TWI720075B - Use of microrna precursors as drugs for inducing cd34-positive adult stem cell expansion - Google Patents

Abstract

This invention generally relates to a composition and method for inducing the expansion and/or regeneration of CD34-positive adult stem cell population, which is useful for developing drugs/vaccines and/or therapies against a variety of ageing-related degenerative diseases in humans. Also, the present invention teaches the production and purification methods required for making high quality and high quantity of small hairpin-like RNA (shRNA) compositions, such as microRNA precursors (pri-miRNA and pre-miRNA) and short interfering RNAs (siRNA), which are useful for treating human ageing-related diseases, such as, but not limited, Alzheimer’s diseases, Parkinson’s diseases, osteoporosis, diabetes, and cancers. The resulting shRNAs, preferably pre-miRNAs, are useful for developing therapeutic drugs against human degenerative diseases, particularly through a mechanism to induce CD34-positive stem cell expansion and/or regeneration.

Description

Use of microribonucleic acid precursors as drugs for inducing the proliferation of CD34-positive adult stem cells Cross-reference of related applications

The present invention claims the priority of U.S. Provisional Application No. 62/262,280 filed on December 2, 2015, entitled "miR-302 Attenuates Aβ-induced Neurotoxicity through Activation of Akt Signaling". In addition, the present invention claims the priority of US Patent Application No. 15/048,964 filed on February 19, 2016, entitled "A Composition and Method of Using miR-302 Precursors as Drugs for Treating Alzheimer's Diseases". In addition, the present invention claims the priority of US Patent Application No. 15/167,226 filed on May 27, 2016, entitled "Use of MicroRNA Precursors as Drugs for Inducing CD34-positive Adult Stem Cell Expansion".

The present invention generally relates to a composition and method for inducing the proliferation and/or regeneration of CD34 cells with small hairpin RNA (shRNA) molecules, preferred microRNA precursors (pre-miRNA) and/or siRNA, and these molecules can be used for Develop drugs for a variety of aging-related degenerative diseases in humans/ Vaccines and/or therapy. In addition, the present invention teaches the manufacturing and purification methods required to manufacture high-quality and large amounts of small hairpin RNA (shRNA) compositions, such as microRNA precursors (pri-miRNA and/or pre-miRNA) and short Interfering RNAs (siRNA) can be used to treat human aging-related diseases, such as but not limited to Alzheimer's disease, Parkinson's disease, osteoporosis, diabetes, and cancer. The resulting shRNAs, preferably pre-miRNAs and siRNAs, can be used to develop therapeutic drugs for human degenerative diseases, specifically through mechanisms that induce the proliferation and/or regeneration of CD34-positive stem cells. In addition, the present invention also discloses a novel pre-miRNA-based pharmaceutical composition that can reprogram the malignant characteristics of high-grade liver cancer to low-grade benign or even relatively normal stages-a mechanism called "Cancer Reversion" . Because cancer reversal is a completely new concept in drug design, the present invention uses this novel mechanism to design the preferred drug for cancer therapy.

Stem cells are like a treasure chest containing many active ingredients, which can be used to stimulate new cell growth/tissue regeneration, repair and/or restore damaged/aging tissues, treat degenerative diseases and prevent tumor/cancer formation/process. Therefore, the inventor can imagine that these stem cells can be used as a tool for screening, identification and manufacturing of novel drugs. Therefore, the drugs thus obtained can be used to develop medical and therapeutic applications, such as biomedical applications, devices and/or equipment and combinations thereof for research, diagnosis, and/or therapy.

MicroRNA (miRNA) is one of the main effective components in human embryonic stem cells (hESCs). The main hESC-specific miRNA types include But not limited to members of the miR-302 family, miR-371 to 373 family, and miR-520 family. Among them, the miR-302 family has been found to play a functional role in tumor suppression (Lin et al., 2008 and 2010). MiR-302 contains eight (8) family members, including four (4) sense miR-302 (a, b, c, and d) and four (4) antisense miR-302* (a*, b*, c* and d*). These sense and antisense members are partially matched and can respectively form a double-stranded duplex. The precursors of miR-302 are composed of miR-302a and a*(pre-miR-302a), miR-302b and b*(pre-miR-302b), miR-302c and c*(miR-302c), and miR-302a, respectively. 302d is formed with d* (pre-miR-302d), with a connecting sequence (stem loop) at one end. In order to activate the function of miR-302, miR-302 precursor (pre-miR-302) is first processed into mature miR-302s by ribonuclease III Dicers (RNase III Dicers) and further formed with certain argonaute proteins. RNA-induced silencing complexes (RISCs) subsequently lead to target gene transcripts (mRNAs), especially RNA interference (RNAi) targeted degradation or translational inhibition of oncogene mRNAs (Lin et al., 2008, 2010 and 2011).

MiR-302 is the most abundant ncRNA species found in hESCs and induced pluripotent stem cells (iPSCs). The inventors’ previous studies have shown that the ectopic overexpression of miR-302 beyond the level seen in hESCs can normalize humans with a relatively slow cell cycle rate (20-24 hours/cycle) similar to the early human fertilized eggs in the morula stage. Both cancer cells are reprogrammed into hESC-like iPSCs (Lin et al., 2008, 2010 and 2011; EP 2198025; US12/149,725; US12/318,806; US12/792,413). Relative quiescence is the definite feature of miR-302 inducing iPSCs, and hESCs and other previously reported four-factor induction ( Oct4-Sox2-Klf4-c-Myc or Oct4-Sox2-Nanog-Lin28 ) iPSCs all show It is similar to the highly proliferative cell cycle rate (12-15 hours/cycle) of tumor/cancer cells (Takahashi et al., 2006; Yu et al., 2007; Wernig et al., 2007; Wang et al., 2008). To reveal this tumor suppressor effect of miR-302, the inventors identified two miR-302 targeting G1-checkpoint regulators, including cyclin-dependent kinase 2 (CDK2) and cyclin D (Lin et al., 2010; US12/792,413; US13/964,705). It is known that cell cycle progression is driven by the activity of cyclin-dependent kinase (CDK), which is related to positive regulatory subunits (positive regulatory subunits), cyclin and negative regulators, CDK inhibitors (CKIs, such as p14/p19Arf, p15Ink4b). , P16Ink4a, p18Ink4c, p21Cip1/Waf1 and p27Kip1) form functional complexes. In mammals, different cyclin-CDK complexes are involved in the regulation of different cell cycle transitions, such as cyclin-D-CDK4/6 for G1 phase progression, cyclin-E-CDK2 for G1-S transition, and cyclin- A-CDK2 is used for S phase progression and Cyclin-A/B-CDC2 (Cyclin-A/B-CDK1) is used to enter M phase. Therefore, the inventor's research shows that the tumor suppressor function of miR-302 is produced by the co-inhibition of the cyclin-E-CDK2 and cyclin-D-CDK4/6 pathways during the G1-S transition period.

Although miR-302 can be used to design and develop novel anti-cancer drugs/vaccines, its production is a problem because natural miR-302 can only be found in human pluripotent stem cells such as hESCs, and its resources are extremely limited. Alternatively, synthetic small interfering RNA (siRNA) can be used to mimic pre-miR-302; however, because the structure of pre-miR-302 is formed by two mismatched miR-302 and miR-302* strands, this type of perfect match The siRNA mimic cannot replace the function of miR-302*, and its sequence is completely different from the antisense strand of siRNA. For example, the antisense strand of the siRNA-302a mimic is 5'-UCACCAAAAC AUGGAAGCAC UUA-3' (SEQ.ID.NO.1), while the natural miR-302a* Line 5'-ACUUAAACGU GGAUGUACUU GCU-3' (SEQ.ID.NO.2). Because miR-302 function is produced by both its sense miR-302 and antisense miR-302* strands, previous reports using such siRNA mimics have shown different results from native miR-302 functions. On the other hand, the inventor's recent iPSCs research can provide an alternative solution for the production of pre-miR-302 (EP 2198025; U.S. 12/149,725; U.S. 12/318,806). Nevertheless, the cost of growing such iPSCs is still too high to be used in industrial manufacturing.

Alternatively, the use of prokaryotic competent cells may be a possible method for the production of human microRNAs and their precursors. However, prokaryotic cells do not have several essential enzymes required for eukaryotic microRNA expression and processing, such as Drosha and Dicer. In addition, prokaryotic RNA polymerase does not efficiently transcribe small RNAs with high secondary structure, such as hairpin pre-miRNAs and shRNAs. In fact, the bacterial genome does not encode real microRNAs and bacteria do not naturally express microRNAs. Therefore, if the inventors can force the expression of human microRNAs in prokaryotes, the resulting microRNAs will most likely remain similar to pri-miRNAs (large primary clusters of multiple pre-miRNAs) and/or pre-miRNAs. -miRNA (a single hairpin RNA) precursor configuration. Despite all the above problems, the real challenge is how to force the expression of human microRNA in prokaryotes. To overcome this major problem, the inventor’s priority application US No. 13/572,263 has established a preliminary method; however, it is currently uncertain whether the microRNAs (pro-miRNA) produced by these prokaryotes will have the same characteristics as their human counterparts. The structure and function. In addition, the pro-miRNAs thus obtained may be contaminated with bacterial endotoxins, which are not suitable for direct use in therapy.

If acquired from current textbooks, there are general The intellectuals are well aware that prokaryotic and eukaryotic transcription mechanisms are different and therefore incompatible with each other. For example, based on current understanding, eukaryotic RNA polymerase does not directly bind to the promoter sequence and requires additional accessory proteins (cofactors) to initiate transcription, while prokaryotic RNA polymerase forms directly binds to the promoter sequence to initiate transcription The whole enzyme. It is generally known that eukaryotic messaging RNA (mRNA) is synthesized in the nucleus by type 2 RNA polymerase (pol-2), and then processed and exported to the cytoplasm for protein synthesis. Prokaryotic RNA transcription and protein translation The same position occurs at the same time by the same piece of DNA. This is because prokaryotes, such as bacteria and archaea, do not have any nuclear-like structures. Therefore, these differences make it difficult or even impossible for prokaryotic cells to use eukaryotic promoters to produce eukaryotic RNAs.

Prior art attempts to produce mammalian peptides and/or proteins in bacterial cells, such as Buechler's US Patent No. 7,959,926 and Mehta's US Patent No. 7,968,311, use bacterial or phage promoters. To initiate expression, the desired gene is cloned into a plastid vector driven by a bacterial or phage promoter. Genes must not contain any non-coding introns, because bacteria do not have any RNA splicing mechanism to handle introns. Subsequently, the thus obtained vector was introduced into competent cells of bacterial strain, such as E. coli (Escherichia coli, E.coli) so that the performance of gene transcripts (mRNAs) and subsequently translated into protein mRNAs. Nevertheless, bacterial and phage promoters, such as Tac, Lac, T3, T7, and SP6 RNA promoters are not pol-2 promoters and their transcriptional activities tend to be error-prone processes that can lead to mutations. In addition, Mehta further teaches that glycerol/glycerin can be used to increase the efficiency of bacterial transformation; however, no teaching relates to the enhancement of RNA transcription, especially prokaryotic RNA transcription driven by the pol-2 promoter. Since there is no possible compatibility between eukaryotic and prokaryotic transcription systems, such prior art is still limited by the use of prokaryotic RNA promoters for gene expression in prokaryotes.

