WO2017095489A1 - Utilisation de précurseurs de micro-arn comme médicaments permettant d'induire l'expansion de cellules souches adultes - Google Patents

Utilisation de précurseurs de micro-arn comme médicaments permettant d'induire l'expansion de cellules souches adultes Download PDF

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WO2017095489A1
WO2017095489A1 PCT/US2016/049583 US2016049583W WO2017095489A1 WO 2017095489 A1 WO2017095489 A1 WO 2017095489A1 US 2016049583 W US2016049583 W US 2016049583W WO 2017095489 A1 WO2017095489 A1 WO 2017095489A1
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mir
rna
cells
seq
cell
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Shi-Lung Lin
Donald Chang
David Ts Wu
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Shi-Lung Lin
Donald Chang
David Ts Wu
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Priority claimed from US15/048,964 external-priority patent/US10519440B2/en
Priority claimed from US15/167,226 external-priority patent/US9879263B2/en
Application filed by Shi-Lung Lin, Donald Chang, David Ts Wu filed Critical Shi-Lung Lin
Priority to EP16763666.1A priority Critical patent/EP3384026A1/fr
Priority to JP2018528746A priority patent/JP2018535684A/ja
Priority to CN201680070742.9A priority patent/CN108431227A/zh
Priority to TW105139971A priority patent/TWI720075B/zh
Publication of WO2017095489A1 publication Critical patent/WO2017095489A1/fr

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    • C12N2310/531—Stem-loop; Hairpin
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    • C12N2501/65—MicroRNA

Definitions

  • the present invention claims priority to the U.S. Provisional Application Serial No. 62/262,280 filed on December 2, 2015, which was entitled “miR-302 Attenuates ⁇ -induced Neurotoxicity through Activation of Akt Signaling”. Also, the present invention claims priority to the U.S. Patent Application Serial No. 15/048,964 filed on February 19, 2016, which was entitled “A Composition and Method of Using miR-302 Precursors as Drugs for Treating Alzheimer's Diseases”. Furthermore, the present invention claims priority to the U.S. Patent Application Serial No. 15/167,226 filed on May 27, 2016, which was entitled “Use of MicroRNA Precursors as Drugs for Inducing CD34-positive Adult Stem Cell Expansion”.
  • This invention generally relates to a composition and method for inducing CD34-cell expansion and/or regeneration with small hairpin RNA (shRNA) molecules, preferably microRNA precursor (pre-miRNA) and/or siRNA, which are useful for developing drugs/vaccines and/or therapies against a variety of ageing-related degenerative diseases in humans.
  • shRNA small hairpin RNA
  • pre-miRNA microRNA precursor
  • siRNA siRNA
  • 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/or 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.
  • shRNA small hairpin-like RNA
  • shRNA small hairpin-like RNA
  • pri-miRNA and/or pre-miRNA pri-miRNA and/or pre-miRNA
  • siRNA short interfering RNAs
  • the resulting shRNAs preferably pre-miRNAs and siRNAs, are useful for developing therapeutic drugs against human degenerative diseases, particularly through a mechanism to induce CD34-positive stem cell expansion and/or regeneration.
  • the present invention also reveals a novel pre-miRNA-based drug composition that is able to reprogram the malignant properties of high-grade liver cancers to a low-grade benign or even relatively normal stage - a mechanism called "Cancer Reversion".
  • cancer reversion is a totally new concept in drug designs, the present invention devises the first drug of its kinds using such a novel mechanism for cancer therapy.
  • Stem cells are like a treasure box containing numerous effective ingredients useful for stimulating new cell growth/tissue regeneration, repairing and/or rejuvenating damaged/aged tissues, treating degenerative diseases, and preventing tumor/cancer formation/progression.
  • these stem cells as a tool for novel drug screening, identification and production.
  • the drugs so obtained will be useful for developing pharmaceutical and therapeutic applications, such as a biomedical utilization, device and/or apparatus for research, diagnosis, and/or therapy, and a combination thereof.
  • MicroRNA is one of the main effective ingredients in human embryonic stem cells (hESCs).
  • Major hESC-specific miRNA species include, but not limited, members of the miR-302 family, miR-371-373 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) familial 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 form double-stranded duplex, respectively.
  • Precursors of miR-302 are formed by miR-302a and a* (pre-miR-302a), miR-302b and b* (pre-miR-302b), miR-302c and c* (miR-302c), and miR-302d and d* (pre-miR-302d) with a link sequence in one end (stem loop), respectively.
  • miR-302 precursors are first processed into mature miR-302s by cellular RNase III Dicers and further form RNA-induced silencing complexes (RISCs) with certain argonaute proteins, subsequently leading to either RNA interference (RNAi)-directed degradation or translational suppression of targeted gene transcripts (mRNAs), in particular oncogene mRNAs (Lin et al., 2008, 2010 and 2011).
  • RISCs RNA-induced silencing complexes
  • mRNAs RNA interference-directed degradation or translational suppression of targeted gene transcripts (mRNAs), in particular 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). Our previous studies have shown that ectopic overexpression of miR- 302 beyond the level found in hESCs is able to reprogram both human normal and cancerous cells to hESC-like iPSCs with a relatively slow cell cycle rate (20-24 hours/cycle) similar to that of a morula-stage early human zygote (Lin et al., 2008, 2010 and 2011; EP 2198025; U.S. 12/149,725; U.S. 12/318,806; U.S. 12/792,413).
  • Relative quiescence is a defined characteristic of these miR-302-induced iPSCs, whereas hESCs and other previously reported four-factor-induced (either Oct4-Sox2-Klf4-c-Myc or Oct4-Sox2-Nanog-Lin28) iPSCs all showed a highly proliferative cell cycle rate (12-15 hours/cycle) similar to that of a tumor/cancer cell (Takahashi et al., 2006; Yu et al., 2007; Wernig et al., 2007; Wang et al., 2008).
  • CDKs cyclin-dependent kinases
  • CKIs CDK inhibitors
  • cyclin-CDK complexes are involved in regulating different cell cycle transitions, such as cyclin-D-CDK4/6 for Gl-phase progression, cyclin-E-CDK2 for Gl-S transition, cyclin-A-CDK2 for S-phase progression, and cyclin-A/B-CDC2 (cyclin-A/B-CDKl) for entry into M-phase.
  • cyclin-D-CDK4/6 for Gl-phase progression
  • E-CDK2 for Gl-S transition
  • cyclin-A-CDK2 for S-phase progression
  • cyclin-A/B-CDC2 cyclin-A/B-CDKl
  • miR-302 is useful for designing and developing novel anti-cancer drugs/vaccines, its production is problematic because natural miR-302 can only be found in human pluripotent stem cells such as hESCs, of which the resource is very limited.
  • synthetic small interfering RNAs may be used to mimic pre-miR-302; yet, since the structure of a pre-miR-302 is formed by two mis-matched strands of miR-302 and miR-302*, those perfectly matched siRNA mimics can not replace the function of miR- 302*, of which the sequence is totally different from the antisense strand of siRNA.
  • siRNA-302a mimic is 5'-UCACCAAAAC AUGGAAGCAC UUA-3' (SEQ.ID.NO.l), whereas native miR-302a* is 5 ' -ACUUAAACGU GGAUGUACUU GCU-3' (SEQ.ID.NO.2).
  • miR-302 function results from both of its sense miR-302 and antisense miR-302* strands
  • previous reports using those siRNA mimics have shown different results from native miR-302 function.
