tTS-miR302s expression vector (Fig. 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- 302s; FIG. 1B). Upon stimulation with doxycycline (Dox), this mir-302s structure is driven and expressed by a cytomegalovirus (CMV) promoter controlled by a tetracycline-responsive element (TRE). Following infection / transfection, mir-302 expression occurs along the natural microribonucleic acid (miRNA) biosynthetic pathway. Among them, the mir-302s structure is co-transcribed with a reporter gene such as red-shifted fluorescent protein (RGFP) and is further spliceosomal components and / or in the cytoplasm The RNA endoribonuclease (RNase III) is processed into a single mir-302 member by Dicers (FIG. 2A) (Lin et al., 2003 (Non-patent Document 15)). The result of this measure was that, in the microribonucleic acid microarray analysis (Example 3), all sense mir-302 members were effectively expressed in the transfected cells after stimulation with doxycycline (FIG. 1C). . The process of transducing human cells with the mir-302 expression vector is illustrated in FIG. 2B.

  The present inventor designed an intron microribonucleic acid expression system, namely SpRNAi-RGFP, to mimic the biosynthetic pathway of natural intronic microribonucleic acid (intronic miRNA) (Figure 2A) and to transcribe the recombinant RGFP gene. did. SpRNAi-RGFP contains artificial / artificial splicing introns (SpRNAi), which intron miribonucleic 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 (Non-Patent Document 16)). SpRNAi and RGFP are co-transcribed by type II RNA polymerase (Pol-II RNA polymerase) and included in the messenger ribonucleic acid precursor / pre-messenger ribonucleic acid (pre-mRNA) of the SpRNAi-RGFP gene, and RGFP is RNA It is excised by RNA splicing components. Spliced SpRNAi is then further processed into mature gene silencing effectors such as natural microribonucleic acids (native miRNAs) and artificial small hairpin ribonucleic acids (shRNAs), resulting in specific ribonucleic acid interference effects on target genes ( Induces RNA interference (RNAi). At the same time, after intron splicing, exons of SpRNAi-RGFP gene transcripts are ligated to form mature messenger ribonucleic acid (mRNA), and then miRNA / shRNA expression Is translated into an RGFP reporter protein used to identify Quantitatively, 1-fold concentration of RGFP corresponds to 4-fold concentration of mir-302 expression. Alternatively, additional gene functions may be provided by selectively using functional protein exons instead of RGFP, for example, hES gene markers in somatic cell reprogramming (SCR). Currently, more than 1000 types of natural microribonucleic acids with unknown functions have been discovered in vertebrates, and more and more new microribonucleic acids have been identified one after the other, so our intron microribonucleic acid expression system is in vitro and in vivo. It can be an advantageous tool for the measurement of the above-mentioned microribonucleic acid function.

  The SpRNAi intron contains a 5 'splice site (5'-splice site), a branch point sequence (Branch-Point, BrP), a polypyrimidine tract, and a 3' splice site (3'-splice site). It contains several common nucleotide compositions. Also included are hairpin microribonucleic acid (miRNA) or small hairpin ribonucleic acid (shRNA) precursors 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 intronic RNA splicing and processing accuracy. This T codon, when expressed by messenger ribonucleic acid (mRNA) in the cytoplasm, generates an activation signal for the nonsense-mediated decay (NMD) system in the cell and prevents the occurrence of cytotoxicity. Decomposes any unstructured ribonucleic acid accumulated within. However, highly structured small hairpin ribonucleic acid (shRNA) and microribonucleic acid precursor (pre-miRNA) are retained and further cleaved by Dicer to produce mature small interfering ribonucleic acid (siRNA) and Forms ribonucleic acid (miRNA). We have manually integrated SpRNAi into the DraII restriction site of the RGFP gene (Lin et al., 2006 and 2008 (Non-Patent Documents 16 and 1)) to express intron miRNA / shRNA, and the recombinant SpRNAi-RGFP gene Formed. Cleavage of RGFP with DraII restriction enzyme results in an AG-GN nucleotide cleavage site with 3 recessed nucleotides at each end, forming a 5 ′ and 3 ′ splice site, respectively, after SpRNAi insertion. This intron insertion destroys the integrity of the RGFP protein, but this can be restored by intron splicing, so we have red red-shifted fluorescent protein (RGFP) appearing in the transfected cells. The expression of mature miRNA / shRNA can be measured. The RGFP gene also has multiple exonic splicing enhancers (ESEs) to improve the accuracy and efficiency of RNA splicing.

  Specifically, the SpRNAi intron is 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)) containing a 5′-splicing site that is homologous to any of the 3 ′ (SEQ.ID.NO.39)), 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'-GATATCCTGC AG-3 '(SEQ.ID.NO.41), 5'-GGCTGCAG- 3 ′ (SEQ.ID.NO.42) and 5′-CCACAG-3 ′ (SEQ.ID.NO.43)) are 3′-splicing sites. In addition, the branch point sequence is located between the 5 ′ end and the 3 ′ end splice site, and 5′-TACTAAC-3 ′ (SEQ.ID.NO.44) and 5′-TACTTAT-3 Highly homologous to 5'-TACTWAY-3 '(SEQ.ID.NO.6) motifs such as' (SEQ.ID.NO.45). The `` A '' nucleotide, which is the adenosine of the branch point sequence, is the 2'-5 'oligoadenylate synthase ((2'-5')-oligoadenylate of the cell in almost all spliceosomal introns. synthetases) and spliceosomes can form part of a (2'-5 ')-linked lasso intron RNA. The polypyrimidine tract is located close to 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' Homologous to any of the (SEQ.ID.NO.8) motifs. Among them, the symbols “m” and “n” indicate a plurality of overlaps, and ≧ 1, and most preferably, m is 1 to 3 and n is 7 to 12. The symbol “-” indicates that there are nucleotides that may be skipped within the sequence. 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), S is cytosine (C) or guanine (G), Y is cytosine (C) or thymine (T) / uracil (U), R is adenine ( A) or guanine (G), the symbol N represents adenine (A), cytosine (C), guanine (G) or thymine (T) / uracil (U).

  In another possible embodiment, the present invention is a direct (exon-type) mir-302 miRNA / shRNA expression system that does not undergo intracellular RNA splicing and / or NMD mechanisms, Silencing effectors can be generated directly. However, the disadvantage of this method is that the expression of mir-302-like gene silencing effectors is not regulated by any intracellular surveillance system such as nonsense-mediated decay (NMD), so Cytotoxicity may be generated by supersaturation of the synthetic pathway (Grimm et al., 2006 (Non-patent Document 13)). The expression system used in this method is a plasmid, viral vector, lentiviral vector, transposon, retrotransposon, jumping gene, protein coding 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. The mir-302-like gene silencing effectors, including microribonucleic acid, small hairpin ribonucleic acid, small interfering ribonucleic acid, and their precursors and homologues / derivatives, are either tissue specific or non-specific ribonucleic acid (RNA) promoters. Expressed by regulation. 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 RNA polymerase (TRE) promoter controlled by tetracycline response element. The viral promoter is a Pol-II-like RNA promoter, cytomegalovirus (cytomegalovirus, CMV), retrovirus long-terminal region (LTR), hepatitis B virus (hepatitis B virus, HBV), adenovirus ( isolated from, but not limited to, adenovirus (AMV), and adeno-associated virus (AAV). As an example, the lentiviral LTR promoter can produce 500,000 pre-messenger ribonucleic acid transcripts (copies) in one cell. It is also possible to control the transcription rate of the gene silencing effector by inserting a drug-sensitive repressor (ie, tTS) in front of the RNA polymerase promoter. The suppressor is a chemical agent or an antibiotic selected from G418, neomycin, tetracycline, doxycycline, ampicillin, kanamycin, puromycin, and derivatives thereof. It is suppressed by.

  On the other hand, a gene silencing effect may be realized for a target gene of mir-302 using a plurality of transgenes and / or vectors expressing several intron gene silencing effectors. Also, multiple gene silencing effectors can be generated by a single intronic insert. For example, ectopic expression of an anti-EGFP intron containing a microribonucleic acid precursor (pre-miRNA) in zebrafish, miR-EGFP, two different sizes of miRNA (282/300) and miR-EGFP (280-302) were reported to be produced. This indicates that one SpRNAi insert can generate multiple gene silencing effectors (Lin et al. (2005) Gene 356: 32-38 (Non-patent Document 17)). In some instances, intron gene silencing effectors hybridize with target gene transcripts (ie, messenger ribonucleic acid, mRNA) to form double-stranded small interfering ribonucleic acids (siRNAs), resulting in ribonucleic acid interference secondary effects. Can cause (RNAi). These gene silencing effectors are constantly generated by the transgene vector, thus reducing the concern for rapid degradation of RNA in vivo. The advantage of this measure is that it can provide a reliable and long-term gene silencing effect by transfection with a vector transgene or viral infection.

Since the stem-loop structure of some natural pre-microribonucleic acids (pre-miRNAs) is oversized and / or complex and incompatible with microribonucleic acid expression systems / vectors, the present inventors Instead of this loop, a manually redesigned methionyl-carrying ribonucleic acid loop (tRNA ^met loop, ie 5 ′-(A / U) UCCAAGGGGG-3 ′ (SEQ.ID.NO.46)) is often used. the tRNA ^{the met} loop natural micro ribonucleic acid and similar transport mechanism, through the Ran-GTP and Exportin down -5 (Exportin-5), transport of the manually re-designed micro-ribonucleic acid (miRNA) into the cytoplasm from the nucleus Can be effectively executed (Lin et al., 2005 (Non-patent Document 17)). The advantages of the present invention include an artificially improved pair of pre-compounds comprising 5′-GCTAAGCCAG GC-3 ′ (SEQ.ID.NO.1) and 5′-GCCTGGCTTA GC-3 ′ (SEQ.ID.NO.2). By using microribonucleic acid loops, manually redesigned microribonucleic acid (pre-miRNA) increases the same nuclear export efficiency as natural pre-microribonucleic acid and interferes with tRNA export. Can not. This improvement can promote the formation of the mir-302a-mir-302a * duplex and the mir-302c-mir-302c * duplex, 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.

  A family cluster of Mir-302 pre-microribonucleic acids is formed by hybridization and linkage / ligation of synthesized mir-302 homologues, mir-302a, mir-302b, mir from 5 ′ end to 3 ′ end It contains four parts of pre-miRNAs (Figure 1B) of -302c and mir-302d. All these manually redesigned mir-302 miRNA / shRNA molecules have the same 17 nucleotides [e.g., 5'-UAAGUGCUUC CAUGUUU-3 '(SEQ.ID.NO.3 )]. As synthetic oligonucleotides used in 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), mir-302b-sense: 5'-ATAGATCTCT CGCTCCCTTG AACTTTAACT TG GT 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- 302c-sense: 5'-CCATATGGCT ACCTTTGCTT TAACATGGAG GTACCTGCTG TGTGAAACAG AAGTAAGTGC TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3 '(SEQ.ID.NO.13), mir-302c-antisense: 5'-ATGTCTAGAC GCCTCCACTGAAGTGGTT GCCATCATGCGT ' (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 Contains '-ATGCGATCGC GAACACTCAA ACATGGAAGC ACTTAGCCTG GCTTAGCTAA GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3' (SEQ.ID.NO.16). Alternatively, manually redesigned small hairpin ribonucleic acid (shRNA) may be used. The manually redesigned small hairpin ribonucleic acid is synthesized miR-302s-sense: 5'-GCAGATCTCG AGGTACCGAC GCGTCCTCTT TACTTTAACA TGGAAATTAA GTGCTTCCAT GTTTGAGTGG TGTGGCGCGA TCGATATCTC TAGAGGATCC ACATC-3 '(SEQ.ID.NO.17) and mir-302s-sense : 5'-GATGTGGATC CTCTAGAGAT ATCGATCGCG CCACACCACT CAAACATGGA AGCACTTAAT TTCCATGTTA AAGTAAAGAG GACGCGTCGG TACCTCGAGA TCTGC-3 '(SEQ.ID.NO.18) hybridization formed, mir-302 pre-miribonucleic acid cluster (mir-302 pre-miRNA cluster Instead of), insert a simple intron. 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 gene. In designing mir-302 homologues, thymine may be used in place of uracil, and vice versa.

  For intronic insertion of mir-302 pre-miRNA / shRNA, there is a PvuI at the 5 'end and an MluI restriction / cloning site at the 3' end on the side of the insertion site of the recombinant SpRNAi-RGFP transgene, respectively. Thus, the first 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 the PvuI and MluI restriction sites. By varying intron inserts that target different gene transcripts, the intronic mir-302s expression system of the present invention can be used in vitro, 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 further introduced into the restriction / cloning site of the pSingle-tTS-shRNA vector induced by doxycycline (i.e., XhoI-HinDIII restriction / cloning site). A pTet-On-tTS-miR302s expression vector that can be inserted and expressed in cells is formed (FIG. 1A).