Due to the problems of system incompatibility and possible endotoxin contamination, there was no previous method for producing human pre-miRNA/shRNA drugs in prokaryotes. In addition, the size of pre-miRNA/shRNA is about 70-85 nucleotides long, which is too large and costly to be prepared by RNA synthesis mechanism. In order to overcome these problems, the present invention provides a novel breakthrough: by adding certain chemicals that mimic some transcription cofactors, the inventors can establish a novel adaptive environment for prokaryotic cells to transform eukaryotic pol-2 and/or pol Type -2 promoters are used to transcribe desired pre-miRNAs and shRNAs without going through error-prone prokaryotic promoters. This advantage is: first, it can be manufactured in large quantities cost-effectively due to the rapid growth of bacteria; second, it is easy to handle because it does not require the growth of dedicated hESCs or iPSCs; third, it is driven by the pol-2 promoter In terms of RNA transcription, there is high fidelity productivity; fourth, due to the lack of real microRNAs in prokaryotes, but high purity of the desired microRNAs; and finally, there is no endotoxin, which can be treated by certain chemical treatments. And further removed. Therefore, a method for producing human pre-miRNAs and/or shRNAs in prokaryotic cells without the problems of system incompatibility and endotoxin contamination is highly ideal. In addition, the drugs thus obtained can exhibit novel therapeutic effects in addition to the currently known functions of microRNA.

The principle of the present invention relies on the different and incompatible characteristics between prokaryotic and eukaryotic RNA transcription systems. Naturally, prokaryotic RNA polymerase does not recognize eukaryotic promoters, and vice versa. However, the present invention has identified that it can act as a transcriptional inducer to trigger and/or enhance eukaryotic promoter-driven prokaryotes in prokaryotes. Chemical agents for RNA transcription. Therefore, the knowledge taught in this invention is a brand new breakthrough beyond all current understandings about the differences between prokaryotic and eukaryotic transcription systems.

The present invention relates to an inducible gene expression composition that uses certain chemical inducers to stimulate and/or enhance RNA transcription driven by eukaryotic promoters in prokaryotes. These chemical inducers have not been used in cell culture media due to their antibacterial and/or bactericidal properties, including 3-morpholinopropane-1-sulfonic acid [or named 3-(N-morpholino)propane Sulfonic acid; MOPS], glycerol and ethanol, and their functional analogs, such as 2-(N-morpholino)ethanesulfonic acid (MES), 4-(2-hydroxyethyl)-1-piperazineethane Sulfonic acid (HEPES) and mannitol (mannitol). It is conceivable that chemicals with similar structures, such as these transcription inducers, can share similar functions. For example, MOPS is often used as a buffer in bacterial cell lysis and therefore is not suitable for growing bacteria. On the other hand, ethanol is a well-known disinfectant and glycerol is often used for bacterial transformation by destabilizing the bacterial cell wall, indicating that glycerol is antibacterial and ethanol is sterilizing. In view of the known functions of MOPS, ethanol and glycerol, those with ordinary knowledge in the art would not expect to use trace amounts (0.001% to 4% volume/volume concentration) without first knowing the knowledge of the present invention. ) These chemicals induce gene expression driven by eukaryotic promoters in prokaryotic cells.

Based on the above knowledge, the present invention is a design and method for using prokaryotic cells to produce human microRNA precursors (pre-miRNAs) and/or shRNAs as therapeutic drugs and/or vaccines for cancer therapy. More specifically, the present invention is a design and method for using prokaryotic cells to produce a type of special pre-miRNA drugs (named pro-miRNA), which can reprogram the malignant properties of human high-grade cancer cells into low-grade benign ones Or even relatively normal appearance state. Preferably, these pro-miRNAs are tumor suppressor microRNAs (TS-miRNAs), similar to miR-302a, b, c, d, e and/or f precursors (pre-miR-302s) and their natural families Clusters and their artificially redesigned small hairpin RNA (shRNA) homologs/derivatives and/or combinations thereof. The design of pro-miRNA shRNA homologs/derivatives includes the imperfect and perfectly matched hairpin composition of pro-miRNA and its homologous small interfering RNA (siRNA), which can be formed into a single unit or multiple unit clusters in. These designs can improve target specificity and/or reduce the number of copies of pro-miR-302 required for effective delivery and therapy. Human cells suitable for such drug treatment include normal, tumor and cancer cells in vitro, in vitro and/or in vivo.

Preferably, the prokaryotic cell line used in the present invention is a competent cell of bacteria, especially E. coli, and the chemical inducer is MOPS, ethanol or glycerol or a mixture thereof. Also preferably, the eukaryotic RNA promoter used is the eukaryotic pol-2 promoter (that is, the EF1α promoter) or the pol-2 compatible (pol-2 type) viral promoter (that is, the cytomegaloviral ). CMV promoter). Genes mediated by eukaryotic RNA promoters can be encoded from microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), messaging RNA (mRNA), their precursors and homologs, and combinations thereof The group of non-coding or protein-coding RNA or both (such as intron-containing gene transcripts). In order to induce gene expression, prokaryotic cells were transfected with genes mediated by eukaryotic RNA promoters, and then grown in a medium similar to Luria-Bertani (LB) medium at 37°C with the addition of chemical inducers for >24 hours .

In order to verify the inducibility of these chemical inducers for the production of human microRNAs in prokaryotes, the inventors gave priority to the lentiviral vector pSpRNAi-RGFP of US Patent Application Nos. 12/149,725 and 12/318,806 from the inventors. -miR302 was modified into a new plastid vector pLenti-EF1a-RGFP-miR302 , whose SpRNAi-RGFP gene expression system is driven by eukaryotic promoters such as EF1α or CMV (Figure 1A). Thereafter, the inventors used it to transform E. coli competent cells, and then used the production of red fluorescent protein (RGFP) as a visible marker for measuring the transcription rate and process of microRNA miR-302, as shown in Figure 1B. Because the miR-302 family cluster has also been modified to encode in the 5'-intron region of the RGFP gene [for example, the 5'-untranslated region (5'-UTR) or the first intron], each RGFP mRNA The transcription leads to a 4-hairpin miR-302 precursor cluster (pri-miR-302) and/or four 1-hairpin miR-302 precursors (pre-miR-302s), as shown in Figures 5 and 6 Shown in. Since there is no RNase III Dicer in prokaryotes, the pri-miR-302 transcript will eventually be decomposed (by some single-stranded RNases in E. coli) into 1 hairpin pre-miR-302s, all of which can be extracted and It is further used as a therapeutic drug in the present invention. Broadly speaking, 5'-UTR and 3'-UTR are regarded as part of introns in the present invention.

All miR-302 members share the exact same sequence 5'-UAAGUGCUUC CAUGUUU-3' (SEQ.ID.NO.3) in the first 5'-17 (17) nucleotides, and the full length of the mature microRNA The 23 nucleotides contain >82% homology. Based on the results predicted by online calculation programs TARGETSCAN (http://www.targetscan.org/) and PICTAR-VERT (http://pictar.mdc-berlin.de/), these miR-302 targets almost the same genes at the same time , Including >600 human genes. In addition, miR-302 also shares multiple overlapping target genes with mir-92, mir-93, mir-200c, mir-367, mir-371, mir-372, mir-373, mir-374 and mir-520 family members. All of them can have similar functions. Most of these target gene lines are involved in the initiation And/or establish developmental signals and transcription factors for the differentiation of certain lineage-specific cells during early embryogenesis (Lin et al., 2008). Many of these target genes are also known oncogenes; therefore, miR-302s is likely to act as a tumor suppressor to prevent normal hESC growth from deviating to tumor/cancer formation.

Induces gene expression driven by eukaryotic promoters in prokaryotes.

E. coli competent cells were transformed with pLenti-EF1α-RGFP-miR302 plastids (Figure 1A) using the z-competent E. coli transformation kit (Zymo Research, Irvine, CA), and at 37°C It was cultured in Luria-Bertani (LB) medium supplemented with a mixture of 0.1% (v/v) MOPS and 0.05% (v/v) glycerol (inducer) under frequent stirring at 170 rpm. After overnight incubation, the transformed E. coli competent cells express highly abundant red RGFP protein, which is clearly visible in the color of the LB medium, while the blank control E. coli does not show RGFP, as shown in Figure 2. The existence of functional RGFP indicates that both the RNA and protein encoded by it are successfully produced and processed in competent cells.

In order to further confirm the specificity of gene expression induced by chemical inducers, two transformed E. coli strains were prepared: a pLVX-Grn-miR302 + carrying a green fluorescent protein (GFP) gene driven by a CMV promoter 367 plastid vector and the other carrying the aforementioned pLenti-EF1α-RGFP-miR302 vector. After incubating overnight with only 0.1% (v/v) MOPS, the Escherichia coli transformed by pLVX-Grn-miR302 + 367 turned green, while the Escherichia coli transformed by pLenti-EF1α-RGFP-miR302 still showed as Red, as shown in Figure 3. This result indicates that chemical inducers such as MOPS can stimulate specific RNA transcription and related protein production via eukaryotic pol-2 or pol-2 virus promoters. In particular, it should be noted that the production of RGFP and GFP is so abundant that E. coli cells are even visually stained with red and green respectively.

Among all the chemicals tested in the present invention, the first three most potent inducers are MOPS, glycerol and ethanol, as shown in Figure 4. The quantitative results of induced RGFP production were further confirmed by Western blot analysis, as shown in Figure 5 and Example 3. The bacterial RuvB protein serves as a housekeeping standard to standardize RGFP expression. It was also found that the inducibility of these identified inducers was dose-dependent in proportion to their concentration. Without any treatment, the negative control E. coli cells showed only their original color in the absence of any fluorescent stain. Therefore, based on all these results, the present invention clearly provides a novel chemically inducible composition and its application for regulating eukaryotic pol-2 or pol-2 virus promoter-driven RNA production in prokaryotic cells. In view of the above arguments, it is very obvious that those with ordinary knowledge in the technical field can use other genes or related cDNAs to replace the RGFP gene to produce functional RNAs and related proteins in prokaryotes.

Induces the expression of microRNA driven by eukaryotic promoters in prokaryotes.

Following the RGFP induction experiment shown above, the inventors further measured the expression of pri-/pre-miR-302s and its mature miR-302s in pLenti-EF1α-RGFP-miR302 transformed cells with or without chemical induction . As shown in Figure 6 and Example 4, the quantitative results produced by induced pri-/pre-miR-302 have been confirmed by Northern blot analysis. Similar to the results of RGFP induction in Figures 4 and 5, the expression of pri-/pre-miR-302 was largely detectable in transformed cells treated with MOPS, glycerol, or ethanol, but it was not detected in the blank control. Measured, indicating that these chemical inducers substantially stimulate the expression of pri-/pre-miRNAs in prokaryotic cells via the eukaryotic pol-2 promoter (Figure 6). Due to the structural similarity between pre-miRNAs and shRNAs, those skilled in the art can obviously use the present invention to produce other types of pri-/pre-miRNA, such as but not limited to miR-34, miR-146, miR-371-373 and miR-520. To clarify, the pri-/pre-miRNAs produced by these prokaryotes are called pro-miRNAs.