  • our recent discovery of iPSCs may provide an alternative solution for pre-miR-302 production (EP 2198025; U.S. 12/149,725; U.S. 12/318,806).
  • prokaryotic competent cells may be a possible approach for producing human microRNAs and their precursors.
  • prokaryotic cells lack several essential enzymes required for eukaryotic microRNA expression and processing, such as Drosha and Dicer.
  • prokaryotic RNA polymerases do not efficiently transcribe small RNAs with high secondary structures, such as hairpin-like pre-miRNAs and shRNAs. In fact, there is no true microRNA encoded in bacterial genomes and bacteria do not naturally express microRNA.
  • prokaryotic and eukaryotic transcription machineries are different and hence not compatible to each other.
  • eukaryotic RNA polymerases do not bind directly to a promoter sequence and require additional accessory proteins (cofactors) to initiate transcription, whereas prokaryotic RNA polymerases form a holoenzyme that binds directly to a promoter sequence to start transcription.
  • RNA messenger RNA
  • pol-2 type- ⁇ RNA polymerases
  • prokaryotic RNA transcription and protein translation take place simultaneously off the same piece of DNA in the same place.
  • prokaryotes such as bacteria and archaea do not have any nucleus-like structure. Accordingly, these differences make a prokaryotic cell difficult or even impossible to produce eukaryotic RNAs using eukaryotic promoters.
  • coli for expressing the transcripts (mRNAs) of the gene and subsequently translating the mRNAs into proteins.
  • the bacterial and bacteriophage promoters such as Tac, Lac, T3, T7, and SP6 RNA promoters, are not pol- 2 promoters and their transcription activities tend to be an error-prone process which causes mutations.
  • Mehta further taught that glycerol/glycerin might be used to increase the efficiency of bacterial transformation; yet, no teaching was related to enhancement of RNA transcription, in particular pol-2 promoter-driven prokaryotic RNA transcription. Due to lack of possible compatibility between eukaryotic and prokaryotic transcription systems, these prior arts were still limited by the use of prokaryotic RNA promoters for gene expression in prokaryotes.
  • a pre-miRNA/shRNA is sized about 70 ⁇ 85-nucleotides in length which is too large and costly to be made by a RNA synthesis machine.
  • the present invention provides a novel breakthrough - By adding some defined chemicals mimicking certain transcriptional cofactors, we can create a novel adaptation environment for prokaryotic cells to use eukaryotic pol-2 and/or pol-2-like promoters for transcribing desired pre-miRNAs and shRNAs without going through error-prone prokaryotic promoters.
  • the advantages are: first, cost-effective mass production due to the fast growth of bacteria; second, easy handling because of no need for growing dedicate hESCs or iPSCs; third, high fidelity productivity in terms of pol-2 promoter-driven RNA transcription; fourth, high purity of desired microRNAs due to lack of true microRNA in prokaryotes; and last, no endotoxin, which can be further removed by certain chemical treatments. 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 desirable. Furthermore, the drugs so obtained may present novel therapeutic effects other than the currently known function of a microRNA.
  • the principle of the present invention is relied on the different and incompatible properties between prokaryotic and eukaryotic RNA transcription systems.
  • prokaryotic RNA polymerases do not recognize eukaryotic promoters and vise versa.
  • the present invention has identified chemical agents that can serve as transcriptional inducers to trigger and/or enhance eukaryotic promoter-driven RNA transcription in prokaryotes.
  • the knowledge taught in the present invention is a totally novel breakthrough beyond all current understandings regarding the differences between prokaryotic and eukaryotic transcription systems.
  • the present invention is related to an inducible gene expression composition using certain chemical inducers to stimulate and/or enhance eukaryotic promoter-driven RNA transcription in prokaryotes.
  • chemical inducers have not been used in a cell culture medium due to their bacteriostatic and/or bactericidal properties, including 3- morpholinopropane-1 -sulfonic acid [or named 3-(N-morpholino)propanesulfonic acid; MOPS], glycerin and ethanol, as well as their functional analogs such as 2-(N- morpholino)ethanesulfonic acid (MES), 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) and mannitol.
  • MES 2-(N- morpholino)ethanesulfonic acid
  • HEPES 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
  • chemicals with a similar structure like these transcriptional inducers may share a similar function.
  • MOPS is frequently used as a buffering agent in bacterial cell lysis and hence is not suitable for growing bacteria.
  • ethanol is a well-known sanitizer and glycerin is frequently used in bacterial transformation by destabilizing the bacterial cell walls, indicating that glycerin is bacteriostatic and ethanol is bactericidal, respectively.
  • the present invention is a design and method for utilizing prokaryotic cells to produce human microRNA precursors (pre-miRNAs) and/or shRNAs as therapeutical drugs and/or vaccines for cancer therapy. More specifically, the present invention is a design and method of utilizing prokaryotic cells to produce a special kind of pre-miRNA-like agents, named pro-miRNA, that are capable of reprogramming the malignant properties of high-grade human cancer cells into a low-grade benign or even relatively normal-like state.
  • these pro-miRNAs are tumor suppressor microRNAs (TS-miRNA) similar to the precursors of miR-302a, b, c, d, e, and/or f (pre-miR- 302s) and their natural familial cluster as well as their manually re-designed small hairpin RNA (shRNA) homologues/derivatives, and/or a combination thereof.
  • TS-miRNA tumor suppressor microRNAs
  • shRNA small hairpin RNA
  • the designs of pro- miRNA-like shRNA homologues/derivatives include imperfectly and perfectly matched hairpin compositions of the pro-miRNA and its homologous small interfering RNA (siRNA), which may be formed in a single unit or in a multiple unit cluster.
  • the human cells suitable for such a drug treatment include normal, tumor, and cancerous cells in vitro, ex vivo and/or in vivo.
  • the prokaryotic cells used for the present invention are bacterial competent cells in particular, Escherichia coli (E. coli), and the chemical inducer is MOPS, ethanol, or glycerin, or a mixture thereof.
  • the eukaryotic RNA promoter used is either a eukaryotic pol-2 promoter (i.e. EF1 alpha promoter) or a pol-2 compatible (pol-2-like) viral promoter (i.e. cytomegaloviral CMV promoter).
  • the gene mediated by the eukaryotic RNA promoter may code for either a non-coding or protein-coding RNA, or both (such as an intron-containing gene transcript), selected from the group consisted of microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), messenger RNA (mRNA), their precursors and homologues, and a combination thereof.
  • miRNA microRNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • mRNA messenger RNA
  • the prokaryotic cells are transfected with the eukaryotic RNA promoter-mediated gene and then grown in a culture medium similar to Luria-Bertani (LB) broth at 37°C with addition of the chemical inducer(s) for >24 hours.
  • LB Luria-Bertani
  • the miR-302 familial cluster was also modified to be encoded in the 5'-intron region [e.g. 5 '-untranslated region (5'-UTR) or the first intron] of the RGFP gene, the transcription of each RGFP mRNA led to the production of one 4-hairpin miR-302 precursor cluster (pri-miR-302) and/or four 1 -hairpin miR-302 precursors (pre-miR-302s), as shown in FIGs. 5 and 6. Due to lack of RNase ⁇ Dicer in prokaryotes, the pri-miR-302 transcripts would be eventually broke down (by certain single-strand RNases in E.