  Methods for delivering pre-mir-302-like (pro-mir-302) or mir-302-expressing nucleic acid compositions to human cells include liposomal / polysomal / chemical transfection, DNA DNA recombination, electroporation, gene gun penetration, transposon / retrotransposon insertion, jumping gene integration, micro-injection (micro- a non-gene transfer method or a gene transfer method selected from injection, viral infection, retrovirus / lentivirus infection, and combinations thereof. In order to prevent the risk of random transgene insertions and cellular mutations, the present invention uses liposome or polysome transfection methods to detect 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 TRE-controlled CMV promoter in the pTet-On-tTS-miR302s vector at the concentration of mir-302-like gene silencing effector or at each concentration of doxycycline . Thus, the present invention provides in vitro, ex vivo, in vivo expression levels of mir-302 by providing a controllable mechanism through the use of known drugs (ie, 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 via a one month activation state in human cells. Its one month activation mechanism is beneficial in the treatment of cancer because it can avoid long-term accumulation or supersaturation of RNA in treated cells.

  In short, the present invention adopts a novel design and measure, 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) homologues / derivatives, as well as combinations thereof. In a preferred embodiment, the present invention provides cell cycle regulators and oncogenes that are processed into a sufficient amount of a mir-302-like gene silencing effector that can be delivered in treated tumor / carcinoma cells and are targeted by mir-302. Provided are designs and methods for treating human tumors and cancers using recombinant nucleic acid compositions that inhibit stagnation 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 (ie CMV) or drug-inducible promoter (ie TRE-CMV) in treated cells Can be made. On the other hand, the recombinant nucleic acid composition is used for the production of pre-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 for treating human tumors / cancers. Preferably, the mir-302-like gene silencing effector is a recombinant mir-302 family cluster (mir-302s; a hybrid of SEQ. ID. NOs. 9-16) or a manually redesigned mir-302 small hairpin ribonucleic acid homolog. Transcribed from (shRNA, ie hybrid of SEQ.ID.NOs.17 and 18). The treated cell matrix is capable of expressing cellular genes targeted by mir-302 in vitro, ex vivo or in vivo. The present invention can suppress the formation of tumor / cancer cells by silencing a cellular gene targeted by mir-302 and reprograms 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 attenuates the cell cycle but does not cause apoptosis or cell death. Second, by transfecting mir-302 into human cells, the cells can be reprogrammed to a stem cell-like state, which is beneficial for the treatment of damaged tissue (FIGS. 4A-4B and FIGS. 5A-5D). Third, these cells are transfected by transfecting mir-302 into human tumor / cancer cells (e.g. prostate cancer cell PC3, skin cancer cell Colo, breast cancer cell MCF7, liver cancer cell HepG2 and teratoma cell 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-inhibits multiple cell cycle regulators such as cyclin-dependent kinase 2 (CDK2), cyclin D1 / D2 (cyclin D1 / D2) and BMI-1, but also tumors Tumor formation can also be suppressed by activating suppressors such as p16INK4a and p14 / p19Arf (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). It should be noted that we have successfully prepared mir-302-like gene silencing effector and / or its expression vector and successfully transfected it into target tumor / cancer cells in vivo, retroviral infection and transgene mutation. The risk of mutation could be avoided (FIGS. 8A-8C and FIG. 15). These findings provided strong evidence that mir-302-like gene silencing effectors could be used as therapeutics and / or vaccines for anti-tumor / cancer therapy. In addition, since mir-302 has a function of restoring and regenerating damaged human tissue (Lin et al., 2008 and 2010 (Non-Patent Documents 1 and 6)), the present invention is directed to antitumor / cancer treatment. In addition to being useful for applications, it is also used for tissue recovery and / or organ regeneration.

  Apparently, the drawings described below are for illustrative purposes only and do not limit the present 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.Method 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 the 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 results of microribonucleic acid microarray analysis of induced mir-302 treated with 10 μM doxycycline (Dox) (n = 3, p <0.01) and 6 hours later. FIG. 5, p <0.01) was analyzed by Northern and Western blot analysis. FIG. 1E 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-hHFC cell clusters. FIG. 1F shows the characteristics of DNA laddering of apoptotic DNA breakage induced by mir-302 induction in each mirPS cell line after treatment with 10 μM doxycycline (Dox). 2A-2C show mir-302s biosynthesis and mirPS cell generation. Fig. 2A shows the biosynthesis mechanism of intron mir-302. The Mir-302 family cluster is transcribed with a gene encoding red fluorescent protein (RGFP), and is further isolated by the spliceosomal components and the dicer of RNA endoribonuclease (RNaseIII), a cytoplasmic enzyme. Spliced to mir-302 members. Of these, red fluorescent protein is used as an indicator of the amount of mir-302 produced. A 1-fold concentration of red fluorescent protein corresponds to a 4-fold concentration of mir-302. FIG. 2B shows the process 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 papilla) were transfected with 10 μg pTet-On-tTs-miR302s per test. After being induced and expressed by doxyxycline (Dox), the biosynthesis of mir-302 was based on the natural intronic miribonucleic acid pathway. FIG. 2C shows the process 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 papilla) were transfected with 10 μg pTet-On-tTs-miR302s per test. After being induced and expressed by doxyxycline (Dox), the biosynthesis of mir-302 was based on the natural intronic miribonucleic acid pathway. 3A-3C show the changes that mir-302 expressed after induction with doxycycline (Dox = 5 or 10 μg) caused to mirPS-hHFC cell properties. FIG. 3A shows changes in cell morphology and cell cycle speed before and after inducing cell reprogramming with doxycycline. The results of flow cytometric analysis of each cell cycle stage and its corresponding DNA content are shown graphically on 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) cells and mitotic (M) cells to all test cell clusters, respectively. Scale = 100 μm. FIG. 3B shows changes in cell morphology and cell cycle speed before and after inducing cell reprogramming with doxycycline. The results of flow cytometric analysis of each cell cycle stage and its corresponding DNA content are shown graphically on 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) cells 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 estimated to gradually accelerate after 72 hours. Scale = 100 μm. 4A-4B show analysis of pluripotency marker expression and pluripotent differentiation and assimilation in vivo. FIG. 4A shows the expression pattern after the human embryonic stem cell (hES) marker gene is induced in mirPS cells by high concentrations of mir-302, and the WA01- in which the human embryonic stem cell (hES) marker gene is a human embryonic stem cell. The result of the comparison by the northern blot analysis with the expression pattern 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 is 30% higher than that in H1 and H9 cells (> 30 times that of untreated hHFC), a 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 mirPS cells were transplanted into immunodeficient SCID-beige mice, tissues differentiated by mirPS cells were assimilated into surrounding 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 mouse uterus and abdominal cavity. We further dissected one week after transplantation and examined the tissue formation around the injection site. The day before dissection, mice were administered 10 μg doxycycline by tail vein injection. We have infused GFP cells that are RGFP positive, including intestinal epithelial tissue formed after intraperitoneal injection, myocardium after heart injection, and skeletal muscle formed after dorsal flank injection. We observed that they differentiated into the same type of cells. Assimilated mirPS cells are also similar to surrounding tissues such as intestinal epithelial MUC2, myocardial troponin T2 (troponin T type 2, cTnT), and skeletal myosin heavy chain (MHC). Tissue specific markers were expressed. FIGS. 5A-5D show that mirPS-hHFCs induced by doxycycline (Dox = 10 μM) have obtained the characteristics of human embryonic-like stem cells (hES-like). FIG. 5A shows the results of analysis of total 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 the appearance and increase of relatively small DNA fragments after being cleaved by the restriction enzyme HpaII is a significant increase in total CpG methylation across the entire genome in mirPS cells after treatment with 10 μM doxycycline (Dox) but not 5 μM. Proven decrease. FIG. 5C is a detailed methylation map obtained by sequencing 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 pluripotently differentiated by mirPS-hHFCs are derived from three embryonic germ layers. FIGS. 6A-6E show in vitro tumorigenicity analysis 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 speed before and after induction of mir-302 expression by doxycycline (Dox). The results of flow cytometric analysis of each cell cycle stage and its corresponding DNA content are shown graphically on the cell morphology (n = 3, p <0.01). FIG. 6B shows changes in cell morphology and cell cycle speed before and after induction of mir-302 expression by doxycycline (Dox). The results of flow cytometric analysis of each cell cycle stage and its corresponding DNA content are shown graphically on the cell morphology (n = 3, p <0.01). Figure 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 of the invasion activity in which suppression of tumors (and / or cancer cells) by Mir-302 appeared in the Matrigel chamber (n = 4, p <0.05). FIG. 6E shows a comparison result of the ratio of cells adhering to human bone marrow endothelial cells (hBMECs) monolayer before and after doxycycline-induced mir-302 expression (n = 4, p <0.05). Figures 7A-7D show the genes induced by mir-302 in G1-checkpoint regulators targeted by the luciferase 3'-UTR reporter gene assay Shows silencing effect. FIG. 7A shows the structure of the luciferase 3 ′ end untranslated region reporter gene with two normal (T1 + T2), or two mutations (M1 + M2), or normal and mutant There are mir-302 target sites with a mixture of (T1 + M2 or M1 + T2). The mutation site contains a mismatched TCC motif instead 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 that the G1 checkpoint regulator that is the main target of mir-302 is mirPS cells in the induction of mir-302 at high concentration (10 μM Dox) and low concentration (5 μM Dox) by Western blot analysis. 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 the in vivo tumorigenicity analysis, mir-302s (NTera2 + mir-302s) or mir-302d * (NTera2 + mir-302d *) in which tumor NTera2 cells were expressed by the vector. ) (n = 3, p <0.05). Mir-302s and mir-302d * in 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 after 3 weeks from the in-situ injection (post-is). All tumors were located at the original transplant site (shown by black arrows). No signs of cachexia or tumor metastasis were observed in any of the test mice. Figure 8B shows that cyclin-dependent kinase 2 is a G1 checkpoint regulator that targets mir-302 to the core reprogramming transcription factors Oct3 / 4-Sox2-Nanog and mir-302 in vivo by Northern and Western blot analysis. (CDK2), Cyclin D1 / D2 (cyclins D1 / D2), BMI-1, and the effect on the expression pattern of p16Ink4a and p14Arf. FIG. 8C shows cyclin-dependent kinase 2 (CDK2), a G1 checkpoint regulator that targets mir-302 to core reprogramming transcription factors Oct3 / 4-Sox2-Nanog and mir-302 in vivo by immunohistochemical staining analysis. ), Cyclins D1 / D2 (cyclins D1 / D2), BMI-1, and the effect on the expression pattern of p16Ink4a and p14Arf. FIGS. 9A-9C show mirPS-hHFC and telomerase activity analysis of 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 telomeric repeat amplification protocol test (TRAP) (n = 5, p <0.01). Telomerase activity is affected by RNase treatment (hHFC + RNase). Figure 9B shows that analysis of Western blots showed consistent increases in human telomerase reverse transcriptase (hTERT) expression in various mirPS cell lines, but decreased expression in AOF2 and HDAC2 (n = 5 , P <0.01). FIG. 9C measured telomerase activity in telomerase PCR ELISA test (OD470-OD680; n = 3, p <0.01). 10A to 10D show the silencing effect induced by mir-302 on the target epigenetic gene. FIG. There are multiple (T1 + M2 or M1 + T2) mir-302 target sites. 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 induction with doxycycline (Dox) on luciferase expression (n = 5, p <0.01). Figures 10C and 10D show the epigenetic gene that is the main target of mir-302 in the induction of mir-302 at high (10 μM Dox) and low (5 μM Dox) concentrations by Western blot analysis. Is a comparison result of expression change 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 cell cycle regulation mechanism. Based on previous and current studies, we have discovered two parallel events. First, mir-302 initiates reprogramming with strong silencing effects on multiple epigenetic regulators, AOF1 / 2, MECP1 / 2, and HDAC2, while degrading total genomic DNA. Inducing methylation activates the cell marker gene of human embryonic stem cells that is further required to induce SCR (gray marker). Second, the G1 checkpoint regulator cyclin-dependent kinase (CDK2), cyclins D1 / D2 (cyclins D1 / D2) and BMI-1 co-suppression and activation of p16Ink4a and p14 / p19Arf attenuate the cell cycle. By causing it, it suppresses the whole cell activity to prepare for SCR (black marker). Prevents random growth and / or tumor-like transformation of reprogrammed pluripotent stem cells that can also be dormant (G0 / G1) pauses. In short, the result of the cooperative effect of these two events is that a more accurate and safe reprogramming process can be simultaneously suppressed with pre-mature and tumor formation. Figures 12A-12C show cell morphology and proliferation after transgene expression of mir-302-like RNA molecules in human epithelial primary cultured cells (hpESC), human prostate cancer cells (PC3) and human primary melanoma cells (Colo) Shows changes. After mir-302 transfection, cells that are positive for mir-302 expression are hpESC + mir-302, PC3 + mir-302, and Colo + mir-302, respectively. FIG. 13 schematically shows the natural biosynthesis mechanism of intron miRNA miR-302. Mir-302 is a natural intron miRNA encoded by the intron region of the La ribonucleoprotein domain family member 7 gene (LARP7). In the biosynthesis of mir-302, the primary miRNA precursor (pri-miRNA) is first transcribed with a transcript of the LARP7 gene by type II RNA polymerase (Pol-II), and then the hairpin-like miRNA by the cell spliceosome Modified to 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, along 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 preclinical study (pre-IND) in which mouse skin open wounds are treated with pro-mir-302. The test included the upper blank group, ie treatment with ointment alone, the middle pro-mir-434 concentration 10 μg / mL ointment (negative control group), the lower pro-mir-302 concentration 10 μg / mL ointment Including three different treatments. FIG. 15 shows the results of an in vivo new preclinical study (pre-IND) for treating human liver cancer cells xenografts in SCID nude mice using pro-mir-302 as an injection drug. After 3 treatments (once a week), pro-mir-302 (pre-mir-302) drugs removed tumors from 728 ± 328 mm ³ (untreated blank group, control group C) to 75 ± 15 mm ³ (Treatment with pro-mir-302, group T) was successful and showed a reduction rate close to 90% of the average tumor size. After being treated with a mimetic such as synthetic siRNA (siRNA-302) in the same process, no significant therapeutic effect was found. In a further histological study (rightmost) normal xenografted liver cancer cells / tumors treated with pro-mir-302, a normal liver lobule-like structure (circles indicated by black arrows) was observed, It was shown not observed in other treatment groups or control groups. This result suggested that the reprogramming mechanism can restore malignant cancer cell properties to a relatively normal state (cancer reversion). FIG. 16 shows the 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), pro-mir-302 (pre-mir-302) drugs are treated with advanced (grade IV) xenograft human liver cancer at a relatively benign (grade II Successfully reprogrammed to a lower) state. Treated xenograft cancer (tumor), like normal liver tissue (above), has a central vein (CV) -like and portal vein three-pipe (PT) -like structure (shown by black arrows) A typical liver lobule can be formed. In general, cancer cells are more acidic than normal hepatocytes, and hematoxylin-eosin (H & E) staining results in a relatively purple cancer cell color and a comparison of normal hepatocyte color. Indicates red. Figure 17 shows the histopathological comparison of untreated, siRNA-treated xenografted human liver cancer (tumor) and normal liver tissue in SCID-beige nude mice. Indicates. Untreated (top), xenografted human liver cancer (tumor) invasively invades normal tissues such as muscle and blood vessels to form large cell-cell and cancer-tissue fusion structures. Its highly malignant and metastatic. Treatment with a mimetic such as siRNA (siRNA-302) did not significantly reduce the malignancy of the xenografted liver tumor (middle figure). The reason may be 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 to a normal hepatocyte-like form (unfused), but also cancer (cells) We succeeded in suppressing the invasion of normal tissues around the (Fig. Compared with normal liver tissue (below), malignant tumors treated with pro-mir-302 have a hepatic lobule-like structure, normal glandular-like arrangement, and cell-cell and cancer-tissue junction (black arrow) Showed a very clear boundary. This proved that the treated cancer (tumor) was in a relatively benign state.