Because pLenti-EF1α-RGFP-miR302 contains the miR-302 family cluster located in the 5'-UTR of the RGFP gene (Figure 1A and Figure 1B), the induced expression of the RGFP gene will also produce the miR-302 cluster (pri-miR- 302) and its derivatives pre-miR-302a, b, c and d (pre-miR-302s), as shown in Figure 1B. Since there is no RNase III Dicer in prokaryotes, it is found that the pri-miR-302 and pre-miR-302s obtained can be kept as hairpin microRNA precursors, which can be used to develop therapeutic drugs. In human cells, these pre-miR-302s and pri-miR-302 can be processed into mature miR-302 to trigger its tumor suppressor function. Similarly, the present invention can also be used to produce other types of TS-miRNA species and their precursors, such as miR-34a, miR-146a, miR-373 and miR-520 families.

The resulting pro-miRNAs can be easily extracted from competent E. coli cells (Examples 5 and 6) and further purified by high performance liquid chromatography (HPLC) (Figures 10A and 10B). In the purified pro-miR-302s, the inventors have used microRNA microarray analysis (Figure 11B and 12) and RNA sequencing [Figure 13A (pri-miR-302) and 13B (pre-miR-302s)] to identify All miR-302 family members (miR-302a, a*, b, b*, c, c*, d, and d*). In particular, the sequencing results showed that these pro-miR-302s share exactly the same sequence with their natural pre-miR-302 counterparts (Figure 13B). In addition, the inventors have formulated these pro-miR-302s as soluble drugs for IV/in vivo injection for testing Its therapeutic effect on human liver cancer in vivo (Example 11). As shown in Figure 14, after 3 injections, the pro-miR-302 drug successfully reduced the volume of human liver cancer transplanted in vivo by >90%, reducing the average cancer size to less than that of untreated cancer. <10%. In addition, the histological examination of hematoxylin and eosin (H&E) staining further demonstrated that this significant therapeutic effect not only comes from the reported tumor suppressor function of miR-302 (Lin et al., 2010), but also From another novel reprogramming function that has not been observed before. For example, Figure 15 clearly shows that the pro-miR-302 drug can reprogram the malignant characteristics of human high-grade liver cancer to a benign stage that is almost similar to normal liver tissue in vivo. These treated cancers can even form normal liver-like structures, such as classic liver lobules (liver lobules), central veins (CV) and portal triads (PT). Therefore, these evidences strongly indicate that pro-miR-302 can not only inhibit the growth of tumor/cancer cells, but also reset the malignant human cancer to a relatively benign or normal state in vivo, leading to a new therapeutic effect for cancer drug design.

In the present invention, both the plastid vector and its encoded non-coding RNAs (ie pre-miRNA/shRNA) can be simultaneously amplified in prokaryotic cells, preferably E. coli DH5α competent cells (Examples 1, 5 and 6 ). The methods used to isolate amplified pLenti-EF1α-RGFP-miR302 plastid DNA and transcribed pri-/pre-miR-302s are described in Examples 5 and 6. The technique of delivering plastid vector (ie pLenti-EF1α-RGFP-miR302 ) into prokaryotic cells is called cell transformation, and delivery of amplified non-coding RNAs (ie pro-/pri-/pre-miR-302s) The method to eukaryotic cells can be selected from endocytosis, chemical/glycerol infusion, peptide/liposome/chemically mediated transfection, electroporation, gene gun penetration, microinjection , A group consisting of transposon/retrotransposon insertion and/or adenovirus/retrovirus/lentivirus infection.

Pre-miR-302 induced pluripotent stem cell derived.

MiR-302 has been reported to reprogram mammalian somatic cells into human embryonic stem cells (hESC)-like induced pluripotent stem cells (iPSCs), such as the inventor’s priority US Patent Application Nos. 12/149,725 and 12/318,806 Demonstrated in the number. Many stem cell applications and therapies have been designed and developed using these iPSCs. Nevertheless, it is difficult and inefficient to collect miR-302 and its precursors from such pluripotent stem cells because of the extremely high cost and laborious cultivation of such iPSCs and hESCs. On the other hand, the manufacturing of synthetic shRNA mimics, pre-miR-302, is another possible alternative; however, the cost is still extremely high. In addition, the similarity between synthetic shRNA and natural pre-miR-302 is very questionable. To solve these problems, the present invention provides a simple, cheap and efficient method for mass production of pre-miR-302 in prokaryotes. In addition, the extraction and purification of pre-miR-302s (pro-miR-302s) produced by these prokaryotes is relatively simple and cost-effective, as shown in Figure 6 and Example 6 of the present invention.

The inventors have used pLenti-EF1α-RGFP-miR302 to transform E. coli cells to produce and isolate a large number of high-quality pLenti-EF1α-RGFP-miR302 vectors and pro-miR-302s, as shown in Examples 5 and 6. Both pLenti-EF1α-RGFP-miR302 and pro-miR-302s can be used to generate iPSCs. Following Example 2, when the pro-miR-302s produced by the present invention were transduced into primary keratinocytes of human skin, the transfected keratinocytes were reprogrammed into hESC-like iPSCs that exhibit a strong hESC marker Oct4 (Figure 7). In Figure 8 and Example 8, the inventors further performed sulfite DNA sequencing analysis to show that the overall DNA demethylation did occur in the promoters of both Oct4 and Sox2 genes (two key reprogramming factors) and hESC markers. . Since global DNA demethylation and Oct4 expression are known to be the first step in somatic cell reprogramming to form hESC-like iPSCs (Simonsson and Gurdon, Nat Cell Biol. 6: 984-990, 2004), it is derived from MOPS-induced E. coli The pro-miR-302s isolated from cell extracts proved to be as effective as the natural pre-miR-302s that can be used for iPSC derivation. Therefore, pro-miR-302 and pre-miR-302 are likely to have the same function in stem cell induction.

Proliferation and/or regeneration of CD34-positive adult stem cells induced by Pre-miR-302.

It has been found that MicroRNA miR-302 can reprogram mammalian somatic cells into embryonic stem cells (ESC)-like induced pluripotent stem cells (iPSC) (Lin, 2008, 2010, 2011; Lin's U.S. Patent Application No. 12/149,725 and No. 12/318, No. 806). Using these iPSCs, a variety of stem cell-related applications and therapies have been developed to advance modern regenerative medicine. However, miR-302 is only enriched in human ESCs and not differentiated tissue cells. In addition, the isolation of miR-302 from human ESCs is very controversial, costly and cumbersome. In order to solve these problems, the present invention provides a simple, cheap, fast and inducible composition and method to facilitate the mass production of hairpin miR-302 molecules and/or their precursors/homologs in prokaryotes. In addition, the isolation of miR-302 and/or its precursors from prokaryotic cells is relatively simple and cost-effective, as shown in Figure 6 and Example 6 of the present invention.

The inventors have used pLenti-EF1α-RGFP-miR302 to transform E. coli cells to produce and isolate a large number of high-quality pLenti-EF1α-RGFP-miR302 vectors and pre-miR-302s, as shown in Examples 5 and 6. In view of the inventors' previous US Patent Application Nos. 12/149,725 and 12/318,806, the use of pLenti-EF1α-RGFP-miR302 has been shown to produce human ESC-like iPSCs. In addition, the iPSCs thus obtained can be further differentiated into various somatic tissue cells, as demonstrated in the inventors' previous studies (Lin et al., 2008, 2010 and 2011).

40倍之增加。當前已知之CD34陽性成體幹細胞類型包括但不限於皮膚、毛髮、肌肉、血液(造血)、間質細胞(mesenchymal)及神經幹細胞。因此，因為miR-302可用以活體內誘導CD34陽性成體幹細胞增殖及/或再生，所以此治療效果亦可幫助使功能性成體幹細胞再生長及/或複生以便治療人類之退化性疾病，諸如但不限於阿茲海默症、帕金森氏症、骨質疏鬆症、糖尿病及癌症。 The application of the isolated miR-302 and/or pre-miR-302 molecules may further include the induction and proliferation of CD34-positive adult stem cells. As shown in Figures 17A and 17B, recent studies by the inventors in wound healing and cancer therapy using novel miR-302 formulations show that relatively low concentrations (50-500μg/mL) of isolated miR-302/pre -The treatment of miR-302 molecules not only greatly enhances scarless wound healing but also induces the proliferation of CD34-positive adult stem cells around the injured area in pig skin in vivo. Based on the comparison of the results of miR-302 treatment (miR-302s/pre-miR-302s+antibiotic ointment) in Figure 17B with the results of the control (antibiotic ointment only) in Figure 17A, it is obvious that CD34-positive adults in vivo after miR-302 treatment Stem cell population (labeled with green fluorescent antibody)

An increase of 40 times. The currently known CD34-positive adult stem cell types include but are not limited to skin, hair, muscle, blood (hematopoietic), mesenchymal and neural stem cells. Therefore, because miR-302 can be used to induce the proliferation and/or regeneration of CD34-positive adult stem cells in vivo, this therapeutic effect can also help regenerate and/or regenerate functional adult stem cells to treat degenerative diseases in humans, such as But not limited to Alzheimer’s disease, Parkinson’s disease, osteoporosis, diabetes and cancer.

Use pro-miR-302 for in vivo tumor/cancer therapy.

The inventors’ previous studies have demonstrated that this method can treat human hepatocellular carcinoma (hepatocellular carcinoma) HepG2 cells in vitro rather than in vivo. The viability of the cell (Lin et al., 2010). As shown in Figure 9, the treated tumors/cancer cells were reprogrammed into iPSCs (labeled as mirPS-HepG2) and formed embryoid body-like cell colonies. In addition, it was also found that miR-302 can induce apoptosis in >95% of the treated cancer cell population. The top panel of Figure 9 further shows the flow cytometry analysis of DNA content in response to cell cycle stages, showing a significant reduction in the mitotic cell population (from 45.6% to 17.2%) after miR-302 treatment. These results indicate that miR-302 can effectively slow down the rapid cell cycle rate of human liver cancer cells and thus cause significant apoptosis of these cancer cells.

The process of cancer progression is considered irreversible due to accumulated gene mutations; however, the present invention reveals a novel pre-miRNA (pro-miR-302) function, which can reprogram highly malignant cancers back to low-grade benign in vivo Or even the normal-like stage, the mechanism may be related to the very rare natural healing process called spontaneous cancer regression. Spontaneous cancer regression rarely occurs at a rate of less than 1 in 100,000 cancer patients. The inventor found that pro-miR-302 treatment can increase this rare healing rate to >90% in human liver cancer. As shown in Figure 14, the treatment results of using pro-miR-302s as a drug to treat human liver cancer xenografts in SCID-beige nude mice (n=6) confirmed that this pro-miR-302 drug successfully reduced the size of the cancer. Decreased from 728±328mm ³ (untreated blank control, C) to 75±15mm ³ (treated with pro-miR-302, T), indicating that the average cancer size is reduced by about 90%, while other synthetic siRNA mimics (SiRNA-302) treatment does not provide any similar therapeutic effects.

Further histological examination (the rightmost image in Figure 14) showed that normal liver lobule-like structures (circled and indicated by black arrows) were only observed in cancer grafts treated with pro-miR-302 but not in other treatments or treatments. It was not observed in the photo, indicating that the reprogramming mechanism occurred to reset the characteristics of the malignant cancer cells back to a relatively normal-like state (cancer reversal). This novel reprogramming mechanism is likely to be caused by the gene silencing effect of miR-302 on human oncogenes, especially mutated oncogenes involved in cancer progression. By silencing these mutated oncogenes, pro-miR-302 can reset the oncogene expression pattern back to a normal-like state, thereby producing cancer reversal treatment results. Nevertheless, this in vivo reprogramming mechanism is significantly different from previously reported somatic cell reprogramming (Lin et al., 2008 and 2011) because Oct4-positive pluripotent stem cells have not been identified.