  • 5'-intron region e.g. 5 '-untranslated region (5'-UTR) or the first intron
  • miR-302 also shares many overlapping target genes with mir-92, mir-93, mir-200c, mir-367, mir-371, mir-372, mir-373, mir-374, and mir-520 familial members, all of which may possess similar functions. Most of these target genes are developmental signals and transcriptional factors involved in initiating and/or establishing certain lineage- specific cell differentiation during early embryogenesis (Lin et al., 2008). Many of these target genes are also well-known oncogenes; as a result, miR-302s likely functions as a tumor suppressor to prevent the deviation of normal hESC growth into tumor/cancer formation.
  • Escherichia coli (E. coli) competent cells were transformed by the pLenti-EFlalpha- RGFP-miR302 plasmid (FIG. 1A) using a z-competent E. coli transformation kit (Zymo Research, Irvine, CA) and cultivated in Luria-Bertani (LB) broth supplemented with a mixture of 0.1% (v/v) MOPS and 0.05% (v/v) glycerin (inducers) at 37°C with frequent agitation at 170 rpm. After overnight incubation, the transformed E. coli competent cells expressed highly abundant red RGFP proteins that could be clearly seen in the color of the LB broth, whereas the blank control E. coli presented no RGFP, as shown in FIG. 2. The presence of functional RGFP indicated that both of its encoded RNA and protein are successfully produced and processed in the competent cells.
  • two transformed E. coli strains were prepared: one carried a pLVX-Grn-miR302+367 plasmid vector containing a CMV promoter-driven green fluorescent protein (GFP) gene and the other carried the aforementioned pLenti-EFlalpha-RGFP-miR302 vector.
  • GFP green fluorescent protein
  • the E. coli transformed with pLVX-Grn-miR302 +367 were changed to green color while the other with pLenti-EFlalpha-RGFP-miR302 still showed red color, as shown in FIG. 3.
  • the top three most potent inducers are MOPS, glycerin and ethanol, as shown in FIG. 4.
  • the quantitative result of the induced RGFP production was further confirmed by Western blot analysis, as shown in FIG. 5 and Example 3.
  • Bacterial RuvB protein was served as a house-keeping standard to normalize RGFP expression.
  • the inducibility of these identified inducers was also found to be dose- dependent in proportional to their concentrations. Without any treatment, negative control E. coli cells just showed their original color in absence of any fluorescent stain.
  • the present invention clearly provides a novel chemical- inducible composition and its application for modulating eukaryotic pol-2-driven or pol-2- like viral promoter-driven RNA production in prokaryotic cells.
  • the pri-/pre-miR-302 expression was strongly detected in transformed cells treated with MOPS, glycerin or ethanol, but not blank control, indicating that these chemical inducers indeed stimulated the expression of the encoded pri-/pre-miRNAs in prokaryotic cells through a eukaryotic pol-2 promoter (FIG. 6).
  • a eukaryotic pol-2 promoter FOG. 6
  • pri-/pre-miRNA species such as but not limited miR-34, miR-146, miR- 371-373 and miR-520.
  • pro-miRNAs pro-miRNAs.
  • pLenti-EFl alpha-RGFP-miR302 contains a miR-302 familial cluster located in the 5' -UTR of the RGFP gene (FIGs. 1A and IB)
  • the induced RGFP gene expression will also generate the miR-302 cluster (pri-miR-302) and its derivative pre-miR-302a, b, c and d (pre-miR-302s) as demonstrated in FIG. IB.
  • the pri-miR-302 and pre-miR-302s so obtained were found to remain as hairpin-like microRNA precursors, which are useful for developing therapeutic drugs.
  • these pre-miR-302s and pri-miR-302 can be processed into mature miR-302 for eliciting its tumor suppression function.
  • the present invention can also be used to produce other kinds of TS-miRNA species and their precursors, such as the miR-34a, miR-146a, miR- 373 and miR-520 family.
  • pro-miRNAs can be easily extracted from competent E. coli cells (Examples 5 and 6) and further purified by high-performance liquid chromatography (HPLC) (FIGs. 10A and 10B).
  • HPLC high-performance liquid chromatography
  • pro-miR-302s we have identified all of the miR- 302 familial members (miR-302a, a*, b, b*, c, c*, d, and d*) using analyses of microRNA microarrays (FIGs. 11B and 12) and RNA sequencing [FIGs. 13A (pri-miR-302) and 13B (pre-miR-302s)].
  • pro-miR-302s all share exactly the same sequences as their natural pre-miR-302 counterparts (FIG. 13B). Furthermore, we have formulated these pro-miR-302s into a soluble drug for IV/in-vivo injection in order to test their therapeutic effects on human liver cancers in vivo (Example 11). As shown in FIG.14, after 3 injection treatments, the pro-miR-302 drug successfully reduced >90% volume of the engrafted human liver cancers in vivo, shirking the average cancer size to ⁇ 10% compared to the untreated cancers.
  • FIG.15 clearly showed that the pro-miR-302 drug can reprogram the malignant properties of high-grade human liver cancers in vivo to a much more benign stage almost similar to that of normal liver tissues!
  • These treated cancers can even form normal liver-like structures, such as classical liver lobules, central veins (CV) and portal triads (PT).
  • pro-miR-302 is able to not only inhibit tumor/cancer cell growth but also reset the malignancy of human cancers to a relatively benign or normal state in vivo, leading to a totally novel therapeutic effect for cancer drug design.
  • both of the plasmid vector and its encoded non-coding RNAs can be simultaneously amplified in the prokaryotic cells, preferably E. coli DHSalpha competent cells (Examples 1, 5 and 6).
  • the method for isolating the amplified pLenti-EFlalpha-RGFP-miR302 plasmid DNA and the transcribed pri-/pre-miR- 302s is described in Examples 5 and 6.
  • the technology for delivering plasmid vectors i.e.
  • pLenti-EFl alpha-RGFP-miR302 into prokaryotic cells is called cell transformation, while the method for delivering the amplified non-coding RNAs (i.e. pro-/pri-/pre-miR-302s) into eukaryotic cells can be selected from the group of endocytosis, chemical/glycerol infusion, peptide/liposomal/chemical-mediated transfection, electroporation, gene gun penetration, micro-injection, transposon/retrotransposon insertion and/or adenoviral/retroviral/lentiviral infection.
  • MiR-302 has been reported to reprogram mammalian somatic cells to human embryonic stem cell (hESC)-like induced pluripotent stem cells (iPSCs) as demonstrated in our priority U.S. Patent Applications No. 12/149,725 and No. 12/318,806. Numerous stem cell applications and therapies have been designed and developed using these iPSCs. Nevertheless, since cultivating these iPSCs and hESCs is very costly and laborious, it is difficult and inefficient to collect miR-302 and its precursors from these pluripotent stem cells. On the other hand, making synthetic shRNA mimics is another possible alternative for pre-miR-302 production; yet, the cost is still very expensive.
  • hESC human embryonic stem cell
  • iPSCs induced pluripotent stem cells
  • the present invention provides a simple, cheap and efficient method for mass production of pre-miR-302 in prokaryotes. Moreover, the extraction and purification of these prokaryote- produced pre-miR-302s (pro-miR-302s) is relatively easy and cost-effective, as shown in FIG. 6 and Example 6 of the present invention.