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

  The present invention provides a novel nucleic acid composition and a method for inhibiting human tumor / cancer cell growth and tumor formation using a recombinant mir-302-like gene silencing effector. Unlike conventional small hairpin ribonucleic acid (shRNA) designs, the small hairpin ribonucleic acid according to the present invention contains 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 is similar to natural microribonucleic acid precursors (pre-miRNAs) into the nucleus without interfering with transport of transporting ribonucleic acid (tRNA). It can provide transportation export efficiency. Without being limited to any particular theory, the anti-proliferative, anti-tumorogenic effects of the present invention can express newly discovered mir-302 family clusters (mir-302s) or small hairpin ribonucleic acids homologous to mir-302 FIG. 5 shows a mir-302 mediated gene silencing mechanism caused by transfection with a recombinant nucleic acid composition. All manually redesigned miRNA / shRNA molecules have 5'-UAAGUGCUUC CAUGUUU-3 '(SEQ.ID.NO.3), a similar sequence to the first 17 nucleotides of its 5' end 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).

  In order to position the role of mir-302 in the human cell cycle, we designed an inducible pTet-On-tTS-miR302s expression vector (Figure 1A, Example 2) in normal human cells and human cancer cells. Transfected. Mir-302s is a hES-specific microribonucleic acid (miRNA) family with four small non-coding families: 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 cytomegaloviral (CMV) promoter controlled by tetracycline-response-element (TRE) under the stimulation of doxycycline (Dox). The After transfection, mir-302 biosynthesis is based on the natural intron microribonucleic acid biosynthetic pathway, of which mir-302 is transcribed with a reporter gene (RGFP) that encodes a red fluorescent protein, and then spliced. The spliceosomal components and the cytoplasmic RNA endoribonuclease RNase III, Dicers, are spliced to a single mir-302 member (FIG. 2A) (Lin et al., 2008). For quantification, 1-fold concentration of RGFP corresponds to 4-fold concentration of mir-302. Microarray analysis of microribonucleic acid confirmed that all mir-302 members except mir-302b * were effectively expressed in transfected cells under the stimulation of doxycycline (Dox) (Fig. Example 3). The process of transfecting cells with the pTet-On-tTS-miR302s expression vector is outlined in FIGS. 2B-2C.

  In addition, mir-302-expressing nucleic acid compositions such as pTet-On-tTS-miR302s express mir-302, a Kozak consensus translation initiation site that improves translation efficiency in eukaryotic cells. Multiple SV40 polyadenylation signals (SV40 polyadenylation signals) located downstream of the mir-302 construct, the pUC replication origin for prokaryotic proliferation, and the mir-302 expression construct (i.e., SpRNAi- RGFP) into the nucleic acid composition at least two restriction sites, a selective SV40 origin of replication required for replication in mammalian cells expressing the SV40 T antigen, and an antibiotic resistance gene in a replicable prokaryotic cell. Contains a selective SV40 early promoter for expression. The expression of the antibiotic resistance gene is used as a selective marker to isolate the positive cloning in which the transgene is expressed. These antibiotics include G418, neomycin, puromycin, penicillin G, ampicillin, kanamycin, streptomycin, erythromycin, spectromycin ), Fophomycin, tetracycline, doxycycline, rifapicin, amphotericin B, gentamycin, chloramphenicol, cephalothin, tyrosine (cephalothin) tylosin), and combinations thereof.

In our previous study, Mir-302 attenuates the cell cycle of normal cells without causing apoptosis . In human melanoma cells Colo-829 and prostate cancer cells PC3, increased mir-302 expression in these malignant cancer cells Have been shown to be reprogrammed to a pluripotent state similar to hES (Lin et al., 2008). In the process of somatic cell reprogramming (SCR), mir-302 causes apoptosis of more than 98% (> 98%) of cancer cells and further increases the proliferation rate of the remaining (<2%) reprogrammed cells. Significantly reduced. Certainly these properties are beneficial for the treatment of cancer, but mir-302 is not yet sure to function in normal human cells. To evaluate its effect, we introduced an inducible pTet-On-tTs-miR302s expression vector into normal human hair follicle cells (hHFCs). The reason for choosing hHFCs is that the amount is sufficient, reachable, and fast growing. As the concentration of doxycycline (Dox) increases to 10 μM, we have core reprogramming factors at the threshold of doxycycline concentrations higher than 7.5 μM (> 7.5 μM) (Figure 1D, Example 5). ) Oct4-Sox2-Nanog was promoted all at once, and proliferative cell clusters were reduced by 70%, i.e. from the original 37% ± 2% to 11% ± 2% (FIG. 1E, M phase, Example 7) was observed. Correspondingly, the dormant cell cluster increased by 41%, i.e., increased from the original 56% ± 3% to 79% ± 5% (FIG. 1E, G0 / G1 phase, Example 7). (MirPS cells; Lin et al., 2008), which was found in mir-302-reprogrammed pluripotent stem cells reprogrammed by -302, suggested a similarity to a potent antiproliferative effect. However, hHFC cells reprogrammed with mir-302 (mirPS-hHFC) show no signs of DNA laddering or cell death that can measure apoptosis (Figure 1F, Example 6), Compared with tumor / cancer cells, normal cells were more tolerated by mir-302. Tumor / cancer cells are considered to be extremely difficult to survive in such a dormant state due to vigorous metabolism and rapid growth expansion.

  It should be noted that after treatment with doxycycline at a concentration higher than 7.5 μM, the morphology of mirPS-hHFC changed from spindle-shaped wrinkles 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). Under 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 changes from a hHFC somatic pattern to an hES-like expression pattern and is higher than 93% with H1 / H9 cells (> 93%) It was further shown to have similarity (FIG. 5A, Example 8). Demethylation of total genomic DNA, the first sign of somatic cell reprogramming (SCR) initiation, can be clearly measured in these mirPS-hHFCs, and the pattern of demethylation in H1 / H9 cells The same (FIGS. 5B and 5C, Example 9). In addition, each mirPS-hHFC cell grows into a single embryoid-like colony, and the cell division rate of 20-24 hours per cycle is consistent with the anti-proliferative effect of mir-302 (Figure 3C). ). We find that these mirPS-hHFCs are pluripotent because they form teratoid tissue cysts only in the uterus and abdominal cavity of pseudopregnant immunodeficient SCID-beige mice Special attention was paid to the lack of tumoregenetic. These teratoid cysts include a variety of tissues derived from three embryonic germ layer tissues: ectoderm, mesoderm, and endoderm (FIG. 5D, Example 10). In addition, when mirPS-hHFCs were xenografted into normal male mice, mirPS-hHFCs were assimilated into the surrounding tissues and exhibited similar tissue markers. This also indicated that mir-302 could be used to heal damaged cells (FIG. 4B). In summary, mir-302 suggests that somatic cell hHFCs can be reprogrammed into hES-like iPS cells (induced pluripotent stem cells). Transcription factors such as Oct3 / 4 and Sox2 are important for the expression of mir-302 (Marson et al., 2008; Card et al., 2008), and mir-302 replaces Oct3 / 4-Sox2 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 initiates the two events. It was shown to be a threshold value. In previous experiments, the event could not be triggered when using a single mir-302 member or a concentration of mir-302 corresponding to a relatively low concentration in H1 / H9 cells. We also demonstrated that mir-302 concentrations induced by a relatively low concentration of 5 μM doxycycline cannot silence the reporter gene target site or target G1 checkpoint regulator. Compared to natural development, embryonic cells before the morula stage (32-64 cell stage) often show a very slow cell cycle rate similar to that of mirPS cells. Such cell cycle regulatory effects were not found in blastocyst-derived hES cells. From this, it can be inferred that mir-302 at a relatively low concentration in the hES cytoplasm is insufficient to silence target G1 checkpoint regulators and oncogenes. This may explain why blastocyst-derived hES has a dramatic ability to grow and a tendency to form tumors. Therefore, the present invention can also be applied to reduce 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 induce cancer cell apoptosis 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 that breast cancer cells MCF7, liver cancer cells Hep G2, and embryonic The possibility in the teratoma cell NTera-2 was further tested. As shown in FIGS. 6A-6B, three tumor / cancer cells were transfected with the pTet-On-tTS-miR302s vector and all were reprogrammed into dormant mirPS cells under stimulation with 10 μM doxycycline, and Embryoid body-like colonoes were formed. In all three tumor / cancer cells, the concentration level of mir-302 also caused significant apoptosis (> 95%) (FIG. 1F, Example 6). Furthermore, as a result of analyzing the DNA content at each stage of the cell cycle by flow cytometry, it was shown that the mitotic cell cluster in all mirPS cells was 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, there was a 62% reduction from 50% ± 6% to 19% ± 4%. Conversely, the resting / dormant cell cluster (G0 / G1 phase) is from 41% ± 4% to 74% ± 5% in mirPS-MCF7 cells in mirPS-MCF7, mirPS-HepG2 and mirPS-NTera2 cells, There was an increase of 80%, 65% and 72% in mirPS-HepG2 cells from 43% ± 3% to 71% ± 4% and in mirPS-NTera2 cells from 40% ± 7% to 69% ± 8%, 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 Matrigel chambers) and cell adhesion tests (cells adhere to a single cell layer of human bone marrow endothelial cells (hBMEC)), mir-302 Have anti-proliferative properties and two other anti-tumor effects. Cell invasion assay showed that all three dormant mirPS-tumor / cancer cells lost their ability to migrate (reduced to <1%), but the original tumor / cancer cells had higher nutrients. Forcibly invade the supplemented compartment area and occupy more than 9% ± 3% in MCF7 cells, 16% ± 4% in Hep G2 cells, and 3% ± 2% cell clusters in NTera-2 cells (FIG. 6D, Example 11). Cell adhesion assay consistently showed that these mirPS-tumor / cancer cells could not adhere to the hBMEC single cell layer, but after the original MCF7 and Hep G2 cells had been cultured for 50 minutes, A significant number of cell clusters (MCF7 7% ± 3%, Hep G2 20% ± 2%) rapidly transferred to the hBMEC single cell layer (FIG. 6E, Example 12). In summary, all findings so far show that mir-302 is a human tumor suppressor and can slow down rapid cell growth, induce tumor / cancer cell apoptosis, tumor / cancer cell invasion and Again, it was strongly shown that metastasis can be suppressed. Most importantly, this novel function of mir-302 is a diverse human tumor not limited to malignant skin cancer (Colo-829), prostate cancer (PC-3), breast cancer (MCF7), and liver cancer (HepG2) / Can provide general-purpose treatment for various types of tumors from the viewpoint of being able to combat cancer and containing various different tissues in teratomas (NTera-2).