More detailed histological examination (Figure 15) further confirmed that the pro-miR-302 drug did indeed reprogram high-grade (IV degree) human liver cancer grafts to a more benign low-grade (less than II degree) state. As shown in Figure 15, the treated cancer grafts formed classic liver lobules containing central vein (CV)-like and portal triplet (PT)-like structures (indicated by black arrows), which are very similar to normal liver tissue structures ( Top image). The histological comparison between untreated, siRNA-treated, pro-miR-302-treated human liver cancer transplants and normal liver tissues in vivo (Figure 16) also showed that untreated human liver cancer transplants (top image) ) Invasively invade surrounding normal tissues (such as muscles and blood vessels) and form large-scale cell-cell and cancer-tissue fusion structures, confirming its high malignancy and metastasis. siRNA mimic (siRNA-302) treatment does not significantly reduce the malignancy of transplanted liver cancer (upper middle), which is probably caused by the short half-life of siRNA in vivo. In contrast, pro-miR-302 treatment not only reprogrammed the transplanted cancer cells to a normal hepatocyte-like morphology (without fusion), but also successfully inhibited the invasion of any cancer into surrounding tissues (bottom middle panel). Compared with normal liver tissue (bottom image), cancers treated with pro-miR-302 clearly show a lobular-like structure, a normal glandular cell-like configuration, and the cell-cell and cancer-tissue junctions are very clear The border (black arrow) indicates that these treated cancers have been greatly degraded to an extremely benign state. The pro-miR-302 drug was further processed for six to ten consecutive times to completely eliminate cancer xenografts in all six samples (n=6).

A. Definition

To facilitate the understanding of the present invention, a number of terms are defined as follows: Nucleotide: DNA or DNA composed of sugar moiety (pentose), phosphate and nitrogenous heterocyclic base The monomeric unit of RNA. The base is linked to the sugar moiety through a glycosidic carbon (the No. 1 carbon of pentose), and the combination of the base and the sugar is a nucleoside. Nucleosides containing at least one phosphate group bonded to the three-terminal or five-terminal position of the pentose are referred to as nucleotides. Deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) is composed of different types of nucleotide units, namely deoxyribonucleotides and ribonucleotides.

Oligonucleotide (Oligonucleotide): A molecule composed of two or more than two, preferably more than three and usually more than ten deoxyribonucleic acids (DNAs) and/or ribonucleotides (RNAs). Oligonucleotides longer than 13 nucleotide monomers are also called polynucleotides. The exact size of the oligonucleotide depends on many factors and depends on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced in any manner, including chemical synthesis, DNA replication, RNA transcription, reverse transcription, or a combination thereof.

Nucleotide analogues: purine or pyrimidine nucleotides, which are structurally different from adenine (A), thymine (T), guanine (G), cytosine (C) or uracil (U), but sufficient Similar to normal nucleotides in nucleic acid molecules.

Nucleic acid composition: nucleic acid composition refers to oligonucleotides or polynucleosides Acids, such as DNA or RNA sequences in single-stranded or double-stranded molecular structures, or mixed DNA/RNA sequences.

Gene: a nucleic acid composition whose oligonucleotide or polynucleotide sequence encodes RNA and/or polypeptide (protein). The gene can be RNA or DNA. Genes can encode non-coding RNA, such as small hairpin RNA (shRNA), microRNA (miRNA), rRNA, tRNA, snoRNA, snRNA and their RNA precursors and derivatives. Alternatively, genes can encode protein-encoding RNA necessary for protein/peptide synthesis, such as messaging RNA (mRNA) and its RNA precursors and derivatives. In some cases, the gene can encode protein-coding RNA that also contains at least one microRNA or shRNA sequence.

Primary RNA transcripts: RNA sequences directly transcribed from genes without any RNA treatment or modification, which can be selected from the group consisting of mRNA, hnRNA, rRNA, tRNA, snoRNA, snRNA, pre-microRNA, viral RNA and Its RNA precursors and derivatives.

Pre-mRNA (pre-mRNA): The primary RNA transcript of protein-coding genes, which is produced in eukaryotes through an intracellular mechanism called transcription by the mechanism of eukaryotic type II RNA polymerase (Pol-II). The pre-mRNA sequence contains 5'untranslated region (UTR), 3'-UTR, exons and introns.

Intron: A part or multiple parts of a gene transcription sequence that encodes a non-protein reading frame, such as in-frame introns, 5'-UTR and 3'-UTR.

Exon: A part or multiple parts of a gene transcription sequence (cDNA) encoding a protein reading frame, such as cDNA for cell genes, growth factors, insulin, antibodies and their analogs/homologs and derivatives.

Signaling RNA (mRNA): A collection of pre-mRNA exons, which are formed after the introns are removed by the intracellular RNA splicing mechanism (splicesome) and Serves as protein coding RNA for peptide/protein synthesis. Peptides/proteins encoded by mRNAs include but are not limited to enzymes, growth factors, insulin, antibodies and their analogs/homologs and derivatives.

Complementary DNA (cDNA): Single-stranded or double-stranded DNA that contains a sequence complementary to the mRNA sequence and does not contain any intron sequences.

Sense: A nucleic acid molecule with the same sequence order and composition as homologous mRNA. The justice configuration is indicated by the symbol "+", "s" or "justice".

Antisense: nucleic acid molecules that are complementary to individual mRNA molecules. The antisense configuration indicator is indicated by "-" or "*" or "a" or "antisense" in front of DNA or RNA, such as "aDNA" or "aRNA".

Base pair (bp): The partnership of adenine (A) and thymine (T) or cytosine (C) and guanine (G) in a double-stranded DNA molecule. In RNA, uracil (U) replaces thymine (T). Generally speaking, partnership is achieved through hydrogen bonding.

Base pair (bp): the paired combination of adenine (A) and thymine (T) or cytosine (C) and guanine (G) in double-stranded DNA molecules (partnership) . In RNA, uracil (U) replaces thymine (T). Generally speaking, partnership is achieved through hydrogen bonding. For example, the sense nucleotide sequence "5'-A-T-C-G-U-3'" can form a complete base pairing with its antisense sequence "5'-A-C-G-A-T-3'".

5'-end: there is no nucleotide end at the 5'position of consecutive nucleotides, and the 5'-hydroxyl group of one nucleotide is joined to the 3'of the next nucleotide by a phosphodiester bond -Hydroxyl. Other groups, such as one or more phosphate esters can be present On the end.

3'-end: There is no nucleotide end at the 3'position of consecutive nucleotides, and the 5'-hydroxyl group of one nucleotide is joined to the 3'of the next nucleotide by a phosphodiester bond -Hydroxyl. There may also be other functional groups at this end, the most common being a hydroxyl group. .

Template: A nucleic acid molecule replicated by nucleic acid polymerase. The template may be single-stranded, double-stranded or partially double-stranded depending on the polymerase. The synthesized copy is complementary to the template or to at least one strand of the double-strand or part of the double-strand template. Both RNA and DNA are synthesized in the 5'to 3'direction. The two strands of the nucleic acid duplex are always aligned so that the 5'ends of the two strands are at the opposite ends of the duplex (and necessarily the 3'ends).

Nucleic acid templates: double-stranded DNA molecules, double-stranded RNA molecules, hybrid molecules, such as DNA-RNA or RNA-DNA hybrids, or single-stranded DNA or RNA molecules.

Conserved: If the nucleotide sequence is non-randomly heterogeneous with the exact complement of a preselected (reference) sequence, the nucleotide sequence is conserved with the preselected sequence.

Homologous or homology: a term indicating the similarity between a polynucleotide and a gene or mRNA sequence. For example, the nucleic acid sequence may be partially or completely homologous to a specific gene or mRNA sequence. Homology can also be expressed as a percentage of the number of similar nucleotides in the total number of nucleotides.

Complementary (Complementary or Complementarity or Complementation): used to indicate the matching base pairing between two polynucleotides (that is, mRNA And cDNA sequence). For example, the sequence "5'-A-G-T-3'" and the sequence "5'-A-C-T-3'" and "5'-A-C-U-3'" interact with each other Make up. Complementarity can be between two DNA strands, one DNA and one RNA strand, or two RNA strands. Complementarity can be "partial" or "complete" or "total". Partial complementarity (complementation) occurs when only some nucleic acid bases are matched according to the base pairing rules. When the bases between nucleic acid strands are completely or perfectly matched, complete or total complementarity or complementation occurs. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important for amplification reactions and detection methods that depend on the binding between nucleic acids. Percent complementarity (or complementation) refers to the proportion of mismatched bases in a strand of the nucleic acid among all bases. Therefore, 50% complementation means that half of the bases are mismatched and half of the bases are matched. Even if the number of bases in the two strands of nucleic acid is different, the two strands can be complementary. In this case, complementation occurs between a part of the longer strand and the shorter strand, wherein the strand of the longer strand has the base paired with the base of the shorter strand.

Complementary bases: Nucleotides that normally pair when DNA or RNA is in a double-stranded configuration.

Complementary Nucleotide Sequence: A nucleotide sequence of a single-stranded DNA or RNA molecule that is sufficiently complementary to the nucleotide sequence of another single-stranded molecule, so that the two strands correspond to each other Hydrogen bonding and exclusive hybridization.

Hybridization (Hybridize and Hybridization): The formation of a duplex between nucleotide sequences that are sufficiently complementary to form a complex via base pairing. When the primer (or splicing template) is "hybridized" with the target (template), the complexes (or hybrids) are sufficiently stable to provide DNA polymerase to initiate DNA synthesis The required priming function. There is a special feature between two complementary polynucleotides that can be competitively inhibited, that is, non-random interaction.

Posttranscriptional Gene Silencing: The knockout or knockdown effect of the target gene in the mRNA degradation or translation inhibition stage, which is usually caused by foreign/viral DNA or RNA transgenes or small Triggered by any inhibitory RNAs.

RNA interference (RNAi): The mechanism of post-transcriptional gene silencing in eukaryotes, which can be triggered by small inhibitory RNA molecules such as microRNA (miRNA), small hairpin RNA (shRNA), and small interfering RNA (siRNA). These small RNA molecules usually act as gene silencers, interfering with the expression of intracellular genes that are completely or partially complementary to small RNAs.

Gene silencing effect: the cellular response after gene function is inhibited, including but not limited to cell cycle attenuation, G0/G1 checkpoint arrest, tumor suppression, anti-tumorigenicity ( anti-tumorigenecity), cancer cell apoptosis, and combinations thereof.

Non-coding RNA: RNA transcripts that cannot be used to synthesize peptides or proteins via intracellular translation mechanisms. Non-coding RNA includes long and short regulatory RNA molecules, such as microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), and double-stranded RNA (dsRNA). These regulatory RNA molecules usually act as gene silencers, interfering with the expression of intracellular genes that are completely or partially complementary to non-coding RNA.

MicroRNA (miRNA): Single-stranded RNA that can bind to target gene transcripts (mRNAs) that are partially complementary to the sequence of microRNA. Mature microRNAs are usually about 17-27 oligonucleotides long, and can directly degrade the mRNA target in the cell or inhibit the protein translation of the target mRNA depending on the complementarity between the microRNA and its target mRNA(s). Natural microRNAs are found in almost all eukaryotes and act as defenses against viral infections and can regulate specific gene expression during plant and animal development. In principle, one microRNA usually targets multiple target mRNAs to achieve all of its functions. On the other hand, multiple miRNAs can target the same gene transcript to enhance the effect of gene silencing.