  • Example 2 when the pro-miR-302s produced by the present invention were transduced into human skin primary keratinocytes, the transfected keratinocytes were reprogrammed to hESC-like iPSCs that expressed strong hESC marker Oct4 (FIG. 7).
  • FIG. 8 and Example 8 we further performed bisulfite DNA sequencing assays to show that global DNA demethylation did occurr in the promoters of both Oct4 and Sox2 genes, two of the key reprogramming factors as well as hESC markers.
  • global DNA demethylation and Oct4 expression are known to be the first step of somatic cell reprogramming to form hESC-like iPSCs (Simonsson and Gurdon, Nat Cell Biol.
  • pro-miR-302s isolated from the MOPS -induced E. coli cell extracts is proven to be as effective as natural pre-miR-302s, which are useful for iPSC derivation.
  • pro-miR-302 and pre-miR-302 likely possess the same function in stem cell induction.
  • MicroRNA miR-302 has been found to reprogram mammalian somatic cells to embryonic stem cell (ESC)-like induced pluripotent stem cells (iPSC) (Lin, 2008, 2010, 2011; U.S. Patent Applications No. 12/149,725 and No. 12/318,806 to Lin). Using these iPSCs, many stem cell-associated applications and therapies have been developed for advancing modern regenerative medicine. Yet, miR-302 is only abundantly found in human ESCs rather than differentiated tissue cells. Also, isolation of miR-302 from human ESCs is highly debatable, costly and tedious.
  • ESC embryonic stem cell
  • iPSC induced pluripotent stem cells
  • the present invention provides a simple, cheap, fast and inducible composition and method for mass production of hairpin-like miR-302 molecules and/or their precursors/homologs in prokaryotes. Moreover, the isolation of miR-302 and/or its precursors from prokaryotic cells is relatively easy and cost-effective, as shown in FIG. 6 and Example 6 of the present invention.
  • the applications of isolated miR-302 and/or pre-miR-302 molecules may further include the induction and expansion of CD34-positive adult stem cells.
  • FIGs. 17A and 17B our recent studies in wound healing therapy and cancer therapy using a novel miR-302-formulated drug revealed that treatments of relatively low concentrations (50 ⁇ 50( g/mL) of the isolated miR-302/pre-miR-302 molecules not only greatly enhance scar-less wound healing but also induce CD34-positive adult stem cell expansion around the wounded area in pig skins in vivo.
  • the miR-302-treated (miR-302s/pre-miR- 302s+antibiotic ointment) result of FIG.
  • CD34-positive adult stem cell populations include, but not limited, skin, hair, muscle, blood (hematopoietic), mesenchymal, and neural stem cells.
  • miR-302 can be used to induce CD34-positive adult stem cell expansion and/or regeneration in vivo, this therapeutic effect may also help to re-grow and/or revive functional adult stem cells for treating degenerative diseases in humans, such as, but not limited, Alzheimer's diseases, Parkinson's diseases, osteoporosis, diabetes, and cancers. Utilization of pro-miR-302 for Tumor/Cancer Therapy in vivo.
  • pro-miR-302 The process of cancer progression was thought to be irreversible due to accumulative gene mutations; yet, the present invention discloses a novel pre-miRNA (pro-miR-302) function that can reprogram high-grade malignant cancers back to a low-grade benign or even normal-like stage in vivo, of which the mechanism may be related to a very rare natural healing process called spontaneous cancer regression. Spontaneous cancer regression occurs rarely at a rate of less than 1 in 100,000 cancer patients. We found that pro-mir-302 treatment is able to increase this rare healing rate to >90% in human liver cancers. As shown in FIG.
  • liver lobule-like structures (circled and pointed by a black arrow) were observed only in pro- miR-302-treated cancer grafts but not other treatments or controls, suggesting that a reprogramming mechanism has occurred to reset the malignant cancer cell property back to a relatively normal-like state (Cancer Reversion).
  • This novel reprogramming mechanism is likely resulted from the gene silencing effect of miR-302 on human oncogenes in particular, those mutated oncogenes involved in cancer progression.
  • pro-miR-302 is able to reset the cancerous gene expression patterns back to a normal-like state, consequently leading to the therapeutic result of cancer reversion. Nevertheless, this in-vivo reprogramming mechanism is clearly different from the previously reported somatic cell reprogramming (Lin et al., 2008 and 2011) because no Oct4-positive pluripotent stem cell has been identified.
  • FIG. 15 More detailed histological examination (FIG. 15) further confirmed that the pro-miR- 302 drug did reprogram high-grade (Grade IV) human liver cancer grafts to a more benign low-grade (less than Grade II) state.
  • the treated cancer grafts formed classical liver lobules containing central vein (CV)-like and portal triad (PT)-like structures (indicated by black arrows), highly similar to normal liver tissue structures (top). Histological comparison among untreated, siRNA-treated, pro-miR-302-treated human liver cancer grafts and normal liver tissues in vivo (FIG.
  • siRNA mimics did not significantly reduce the malignancy of the engrafted liver cancers (upper middle), probably due to the short half-life of siRNA in vivo.
  • treatment of pro- miR-302 not only reprogrammed the engrafted cancer cells to a normal liver cell-like morphology (no fusion) but also successfully inhibited any cancer invasion into the surrounding tissues (lower middle).
  • Nucleotide a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon ( ⁇ carbon of the pentose) and that combination of base and sugar is a nucleoside.
  • a nucleoside containing at least one phosphate group bonded to the 3' or 5' position of the pentose is a nucleotide.
  • DNA and RNA are consisted of different types of nucleotide units called deoxyribonucleotide and ribonucleotide, respectively.
  • Oligonucleotide a molecule comprised of two or more DNAs and/or RNAs, preferably more than three, and usually more than ten. An oligonucleotide longer than 13 nucleotide monomers is also called polynucleotide. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide.
  • the oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, RNA transcription, reverse transcription, or a combination thereof.
  • Nucleotide Analog a purine or pyrimidine nucleotide that differs structurally from adenine (A), thymine (T), guanine (G), cytosine (C), or uracil (U), but is sufficiently similar to substitute for the normal nucleotide in a nucleic acid molecule.
  • nucleic Acid Composition refers to an oligonucleotide or polynucleotide such as a DNA or RNA sequence, or a mixed DNA/RNA sequence, in either a single-stranded or a double-stranded molecular structure.
  • Gene a nucleic acid composition whose oligonucleotide or polynucleotide sequence codes for an RNA and/or a polypeptide (protein).
  • a gene can be either RNA or DNA.
  • a gene may encode a non-coding RNA, such as small hairpin RNA (shRNA), microRNA (miRNA), rRNA, tRNA, snoRNA, snRNA, and their RNA precursors as well as derivatives.
  • a gene may encode a protein-coding RNA essential for protein peptide synthesis, such as messenger RNA (mRNA) and its RNA precursors as well as derivatives.
  • mRNA messenger RNA
  • a gene may encode a protein-coding RNA that also contains at least a microRNA or shRNA sequence.
  • RNA sequence that is directly transcribed from a gene without any RNA processing or modification, which may be selected from the group consisting of mRNA, hnRNA, rRNA, tRNA, snoRNA, snRNA, pre-microRNA, viral RNA and their RNA precursors as well as derivatives.
  • Precursor messenger RNA primary RNA transcripts of a protein-coding gene, which are produced by eukaryotic type-II RNA polymerase (Pol-II) machineries in eukaryotes through an intracellular mechanism termed transcription.