Mir-302-mediated antiproliferative function is achieved by co-suppression against CDK2, cyclin-D1 / D2, and BMI-1
To confirm the actual interaction between mir-302 and its target G1 checkpoint regulator, we conducted a luciferase 3'UTR region reporter test. (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. The target G1 checkpoint regulators that were included caused significantly different inhibitory effects. In the presence of 10 μM doxycycline, mir-302 binds effectively to the target sites of CDK2, cyclins D1 / D2, and BMI-1 transcripts, resulting in greater than 80% (> 80%) reporter luciferase expression. 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 is consistent with the results of the luciferase 3'-terminal untranslated region reporter gene test (Figure 7C, Example 5) . In contrast, the relatively small expression of mir-302 induced by 5 μM doxycycline is prominent for either the reporter gene target site or a target G1 checkpoint regulator other than cyclin D2. No silencing effect can be induced (FIGS. 7B and 7D). This indicates a dose-dependent manner of mir-302 expression and the ability of mir-302 to fine tune cell cycle rate based on the dose dependency. In the mammalian cell cycle, the G1-S phase transition is usually driven by cyclin-D-CDK4 / 6 and cyclin-E-CDK2, two compensatory cyclin-cyclin-dependent kinase complexes (cyclin-CDK complexes). Controlled (Berthet et al., 2006). We reprogrammed, while high concentrations of mir-302 inhibit two pathways controlling G1-S transition by inactivating the two complexes through co-suppression against CDK2 and cyclins D1 / D2. It was found to reduce the cell cycle rate in mirPS cells. In hHFCs and mirPS cells, the finite expression level of cyclin D3 does not compensate for the loss of cyclins D1 / D2 in mirPS cells.

  We further detected that p16Ink4a and p14Arf expression increased slightly with BMI-1 silencing (increase rates in hHFCs were 63% ± 17% and 57% ± 13%, respectively), but p21Cip1 There was no change in the expression of (Fig. 7C). The deficiency of BMI-1, an oncogenic cancer stem cell marker, suppresses the 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). Thus, Rb is prevented from releasing E2F necessary for entering S phase, and further, E2F-dependent transcription is performed (Parry et al., 1995; Quelle et al., 1995). P14Arf also permits p53-dependent transcription (Kamijo et al., 1997), which plays a role in G1 arrest or apoptosis, while preventing the binding of HDM2 to p53. However, as far as is known, embryonic stem cells are not regulated by cyclin-D-dependent CDK (Burdon et al., 2002; Jirmanova et al., 2002; Stead et al. , 2002), it is recognized that CDK2 is a major determinant for inducing the G1-S transition in the cell cycle regulation of hES cells. Therefore, CDK2 silencing is most likely to affect the G1 arrest of mirPS cells (G1-arrest) .Co-suppression of cyclin-D and BMI-1 and co-activation of p16Ink4a and p14Arf It additionally suppresses cell growth induced by tumorigenetic signal-induced. Moreover, the loss of cyclin-D and the activation of p16Ink4a can also be interpreted as the lack of cyclin-D-dependent CDK activity in embryonic stem cells.

  Therefore, the stringency of the interaction between the microribonucleic acid (miRNA) and the target gene can determine the original function of the microribonucleic acid. Due to different cellular conditions, microribonucleic acids represent different preferences of gene targets. The present invention has discovered its important details and has disclosed for the first time that mir-302 has very different functions in human and mouse cells. mir-302 strongly targets CDK2, cyclins D1 / D2 and BMI-1 in human cells, but interestingly it does not contain p21Cip1. Unlike mouse p21Cip1, human p21Cip1 does not contain any of the mir-302 target sites. This difference in gene target led to an important schism in the cell cycle regulation of both. In mouse embryonic stem cells (mES), mir-302 silences p21Cip1 and promotes tumor-like cell proliferation (Wang et al., 2008; Judson, 2009), but in human mirPS cells In which p21Cip1 expression is maintained, leading to relatively slow cell growth and relatively low tumorigenicity. In addition, mouse BMI-1 is also not a target gene for mir-302 because it lacks an appropriate target site. We found that silencing of human BMI-1 slightly stimulates p16Ink4a / p14ARF expression and attenuates cell proliferation in mirPS cells, whereas mir-302 silences murine BMI-1 Disclosed that similar effects cannot be induced in mouse cells. In mirPS cells, p16Ink4a / p14ARF expression increases and p21Cip1 is not affected. It should be understood that it is most likely to exert through the p16Ink4a-Rb and / or p14 / 19ARF-p53 pathway other than the repressive pathway. The striking differences in the preference of Mir-302 targeting human and mouse genes suggest that there is a fundamental difference in their cell cycle regulatory mechanisms.

Treatment of Mir-302 reduces tumor cell growth in vivo by more than 90% without altering stem cell pluripotency. Whether 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). The tumor Tera-2 cell line (neoplastic Tera-2, NTera-2) is a pluripotent human embryonal teratocarcinoma cell line, which is a variety of primitive somatic tissue in vivo. ), In particular, can be differentiated into primitive glandular tissue and primitive neural tissue (Andrews et al., 1984). Because of its pluripotency, NTera-2 derived teratomas can serve as models for treating various tumors in vivo. For drug administration, we injected pCMV-miR302s expression vector prepared with polyethylenimine (PEI) by in situ injection as close as possible to the tumor. The pCMV-miR302s vector was constructed by changing the CMV promoter controlled by a tetracycline response element (TRE-controlled) to a common CMV promoter (Example 2), but for DNA methylation, human cells of pCMV-miR302s The onset lasts for about a month. After injection of the pCMV-miR302s vector with an upper limit of 10 μg / mouse body weight (g) (maximum amount of a single injection), no signs of disease or cachexia were observed in the mice. Safety has been proven. Histological examination also showed that no tissue damage was detected in the brain, heart, lung, liver, kidney, and spleen.

In the Examples of the present invention, when treated 5 times (treated every 3 days) with 2 μg / mouse body weight (g) of pCMV-miR302s, there is a remarkable inhibitory effect on the growth of teratomas. I found out. 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) is untreated (104 ± 23 mm ³ , Compared with n = 4), it decreased more than 89% (> 89%). In contrast, administration of an equal amount of antisense-mir-302d (antisense-mir-302d) expression vector (pCMV-miR302d *) prepared with PEI reduced the size of the teratomas to the original 140% (250 ± 73 mm). ³ and n = 3). Based on the above results, the present invention found that NTera-2 cells expressed moderate mir-302 (FIG. 8B). The results of Northern blot analysis also showed that in these differently treated teratomas cells, mir-302 expression was negatively correlated with tumor size (Figure 8B), ie, mir-302 This suggests that modulating the expression can effectively control teratoma growth in vivo. To verify past in vitro findings, we performed Western blot analysis in teratomas treated with mir-302 in the G1 checkpoint regulator CDK2-cyclins-D1 / D2-BMI-1 And co-activation of the core reprogramming factor Oct3 / 4-Sox2-Nanog was confirmed (FIG. 8B). Similar results were demonstrated when these proteins in teratomas were analyzed by immunochemical (IHC) staining (FIG. 8C, Example 14). Most notably, we have found that mir-302 can inhibit teratoma cell growth without affecting natural pluripotency. This suggests that high concentration of mir-302 plays both role of tumor suppressor and reprogramming factor. Based on the dual function of mir-302 and the consistent results obtained in vitro and in vivo, we found that the anti-proliferative mechanism of mir-302 discovered in vitro is responsible for teratoma growth in vivo. It can be applied to the suppression, so it was speculated that there is a possibility of treatment for various tumors.

Mir-302 inhibits human epithelial primary culture cells (hpESC), human prostate cancer cells (PC3) and human melanoma cells Colo-829 (Colo) . To silence the targeted gene by transfection, the linked mir-302a-mir-302b-mir-302c-mir-302d pre-miRNA cluster (SEQ.ID.NOs.9-16) or manually A series of mir-302 configurations, including mimics such as pre-mir-302 redesigned in (e.g., a hairpin-like sequence containing 5'-UAAGUGCUUC CAUGUUU-3 '(SEQ.ID.NO.3)). 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). It should be noted that these homologous mir-302-like gene silencing effectors are highly conserved (100% homology) in the first 17 nucleotides in the 5 ′ end region, ie the sequence 5′-UAAGUGCUUC CAUGUUU. -3 '(SEQ.ID.NO.3) is shared. In these mir-302 homologous sequences, thymine (T) may be used instead of uracil (U).

  In these experiments, we used the mir-302 homologue (SEQ.ID.ID) to redesign the pre-miRNA cluster of mir-302a-mir-302b-mir-302c-mir-302d in hpESC and PC3 cells. .NO.75) were each transfected. After transfection of these mir-302, all cell lines change their cell morphology (below) from a spindle or amoeba shape to a more rounded shape, which can cause cells to lose their cell migration ability, and It suggests having a very slow cell replication rate similar to that of stem cells (FIGS. 12A-12C). Flow cytometric analysis showing the DNA content (y-axis) at different cell cycle stages (x-axis) (upper figure) shows a mitotic cell population decreased by more than 67% and slow cell growth of mir-302 transfected cells Although the rate was confirmed, the number of cell populations is indicated by the DNA content of each cell cycle stage. The first (left) and second (right) peaks indicate the level of the number of cell populations in the G0 / G1 phase of dormancy and the M phase of mitosis in the entire test cell population, respectively. After mir-302 transfection, the mitotic cell population 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 empty. In groups transfected with a vector or a vector expressing a pre-miRNA containing mir-gfp, there is no significant change in either cell morphology or cell proliferation after transfection. Pre-miRNA containing mir-gfp is used to target firefly EGFP genes that are not homologous to human and mouse genes. Based on these findings, we show that transgene expression of mir-302 homologues can transform human primary cultured cells and cancer cells 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).

Cancer progression is considered irreversible because of the accumulation mutation in the gene that reprograms highly malignant human liver cancer xenografted with Mir-302 to a low-grade benign state in vivo . However, the present invention discloses a novel function of the precursor of microribonucleic acid mir-302 (pre-mir-302), that is, reprogramming highly malignant cancer / tumor to a low stage to benign state, It may belong to a rare natural healing process called spontaneous cancer regression. Spontaneous cancer regression occurs very rarely in 1 of approximately 100,000 cancer patients. The inventor has discovered that treatment with pre-mir-302 can improve 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 a therapeutic agent for malignant human liver tumors xenografted in SCID-beige nude mice. -302 drug successfully reduced malignancy from 728 ± 328 mm ³ (untreated blank, control group C) to 75 ± 15 mm ³ (treated with pro-mir-302, group T) With a reduction rate approaching 90% of the average tumor size. In contrast, treatment with other mimics such as synthetic siRNA (siRNA-302) did not show any approximate therapeutic effect. In a further histological study (rightmost), only a group of xenografted malignant tumors treated with pro-mir-302 showed a normal liver lobule-like structure (circles indicated by black arrows) This was not observed in the treatment group or control group, and it was proved that the reprogramming mechanism restored the malignant cancer cell properties to a relatively normal state (cancer reversion). . This novel reprogramming mechanism may be related to the function of mir-302 tumor suppressor. This mechanism silences multiple human oncogenes at the same time, particularly targeting them, and eliminates / prevents activation of tumor signaling pathways. However, this reprogramming mechanism may be different from the previously reported reprogramming function of mir-302 on somatic cells because the appearance of pluripotent Oct4-positive stem cells has not been confirmed (Lin et al., 2008 and 2011). ).

  In more detailed histological studies, pro-mir-302 drug reprograms advanced (grade IV) xenografted human liver cancer cells / tumors to a relatively benign (lower than grade II) state Further disclosed that it was successful in doing. As shown in FIG. 16, the treated xenograft cancer (tumor) is a typical hepatic lobule with central vein (CV) -like and portal vein three-pipe (PT) -like structures (shown by black arrows). It is highly similar to normal liver tissue structure (above). Comparison of untreated, siRNA-treated, pro-mir-302-treated xenograft human liver cancer (tumor) with normal liver tissue in nude SCID-beige mice (Figure 17), Untreated (top), xenografted human liver cancer (tumor) invasively invades surrounding normal tissues, such as muscles and blood vessels, resulting in large cell-cell and cancer-tissue fusion structures. It was found to have formed and showed its high malignancy and metastasis. Treatment with mimetics such as siRNA (siRNA-302) did not significantly reduce the malignancy of xenografted liver cancer cells / tumors (middle panel). The reason may be 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 to a normal hepatocyte-like form (unfused), but also cancer (cells) We succeeded in suppressing the invasion of normal tissues around the (Fig. Compared with normal liver tissue (below), malignant tumors treated with pro-mir-302 have a hepatic lobule-like structure, normal glandular-like arrangement, and cell-cell and cancer-tissue junction (black arrow) Showed a very clear boundary. This proved that the treated cancer (tumor) became relatively benign. Subsequent treatment with drugs containing 6 or more pro-mir-302s completely resolved the xenografted cancer cells / tumors in all 6 samples.