MicroRNA precursor (Pre-miRNA): Hairpin single-stranded RNA containing stem-arm and stem-loop regions, used to interact with intracellular RNase III Dicer endonuclease to produce one or more mature microRNAs (miRNAs), microRNAs (miRNAs) can silence target genes or specific groups of target genes that are completely or partially complementary to mature microRNA sequences. The stem-arm of pre-miRNA can form a perfect (100%) or partially (mismatched) heterozygous duplex, and the stem-loop connects one end of the stem-arm duplex to form a combination with some Alguin (AGO) ) Assemble into the circle or hairpin-loop configuration required in RNA-induced silencing complex (RISC).

MicroRNA precursors (Pro-miRNA) produced by prokaryotes: similar to natural microRNA precursors (pre-miRNA) but in prokaryotic competent cells driven by eukaryotic promoters from artificial recombinant microRNA expression plastid-transcribed small hairpin RNA . For example, pro-miR-302 is structurally the same as pre-miR-302 (Figure 13A and Figure 13B), but is derived from pLVX-Grn-miR302 + 367 or pLenti-EF1α-RGFP-miR302 in E. coli DH5α competent cells Vector transcription (Example 1). Because prokaryotic cells usually do not exhibit short RNAs with high secondary structure, such as eukaryotic pre-miRNA, the production of pro-miRNA in prokaryotes usually requires the addition of chemical inducers to stimulate the transcription of pre-miRNA driven by eukaryotic promoters (Figure 2-4).

Small interfering RNA (siRNA): A short double-stranded RNA with a size of about 18-27 perfect base pairing ribonucleotide duplexes and capable of degrading target gene transcripts with almost perfect complementarity.

Small or short hairpin RNA (shRNA): A single-stranded RNA containing a pair of partially or completely matched stem-arm nucleotide sequences separated by mismatched circular oligonucleotides to form a hairpin-type structure. A variety of natural miRNAs are derived from hairpin RNA precursors, that is, precursor microRNA (pre-miRNA).

Vector: A recombinant nucleic acid composition capable of moving and staying in different genetic environments, such as recombinant DNA (rDNA). Generally speaking, another nucleic acid is operably linked to it. The vector can replicate autonomously in the cell, in this case the vector and the connected segments replicate. One type of preferred carrier system is episomes, that is, nucleic acid molecules capable of extrachromosomal replication. The preferred carrier system is a vector capable of autonomous replication and expression of nucleic acid. Vectors capable of directing the expression of genes encoding one or more polypeptides and/or non-coding RNAs are referred to herein as "expression vectors" or "expression competent vectors". Particularly important vectors can be used to clone cDNA from mRNAs produced by reverse transcriptase. The vector may comprise the following components: viral promoter or type 2 RNA polymerase (Pol-II or pol-2) promoter or both, Kozak consensus translation initiation site (Kozak consensus translation initiation site) , Polyadenylation signals, multiple restriction/cloning sites, pUC origin of replication, used to express at least one antibiotic in replication-competent prokaryotic cells The SV40 early promoter of the drug resistance gene, the selective SV40 starting point for replication in mammalian cells, and/or the tetracycline responsive element. The structure of the vector can be linear or circular single-stranded or double-stranded DNA, and is selected from the group consisting of plastids, viral vectors, transposons, reversal positions, DNA transgenes, jumping genes, and combinations thereof The group formed.

Promoter: A nucleic acid that is recognized by a polymerase molecule, or binds to it and initiates RNA transcription. According to the purpose of the present invention, the promoter can be a conventional polymerase binding site, enhancer and the like, and any sequence that can use the required polymerase to initiate the synthesis of RNA transcripts.

Eukaryotic promoter: required for RNA and/or gene transcription and compatible with eukaryotic type II RNA polymerase (pol-2), pol-2 equivalent and/or pol-2 (pol-2 type) The viral polymerase recognizes the sequence of the nucleic acid motif used to initiate RNA/gene transcription.

The second type of RNA polymerase (Pol-II or pol-2) promoter: can be recognized by the second type of eukaryotic RNA polymerase (Pol-II or pol-2) and can therefore initiate eukaryotic signaling RNAs (mRNAs) and / Or RNA promoters for transcription of microRNAs (miRNAs). For example, but not limited to, the pol-2 promoter may be a mammalian RNA promoter or a cytomegalovirus (CMV) promoter.

Type II RNA polymerase (Pol-II or pol-2) equivalent: Eukaryotic transcription mechanism selected from the group consisting of: mammalian type II RNA polymerase (Pol-II or pol-2) and Pol-II compatible (pol-2 type) viral RNA polymerase.

Pol-II compatible (pol-2 type) virus promoter: can use eukaryotic pol-2 or pol-2 equivalent transcription mechanism to initiate gene and/or RNA expression Viral RNA promoter. For example, but not limited to, the pol-2 virus promoter may be a cytomegalovirus (CMV) promoter or a retrovirus long terminal repeat (LTR) promoter.

Cistron: A nucleotide sequence that encodes amino acid residue sequences in a DNA molecule and includes upstream and downstream DNA expression control elements.

Intron excision: The cellular mechanism responsible for RNA processing, maturation and degradation, including RNA splicing, exosomal digestion, nonsense-mediated degradation (NMD) processing and combinations thereof.

RNA processing: an intracellular mechanism responsible for the maturation, modification, and degradation of RNA, including RNA splicing, intron excision, exosome digestion, and nonsense-mediated decay , NMD), RNA editing, RNA processing and combinations thereof.

Target cells: single or plural human cells selected from the group consisting of somatic cells, tissues, stem cells, germline cells, teratoma cells, tumor cells, cancer cells and combinations thereof.

Cancer tissue: neoplastic tissue derived from the group consisting of skin cancer, prostate cancer, breast cancer, liver cancer, lung cancer, brain tumor/brain cancer, lymphoma, leukemia, and combinations thereof.

Expression competent vector: linear or round single-stranded or double-stranded DNA selected from the group consisting of plastids, viral vectors, transposons, reverse transcription transposons, DNA transgenes, jumping genes, and combinations thereof.

Antibiotic resistance genes: genes capable of degrading antibiotics selected from the following group: penicillin G (penicillin G), streptomycin, ampicillin (Amp), neomycin (neomycin), G418, kanamycin (kanamycin), erythromycin (erythromycin), paromycin (paromycin), phophomycin (phophomycin), spectromycin (spectromycin), tetracycline ( tetracycline (Tet)), doxycycline (Dox), rifampicin (rifapicin), amphotericin B (amphotericin B), gentamycin (gentamycin), chloramphenicol (chloramphenicol), pioneer mold Cephalothin, tylosin and combinations thereof.

Restriction/selection sites: DNA motifs used for restriction enzyme cleavage, including but not limited to 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, 10PmlI, PpuI , PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI, XmaI cleavage sites.

Gene delivery: a genetic engineering method selected from the group consisting of: polysomal transfection, liposomal transfection, chemical transfection, electroporation, viral infection, DNA recombination, translocation insertion , Jumping gene insertion, microinjection, gene gun penetration, and combinations thereof.

Genetic engineering: DNA recombination method selected from the group consisting of DNA restriction enzyme reaction and conjugation reaction, homologous recombination, transgenic incorporation, translocation insertion, jumping gene incorporation, retrovirus infection, and Its combination.

Cell cycle regulators: cell genes involved in the control of cell division and proliferation rates, including but not limited to: CDK2, CDK4, CDK6, cyclin, BMI-1, p14/p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1/Waf1, and p27Kip1 and Its combination.

Tumor inhibitory effect: cellular anti-tumor and/or anti-cancer mechanisms and responses, including but not limited to: cell cycle reduction, cell cycle arrest, tumor cell growth inhibition, cell tumorigenicity inhibition, tumor/cancer transformation inhibition, tumor /Induction of apoptosis of cancer cells, induction of normal cell recovery, reprogramming of highly malignant cancer cells to a more benign low-grade state (tumor regression) and combinations thereof.

Cancer treatment effect: the cellular response and/or cellular mechanism produced by drug treatment, including but not limited to: inhibition of oncogene expression, inhibition of cancer cell proliferation, inhibition of cancer cell invasion and/or migration, inhibition of cancer metastasis, cancer Induction of cell death, prevention of tumor/cancer formation, prevention of cancer recurrence, inhibition of cancer progression, repair of damaged tissue cells, reprogramming of high-malignant cancers to a more benign low-grade state (cancer regression/remission) and combinations thereof.

Gene silencing effect: the cellular response after gene function is inhibited, including but not limited to: inhibition of oncogene expression, inhibition of cell proliferation, cell cycle arrest, tumor suppression, cancer regression, cancer prevention, cell apoptosis, cell Repair and/or recovery, cell reprogramming, diseased cells reprogramming to a relatively normal state (spontaneous healing), and combinations thereof.

Cancer reversal: A reprogramming mechanism that resets the malignant characteristics of high-grade cancer to a relatively normal low-grade state in vitro, in vitro or in vivo.

Target cells: selected from body cells, tissues, stem cells, germline cells, teratoma cells, tumor cells, cancer cells and combinations thereof A group of single or multiple human cells.

Cancer tissue: neoplastic tissue derived from the group consisting of skin cancer, prostate cancer, breast cancer, liver cancer, lung cancer, brain tumor/brain cancer, lymphoma, leukemia, and combinations thereof.

Transcription inducer: a chemical agent that can induce and/or enhance eukaryotic RNA and/or gene transcription from pol-2 or pol-2 promoters in prokaryotic cells. For example, transcription inducers contain, but are not limited to, chemical structures similar to the following: MOPS, ethanol, glycerol, and their functional analogs, such as 2-(N-morpholino)ethanesulfonic acid (MES), 4-( 2-Hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES) and mannitol or mixtures thereof.

Antibody: A peptide or protein molecule that has a preselected conserved domain structure and encodes a receptor that can bind to a preselected ligand.

Medical and/or therapeutic applications: can be used for diagnosis, stem cell generation, stem cell research and/or therapy development, tissue/organ repair and/or restoration, wound healing treatment, tumor suppression, cancer treatment and/or prevention, disease treatment, drug manufacturing And its combination of biomedical uses, devices and/or equipment.

B. Composition and application

A composition and method for producing new types of microRNA precursors (pro-miRNAs) produced by prokaryotes that can reprogram the malignant characteristics of human cancer to a low-grade benign or normal-like state in vitro, in vitro and in vivo, which Containing: (a) containing at least one chemical inducer similar to the structure of 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol or glycerol or a mixture thereof; and (b) containing eukaryotic pol-2 and / Or a plurality of prokaryotic cells of at least one pre-miRNA encoding gene driven by transcription driven by a pol-2 promoter; wherein (a) and (b) are mixed together under the condition of inducing the expression of the gene to facilitate the prokaryotic In the cell Generate the encoded pre-miRNA. It is worth noting that chemical inducers can stimulate RNA transcription driven by eukaryotic promoters in prokaryotes.

In principle, the present invention provides a novel composition design and applicable strategies for inducing rapid adaptation of prokaryotes to use eukaryotic pol-2 and pol-2 promoters to directly express specific required microRNA precursors ( pre-miRNA) without using error-prone prokaryotic promoters or cultivating laborious and costly fusion tumors or mammalian cells.