  • a pre-mRNA sequence contains a 5 ' -untranslated region (UTR), a 3'-UTR, exons and introns.
  • Intron a part or parts of a gene transcript sequence encoding non-protein-reading frames, such as in- frame intron, 5' -UTR and 3' -UTR.
  • cDNA protein-reading frames
  • mRNA Messenger RNA
  • assembly of pre-mRNA exons which is formed after intron removal by intracellular RNA splicing machineries (spliceosomes) and served as a protein-coding RNA for peptide/protein synthesis.
  • the peptides/proteins encoded by mRNAs include, but not limited, enzymes, growth factors, insulin, antibodies and their analogs/homologs as well as derivatives.
  • cDNA Complementary DNA
  • Sense a nucleic acid molecule in the same sequence order and composition as the homologous mRNA. The sense conformation is indicated with a "+”, “s” or “sense” symbol.
  • Antisense a nucleic acid molecule complementary to the respective mRNA molecule.
  • the antisense conformation is indicated as a "-” or “*” symbol or with an "a” or “antisense” in front of the DNA or RNA, e.g., "aDNA” or "aRNA”.
  • Base Pair (bp) a partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule.
  • uracil (U) is substituted for thymine.
  • the partnership is achieved through hydrogen bonding.
  • Base Pair (bp) a partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule.
  • uracil (U) is substituted for thymine.
  • the partnership is achieved through hydrogen bonding.
  • a sense nucleotide sequence "5'-A-T-C-G-U-3"' can form complete base pairing with its antisense sequence "5'-A-C-G-A-T-3"'.
  • 5 '-end a terminus lacking a nucleotide at the 5' position of successive nucleotides in which the 5 '-hydroxyl group of one nucleotide is joined to the 3 '-hydroyl group of the next nucleotide by a phosphodiester linkage.
  • Other groups, such as one or more phosphates, may be present on the terminus.
  • 3 '-end a terminus lacking a nucleotide at the 3' position of successive nucleotides in which the 5 '-hydroxyl group of one nucleotide is joined to the 3 '-hydroyl group of the next nucleotide by a phosphodiester linkage.
  • Other groups, most often a hydroxyl group, may be present on the terminus.
  • Template a nucleic acid molecule being copied by a nucleic acid polymerase.
  • a template can 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 a double-stranded or partially double- stranded template.
  • Both RNA and DNA are synthesized in the 5' to 3' direction.
  • the two strands of a nucleic acid duplex are always aligned so that the 5' ends of the two strands are at opposite ends of the duplex (and, by necessity, so then are the 3' ends).
  • Nucleic Acid Template a double-stranded DNA molecule, double stranded RNA molecule, hybrid molecules such as DNA-RNA or RNA-DNA hybrid, or single-stranded DNA or RNA molecule.
  • nucleotide sequence is conserved with respect to a pre-selected (referenced) sequence if it non-randomly hybridizes to an exact complement of the preselected sequence.
  • Homologous or Homology a term indicating the similarity between a polynucleotide and a gene or mRNA sequence.
  • a nucleic acid sequence may be partially or completely homologous to a particular gene or mRNA sequence, for example.
  • Homology may be expressed as a percentage determined by the number of similar nucleotides over the total number of nucleotides.
  • Complementary or Complementarity or Complementation a term used in reference to matched base pairing between two polynucleotides (i.e. sequences of an mRNA and a cDNA) related by the aforementioned "base pair (bp)" rules.
  • sequence “5'-A-G-T- 3"' is complementary to the sequence "5'-A-C-T-3"', and also to "5'-A-C-U-3"'.
  • Complementation can be between two DNA strands, a DNA and an RNA strand, or between two RNA strands.
  • Complementarity may be "partial” or “complete” or “total”. Partial complementarity or complementation occurs when only some of the nucleic acid bases are matched according to the base pairing rules. Complete or total complementarity or complementation occurs when the bases are completely or perfectly matched between the nucleic acid strands.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as in detection methods that depend on binding between nucleic acids. Percent complementarity or complementation refers to the number of mismatch bases over the total bases in one strand of the nucleic acid. Thus, a 50% complementation means that half of the bases were mismatched and half were matched. Two strands of nucleic acid can be complementary even though the two strands differ in the number of bases. In this situation, the complementation occurs between the portion of the longer strand corresponding to the bases on that strand that pair with the bases on the shorter strand.
  • Complementary Bases nucleotides that normally pair up when DNA or RNA adopts a double stranded configuration.
  • Complementary Nucleotide Sequence a sequence of nucleotides in a single-stranded molecule of DNA or RNA that is sufficiently complementary to that on another single strand to specifically hybridize between the two strands with consequent hydrogen bonding.
  • Hybridize and Hybridization the formation of duplexes between nucleotide sequences which are sufficiently complementary to form complexes via base pairing.
  • a primer or splice template
  • target template
  • complexes or hybrids
  • Posttranscriptional Gene Silencing a targeted gene knockout or knockdown effect at the level of mRNA degradation or translational suppression, which is usually triggered by either foreign/viral DNA or RNA transgenes or small inhibitory RNAs.
  • RNA Interference a posttranscriptional gene silencing mechanism 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 function as gene silencers, interfering with expression of intracellular genes containing either completely or partially complementarity to the small RNAs.
  • miRNA microRNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • Gene Silencing Effect a cell response after a gene function is suppressed, consisting but not limited of cell cycle attenuation, G0/G1 -checkpoint arrest, tumor suppression, anti- tumorigenecity, cancer cell apoptosis, and a combination thereof.
  • Non-coding RNA an RNA transcript that cannot be used to synthesize peptides or proteins through intracellular translation machineries.
  • Non-coding RNA includes long and short regulatory RNA molecules such as microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) and double strand RNA (dsRNA). These regulatory RNA molecules usually function as gene silencers, interfering with expression of intracellular genes containing either completely or partially complementarity to the non-coding RNAs.
  • miRNA microRNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • dsRNA double strand RNA
  • MicroRNA single-stranded RNA capable of binding to targeted gene transcripts (mRNAs) that have partial complementarity to the sequence of microRNA.
  • Mature microRNA is usually sized about 17-27 oligonucleotides in length and is able to either directly degrade its intracellular mRNA target(s) or suppress the protein translation of its targeted mRNA(s), depending on the complementarity between the microRNA and its target mRNA(s).
  • Native microRNAs are found in almost all eukaryotes, functioning as a defense against viral infections and allowing regulation of specific gene expression during development of plants and animals. In principle, one microRNA often target multiple target mRNAs to fulfill its full functionality while on the other hand multiple miRNAs may target the same gene transcripts to enhance the effect of gene silencing.
  • Pre- miRNA hairpin-like single-stranded RNA containing stem-arm and stem-loop regions for interacting with intracellular RNase III Dicer endoribonucleases to produce one or multiple mature microRNAs (miRNAs) capable of silencing a targeted gene or a specific group of targeted genes that contain full or partial complementarity to the mature microRNA sequence(s).
  • the stem-arm of a pre-miRNA can form either a perfectly (100%) or a partially (mis-matched) hybrid duplexes, while the stem- loop connects one end of the stem-arm duplex to form a circle or hairpin-loop conformation required for being assembled into an RNA-induced silencing complex (RISC) with some argonaute proteins (AGO).