Mir-302 does not shorten telomere but activates p16Ink4a / p14ARF to accelerate senescence and limit expansion in human induced pluripotent stem cells (iPS) that induce cell senescence (Banito et al., 2009; Feng et al., 2010). Normal mature cells undergo a finite number of divisions and eventually reach a quiescence state called replicative senescence. Since cells that have avoided replication senescence usually become immortal cells such as tumor / cancer cells, replication senescence is the normal defense mechanism against tumor / cancer cell formation. In the present invention, we have found that mir-302 can cause p16Ink4a / p14ART-related cell cycle regulation by directly silencing BMI-1. In other studies, BMI-1 activates the transcriptional action of human telomerase reverse transcriptase (hTERT), which increases 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 telomeric repeat amplification protocol (TRAP) (Example 16) and measured the activity of telomerase. As shown in FIG. 9A, all mirPS cells treated with 10 μM doxycycline surprisingly resemble their original tumor / cancer cells and human stem cells H1 / H9 cells and showed intense telomerase activity. Western blot analysis also showed that hTERT expression increased rather than decreased in these mirPS cells (FIG. 9B). Telomerase PCR ELISA test demonstrated increased relative activity of telomerase (FIG. 9C). In addition, we could further detect silencing of lysine-specific histone demethylase (lysine-specific histone demethylase AOF2 or KDM1 / LSD1) and histone deacetylase (histone deacetylase HDAC2) in mirPS cells (Figure 9B). ). Previous studies have indicated that AOF2 is required to repress hTERT transcription, and that deficiencies in both AOF2 and HDAC2 cause overexpression of hTERT (Won et al., 2002; Zhu et al., 2008). We have also found in the present invention that AOF2 and HDAC2 are strong targets of mir-302 and were both silenced in mirPS cells (FIG. 10). Therefore, in mirPS cells, mir-302 does not actually induce hTERT-associated senescence, but enhances mirPS cell telomerase activity. However, the effect of increasing hTERT activity is neutralized by the suppression of BMI-1 caused by mir-302 and the activation of p16Ink4a / p14ARF, reaching a balance that prevents the formation of tumor / cancer cells in mir-302 cells Yes.

  In short, in the present invention, mir-302 having a novel tumor suppressor function is used as a cancer therapeutic agent. We have 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 occur 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 massively demethylated and more than 91% of the cell transcriptome is reprogrammed to an hES-like gene expression pattern. We were able to recognize the complex mechanism of cell cycle regulation associated with mir-302 during SCR by elucidating the interaction between mir-302 and its target gene (Figure 11). In our past studies, mir-302 silenced its target epigenetic regulators while activating Oct3 / 4-Sox2-Nanog co-expression, followed by 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 the cell cycle rate has biological significance in preventing the oncogenic ability of oncogenes that are well activated during the SCR period. In this regard, mir-302 silences CDK2 and cyclins D1 / D2 and inhibits G1-S phase transition. At the same time, inhibition of BMI-1 further increases the activity of p16Ink4a and p19Arf tumor suppressors. Through coordinated regulation of these cell cycle pathways, mir-302 can initiate the progression of somatic cell reprogramming (SCR) without deteriorating cellular tumorigenecity.

  The present invention has at least five frontier innovations. First, the mir-302-like gene silencing effector replaces all four core reprogramming transcription factors Oct-Sox2-Klf-c-Myc and Oct4-Sox2-Nanog-Lin28 to make human cells hES-like Can be reprogrammed into stem cells. These reprogrammed cells are useful for stem cell therapy. Second, since mir-302-like gene silencing effectors have a small size (including about 23 ribonucleotides), the expression vector for 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, not normal human cells. Finally, the present invention uses a transfection by polysomes, liposomes, and electroporation instead of retrovirus / lentivirus infection to transform a nucleic acid composition 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 therapy. In summary, these innovations show the potential to treat tumors / cancers by using mir-302-like gene silencing effectors and compositions that express the mir-302-like gene silencing effectors, Provided a new design that could be developed into therapeutic drugs and / or vaccines.

A. Definitions In order to make the present invention easier to understand, several terms are defined as follows.
Nucleotide : A monomolecular deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) containing a sugar component (pentose), a phosphate group, and a nitrogenous heterocyclic base. The base is bound to the sugar component by glycoside carbon (1 'carbon of the pentose), and the combination of the base and sugar is a nucleoside. A nucleoside having at least one phosphate group bonded to the 3 ′ end or 5 ′ end of the pentose is a nucleotide. That is, DNA and RNA are composed of different nucleotide units, that is, deoxyribonucleotides and ribonucleotide units, respectively.

Oligonucleotide : A molecule containing 2 or more, preferably 3 or more, and usually 10 or more deoxyribonucleotides (DNAs) or ribonucleotides (RNAs). Oligonucleotides that are longer than 13 nucleotide monomers in length are also called polynucleotides. The exact length depends on many factors, and further on the optimal function or use of the oligonucleotide. Oligonucleotides may be generated by any method including chemical synthesis, DNA replication, RNA transcription, reverse transcription, or a combination thereof.

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

Nucleic Acid Composition : Deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a mixture of deoxyribonucleic acid and ribonucleic acid (DNA / RNA) present in the form of a single-stranded or double-stranded molecular structure 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 RNA, such as small hairpin RNA (shRNA), micro RNA (miRNA), ribosomal RNA (rRNA), transport RNA (tRNA), small nucleolar RNA (snoRNA), low nuclear It may encode molecular RNA (snRNA) and their RNA precursors and derivatives. On the other hand, the gene may also encode protein-coding RNA, such as messenger RNA (mRNA) and its RNA precursors and derivatives essential for the synthesis of proteins / peptides. In some examples, the protein-encoding RNA may comprise at least one miRNA or shRNA sequence.

Primary RNA Transcript : An RNA sequence that has been transcribed by a gene and has not undergone any RNA treatment or modification. Primary ribonucleic acid transcripts include mRNA, heteronuclear RNA (hnRNA), rRNA, tRNA, snoRNA, snRNA pre-microRNA, viral RNA, and their RNA precursors and derivatives.

Premessor 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 pre-messenger ribonucleic acid sequence includes a 5 ′ terminal untranslated region (5′-untranslated region, 5′-UTR), a 3 ′ terminal untranslated region (3′-untranslated region, 3-UTR), an exon and Intron is included.

Intron : A portion or portions of a gene transcript sequence that encodes a non-protein reading frame. For example, in-frame intron, 5 ′ end untranslated region (5′-UTR) and 3 ′ end untranslated region (3′-UTR).

Exon : A portion or portions of a gene transcript sequence that encodes a protein reading frame (cDNA). The cDNA is, for example, a cellular gene, growth factor, insulin, antibody, and analogs / homologs and derivatives thereof.

Messenger ribonucleic acid (Messenger RNA, mRNA) : A combination of exons of pre-messenger ribonucleic acid. A pre-messenger ribonucleic acid is formed after intron removal by an intracytoplasmic RNA splicing mechanism (intracellular RNA splicing machines, spliceosomes) and functions as a RNA encoding a protein in peptide / protein synthesis. Peptides / proteins encoded by mRNA include, but are not limited to, enzymes, growth factors, insulin, antibodies and analogs / homologs and derivatives thereof.

Complementary deoxyribonucleic acid (cDNA) : A single-stranded 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 having the same sequence and composition as mRNA. The symbol “+”, “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 added in front of DNA or RNA, such as "aDNA" or "aRNA".

Base pair (bp) : A match between adenine (A) and thymine (T) or cytosine (C) and guanine (G) in a double-stranded DNA molecule. is there. In RNA, uracil (U) is used instead of thymine (T). In RNA, uracil may match guanine. In general, the matches are linked through hydrogen bonding. As an example, the sense nucleic acid sequence “5′-ATCGU-3 ′” and its antisense nucleic acid sequence “5′-ACGAT-3 ′” or “5′-GCGAT-3 ′” are complementary.

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

3'-end : The end lacking one nucleotide at the 3'-end position of consecutive nucleotides. In the consecutive nucleotides, the 5 ′ hydroxyl group of one nucleotide is linked to the 3 ′ hydroxyl group of the next nucleotide by a phosphodiester bond. There may be other groups at the end, usually a hydroxyl group.

Template : A nucleic acid molecule that can be copied by a nucleic acid polymerase. Depending on the different polymerase, 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 the nucleic acid duplex are always aligned, the 5 'ends of these duplexes are located at the opposite ends of the duplex (if necessary, the two strands of these duplexes The same applies to the 3 ′ end).

Nucleic acid template : a double-stranded DNA molecule, a double-stranded RNA molecule, a hybrid molecule (eg, DNA-RNA or RNA-DNA hybrid), or a single-stranded DNA or RNA molecule.
Conserved : When a nucleotide sequence hybridizes non-randomly with a precisely complementary sequence (reference sequence), both sequences are conserved.

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

Complementary (or complementarity or complementation) : two polynucleotides (ie, mRNA and cDNA sequences) in which a base match occurs according to a base pair (bp) rule. For example, the sequence “5′-AGT-3 ′” is complementary to the sequences “5′-ACT-3 ′” and “5′-ACU-3 ′”, respectively. Complementation may occur between two DNA strands, between one DNA strand and one RNA strand, or between two RNA strands. Complementation may be “partial” or “complete” or “entire”. When only some nucleobases are matched according to the base pairing rule, partial complementarity or complementation is obtained. When the bases are completely or perfectly matched between these nucleic acid strands, it becomes complete or total complementarity or complementation. The degree of complementarity between nucleic acid strands greatly affects the efficiency and strength in hybridization between nucleic acid strands. This is very important for detection methods that rely on amplification reactions as well as binding between nucleic acids. The complementarity (Percent complementarity or complementation) is the ratio between the number of mismatched bases and the total number of bases in a single strand of the nucleic acid. Therefore, 50% complementation means that half of the bases are mismatched and half are matched. The two strands of nucleic acid can be complemented 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 base of the shorter strand.

Complementary base : A nucleotide that is normally matched 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 other single-stranded nucleotide sequence, and is unique between the two strands by hydrogen bonding Hybridize.

Hybridization (Hybridize 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 formed by hybridization (or hybrids) is sufficiently stable that DNA synthesis is initiated by DNA polymerase. The priming function necessary for this is provided. There is a specific interaction (ie non-random) between two complementary polynucleotides that is competitively inhibited.

Posttranscriptional Gene Silencing : The effect of target gene knockout or knockdown in mRNA degradation or translational suppression, generally foreign / viral DNA or RNA transgenes or small suppressions Caused by any of the sex RNAs.

RNA interference (RNAi) : A post-transcriptional gene silencing mechanism in eukaryotic cells, such as microribonucleic acid (miRNA), small hairpin ribonucleic acid (shRNA) and small interference 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 fully or partially complementary to these small RNAs in the cell.

Non-coding ribonucleic acid (Non-coding RNA) : An RNA transcript that cannot be used to synthesize peptides or proteins by an intracellular translation mechanism. Non-coding RNA includes long and short regulatory RNA molecules such as miRNA, small hairpin RNA, small interfering RNA and double stranded RNA. These regulatory RNA molecules generally function as gene silencers, interfering with the expression of genes that are fully or partially complementary to non-coding RNA in cells.

Microribonucleic acid (MicroRNA, miRNA) : A single-stranded RNA capable of binding 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, the intracellular mRNA target is directly degraded or protein translation of the target mRNA is suppressed. can do. Natural miRNAs are found in almost all eukaryotic cells, and their role is like a defense against viral infection and can regulate gene expression during animal and plant development. In principle, one miRNA targets multiple target RNAs to fully achieve its function, or targets the same gene that is the target of multiple miRNAs to promote complete gene silencing.

Microribonucleic acid precursor (MicroRNA Precursor, Pre-miRNA) : One hairpin-like strand containing stem-arm and stem-loop regions that interact with the intracellular RNA endoribonuclease RNaseIII Dicer Generating one or more mature microribonucleic acids (miRNAs) that are strand ribonucleic acids and silencing target genes of these microribonucleic acids, or genes complementary to these microribonucleic acid sequences Can do. The stem arm region of pre-microribonucleic acid (pre-miRNA) forms a fully (100%) or partial (mismatch) hybrid 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 structure with a circular or hairpin loop.

Prokaryote-produced MicroRNA Precursor (Pro-miRNA) : A small hairpin-like ribonucleic acid that resembles a natural ribonucleic acid precursor (pre-miRNA), but has been artificially used as a miRNA expression plasmid for prokaryotic competent cells. Introduce and transfer. As an example, pro-miRNA is structurally similar to pre-mir-302 but is transcribed from the pLVX-Grn-miR302 + 367 or pLenti-EF1alpha-RGFP-miR302 vectors of competent E. coli DH5α cell lines. Example 18). Since prokaryotic cells usually do not express advanced secondary structures like eukaryotic pre-miRNAs, the production of pro-miRNAs in prokaryotic cells usually requires chemical stimulation to stabilize the generation of RNA secondary structures. Necessary (Lin, UA 13 / 572,263).