Preferably, the prokaryotes are bacterial cell strains, especially E. coli, and the chemical inducer is 3-morpholinopropane-1-sulfonic acid (MOPS), ethanol or glycerol or a mixture thereof. Also preferably, the eukaryotic promoter is a eukaryotic pol-2 promoter, such as EF1α; or a pol-2 compatible (pol-2 type) viral promoter, such as a cytomegalovirus (CMV) promoter or reverse transcription Viral long terminal repeat (LTR) promoter. The pre-miRNA encoding gene mediated by the eukaryotic promoter encodes non-coding or protein-coding RNA transcripts or both (such as gene transcripts containing introns), wherein non-coding or protein-coding RNA transcripts or both (Such as gene transcripts containing introns) selected from microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), messaging RNA (mRNA) and its precursors, and shRNA/siRNA homologs and The group formed by its combination. The protein-encoding RNA can be selected from, but not limited to, the group consisting of gene-encoding enzymes, growth factors, antibodies, insulin, botox, functional proteins and/or their analogs and combinations thereof. Preferably, the conditions for inducing the expression of the pre-miRNA encoding gene are bacterial culture conditions, such as Luria-Bertani (LB) culture medium added with the chemical inducer at 37°C.

This patent or application file contains at least one color drawing. After applying and paying the necessary fees, the USPTO will provide a copy of the patent or patent application publication with color pictures.

For the purpose of illustration and not limitation, the drawings are specifically referred to, which illustrate: Figure 1A and Figure 1B show a eukaryotic promoter-driven expression vector composition (1A) and its use in prokaryotes for RNA transcripts and/or The expression mechanism of protein production (1B). In order to verify the present invention, the new pLenti-EF1α-RGFP-miR302 vector (Figure 1A) is used to transform E. coli DH5α competent cells under the stimulation of MOPS, glycerol and/or ethanol to produce RGFP protein and miR-302s and its An example composition of precursors (pre-miR-302s). pLenti-EF1α-RGFP-miR302 is a lentiviral plastid vector designed by the present inventors to express various microRNAs/shRNAs, mRNAs and/or proteins/peptides in both prokaryotes and eukaryotes. According to the disclosed mechanism (1B), those skilled in the art can easily use any microRNA/shRNA to replace miR-302 or any mRNA/protein to replace RGFP, as described in the present invention. Black arrows indicate paths that occur in both prokaryotic and eukaryotic cells, while blank arrows indicate steps that occur only in eukaryotic cells.

Figure 2 depicts the results of bacterial culture broth treated (left) or untreated (right) with a mixture of 0.1% (v/v) MOPS and 0.05% (v/v) glycerol. E. coli competent cells have been transformed with pLenti-EF1α-RGFP-miR302 before treatment with chemical inducers.

Figure 3 shows the results of different bacterial pellets after treatment with 0.1% (v/v) MOPS. E. coli competent cells have been transformed by pLVX-Grn-miR302 + 367 (green) or pLenti-EF1a-RGFP-miR302 (red) before MOPS treatment.

Figure 4 shows the inducibility of various chemical inducers to induce gene expression driven by the pol-2 promoter in competent E. coli cells. Among all the tested chemicals, the first three most potent inducers are MOPS, glycerol and ethanol. The concentration of the chemicals used can range from about 0.001% to 4%, preferably 0.01% to 1%.

Figure 5 shows the results of the Western blot method for the expression of red RGFP protein induced by MOPS, glycerol and ethanol, respectively. The bacterial RuvB protein was used as a housekeeping standard to normalize the detected RGFP expression. The protein extracted from blank E. coli cells (that is, without vector transformation) was used as a negative control.

Figure 6 shows the results of the northern blot method of the expression of the miR-302 family cluster (about 700 nt) and its derivative precursors (pre-miR-302s with 1 to 4 hairpins) induced by MOPS, glycerol, and ethanol, respectively. RNA extracted from blank E. coli cells was used as a negative control.

FIG. 7 shows the iPSC generation using miR-302 and/or pre-miR-302 isolated from bacterial competent cell extract (BE), which was confirmed by the northern blot analysis as shown in FIG. 6. As reported, miR-302 reprogrammed iPSCs (or called mirPSCs) to form globular cell populations and expressed strong Oct4 as a standard hESC marker.

Figure 8 shows the overall DNA demethylation of Oct4 and Sox2 gene promoters induced by miR-302 and/or pre-miR-302 isolated from bacterial competent cell extracts (BE), as shown in Figure 6 Verification by the northern ink dot analysis. As demonstrated by Simonsson and Gurdon ( Nat Cell Biol. 6,984-990, 2004), both signs of global DNA demethylation and Oct4 expression are required for reprogramming of somatic cells to form iPSCs.

Figure 9 shows the in vitro tumorigenicity analysis of human liver cancer cell line HepG2 in response to miR-302 transfection. The cells obtained after miR-302 transfection are labeled mirPS-HepG2, indicating that their cancer cell characteristics have changed to an induced pluripotent stem cell (iPSC)-like state. Compare the morphology and cell cycle rate of miR-302 before and after transfection. The peak chart (n=3, p <0.01) analyzed by flow cytometry of cell morphology shows the DNA content of each cell relative to the cell cycle stage.

Figures 10A and 10B show the results of HPLC purification and analysis using synthetic standard uDNA (from Sigma-Genosys) and freshly extracted pro-miR-302s isolated from pLenti-EF1α-RGFP-miR302 transformed E. coli cells. The standard uDNA is designed to be equivalent to the natural pre-miR-302a as: 5'-CCACCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC UUCCAUGUUU UGGUGAUGG-3' (SEQ. ID. NO. 4).

Figures 11A and 11B show the results of microRNA (miRNA) microarray analysis using small RNA extracted from blank E. coli competent cells or pLenti-EF1α-RGFP-miR302 ( RGFP-miR302) transfected cells. The extracted small RNA was further purified by HPLC as shown in the green marked area in Figure 10B. Figure 11A shows that the RNA from blank E. coli cells hardly presents microRNA (green dots mean not statistically significant, and red dots indicate a positive result). This is because prokaryotes do not have several essential enzymes required for microRNA expression and processing, such as Pol-2, Drosha, and RNase III Dicer. In addition, prokaryotic RNA polymerase does not efficiently transcribe small RNAs with high secondary structure, such as hairpin pre-miRNAs and shRNAs. Therefore, using only the present invention, the inventors can stimulate the expression of specific microRNAs in prokaryotic cells, such as miR-302a, a*, b, b*, c, c*, d, and d*, as shown in Figure 11B. Because prokaryotic cells do not have Dicer, all microRNAs maintain their precursor configuration, such as pri-miRNA (4 hairpin cluster) and/or pre-miRNA (1 hairpin precursor). Taken together, the results of Figures 10B and 11B have confirmed two facts: (1) the small RNA extracted from RGFP-miR302 transfected cells mainly contains pure miR-302 precursors, and (2) there are almost none in E. coli competent cells. There are other types of microRNA contamination.

Figure 12 shows a list of expressed microRNAs extracted from blank E. coli competent cells (group 1, as shown in Fig. 11A) or pLenti-EF1α-RGFP-miR302 transfected cells (group 2, as shown in Fig. 11B) . Signals less than 500 are not statistically significant (shown in green in Figures 11A and 11B), which may be caused by low copy number expression or high background.

Figures 13A and 13B show the sequencing results of the miR-302 family cluster (13A) and individual pro-miR-302a, pro-miR-302b, pro-miR-302c and pro-miR-302d sequences (13B). miR-302 family cluster (= pri-miR-302) of the result based 5'-AAUUUUUUUC UUCUAAAGUU AUGCCAUUUU GUUUUCUUUC UCCUCAGCUC UAAAUACUCU GAAGUCCAAA GAAGUUGUAU GUUGGGUGGG CUCCCUUCAA CUUUAACAUG GAAGUGCUUU CUGUGACUUU AAAAGUAAGU GCUUCCAUGU UUUAGUAGGA GUGAAUCCAA UUUACUUCUC CAAAAUAGAA CACGCUAACC UCAUUUGAAG GGAUCCCCUU UGCUUUAACA UGGGGGUACC UGCUGUGUGA AACAAAAGUA AGUGCUUCCA UGUUUCAGUG GAGGUGUCUC CAAGCCAGCA CACCUUUUGU UACAAAAUUU UUUUGUUAUU GUGUUUUAAG GUUACUAAGC UUGUUACAGG UUAAAGGAUU CUAACUUUUU CCAAGACUGG GCUCCCCACC ACUUAAACGU GGAUGUACUU GCUUUGAAAC UAAAGAAGUA AGUGCUUCCA UGUUUUGGUG AUGGUAAGUC UUCUUUUUAC AUUUUUAUUA UUUUUUUAGA AAAUAACUUU AUUGUAUUGA CCGCAGCUCA UAUAUUUAAG CUUUAUUUUG UAUUUUUACA UCUGUUAAGG GGCCCCCUCU ACUUUAACAU GGAGGCACUU GCUGUGACAU GACAAAAAUA AGUGCUUCCA UGUUUGAGUG UGGUGGUUCC UACCUAAUCA GCAAUUGAGU UAACGCCCAC ACUGUGUGCA GUUCUUGGCU ACAGGCCAUU ACUGUUGCUA- 3'(SEQ.ID.NO.5), and the individual sequences of pro-miR-302a, pro-miR-302b, pro-miR-302c and pro-miR-302d are as follows: 5'-CCACCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC UUCCAUGUUU UGGUGAUGG-3'(SEQ.ID.NO.6), 5'-GCUCCCUUCA ACUUUAACAU GGAAGUGCUU UCUGUGACUU UAAAAGUAAG UGCUUCCAUG UUUUAGUAGG AGU-3'(SEQ.ID.NO.7), 5'-CCUUCUUGCU GUGUA ACCAUGAAA UCCAUGUUUC AGUGGAGG-3'(SEQ.ID.NO.8) and 5'-CCUCUACUUU AACAUGGAGG CACUUGCUGU GACAUGACAA AAAUAAGUGC UUCCAUGUUU GAGUGUGG-3'(SEQ.ID.NO.9).

Figure 14 shows the in vivo treatment results of the pre-inspected new drug (pre-IND) trial of using pro-miR-302 as an injection drug to treat human liver cancer xenografts in SCID-beige nude mice. After three treatments (once a week), pro-miR-302 drug (=pre-miR-302) successfully reduced the size of the cancer from 728±328mm ³ (untreated blank control, C) to 75±15mm ³ (treated with pro-miR-302, T), indicating that the average cancer size is reduced by about 90%. No significant therapeutic effect was found in the treatment of synthetic siRNA mimic (siRNA-302). Further histological examination (far right) revealed that normal liver lobule-like structures (circles indicated by black arrows) were formed only in cancers treated with pro-miR-302 but not in other treatments or controls, indicating reprogramming The mechanism can occur to reset the characteristics of malignant cancer cells back to a relatively normal-like state (called "cancer reversal").

Figure 15 shows the histological similarity between normal liver tissue in vivo and human liver cancer xenografts treated with pro-miR-302. After three treatments (once a week), the pro-miR-302 drug successfully reprogrammed high-grade (IV-degree) human liver cancer grafts to a more benign low-grade (less than II-degree) state. Similar to normal liver tissue (top image), treated cancer grafts can form classic liver lobules containing central vein (CV)-like and portal triplet (PT)-like structures (indicated by black arrows). Because cancer cells are usually more acidic than normal liver cells, the results of hematoxylin and eosin (H&E) staining show more purple in cancer cells and more red in normal liver cells.