  • RISC RNA-induced silencing complex
  • AGO argonaute proteins
  • Prokarvote-produced MicroRNA Precursor small hairpin-like RNA similar to natural microRNA precursor (pre-miRNA) but transcribed from an artificially recombinant microRNA-expressing plasmid driven by a eukaryotic promoter in prokaryotic competent cells.
  • pro-miR-302 is structurally as same as pre-miR-302 (FIGs. 13A and 13B) but transcribed from either a pLVX-Grn-miR302+367 or pLenti-EFlalpha- RGFP-miR302 vector in E. coli DHSalpha competent cells (Example 1).
  • prokaryotic cells normally do not express short RNAs with high secondary structures such as eukaryotic pre-miRNA
  • the production of pro-miRNA in prokaryotes usually requires the addition of chemical inducer(s) in order to stimulate the eukaryotic promoter-driven pre-miRNA transcription (FIGs. 2-4).
  • siRNA small interfering RNA: short double-stranded RNA sized about 18-27 perfectly base-paired ribonucleotide duplexes and capable of degrading target gene transcripts with almost perfect complementarity.
  • small or short hairpin RNA single- stranded RNA that contains a pair of partially or completely matched stem-arm nucleotide sequences divided by an unmatched loop oligonucleotide to form a hairpin-like structure.
  • Many natural miRNAs are derived from hairpin-like RNA precursors, namely precursor microRNA (pre-miRNA).
  • Vector a recombinant nucleic acid composition such as recombinant DNA (rDNA) capable of movement and residence in different genetic environments. Generally, another nucleic acid is operatively linked therein.
  • the vector can be capable of autonomous replication in a cell in which case the vector and the attached segment is replicated.
  • One type of preferred vector is an episome, i.e., a nucleic acid molecule capable of extrachromosomal replication.
  • Preferred vectors are those capable of autonomous replication and expression of nucleic acids.
  • Vectors capable of directing the expression of genes encoding for one or more polypeptides and/or non-coding RNAs are referred to herein as "expression vectors" or "expression-competent vectors”.
  • a vector may contain components consisting of a viral or a type-II RNA polymerase ( ⁇ - ⁇ or pol-2) promoter, or both, a Kozak consensus translation initiation site, polyadenylation signals, a plurality of restriction/cloning sites, a pUC origin of replication, a SV40 early promoter for expressing at least an antibiotic resistance gene in replication-competent prokaryotic cells, an optional SV40 origin for replication in mammalian cells, and/or a tetracycline responsive element.
  • the structure of a vector can be a linear or circular form of single- or double- stranded DNA selected form the group consisting of plasmid, viral vector, transposon, retrotransposon, DNA transgene, jumping gene, and a combination thereof.
  • Promoter a nucleic acid to which a polymerase molecule recognizes, or perhaps binds to, and initiates RNA transcription.
  • a promoter can be a known polymerase or its cofector binding site, an enhancer and the like, any sequence that can initiate synthesis of RNA transcripts by a desired polymerase.
  • Eukaryotic Promoter a sequence of nucleic acid motifs which are required for RNA and/or gene transcription and can be recognized by eukaryotic type II RNA polymerases (pol- 2), pol-2 equivalent, and/or pol-2 compatible (pol-2-like) viral polymerases for initiating the RNA/gene transcription.
  • Tvpe- ⁇ RNA Polymerase (Pol-II or pol-2) Promoter an RNA promoter that can be recognized by eukaryotic type- ⁇ RNA polymerases ( ⁇ - ⁇ or pol-2) and hence is able to initiate the transcription of eukaryotic messenger RNAs (mRNAs) and/or microRNAs (miRNAs).
  • a pol-2 promoter can be a mammalian RNA promoter or a cytomegalo viral (CMV) promoter.
  • Type-II RNA Polymerase (Pol-II or pol-2) Equivalent: a eukaryotic transcription machinery selected from the group consisting of mammalian type- II RNA polymerases ( ⁇ - ⁇ or pol-2) and Pol-II compatible (pol-2-like) viral RNA polymerases.
  • Pol-II Compatible (pol-2-like) Viral Promoter a viral RNA promoter capable of using the eukaryotic pol-2 or pol-2 equivalent transcription machineries for initiating gene and/or RNA expression.
  • a pol-2-like viral promoter can be a cytomegaloviral (CMV) promoter or a retroviral long terminal repeat (LTR) promoter.
  • CMV cytomegaloviral
  • LTR retroviral long terminal repeat
  • Cistron a sequence of nucleotides in a DNA molecule coding for an amino acid residue sequence and including upstream and downstream DNA expression control elements.
  • Intron Excision a cellular mechanism responsible for RNA processing, maturation and degradation, including RNA splicing, exosome digestion, nonsense-mediated decay (NMD) processing, and a combination thereof.
  • RNA Processing a cellular mechanism responsible for RNA maturation, modification and degradation, including RNA splicing, intron excision, exosome digestion, nonsense- mediated decay (NMD), RNA editing, RNA processing, and a combination thereof.
  • Targeted Cell a single or a plurality of human cells selected from the group consisting of a somatic cell, a tissue, a stem cell, a germ-line cell, a teratoma cell, a tumor cell, a cancer cell, and a combination thereof.
  • Cancerous Tissue a neoplastic tissue derived from the group consisting of skin cancer, prostate cancer, breast cancer, liver cancer, lung cancer, brain tumor/cancer, lymphoma, leukemia and a combination thereof.
  • Expression-Competent Vector a linear or circular form of single- or double- stranded DNA selected form the group consisting of plasmid, viral vector, transposon, retrotransposon, DNA transgene, jumping gene, and a combination thereof.
  • Antibiotic Resistance Gene a gene capable of degrading antibiotics selected from the group consisted of penicillin G, streptomycin, ampicillin (Amp), neomycin, G418, kanamycin, erythromycin, paromycin, phophomycin, spectromycin, tetracycline (Tet), doxycycline (Dox), rifapicin, amphotericin B, gentamycin, chloramphenicol, cephalothin, tylosin, and a combination thereof.
  • Restriction/Cloning Site a DNA motif for restriction enzyme cleavage including but not limited Aatll, Accl, Aflll/III, Agel, Apal/LI, Asel, Asp718I, BamHI, Bbel, BclI/II, Bglll, Bsml Bspl20I, BspHI/LUl 11/1201, Bsrl/Bl/Gl, BssHll/Sl, BstBI/Ul/XI, CM, Csp61, Dpnl, Dral/II, EagI, Ecll3611, EcoRl/Rll/47111/RV, Ehel, Fspl, Haelll, Hhal, HinPI, Hindlll, Hinfl, Hpal/II, KasI, Kpnl, Maell/III, Mfel, MM, MscI, Msel, Nael, Narl, Ncol, Ndel, NgoMI, Not
  • Gene Delivery a genetic engineering method selected from the group consisting of polysomal transfection, liposomal transfection, chemical transfection, electroporation, viral infection, DNA recombination, transposon insertion, jumping gene insertion, microinjection, gene-gun penetration, and a combination thereof.
  • Genetic Engineering a DNA recombination method selected from the group consisting of DNA restriction and ligation, homologous recombination, transgene
  • Cell Cycle Regulator a cellular gene involved in controlling cell division and proliferation rates, consisting but not limited of CDK2, CDK4, CDK6, cyclins, BMI-1, pl4/pl9Arf, pl5Ink4b, pl6Ink4a, pl8Ink4c, p21Cipl/Wafl, and p27Kipl, and a combination thereof.