Small interfering RNA (siRNA) : A short double-stranded ribonucleic acid, a ribonucleotide duplex having about 18 to 27 complete base pairs, which is almost perfectly complementary 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 stem arm nucleotide sequences that partially or completely match, the stem arm sequences being mismatched Separated by looped oligonucleotides to form a hairpin-like structure. Many natural microribonucleic acids (miRNAs) are derived from hairpin-like ribonucleic acid precursors, immediately pre-miribonucleic acids (pre-miRNA).

Vector : A recombinant nucleic acid composition that can move or settle in different genetic environments, for example, recombinant DNA (recombinant DNA, rDNA). In general, other nucleic acid molecules are operatively linked thereto. The vector is capable of self-replication in the cell, and the vector and its linked fragment are also replicated. Preferred vectors are episomes, ie, nucleic acid molecules that can replicate extrachromosomally. Preferred vectors are nucleic acids capable of self-replication and expression. A vector capable of inducing the expression of a gene encoding one or more polypeptides and / or non-coding RNA, referred to herein as an “expression vector” or “expression-competent” vector) ”. A particularly important vector is the ability to clone cDNA from mRNAs using reverse transcriptase. The components of the vector are a viral promoter, type II (Type-II) RNA polymerase (Pol-II or pol-2) promoter or both, Kozak consensus translation initiation site, polyadenylation signal (polyadenylation signal) signals), multiple restriction / cloning sites, pUC origin of replication, and SV40 early promoter for expressing at least one antibiotic resistance gene in replication competent prokaryotic cells (SV40 early promoter), a selective SV40 origin of replication (SV40 origin) for replication in mammalian cells, and / or a tetracycline response element. The vector structure may be a linear or circular 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, the promoter may be a known polymerase binding site, enhancer or analog thereof, or a sequence that initiates synthesis of an RNA transcript by the required polymerase.

Eukaryotic Promoter : A nucleic acid motif sequence required for gene transcription, which sequence is a type II eukaryotic RNA polymerase (type-2 RNA polymerase (pol-2)), pol-2 equivalent and / or pol Recognized by -2 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 type II eukaryotic RNA polymerase (Pol-II or pol-2) And for transcribing 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 mechanism, compatible with mammalian type II RNA polymerase (Pol-II or pol-2) and Pol-II Selected from the group consisting of sex virus RNA polymerase.

Type II RNA polymerase compatible viral promoter (Pol-II (pol-2) Compatible Viral Promoter) : A viral RNA promoter capable of performing gene expression using eukaryotic pol-2 or eukaryotic transcription mechanism. 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 includes upstream and downstream DNA expression control elements.
Intron Excision : A cellular mechanism responsible for RNA processing, maturation, and degradation, including RNA splicing, exosome digestion, nonsense-mediated decay (NMD), and These combinations are included.

Ribonucleic acid processing (RNA Processing) : Cellular mechanisms involved in 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 : Contains SEQ.ID.NO.4 sequence, sequence homologous to SEQ.ID.NO.4, or 5'-GTAAG-3 '(SEQ.ID.NO.47) sequence Nucleic acid sequence.
Acceptor Splice Site : Contains the SEQ.ID.NO.5 sequence, the sequence homologous to SEQ.ID.NO.5, or the 5'-CTGCAG-3 '(SEQ.ID.NO.48) sequence Nucleic acid sequence.

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

Poly-Pyrimidine Tract : A nucleic acid sequence containing a high proportion of thymidylate and cytidylate, which sequence is SEQ.ID.NO.7, SEQ.ID.NO.8 or homologous to them. Contain

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.
Cancer tissue (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 : Linear or circular single-stranded or double-stranded DNA, including plasmid, viral vector, transposon, retrotransposon, DNA recombination gene, jumping gene, and these Selected from combinations.

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

Restriction / Cloning Site : DNA motif that is a restriction enzyme cleavage site, 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, NarI, NcoI , NdeI, NgoMI, NotI, NruI, NsiI, PmlI, Ppu10I, PstI, PvuI / II, RsaI, SacI / II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI, XhoI However, it is not restricted to these.

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 : DNA recombination method, selected from DNA restriction enzyme reaction and ligation reaction, homologous recombination, transgene integration, transposon insertion, jumping gene insertion, retroviral infection, and combinations thereof .

Cell cycle regulator : A cellular gene involved in controlling the rate of cell division and proliferation, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin-dependent Sex kinase 6 (CDK6), cyclins, BMI-1, p14 / p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1 / Waf1, p27Kip1, and combinations thereof.

Tumor Suppression Effect : Cell antitumor and / or anticancer mechanism and reaction, cell cycle attenuation, cell cycle arrest, tumor cell growth suppression, cell tumor formation 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 to these.

Cancer Therapy Effect : Cell response and / or mechanism by drug treatment, suppression of oncogene expression, suppression of cancer cell proliferation, suppression of cancer cell invasion and / or migration, cancer Inhibition of cell metastasis, induction of cancer cell death, avoidance of tumor / cancer development, avoidance of cancer recurrence, inhibition of cancer progression, recovery of damaged tissue cells, relatively benign and relatively malignant tumor Includes, but is not limited to, reprogramming to a low stage state (cancer regression / remission) and combinations thereof.

Gene silencing effect : Cell reaction after gene function is suppressed, including suppression of oncogene expression, cell proliferation, cell cycle arrest, G0 / G1 checkpoint halt, cell cycle attenuation , Tumor suppression, anti-tumor formation, cancer regression, cancer avoidance, apoptosis of cancer cells, cell recovery / regeneration, cell reprogramming, reprogramming of diseased cells to a relative normal state (spontaneous treatment) and these Including, but not limited to, combinations.

Transcription Inducer : A chemical agent that can induce and / or enhance transcription of eukaryotic genes against pol-2 or pol-2-like promoters in prokaryotic cells. By way of example, a transcriptional derivative contains a chemical structure similar to 3- (N-Morpholino) propanesulfonic acid (MOPS), ethanol, glycerin or mixtures thereof, but Not exclusively.

Antibody : Peptides or protein molecules with a preselected conserved domain structure, which can be receptors that bind to a preselected ligand.
Pharmaceutical and / or therapeutic application : biomedical use, device and / or equipment, diagnosis, stem cell generation, stem cell research and / or therapeutic development, tissue / organ recovery and / or regeneration, wound 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 that induce full tumor suppression and / or cancer therapeutic effects, in which nucleic acid compositions are used. The nucleic acid composition is delivered and processed into a mir-302-like gene silencing effector in the human cell matrix to suppress tumor cycle genes and / or oncogenes that are targets of mir-302 in cells. / Suppress and / or avoid cancer cell growth. Among them, the mir-302-like gene silencing effector comprises a SEQ.ID.NO.3 sequence, and the human cell matrix may be normal, and may comprise at least one tumor and / or cancer cell. .

  Preferably, the present invention delivers mir-302-like gene silencing effectors that are induced or continuously expressed in transfected cells using novel designs and strategies. mir-302-like gene silencing effectors are mir-302a, mir-302b, mir-302c, mir-302d, their hairpin-like microribonucleic acid precursors (pre-miRNAs), and manual redesign Mimetics such as small hairpins (shRNA), pro-miRNAs and / or small interfering RNAs (siRNAs) and homologues / derivatives thereof, and combinations thereof. Transfection of the mir-302-like gene silencing effector is accomplished by vector or non-vector gene transfer systems. Among them, this transfer method includes polysome transfection, liposomal transfection, chemical transfection, electroporation, viral infection, DNA recombination, and transposon insertion. (transposon insertion), jumping gene insertion, microinjection, gene gun penetration and combinations thereof. Due to the vector delivery system, the expression of the mir-302-like gene silencing effector is driven by a persistently expressed promoter (ie CMV) or a drug-inducible (ie TRE-CMV) promoter. The drug-inducible recombinant nucleic acid composition comprises 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. A Tet-On vector containing a recombinant transgene inserted with a hybrid of SEQ.ID.NOs.17 and 18) is preferred. The cell matrix expresses the target gene of mir-302 in vitro, ex vivo or in vivo. By silencing this mir-302 target cell cycle regulator and oncogene, the present invention suppresses the tumorigenicity of cells and reprograms treated cells to non-tumor / cancer cells Can do.

The following examples are given by way of illustration of certain preferred specific examples and embodiments of the invention, but are not intended to limit the scope of the invention.
In the experimental file disclosed below, the following abbreviations apply: M (molar, millilar), mM (millimolar), μm (micromolar, micromolar), mol (mole, moles), pmol (picomole, picomole), gm (grams, grams), mg (milligrams, milligrams), μg (micrograms), ng (nanograms, nanograms), L (liters, liters), ml (milliliters, milliliters), μl (microliters, microliters), ° C (degrees Centigrade), cDNA (copy) Or complementary DNA, copy or complementary DNA), DNA (deoxyribonucleic acid), ssDNA (single-stranded DNA), dsDNA (double-stranded DNA), dNTP (deoxyribonucleoside- 3 Phosphate, deoxyribonucleotide triphosphate), RNA (ribonucleic acid), PBS (phosphate buffered saline), NaCl (sodium chloride), HEPES (N-2-hydroxyethylpiperazine- N-2-ethanesulfonic acid, N-2-hydroxyethylpi perazine-N-2-ethanesulfonic acid), HBS (HEPES buffered saline), SDS (sodium dodecyl sulfate), Tris-HCl (tris-hydroxymethylaminomethane-hydrochloride, tris-hydroxymethylaminomethane) -hydrochloride), ATCC (American Cell Culture Collection, 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 are obtained from the U.S. Cultured Cell Line Conservation Agency (ATCC, Rockville, MD), and human hair follicle cells (hHFCs) contain at least two hairs. Obtained by digesting hair dermal papillae with 4 mg / ml collagenase, of which collagenase digestion works in fresh RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS) for 45 minutes I let you. 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 the cells grow to 70-80% confluency, the cells are isolated by exposure in trypsin-EDTA solution for 1 minute, and phenol red-free DMEM medium (phenol red-free DMEM After rinsing once with medium, Invitrogen), these isolated cells were diluted 1:10 and then subcultured in fresh 254 culture medium containing HMGS-2 supplement. For 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 the low osmotic pressure buffer solution. In addition to isolated cells (20,000-50,000) in (200 μl, Eppendorf, Westbury, NY), electroporation was performed at 300-400 V for 150 μsec using an electroporation machine, and these vectors were Delivered into these cells. Cells after electroporation are initially 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 (Invitrogen) containing sodium for 24 hours at 37 ° C. and 5% CO ₂ . Subsequently, 850 μg / ml G418 and a concentration of tetracycline (Dox) higher than 3.75 μg / ml were added and continued for 3 to 5 days until the cells expressed intense red fluorescence (RGFP), replacing each day. . Next, individual red fluorescent cells (mirPS) were monitored with a TE200 inverted microscope system (Nikon, Melville, NY) and collected individually in 96-well plates with a MO-188NE 3D micromanipulation system (Nikon). 20% Serum Replacement (Knockout serum), 1% MEM non-essential amino acids, 100 μM β-mercaptoethanol, 1 mM GlutaMax, 1 mM sodium pyruvate, 10 ng / ml basic fibroblasts under doxycycline (Dox) deficiency conditions 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, Calif.) The mirPS cells were cultured in 12 medium under the conditions of 37 ° C. and 5% CO ₂ . On the other hand, mirPS cells were cultured under similar feeder-free culture conditions in the presence of doxycycline (Dox, 3.75-5 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 cause 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 described above (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 a pre-made SpRNAi-RGFP recombinant gene (Lin et al., 2006 and 2008), and by MluI / PvuI restriction enzyme The mixture was digested for 4 hours at 37 ° C. The digested mixture is then purified with a gel extraction kit (Qiagen, CA) and collected in 30 μl double-distilled water (ddH ₂ O) and used at 8 ° C with T4 DNA ligase (T4 ligase). The mixture was ligated by acting for 16 hours. In this step, a recombinant SpRNAi-RGFP gene expressing mir-302 is formed, further cleaved with XhoI / HindIII restriction enzyme, and then pSingle-tTS-shRNA vector (Clontech, Palo Alto, CA) that can be induced by doxycycline Insertion into it produced an inducible pTet-On-tTS-miR302s expression vector. We then further modified the pTet-On-tTS-miR302s vector, i.e. using the TRE-CMV promoter isolated from the pTRE-Tight plasmid (Clontech), the original U6 of the pTet-On-tTS-miR302s vector. The promoter was replaced. To generate a non-inducible pCMV-miR302s persistent expression vector, we cleave the modified pTet-On-tTS-miR302s vector with EcoRI restriction enzyme and further upstream of tTS-TRE by gel electrophoresis. By removing the sequence (1.5 kb) and obtaining a new vector cleaved from the gel, a DNA ligation step was performed to complete the construction of a non-inducible pCMV-miR302s vector.

  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); -302d-antisense: 5'-ATGCGATCGC GAACACTCAA ACATGGAAGC ACTTAGCCTG GCTTAGCTAA GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3 '(SEQ.ID.NO.16) is mentioned. We also synthesized miR-302s-sense instead of mir-302 pre-microribonucleic acid (pre-miRNA) cluster: 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. When designing mir-302 homologues, 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.