Figure 16 shows the histopathological comparison between untreated, siRNA-treated, and pro-miR-302-treated human liver cancer transplants and normal liver tissues in SCID-beige nude mice. Without treatment (top image), transplanted human liver cancer invasively invades normal tissues, such as muscles and blood vessels, and forms large-scale cell-cell and cancer-tissue fusion structures, indicating its malignancy and high metastasis . siRNA mimic (siRNA-302) treatment does not significantly reduce the malignancy of transplanted cancer (upper middle), which is probably due to the short half-life of siRNA. In contrast, pro-miR-302 treatment not only reprograms the transplanted cancer to a relatively normal-like morphology (no fusion), but also greatly inhibits the invasion of the cancer into the surrounding tissues (bottom middle panel). Compared with normal liver tissue (bottom image), cancers treated with pro-miR-302 form a normal-like lobule structure, adenoid cell configuration, and a clear boundary (black arrow) between cell-cell and cancer-tissue junctions, It shows that these treated cancers have been downgraded to a very benign state.

Figures 17A and 17B show a comparison of healing results between wounds that were untreated (17A) and miR-302 treated (17B) in vivo. The isolated miR-302 molecules (20-400μg/mL) are formulated with di-/tri-glycylglycerin (di-/tri-glycylglycerin), delivery reagents and antibiotic ointment to form drug candidates for in vivo testing Local treatment of 2cm×2cm large open wound on pig back skin (n=6 in each group). After about two weeks of treatment (treatment once a day), the healed wound was dissected and further made into tissue sections for histological examination under a microscope. Data show that no scars or very small scars (no scars) can be seen in wounds treated with miR-302 (17B top image, n=6/6), and almost all untreated wounds (treated with antibiotic ointment only) contain large Scar (17A top image, n=5/6). In addition, a significant number of CD34-positive adult stem cell clusters (labeled with green fluorescent antibodies) were seen in miR-302-treated injuries In the mouth (base map 17B, n=6/6), but not seen in untreated control wounds (base map 17A, n=0/6). These results indicate that pre-miR-302 can induce the proliferation and/or regeneration of CD34-positive adult stem cells in order to enhance tissue repair and regeneration. Pathological changes caused by porosity, diabetes and cancer produce extremely beneficial therapeutic effects. The therapeutic effect can also help reprogram highly malignant cancers into low-grade benign or even normal-like tissues, which is a novel mechanism called cancer reversal or cancer regression.

Examples:

In the following experimental disclosures, the following abbreviations apply: M (molar concentration); mM (millimolar concentration); μm (micromolar); mol (mole); pmol (pimol); gm (gram) ; Mg (milligrams); μg (micrograms); ng (naik); L (liters); ml (milliliters); μl (microliters); ℃ (degrees Celsius); RNA (ribonucleic acid); DNA (deoxyribonucleic acid) ); dNTP (deoxyribonucleotide triphosphate); PBS (phosphate buffered saline); NaCl (sodium chloride); HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfon Acid); HBS (HEPES buffered physiological saline); SDS (sodium dodecyl sulfate); Tris-HCl (tris-hydroxymethylaminomethane-hydrochloride); ATCC (American Culture Collection, Rockville, MD ); hESC (human embryonic stem cells); and iPSC (induced pluripotent stem cells).

1. Bacterial cell culture and chemical treatment

Obtain E. coli DH5α competent cells from the z-competent E. coli transformation kit (Zymo Research, Irvine, CA) as part, and then use 5 μg of pre-prepared plastid vectors, such as pLenti-EF1α-RGFP-miR302 or pLVX -Grn-miR302 + 367 ) is mixed and transformed. _{The non-transformed cells were cultured in Luria-Bertani (LB) medium supplemented with 10 mM MgSO 4} and 0.2 mM glucose at 37°C under frequent stirring at 170 rpm, and the transformed cells were further supplemented with an additional 100 μg/ Cultivate in the above LB medium of ml Ambicillin. For chemical induction, 0.5 to 2 ml MOPS, glycerol and/or ethanol are added separately or in combination to _{1 liter of LB culture medium supplemented with 10 mM MgSO 4} and 0.2 mM glucose in the presence of 100 μg/ml ampicillin. For the negative control, the transformed cells were cultured in the above LB medium supplemented with ampicillin but without any chemical inducer. The results are shown in Figures 2-4.

2. Human cell culture and microRNA transfection

The human liver cancer cell line HepG2 was obtained from ATCC and maintained according to the manufacturer's recommendations. For transfection, 15 μg pre-miR-302 was dissolved in 1 ml fresh RPMI medium and mixed with 50 μl X-tremeGENE HP DNA transfection reagent (Roche, Indianapolis, IN). After 10 minutes of incubation, add the mixture to a 100mm cell culture dish containing 50%-60% HepG2 cell fullness. Renew the medium after 12 to 18 hours. After these transfected cells formed a spherical iPSC colony, the medium was changed to supplemented with 20% gene knock-out serum, 1% MEM non-essential amino acid, 100μM ß-mercaptoethanol, 1mM GlutaMax, 1mM sodium pyruvate, 10ng /ml bFGF, 10ng/ml FGF-4, 5ng/ml LIF, 100IU/ml penicillin/100μg/ml streptomycin, 0.1μM A83-01 and 0.1μM valproic acid (Stemgent, San Diego, CA) knock-out DMEM/F-12 medium (Invitrogen), and cells were cultured _{at 37°C under 5% CO 2.} The results are shown in Figure 9.

3. Protein extraction and western ink spot analysis

Follow the manufacturer & recommended, supplemented with protease inhibitors, leupeptin (Leupeptin), TLCK, TAME and PMSF of CelLytic-M lysis / extraction reagent (Sigma) cells (10 ⁶⁾ cracking. The lysate was centrifuged at 12,000 rpm at 4°C for 20 minutes and the supernatant was recovered. The protein concentration was measured on an E-max microdisk reader (Molecular Devices, CA) using a modified SOFTmax protein analysis kit. Add 30μg of cell lysate to SDS-PAGE sample buffer under reducing (+50mM DTT) and non-reducing (without DTT) conditions, and let it boil for 3 minutes, then load it into 6-8% polyacrylamide gel Glue on. The protein was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), electrotransfected onto a nitrocellulose membrane, and incubated in Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) at room temperature 2 hour. Subsequently, the primary antibody was applied to the reagent and the mixture was incubated at 4°C. Primary antibodies include Oct3/4 (Santa Cruz Biotechnology, Santa Cruz, CA), RuvB (Santa Cruz) and RGFP (Clontech). After overnight, the membrane was rinsed three times with TBS-T, and then exposed to a goat anti-mouse IgG-conjugated secondary antibody (1:2,000; Invitrogen-Molecular Probes) labeled with Alexa Fluor 680 reactive dye at room temperature for 1 hour . After three additional TBS-T washes, the Li-Cor Odyssey infrared imager and Odyssey software v.10 (Li-Cor) were used to perform fluorescent scanning and image analysis of immunoblotting. The results are shown in Figure 5.

4. RNA extraction and northern ink spot analysis

The total RNA (10μg) was separated with the mir Vana ^TM miRNA separation kit (Ambion, Austin, TX), and fractionated by 15% TBE-urea polyacrylamide gel or 3.5% low melting point agarose gel electrophoresis , And the electric transfer stains onto the nylon membrane. [LNA]-DNA probe (5'-[TCACTGAAAC] ATGGAAGCAC TTA-3') (SEQ.ID.NO.10) probe was used to detect miR-302s and related pre-miR-302s. The probe was purified by high performance liquid chromatography (HPLC) and ^{tail-labeled with terminal transferase (20 units) in the presence of [32} P]-dATP (>3000 Ci/mM, Amersham International, Arlington Heights, IL) 20 minutes. The results are shown in Figure 6.

5. Plastid amplification and plastid DNA/total RNA extraction

The transformed E. coli DH5α competent cells (from Example 1) were _{cultured in LB broth supplemented with 10 mM MgSO 4} and 0.2 mM glucose at 37° C. under frequent stirring at 170 rpm. In order to induce RNA transcription driven by eukaryotic promoters, 0.5 to 2 ml MOPS, glycerol and/or ethanol were added to 1 liter of LB broth to allow the transformed cells to multiply overnight. Use the HiSpeed plastid purification kit (Qiagen, Valencia, CA) to follow the manufacturer’s protocol but with minor modifications that RNase A is not added to the P1 buffer to separate the amplified plastid DNAs and expressed mRNAs in the transformed cells/ microRNAs. Thereafter, the final extraction product containing both plastids and mRNAs/microRNAs was dissolved in DEPC-treated ddH ₂ O, and stored at -80°C before use. In order to purify only the amplified plastid vector, add RNase A to the P1 buffer and perform the extraction procedure following the manufacturer's protocol.

6. MicroRNA and Pre-miRNA separation/purification

In order to purify microRNAs and pre-miRNAs, the mir Vana ^TM miRNA isolation kit (Ambion, Austin, TX) was used to follow the manufacturer's protocol, and the total RNA isolated from Example 5 was further extracted. The final product thus obtained was dissolved in DEPC-treated ddH ₂ O and stored at -80°C before use. Because bacterial RNAs naturally degrade extremely quickly (within a few hours), the eukaryotic hairpin microRNA precursors (pre-miRNAs and pri-miRNAs) are mostly stable at 4°C (half-life of 3-4 days), Therefore, the inventor can use this half-life difference to obtain relatively pure pri-/pre-miRNAs for other applications. For example, the pre-miR-302s thus obtained can be used to reprogram somatic cells into hESC-like iPSCs, as shown in FIG. 9.

7. Immunostaining analysis

Embedding, sectioning and immunostaining were performed on tissue samples as previously reported (Lin et al., 2008). Primary antibodies include Oct4 (Santa Cruz) and RGFP (Clontech, Palo Alto, CA). Goat anti-rabbit or horse anti-mouse antibodies labeled with fluorescent dyes were used as secondary antibodies (Invitrogen-Molecular Probes, Carlsbad, CA). Use Metamorph imaging program (Nikon) to check and analyze the positive results with 100x or 200x magnification under the fluorescent 80i micro-quantification system. The results are shown in Figure 7.

8. Sulphite DNA Sequencing

A DNA isolation kit (Roche) was used to isolate genomic DNA from approximately 2,000,000 cells, and 1 μg of the isolated DNA was further processed with sulfite (CpGenome DNA Modification Kit, Chemicon, Temecula, CA) following the manufacturer’s recommendations. The sulfite treatment converts all unmethylated cytosine to uracil, while methylated cytosine remains as cytosine. In sulfite DNA sequencing, the inventor used PCR primers to amplify the promoter region of the Oct4 gene. The primers are: 5'-GAGGCTGGAG CAGAAGGATT GCTTTGG-3' (SEQ.ID.NO.11) and 5'-CCCTCCTGAC CCATCACCTC CACCACC -3' (SEQ.ID.NO.12). In PCR, sulfite-modified DNAs (50ng) and primers (100pmol in total) were mixed in 1x PCR buffer, heated to 94°C for 2 minutes, and immediately cooled on ice. Subsequently, using the Expand High Fidelity PCR Kit (Roche), 25 PCR cycles were performed as follows: 94°C for 1 minute and 70°C for 3 minutes. The PCR product with the correct size was further fractionated by 3% agarose gel electrophoresis, purified by a gel extraction filter (Qiagen), and then used in DNA sequencing. Thereafter, by comparing the unaltered cytosine in the transformed DNA sequence with the untransformed DNA sequence, a detailed map of DNA methylation sites is generated, as shown in FIG. 8.