  • Tumor Suppression Effect a cellular anti-tumor and/or anti-cancer mechanism and response consisting of, but not limited, cell cycle attenuation, cell cycle arrest, inhibition of tumor cell growth, inhibition of cell tumorigenecity, inhibition of tumor/cancer cell transformation, induction of tumor/cancer cell apoptosis, induction of normal cell recovery, reprogramming high-grade malignant cancer cells to a more benign low-grade state (tumor regression), and a combination thereof.
  • Cancer Therapy Effect a cell response and/or cellular mechanism resulted from a drug treatment, including, but not limited, inhibition of oncogene expression, inhibition of cancer cell proliferation, inhibition of cancer cell invasion and/or migration, inhibition of cancer metastasis, induction of cancer cell death, prevention of tumor/cancer formation, prevention of cancer relapse, suppression of cancer progression, repairing damaged tissue cells, reprogramming high-grade malignant cancers to a more benign low-grade state (cancer regression/remission), and a combination thereof.
  • a drug treatment including, but not limited, inhibition of oncogene expression, inhibition of cancer cell proliferation, inhibition of cancer cell invasion and/or migration, inhibition of cancer metastasis, induction of cancer cell death, prevention of tumor/cancer formation, prevention of cancer relapse, suppression of cancer progression, repairing damaged tissue cells, reprogramming high-grade malignant cancers to a more benign low-grade state (cancer regression/remission), and a combination thereof.
  • Gene Silencing Effect a cell response after a gene function is suppressed, consisting of, but not limited, inhibition of oncogene expression, inhibition of cell proliferation, cell cycle arrest, tumor suppression, cancer regression, cancer prevention, cell apoptosis, cell repairing and/or rejuvenation, cell reprogramming, reprogramming diseased cells to a relatively normal state (spontaneous healing), and a combination thereof.
  • Cancer Reversion a reprogramming mechanism that resets the malignant properties of high-grade cancers back to a relatively normal-like low-grade state in vitro, ex vivo or in vivo.
  • Targeted Cell a single or a plurality of human cells selected from the group consisting of a somatic cell, a tissue, a stem cell, a germ-line cell, a teratoma cell, a tumor cell, a cancer cell, and a combination thereof.
  • Cancerous Tissue a neoplastic tissue derived from the group consisting of skin cancer, prostate cancer, breast cancer, liver cancer, lung cancer, brain tumor/cancer, lymphoma, leukemia and a combination thereof.
  • Transcriptional Inducer a chemical agent that can induce and/or enhance eukaryotic RNA and/or gene transcription from a pol-2 or pol-2-like promoter in prokaryotic cells.
  • a transcription inducer contains, but not limited, a chemical structure similar to MOPS, ethanol, glycerin, as well as their functional analogs such as 2-(N- morpholino)ethanesulfonic acid (MES), 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) and mannitol, or a mixture thereof.
  • MES 2-(N- morpholino)ethanesulfonic acid
  • HEPES 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
  • mannitol or a mixture thereof.
  • Antibody a peptide or protein molecule having a pre-selected conserved domain structure coding for a receptor capable of binding a pre-selected ligand.
  • compositions useful for diagnosis, stem cell generation, stem cell research and/or therapy development, tissue/organ repair and/or rejuvenation, wound healing treatment, tumor suppression, cancer therapy and/or prevention, disease treatment, drug production, and a combination thereof.
  • a composition and method for producing a new kind of prokaryote-produced microRNA precursors capable of reprogramming the malignant properties of human cancers into a low-grade benign or normal-like state in vitro, ex vivo and in vivo, comprising:: (a) at least a chemical inducer agent containing a structure similar to 3- morpholinopropane-1 -sulfonic acid (MOPS), ethanol, or glycerin, or a mixture thereof; and (b) a plurality of prokaryotic cells that contain at least a pre-miRNA-encoding gene mediated by eukaryotic pol-2 and/or pol-2-like promoter-driven transcription; wherein said (a) and (b) are mixed together under a condition to induce the expression of said gene, so as to generate the encoded pre-miRNA in the prokaryotic cells.
  • the chemical inducer is able to stimulate eukaryotic promoter-driven RNA transcription in prok
  • the present invention provides a novel composition design and its applicable strategy for inducing a quick adaptation of prokaryotes to use eukaryotic pol-2 and pol-2-like promoters for directly expressing certain desired microRNA precursors (pre- miRNA) without the need of using error-prone prokaryotic promoters or growing laborious and costly hybridomas or mammalian cells.
  • said prokaryote is a bacterial cell strain in particular, Escherichia coli (E. coli), and said chemical inducer is 3 -morpholinopropane-1 -sulfonic acid (MOPS), ethanol, or glycerin, or a mixture thereof.
  • said eukaryotic promoter is either a eukaryotic pol-2 promoter, such as EFlalpha, or a pol-2 compatible (pol-2-like) viral promoter, such as cytomegaloviral (CMV) promoter or retroviral long terminal repeat (LTR) promoter.
  • CMV cytomegaloviral
  • LTR retroviral long terminal repeat
  • the pre-miRNA-encoding gene mediated by said eukaryotic promoter is coded for either a non-coding or a protein-coding RNA transcript, or both (such as an intron- containing gene transcript), selected from the group consisted of microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA), messenger RNA (mRNA) and their precursors as well as shRNA/siRNA homologues, and a combination thereof.
  • the protein- coding RNA may be selected from, but not limited to, the group consisted of a gene encoding enzyme, growth factor, antibody, insulin, botulinum toxin (botox), a functional protein and/or its analogs, and a combination thereof.
  • said condition for inducing the expression of said pre-miRNA-encoding gene is a bacterial culturing condition such as Luria- Bertani (LB) broth at 37°C with the addition of said chemical inducer(s).
  • FIGs. 1A and IB show a eukaryotic promoter-driven expression vector composition (1A) and its expression mechanism (IB) for RNA transcript and/or protein production in prokaryotes.
  • a new pLenti-EFl alpha-RGFP- miR302 vector (FIG. 1A) is served as an example composition for transforming E. coli DHSalpha competent cells to produce RGFP proteins as well as miR-302s and their precursors (pre-miR-302s) under the stimulation of MOPS, glycerin and/or ethanol.
  • pLenti- EFlalpha-RGFP-miR302 is a lentiviral plasmid vector that is designed by the inventors to expresses various microRNAs/shRNAs, mRNAs and/or proteins/peptides in both prokaryotes and eukaryotes. According to the disclosed mechanism (IB), it is easy for an ordinary skill in the art to use any microRNA/shRNA in place miR-302 or any mRNA/protein in place of RGFP as described in the present invention. Black arrows indicate the pathways occurring in both prokaryotic and eukaryotic cells, while blank arrows indicate the steps only occurring in the eukaryotic cells.
  • FIG. 2 depicts the results of bacterial culture broths treated with (left) or without (right) the mixture of 0.1% (v/v) MOPS and 0.05% (v/v) glycerin.
  • the E. coli competent cells have been transformed by pLenti-EFl alpha-RGFP-miR302 before the treatment of chemical inducers.
  • FIG. 3 shows the results of different bacterial pellets after treated with 0.1% (v/v) MOPS.
  • the E. coli competent cells have been transformed by either pLVX-Grn- miR302 +367 (green) or pLenti-EFlalpha-RGFP-miR302 (red) before the MOPS treatment.