  The recombined mir-302 family of pre-microribonucleic acid clusters (mir-302s) are mir-302a-sense and mir-302a-antisense, mir-302b-sense and mir-302b-antisense, mir-302c -Formed by ligating 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 are digested with PvuI / XhoI, XhoI / NheI, NheI / XbaI and XbaI / MluI restriction enzymes, respectively, extracted with gel, and then filtered Collected together in 35 μl of sterile double-distilled water on a column (Qiagen, CA). The hybrid mixture was ligated with T4 DNA ligase (Roche, 20U) to form clusters. Finally, the mir-302 family pre-microribonucleic acid cluster was inserted into the SpRNAi-RGFP recombinant gene linearized by PvuI / MluI restriction enzymes. Alternatively, SEQ.ID.NO.17 and SEQ.ID.NO.18 are hybridized, then the hybrid is cleaved using PvuI / MluI restriction enzymes, and the cleaved hybrid is linearized with PvuI / MluI. Mir-302 shRNA was formed by inserting into SpRNAi-RGFP.

  To propagate the pTet-On-tTS-miR302s and CMV-miR302s vectors, E. coli DH5a strains were cultured in LB medium containing ampicillin (Sigma Chemical, St. Louis, MO) at a concentration of 100 μg / ml. Then, using the endotoxin-free maxi-prep plasmid extraction kit (Endo-Free Maxi-Prep Plasmid Extraction Kit, Qiagen, CA), the isolated pTet-On-tTS-miR302s and CMV-miR302s vectors 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 assess the purity and content of isolated small RNAs by 1% formaldehyde-agar gel electrophoresis and spectrometer measurement (Bio-Rad), and then immediately freeze them on dry ice for microarray analysis of microribonucleic acids To LC Sciences (San Diego, Calif.). 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 3 times were listed. The result is 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 Fractionated and the fractionated total RNAs were electroblotted onto a nylon membrane. Subsequently, mir-302 was detected with a [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, IL), terminal transferase, End labeling was performed at 20 units). The hybridization reaction consisted of 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 with 2 × SSC, 0.1% SDS (15 minutes, 25 ° C.), 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
In accordance with the manufacturer's proposal for Western blot analysis , cells were treated with CelLytic-M lysis / extraction reagent (Sigma) supplemented with protease inhibitors, leupeptin, TLCK, TAME and PMSF (CelLytic-M lysis / extraction reagent, Sigma). (1,000,000 pieces) were dissolved. Next, the lysate was centrifuged at 12,000 rpm and 4 ° C. for 20 minutes to obtain a supernatant after centrifugation. Protein concentration was then measured with an E-max microplate reader (microplate reader, Molecular Devices, CA) with the improved SOFTmax protein measurement package software. Under reducing (+50 mM DTT) and non-reducing (no DTT) conditions, add 30 μg of cell lysis product to the SDS-PAGE sample buffer and boil for an additional 3 minutes. From 6% to 8% polyacrylamide gel. Next, after analyzing the protein by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the protein was electroblotted onto a nitrocellulose membrane, and Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) incubated the membranes for 2 hours at room temperature. Thereafter, the primary antibody was added to the reagent, and the mixture was incubated 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 ( Abcam), hTERT (Santa Cruz), s-actin (Chemicon, Temecula, CA), and RGFP (Clontech). After overnight, the membrane was rinsed 3 times with TBS-T and exposed to goat anti-mouse IgG for 1 hour at room temperature. The goat anti-mouse IgG and Alexa Fluor 680 reactive dye (1: 2,000, Invitrogen-Molecular Probes) were bound to form a secondary antibody. After three additional rinses with TBS-T, immunoblotting fluorescence scanning and image analysis were performed using Li-Cor Odyssey Infrared Imager and Odyssey Software V.10 (Li-Cor). went. The results are shown in FIGS. 1D, 4B, 7C to 7D, and FIGS. 8B and 9B.

Example 6
Apoptotic DNA Ladder Kit Isolates and isolates genomic DNA from approximately 2,000,000 cells using the Apoptotic DNA Ladder Kit (Roche Biochemicals, Indianapolis, IA) according to the manufacturer's proposal 2 μg of the prepared DNA was taken and 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, the cells were hydrolyzed with trypsin, centrifuged into granules, and suspended in PBS containing 1 ml 70% methanol pre-cooled. Cells were fixed for 1 hour at -20 ° C. These cells were then centrifuged into granules and washed once with 1 ml PBS. Again centrifuge these cells in a granulate, 30 min in 1 ml PBS solution containing 1 mg / ml propidium iodide (PI), 0.5 μg / ml ribonuclease (RNase) at 37 ° C. Resuspended. Subsequently, approximately 15,000 cells were analyzed by BD FACSCalibur flow cytometry (San Jose, CA). Cell doublets were excluded by drawing a pulse width versus pulse area diagram and 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-3B and FIGS. 6A-6C.

Example 8
Whole genome microarray analysis Human genome GeneChip U133 plus 2.0 array (Affymetrix, Santa Clara, CA) was used to detect changes in expression patterns of more than 47,000 human genes in test cells. 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 was assessed by 1% formaldehyde-agar gel electrophoresis and spectrometer measurements (Bio-Rad). These sample signals were normalized by the overall average difference between the perfect match probe and the mismatch probe. Then, Affymetrix Microarray Suite 5.0 edition, using Expression Console ^TM 1.1.1 Edition (Affymetrix) and Genesprings (Silicon Genetics) software to analyze changes in the whole genome gene expression patterns. If the change in gene expression was greater than 1 fold, it was considered a positive difference gene. A combination of the plug-in program Genetrix (Epicenter Software) and Affymetrix software was used in gene clustering. The signal of the sample was normalized with 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 isolated DNA was taken and subjected to bisulfite treatment (CpGenome DNA modification kit, Chemicon, Temecula, Calif.) According to the manufacturer's suggestion. On the other hand, 2 μg of isolated DNAs was taken and digested with CCGG cleavage restriction enzyme HpaII, and then the demethylation of the whole genome was determined by 1% agar gel electrophoresis (FIG. 5B). Treatment of 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 the amplification of the Oct3 / 4 promoter region are 5′-GAGGCTGGAG CAGAAGGATT GCTTTGG-3 ′ (SEQ.ID.NO.20) and 5′-CCCTCCTGAC CCATCACCTC CACCACC-3 ′ (SEQ.ID.NO.21). )including. The primers used for amplification of the Nanog promoter region were 5′-TGGTTAGGTT GGTTTTAAAT TTTTG-3 ′ (SEQ.ID.NO.22) and 5′-AACCCACCCT TATAAATTCT CAATTA-3 ′ (SEQ.ID.NO.23 )including. First, bisulphite-modified DNAs (50 ng) and these primers (total 100 pmole) were mixed in 1x PCR buffer, heated to 94 ° C, kept for 2 minutes, and immediately cooled with ice did. Then, 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 70 ° C for 3 minutes. After completion of the amplification reaction, the DNA product having the correct length is further fractionated by 3% agar gel electrophoresis, and then the DNA product is purified with a gel extraction kit (Qiagen) for DNA sequencing. It was. A detailed pattern of DNA methylation sites was obtained by comparing unchanged cytosine in a bisulfite modified DNA sequence with unchanged cytosine in a DNA sequence not modified with bisulfite . The result is shown in FIG. 5C.

Example 10
Transplantation and derived embryoid bodies mirPS cells formed about 5-10 teratomas (4-8 cell stage) to 50 [mu] l of DMEM culture medium and Matrigel Matrigel 2: it is suspended in 1 mixture, then these Embryoid bodies derived from mirPS cells were transplanted into the uterus of 6-week-old female pseudopregnant immunodeficient SCID-beige mice. The method of making pseudopregnant mice is to inject 1 IU of human menopausal gonadotrophin (HMG) into the peritoneum for 2 days and another human chorionic gonadotrophin (hCG). Injection for days. The cells and the mice are not treated with doxycycline (Dox) before or after transplantation. During the transplantation period, one mouse was anesthetized with a 2.5% tribromoethanol (Avertin) solution in a volume of 0.4 ml. The Xenografted masses were monitored for 3 to 4 weeks after transplantation or after the transplant cell population had grown to a size greater than 100 mm ³ . After separating the cyst / teratomas, the volume was calculated using the formula (length × width ² ) / 2, and further counting, weighing and further histological analysis were performed. Teratoma-like tissue cysts can usually be observed about 2.5 weeks after transplantation. The result is shown in FIG. 5D.

Example 11
Cell Invasion Test First, use 200 μg / ml Matrigel alone or a chamber insert using Matrigel supplemented with phenol red-free DMEM medium containing 20% FBS and 1% L-Glutamine. (chamber inserts, hole diameter 12 μm, Chemicon) was applied and dried in 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 media. 500 μl of the cell suspension was taken and distributed in the top chamber and 1.5 ml of DMEM conditioned medium was added to the bottom chamber to create a chemotactic gradient. After incubating at 37 ° C overnight for 16 hours, the invasion state was measured. First, wipe the top chamber with cotton wool, fix the invading cells located under the membrane with 100% methanol for 10 minutes, air dry, stain with cresyl violet for 20 minutes, and then with water Gently washed. After drying, the cresyl violet stain on the membrane is washed for 20 minutes with a wash solution containing 1: 1 100% ethanol and 0.2 M sodium citrate (NaCitrate), and the wavelength is set to 570 with a Precision Microplate Reader (Molecular Dynamics). The absorbance at nm was read. The display method of the cell penetration rate was the percentage of the absorbance of the test sample with respect 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 previous reports (Lin et al., 2007). Human bone marrow endothelial cells (hBMECs) were planted in a 96-well plate at a density of 100,000 cells / ml and adhesion medium [RPMI 1640 / 0.1% BSA / 20 mM HEPES (pH 7.4)] before testing Rinse. Isolate the test cells using trypsin (used for tumor / cancer cells) or collagenase (used for mirPS cells), rinse the cells with sterile saline, and then add 10 μM fura-4 acetoxymethyl ester. 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), and then the cells were put into an adhesion culture medium at 37 ° C in a dark environment. Incubate for 20 minutes to activate the intracellular fluorescent probe. 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-adherent cells were washed away by rinsing twice with 250 μl adherent medium. Fluorescence was read at 37 ° C with 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 studies.We used NTera-2 cells (2,000,000 cells in Matrigel-PBS with a total volume of 100 μl) to the flank of 8-week-old male mice (BALB / c-nu / nu species). That is, xenotransplantation was performed on the hind right limb). The tumor was monitored weekly and one week after NTera-2 xenotransplantation, the pCMV-miR302s vector or the pCMV-miR302d * vector was introduced by in situ injection. Five treatments (every 3 days) were performed with 2 μg (per gram 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 suggestion of use. Sampling began 3 weeks after injection or after the non-vector treated tumors grew to an average size of about 100 mm ³ . All major organs such as blood, brain, heart, lung, liver, kidney and spleen, and xenograft tumors were removed for histological evaluation of tumors and immune response cytotoxicity studies. Of these, 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 histological examination was performed with hematoxylin-eosin staining (H & E) and immunostaining tests. Histological examination did not detect any observable 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 with 4% paraformaldehyde at 4 ° C. Before embedding the sample in paraffin wax, it was washed in the order of 1 × PBS, methanol, isopropanol and tetrahydronaphthalene. The embedded sample was then cut to a thickness of 7-10 μm with a microtome and fixed on a clean glass slide coated with TESPA. The glass slide is then dewaxed using xylene, fixed under a cover glass using mounting media (Richard Allan Scientific, Kalamazoo, MI), and hematoxylin and eosin. ) (H & E, Sigma) and the morphology was observed. Immunohistochemistry (IHC) staining kit was purchased from Imgenex (San Diego, Calif.). The antibody dilution and immunostaining procedures were performed according to the manufacturer's proposal. 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 third antibody was added. After the slide glass was washed with PBT three times, the bound antibody was detected with a DAB substrate. A positive result was observed with a 100 × microscope equipped with full-field scanning, and a quantitative analysis was performed by measuring at a magnification of 200 × with a Metamorph image processing program (Nikon 80i microscope quantitative analysis system). The result is 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 (normal and / or mutation) of Mir-302 was inserted into the cloning site of the 3 ′ end 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-Report β-gal Control vector was used as a no reporter control vector. 50,000 mirPS cells were transfected with 200 ng reporter vector using FuGene HD reagent (Roche) with or without treatment with doxycycline (Dox) according to the manufacturer's suggestion. Cell lysates were collected 48 hours after transfection. The degradation level of luciferase is normalized and expressed as relative luciferase activity (RLA), and the calculation method is based on the level of luciferase activity in dox-on mirPS cells treated with doxycycline (Dox-on). Divide by the level of luciferase activity in (Dox-off) mirPS cells not treated with doxycycline. In addition, cells expressing mir-434 were generated by introducing pTet-On-tTS-miR434-5p into hHFCs by electroporation, and used as a negative control group. The results are shown in FIGS. 7B and 10B.