9. DNA density flow cytometry

The cells were trypsinized, clumped, and fixed by resuspending in 1 ml of PBS containing pre-cooled 70% methanol at -20°C for 1 hour. The cells were aggregated into clumps and washed once with 1ml PBS, and then aggregated into clumps again and resuspended in 1ml PBS containing 1mg/ml propidium iodide and 0.5μg/ml RNase at 37°C 30 minutes. Thereafter, about 15,000 cells were analyzed on BD FACSCalibur (San Jose, CA). The cell doublet is eliminated by plotting the pulse width against the pulse area and gating on the single cell. Use the packaged software Flowjo to analyze the collected data using the "Watson Pragmatic" algorithm. The results are shown in the top panel of Figure 9.

10. MicroRNA (miRNA) microarray analysis

At about 70% cell fullness, use the mir Vana ^TM miRNA isolation kit (Ambion) to separate small RNA from each cell culture. Use 1% formaldehyde-agarose gel electrophoresis and spectrophotometric measurement (Bio-Rad) to evaluate the purity and amount of the separated small RNAs, and then immediately freeze them in dry ice and submit to LC Sciences (San Diego, CA ) For miRNA microarray analysis. The microarray wafers are hybridized, and a single sample is labeled with Cy3 or Cy5, or a pair of samples is labeled with Cy3 and Cy5, respectively. Perform background deduction and standardization according to the manufacturer's recommendations. In a two-sample analysis, a p- value calculation is performed and a list of differentially expressed transcripts (yellow-red signal) greater than 3 times is generated. The final microarray results are shown in Figures 11A and 11B, and a list of differentially expressed microRNAs is shown in Figure 12. It combines small RNAs extracted from blank E. coli cell lysates (group 1) and pLenti-EF1α -Comparison of small RNA extracted from RGFP-miR302 transformed cell lysate (group 2).

11. Liver cancer therapy trial in vivo

Xenotransplantation of human liver cancer into immunocompromised SCID-beige mice is an effective animal model for studying liver cancer metastasis and therapy. To establish this model, the inventors mixed 5 million human liver cancer (HepG2) cells with 100 μL of Matrigel, and transplanted the mixture subcutaneously into each flank of the hind limb of the mouse. Therefore, both sides of the hind limbs of the mice undergo approximately the same amount of cancer cell transplantation. The cancer was observed about two weeks after transplantation, and its average size was about 15.6±8 mm ³ (the initial cancer size before treatment). For each mouse, the inventors selected the side with larger cancer as the treatment group and the other side with smaller cancer as the control group. Because the same mouse was treated with a blank formulation reagent (negative control) on one side and a formulated drug (pro-miR-302) on the other side, the results obtained can minimize any differences due to individual differences Possible changes.

In order to deliver pro-miR-302 to the target cancer area in vivo, the inventors contracted a professional blending company Latitude (San Diego, CA) to encapsulate pro-miR-302s into a nanometer with a diameter of 160-200nm via liposomes. In the particles. These nanoparticles containing pro-miR-302 have been tested to be almost 100% stable for more than two weeks at room temperature and almost 100% stable for more than one month at 4°C, while other synthetic siRNA mimics (siRNA-302) are 3 to 5 under the same conditions It degrades rapidly by more than 50% within a day, indicating that pro-miRNA rather than siRNA is stable enough to be used as a therapeutic drug. In the toxicity analysis, the inventors have further injected 300μL of the formulated pro-miR-302 (1mg/mL) into the tail vein of mice (n=8) to the maximum extent, and it has been in all tested mice after six months. No detectable side effects were observed. Generally speaking, unmodified ribonucleic acid is relatively non-immunogenic and can be easily metabolized by tissue cells, providing a safe tool for in vivo therapy.

To test the efficacy of the drug, the inventors subcutaneously injected 200 μL of formulated pro-miR-302 on one side of the mouse and 200 μL of blank formulated reagent on the other side, and continued the same injection mode three times (injection once a week). Drugs and reagents are applied to the area around the cancer site and absorbed by the cancer and surrounding tissues within 18 hours. Samples were collected one week after the third injection. Remove heart, liver, kidney and transplanted cancers for further histological examination. Tumor formation was monitored by palpation, and the tumor volume was calculated ^{using the equation (length×width 2 )/2.} The tumor lesions were counted, dissected, weighed, and subjected to histological examination using H&E and immunostaining analysis. Histological examination showed no detectable tissue lesions in the heart, liver and kidney. The results are shown in Figures 14, 15 and 16.

12. Statistical analysis

Any change in the signal intensity of more than 75% in the analysis of immunostaining, western blotting and northern blotting is regarded as a positive result, which is analyzed again and presented as the mean ± SE. Perform statistical analysis on the data by one-way ANOVA. When the main effect is significant, Dunnett's post-hoc test is used to identify groups that are significantly different from the control. For pairwise comparison (pairwise comparison), using the two-tailed Student t-test between the two treatment groups (two-tailed student t test) . For experiments involving more than two treatment groups, perform ANOVA followed by post hoc multi-range testing. The probability value of p <0.05 is considered significant. All p- values are determined by a two-tailed test.

references:

1. Lin SL and Ying SY. (2006) Gene silencing in vitro and in vivo using intronic microRNAs. Ying SY. (Ed.) MicroRNA protocols. Humana press, Totowa, New Jersey, pages 295-312.

2. Lin SL, Chang D and Ying SY. (2006) Transgene-like animal models using intronic microRNAs. Ying SY. (Ed.) MicroRNA protocols. Humana press, Totowa, New Jersey, pp. 321-334.

3. Lin SL, Chang D, Chang-Lin S, Lin CH, Wu DTS, Chen DT and Ying SY. (2008) Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state. RNA 14, 2115 -2124.

4. Lin SL and Ying SY. (2008) Role of mir-302 microRNA family in stem cell pluripotency and renewal. Ying SY. (Ed.) Current Perspectives in MicroRNAs. Springer Publishers press, New York, pages 167-185.

5. Lin SL, Chang D, Ying SY, Leu D and Wu DTS. (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.

6. Lin SL, Chang D, Lin CH, Ying SY, Leu D and Wu DTS. (2011) Regulation of somatic cell reprogramming through inducible mir-302 expression. Nucleic Acids Res : 39, 1054-1065.

7. Simonsson S and Gurdon J. (2004) DNA demethylation is necessary for the epigenetic reprogramming of somatic cell nuclei. Nat Cell Biol. 6, 984-990.

8. Takahashi et al. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126 , 663-676.

9. Wang et al. (2008). Embryonic stem cell-specific microRNAs regulate the G1-S transition and promote rapid proliferation. Nat. Genet. 40 , 1478-1483.

10. Wernig et al. (2007). In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448 , 318-324.

11. Yu et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science 318 , 1917--1920.

12. Buechler's US Patent No. 7,959,926.

13. Mehta's US Patent No. 7,968,311.

14. Lin's European Patent No. EP 2198025.

15. Lin's US Patent Application No. 12/149,725.

16. Lin's US Patent Application No. 12/318,806.

17. Lin's US Patent Application No. 12/792,413.

18. Lin's US Patent Application No. 13/572,263.

19. Lin's US Patent Application No. 13/964,705.

Sequence Listing

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<110> Lin Xilong (Lin, Shi-Lung) Wu Tangxi (Wu, David TS) Zhang Qinci (Chang, Donald)

<120> Use microribonucleic acid precursors as drugs to induce the proliferation of CD34-positive adult stem cells

<150> US 62/262,280

<151> 2015-12-02

<150> US 15/048,964

<151> 2016-02-19

<150> US 15/167,226

<151> 2016-05-27

<150> PCT/US16/49583

<151> 2016-08-31

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<170> PatentIn version 3.5

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<210> 2

<211> 23

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 2

<210> 3

<211> 17

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 3

<210> 4

<211> 69

<212> RNA

<213> Homo sapiens

<400> 4

<210> 5

<211> 720

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 5

<210> 6

<211> 69

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 6

<210> 7

<211> 73

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 7

<210> 8

<211> 68

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 8

<210> 9

<211> 68

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 9

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 10

<210> 11

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 11

<210> 12

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 12

Claims (15)

Use of a microRNA precursor (pre-miRNA) in the preparation of a medicine for inducing the proliferation or regeneration of CD34-positive adult stem cells, wherein: the microRNA precursor (pre-miRNA) comprises SEQ.ID.NO.3; and used The drug prepared by the microRNA precursor (pre-miRNA) comprises contacting the microRNA precursor (pre-miRNA) with a cell matrix containing at least one CD34-positive adult stem cell to induce the population of the CD34-positive adult stem cell Proliferation or regeneration. The use according to claim 1, wherein the microRNA precursor is produced by driving RNA transcription by a eukaryotic promoter in a prokaryotic cell. The use according to claim 2, wherein the eukaryotic promoter drives RNA transcription by a chemical agent containing the molecular structure of 3-morpholinopropane-1-sulfonic acid (MOPS) and carries the encoding SEQ.ID. NO.6, SEQ.ID.NO.7, SEQ.ID.NO.8, or SEQ.ID.NO.9 sequence of at least one expression vector of at least one transformed prokaryotic cell contact to induce. The use according to claim 3, wherein the expression vector is a recombinant plastid encoding the sequence of SEQ.ID.NO.5. The use according to claim 3, wherein the expression vector is pLenti-EF1alpha-RGFP-miR302 containing cytomegalovirus (CMV) or mammalian EF1α (EF1alpha) promoter or both. The use according to claim 2, wherein the prokaryotic cell is a competent cell of Escherichia coli. The use according to claim 1, wherein the microRNA precursor comprises SEQ.ID.NO.6, SEQ.ID.NO.7, SEQ.ID.NO.8, or At least one hairpin sequence of SEQ.ID.NO.9. The use according to claim 1, wherein the microRNA precursor is a miR-302 precursor (pre-miR-302) produced by prokaryotes. The use according to claim 8, wherein the pre-miR-302 includes at least one sequence of miR-302a, miR-302b, miR-302c, or miR-302d. The use according to claim 8, wherein the pre-miR-302 is used as a part of a pharmaceutical ingredient for medical and therapeutic applications. The use according to claim 1, wherein the microRNA precursor is used as a part of pharmaceutical ingredients for medical and therapeutic applications. The use according to claim 1, wherein the proliferation of the CD34-positive adult stem cells is used for medical and therapeutic applications. The use according to claim 12, wherein the proliferation of CD34-positive adult stem cells is used to promote scarless wound healing in vivo. The use according to claim 1, wherein the CD34-positive adult stem cells comprise skin, hair, muscle, hematopoietic cells, mesenchymal cells, and neural stem cells, or a combination thereof. A method for using microRNA precursors (pre-miRNA) to induce the proliferation or regeneration of CD34-positive adult stem cells in vitro, which comprises: (a) providing at least one microRNA precursor comprising SEQ.ID.NO.3; (b) Provide a cell matrix containing at least one CD34-positive adult stem cell; and (c) contact the microRNA precursor of (a) with the cell matrix of (b) in vitro to induce a population of the CD34-positive adult stem cells The proliferation or regeneration.

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