Example 16
According to the telomere repeat amplification protocol test manufacturer's suggestion, approximately 1,000,000 cells were removed with a cell lysis / extraction reagent (CelLytic-Mlysis / 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 rotational speed of 12,000 rpm, and the lysate after centrifuging (supernatant) was collected. Subsequently, the protein concentration was measured with an E-max microplate reader (microplate reader, Molecular Devices, CA) with the improved SOFTmax protein measurement package software. Infrared Alexa Fluor 680 dye, TS Primer, Sigma-Genosys labeled oligonucleotides 5'-AATCCGTCGAGCAGAGTT-3 '(SEQ.ID.NO.24) and 5'-GTGTAACCCTAACCCTAACCC-3' (CX primer , 30 μM) (SEQ. ID. NO. 25) was used to detect the polymerase chain reaction (PCR) product. Telomerase inhibitors were added directly to the main mixture. For all test cell lines, optimal results were achieved using 50 ng protein per reaction. After incubating at 30 ° C for 30 minutes, 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 at 57 ° C The synthesis was performed for 30 cycles of PCR for 30 seconds. Finally, the post-single synthesis step at 57 ° C was performed for 30 seconds. Next, the PCR product was fractionated by 6% nondenaturing polyacrylamide gel electrophoresis, and the Li-Cor Odyssey Infrared Imager and Odyssey Software V.10 ( 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% is considered a positive result, and after these results are analyzed, mean ± standard deviation (mean ± SE) It was expressed by Statistical analysis of the data was calculated by one-way ANOVA. When the main effect was significant, Dunnett's post-hoc test was used to determine a group significantly different from the control group. Two-tailed student t-test was used when matching between two treatment groups. In experiments involving more than two treatment groups, ANOVA was performed followed by a post-hoc multiple range test. Probability p <0.05 was considered statistically significant. All p's were determined from a two-sided test.

Example 18
Induction of cell transformation and pre-miRNA expression
Competent E. coli DH5α cell line obtained from 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. Non-transformed bacterial cells were shaken frequently 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, 0.5 to 2 ml of MOPS was added for chemical induction per liter in LB medium supplemented with 10 mM magnesium sulfate and 0.2 mM glucose and supplemented with 100 μg / ml ampicillin. In addition, transformed bacterial cells were used as a negative control group and cultured in LB medium to which ampicillin was added but no chemical derivative was added.

Example 19
Human cell culture and intracellular Mir-302 transmission Human primary epidermal skin cells (hpESCs) were prepared in fresh RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS) 4 It was obtained by treatment with mg / ml collagenase I at 37 ° C for at least 2 mm ³ volume for 35 minutes. Isolated keratinocytes are supplemented with human keratinocyte growth supplement (HKGS, Invitrogen, Carlsbad, CA) and antibiotics at 37 ° C and 5% carbon dioxide. Not cultured in EpiLife serum-free cell culture medium. When cells have grown to 50% -60% confluency, the cells are exposed in trypsin / EDTA solution for 1 minute and then added to phenol red-free DMEM medium (Invitrogen). Rinse once and further, these isolated cells were diluted 1:10 and subcultured in fresh EpiLife broth containing HKGS supplement. Human cancer / tumor cell lines Colo-829, PC3, MCF7, HepG2 and Tera-2 were obtained from the American Cultured Cell Line Preservation Agency (ATCC, Rockville, MD) and cultured under conditions suggested by the manufacturer. For microRNA transfection, 15 μg of isolated miR-302 and / or its precursor was dissolved in EpiLife medium mixed with 50 μl liposome / polysome DNA transfection agent (X-tremeGENE HP DNA transfection reagent). After culturing for 10 minutes, this mixture was added to hpESCs or cancer / tumor cells that reached 50-60% confluency in a 100 mm cell culture dish, respectively. After 12-18 hours, the original broth was replaced with a fresh EpiLife broth containing HKGS supplement or a broth suggested by ATCC. Transfection efficiency may be improved by repeating the transfection process 3-4 times every 3-4 days. After cell shape changes to ball shape, 20% serum replacement (Knockout serum), 1% MEM non-essential amino acid, 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, San These cells (mirPSCs) were cultured and passaged in a knockout DMEM / F-12 culture medium supplemented with Diego, CA) at 37 ° C. and 5% CO ₂ .

Example 20
Competent E. coli DH5α cells transformed with plasmid DNA / total RNA / microRNA extraction plasmid (Example 18) were cultured overnight in LB medium supplemented with 10 mM magnesium sulfate and 0.2 mM glucose at 37 ° C., 170 Shake frequently at rpm. In addition to bacterial culture and amplification, addition of 0.5-2 ml MOPS per liter of the medium induced the production of ribonucleic acid and / or protein driven by a eukaryotic promoter. All amplified plasmid DNAs and expressed mRNA / microRNAs precursors (pre-miRNAs) were isolated together using a plasmid purification kit (HiSpeed plasmid purification kit, Qiagen, Valencia, CA). Among them, the isolation process was in accordance with the process suggested by the manufacturer but slightly adjusted, that is, ribonuclease RNase A was not added to the P1 buffer. The final extract product containing both plasmids and mRNAs / pre-miRNAs was dissolved in double distilled water (ddH ₂ O) treated with DEPC and stored at −80 ° C. until used. If only the amplified plasmid vector is to be purified, ribonuclease RNase A is added to the P1 buffer according to the manufacturer's suggested process.

Example 21
Purification of Mir-302 and its precursor Furthermore, according to the manufacturer's suggestion, the whole RNA isolated by the method in Example 20 was purified using the mirVana ^™ miRNA isolation kit (Ambion, Austin, TX). 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. We can take advantage of this difference to obtain pure mRNAs and / or pre-miRNAs / pro-miRNAs for later application as they are (half-life can reach 3-4 days) It was. For example, RGFP mRNA is 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 human Used to treat tumor / cancer cells. Purified pre-miR-302s / pro-mir-302s can also be used to reprogram advanced malignant cancer cells to a relatively benign low stage state. This is a beneficial result for cancer treatment.

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. The isolated pro-miR-302s was then prepared with an ointment base containing cocoa butter, cottonseed oil, olive oil, sodium pyruvate, and white petrolatum prepared in advance. The concentration of pro-mir-302 in the prepared ointment base was about 10 μg / mL. The skin was cut open with a scalpel to form a wound of about 0.5 × 0.5 square centimeter, which is an open wound of the skin. An ointment (about 0.3 mL) was applied directly to the wound to cover the entire wound. Subsequently, the treated area was further sealed with liquid bandage.

Example 23
In Vivo Liver Cancer Treatment Testing We have established an effective animal model to study liver cancer metastasis and treatment by xenografting malignant human liver tumors into immunodeficient SCID-beige mice. To build this model, we mixed 5 million human liver cancer cells (HepG2) with 100 μL matrix gel and implanted this mixture subcutaneously on the flank of the mouse hind limb. Therefore, there are approximately equal amounts of cancer cell transplants on the hind limbs on both sides of the mouse. The malignant tumor was observed about 2 weeks after transplantation. Among them, the average size of the tumor was about 15.6 ± 8 mm ³ (ie, the initial size of the tumor before treatment). For each mouse, we selected the side with the relatively large tumor as the experimental group and the other side with the relatively small 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 differences between individuals.

  In order to deliver pro-mir-302 to the targeted cancer area in vivo, we adopted the method and product of Latitude (San Diego, CA), a specialized pharmaceutical company, encapsulating pro-mir-302 with liposomes, Granules with a diameter of 160-200 nm were formed. Tests showed that these nanogranules containing pro-mir-302 were almost 100% stable after 2 weeks at room temperature and 1 month at 4 ° C, whereas other syntheses under the same conditions The siRNA and other mimetics (siRNA-302) rapidly superdegraded by 50% in 3-5 days, indicating that pro-miRNA rather than siRNA is more stable as a therapeutic agent. In order to conduct toxicity tests, we further injected pro-mir-302 (1 mg / mL), prepared to a maximum volume of 300 μL, into each mouse's tail vein (n = 8). In a period exceeding 6 months, we observed no measurable side effects in all of the tested mice. In general, unmodified nucleic acids are relatively immune to immunity and can be easily metabolized from tissue cells, making them safe tools for treatment in vivo.

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

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The examples and embodiments described herein are merely used for illustration purposes, and various modifications and changes based thereon are conceivable to those skilled in the art, and the appended claims It is understood that it should be included within the spirit and scope of the invention as described in the scope. All publications and patent documents cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims (34)

A composition used in the development of a therapeutic anticancer drug by reprogramming malignant human cancer to benign or normal state at a relatively low stage,
(a) at least one chemical inducer having the molecular structure of 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol, glycerin, or mixtures thereof;
(b) at least one transformed prokaryotic cell line having at least one expression vector that expresses the microribonucleic acid precursor by a eukaryotic promoter;
A microribonucleic acid precursor (pre-miRNA) that induces transcription of the microribonucleic acid precursor driven by the eukaryotic promoter by mixing (a) and (b) under certain conditions Composition.   2. The composition of claim 1, wherein the chemical inducer is a transcriptional inducer that stimulates transcription of ribonucleic acid driven by the eukaryotic promoter in prokaryotic cells.   The prokaryotic cells are present in 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 claim 1.   4. The composition according to claim 3, wherein the bacterial medium is an LB medium.   2. The composition of claim 1, wherein the prokaryotic cell is an E. coli DH5α competent cell.   The composition according to claim 1, wherein the expression vector is a recombinant plasmid encoding the sequence shown in SEQ.ID.NO.3.   2. The composition of claim 1, wherein the expression vector is pLenti-EF1α-RGFP-miR302.   2. The composition of claim 1, wherein the microribonucleic acid precursor contains at least the sequence shown in SEQ.ID.NO.3.   2. The composition according to claim 1, wherein the microribonucleic acid is a microribonucleic acid (pro-miRNA) produced by a prokaryotic cell.   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. 10. A composition according to claim 9, comprising the sequence shown in 17.   2. The composition of claim 1, wherein the microribonucleic acid is used for pharmaceutical or therapeutic applications.   2. The composition of claim 1, wherein the eukaryotic promoter is a type II ribonucleic acid polymerase (pol-2) promoter or a pol-2-like promoter.   13. The composition according to claim 12, wherein the pol-2 promoter is an EF1α promoter.   13. The composition of claim 12, wherein the pol-2-like promoter is a CMV promoter.   The composition according to claim 1, wherein the condition is LB medium that is frequently shaken at 37 ° C.   2. The composition of claim 1, wherein the anti-cancer mechanism of the anti-cancer drug comprises reversal of cancer that reprograms malignant and advanced human cancer to a low stage or normal state in vitro, ex vivo or in vivo.   2. The composition of claim 1, wherein the human cancer comprises a tumor or cancer cell. A method for producing a microribonucleic acid precursor used in the development of a therapeutic anticancer drug by reprogramming malignant human cancer to a benign or normal state at a low stage,
(a) providing at least one chemical inducer 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 by a eukaryotic promoter;
(c) mixing (a) and (b) under certain conditions to induce transcription of the microribonucleic acid precursor driven by the eukaryotic promoter;
A method for producing a microribonucleic acid precursor (pre-miRNA), comprising:   19. The method of claim 18, wherein the chemical inducer is a transcriptional inducer that stimulates transcription of ribonucleic acid driven by the eukaryotic promoter in prokaryotic cells.   The step of providing the prokaryotic cell further comprises providing the prokaryotic cell in a bacterial medium, wherein the step of mixing (a) and (b) comprises 0.05% (v of the chemical derivative in the bacterial medium. 19. The method of claim 18, further comprising adding to a final concentration of / v) to 0.2% (v / v).   21. The method of claim 20, wherein the bacterial medium is LB medium.   19. The method of claim 18, wherein the prokaryotic cell is an E. coli DH5α competent cell.   19. The method of claim 18, wherein the expression vector is a recombinant plasmid that encodes the sequence shown in SEQ.ID.NO.3.   19. The method of claim 18, wherein the expression vector is pLenti-EF1α-RGFP-miR302.   19. The method of claim 18, wherein the microribonucleic acid precursor contains at least the sequence shown in SEQ.ID.NO.3.   19. The method of claim 18, wherein the microribonucleic acid is a microribonucleic acid (pro-miRNA) produced by a prokaryotic cell.   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 of claim 26, comprising the sequence shown in 17.   19. The method of claim 18, wherein the microribonucleic acid is used for pharmaceutical or therapeutic applications.   19. The method of claim 18, wherein the eukaryotic promoter is a type II ribonucleic acid polymerase (pol-2) promoter or a pol-2-like promoter.   28. The method of claim 27, wherein the pol-2 promoter is an EF1α promoter.   28. The method of claim 27, wherein the pol-2-like promoter is a CMV promoter.   19. The method of claim 18, wherein the condition is LB medium that is shaken frequently at 37 ° C.   19. The method of claim 18, wherein the anti-cancer mechanism of the anti-cancer drug comprises reversal of cancer that reprograms malignant and advanced human cancer to a low stage or normal state in vitro, ex vivo or in vivo.   19. The method of claim 18, wherein the human cancer comprises a tumor or cancer cell.