  • FIG. 4 shows the inducibility of various chemical inducers for inducing pol-2 promoter-driven gene expression in E. coli competent cells.
  • the top three most potent inducers are MOPS, glycerin and ethanol.
  • the chemical concentration used can be ranged from about 0.001% to 4%, most preferably, from 0.01% to 1%.
  • FIG. 5 shows the Western blotting results of red RGFP protein expression induced by MOPS, glycerin and ethanol, respectively.
  • Bacterial RuvB protein was used as a housekeeping standard to normalize the detected RGFP expression. Proteins extracted from blank E. coli cells, i.e. transformed with no vector, were used as a negative control.
  • FIG. 6 shows the Northern blotting results of the expression of the miR-302 familial cluster ( ⁇ 700nt) and its derivative precursors (pre-miR-302s with 1 to 4 hairpins) induced by MOPS, glycerin and ethanol, respectively.
  • RNAs extracted from blank E. coli cells were used as a negative control.
  • FIG. 7 shows iPSC generation using miR-302 and/or pre-miR-302 isolated from bacterial competent cell extracts (BE), which is confirmed by Northern blot analysis as shown in FIG. 6.
  • miR-302 -reprogrammed iPSCs or called mirPSCs
  • FIG. 7 shows iPSC generation using miR-302 and/or pre-miR-302 isolated from bacterial competent cell extracts (BE), which is confirmed by Northern blot analysis as shown in FIG. 6.
  • miR-302 -reprogrammed iPSCs or called mirPSCs
  • FIG. 8 shows the global DNA demethylation of Oct4 and Sox2 gene promoters induced by the miR-302 and/or pre-miR-302 isolated from bacterial competent cell extracts (BE), which is confirmed by Northern blot analysis as shown in FIG. 6. 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 somatic cell reprogramming to form iPSCs.
  • FIG. 9 shows the in vitro tumorigenicity assays of human liver cancer cell line HepG2 in response to miR-302 transfection.
  • FIGs. 10A and 10B show the results of HPLC purification and analysis using a synthetic standard uDNA (by Sigma-Genosys) and freshly extracted pro-miR-302s isolated from pLenti-EFlalpha-RGFP-miR302-trsLnsior ed E. coli cells.
  • the standard uDNA was designed to be equal to a natural pre-miR-302a as: 5'-CCACCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC UUCCAUGUUU UGGUGAUGG-3 ' (SEQ.ID.NO.4).
  • FIGs. 11A and 11B show the results of microRNA (miRNA) microarray analyses using small RNAs extracted from either blank E. coli competent cells or pLenti-EFlalpha- RGFP-miR302 (RGFP-miR302)-tiansiected cells.
  • the extracted small RNAs were further purified by HPLC as shown in the green-labeled area of FIG. 10B.
  • FIG. 11A shows that RNAs from blank E.coli cells present almost no microRNA (green dots mean non-statistically significant whereas red dots indicate positive results). This is because prokaryotes lack several essential enzymes required for microRNA expression and processing, such as Pol-2, Drosha and RNase ⁇ Dicer.
  • prokaryotic RNA polymerases do not efficiently transcribe small RNAs with high secondary structures, such as hairpin-like pre-miRNAs and shRNAs.
  • specific microRNAs such as miR-302a, a*, b, b*, c, c*, d and d* as shown in FIG. 11B, in prokaryotic cells. Since prokaryotic cells possess no Dicer, all microRNAs remain in their precursor conformations, such as pri-miRNA (4-hairpin cluster) and/or pre-miRNA (1 hairpin precursor). Taken together, the results of FIGs.
  • FIG. 12 shows the lists of expressed microRNAs extracted from either blank E. coli competent cells (Group 1 as shown in FIG. 11A) or pLenti-EFlalpha-RGFP-miR302- transfected cells (Group 2 as shown in FIG. 11B). Signals less than 500 are not statistically significant (as shown in green in FIGs. 11A and 11B), which may be caused by either low copy number expression or high background.
  • FIGs. 13A and 13B show the sequencing results of the miR-302 familial cluster (13A) and the individual pro-miR-302a, pro-miR-302b, pro-miR-302c, and pro-miR-302d sequences (13B).
  • the result of the miR-302 familial cluster is 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 CACCUUUGU UACAAAAUUU UUUGUUAUU GUGUUUAAG GUUACUAAGC UUGUUACAGG UUAAAGGAUU CUUUUUAAG GUUACUAAGC UUGUUACA
  • FIG. 14 shows the in vivo therapeutic results of a pre-investigational new drug (pre- IND) trial using pro-miR-302 as an injection drug to treat human liver cancer xenografts in SCID-beige nude mice.
  • pre-IND pre-investigational new drug
  • No significant therapeutic effect was found in the treatments of synthetic siRNA mimics (siRNA-302).
  • FIG. 15 shows the histological similarity between normal liver tissues and pro-mir- 302-treated human liver cancer xenografts in vivo.
  • the pro-mir-302 drug successfully reprogrammed high-grade (grade IV) human liver cancer grafts to a more benign low-grade (less than grade ⁇ ) state.
  • the treated cancer grafts could form classical liver lobules, containing central vein (CV)-like and portal triad (PT)-like structures (indicated by black arrows).
  • CV central vein
  • PT portal triad
  • FIG. 16 shows the patho-histological comparison among untreated, siRNA-treated, pro-mir-302-treated human liver cancer grafts and normal liver tissues in SCID-beige nude mice.
  • top the engrafted human liver cancer aggressively invaded into normal tissues, such as muscles and blood vessels, and formed massive cell-cell and cancer- tissue fusion structures, indicating its malignancy and high metastasis.
  • Treatment of siRNA mimics did not significantly reduce the malignancy of the engrafted cancer (upper middle), probably due to the short half-life of siRNA.
  • pro-miR-302 treatment not only reprogrammed the engrafted cancer to a relatively normal-like morphology (no fusion) but also greatly inhibited cancer invasion into the surrounding tissues (lower middle).
  • pro-miR-302-treated cancers formed normal-like lobule structures, gland-like cell arrangements, and clear boundaries between cell-cell and cancer-tissue junctions (black arrows), indicating that these treated cancers have been downgraded to a very benign state.
  • FIGs. 17A and 17B show comparison of the healing results between untreated (17A) and miR-302-treated (17B) wounds in vivo.
  • pre-miR-302 is able to induce CD34-positive adult stem cell expansion and/or regeneration, so as to enhance tissue repairing and regeneration, leading to a very beneficial therapeutic effect on lesions caused by human degenerative diseases, such as Alzheimer's diseases, Parkinson's diseases, osteoporosis, diabetes, and cancers.
  • Such therapeutic effect may also help to reprogram high-grade malignant cancers into low-grade benign or even normal-like tissues, a novel mechanism called Cancer Reversion or Cancer Regression.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • dNTP deoxyribonucleotide triphosphate
  • PBS phosphate buffered saline
  • NaCl sodium chloride
  • HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
  • HBS HBS buffered saline
  • SDS sodium dodecylsulfate
  • Tris-HCl tris- hydroxymethylaminomethane-hydrochloride
  • E. coli DHSalpha competent cells were acquired as a part from the z-competent E. coli transformation kit (Zymo Research, Irvine, CA) and then transformed by mixing with 5 ⁇ g of a pre-made plasmid vector such as pLenti-EFl alpha-RGFP-miR302 or pLVX-Grn- miR302+367.
  • Non-transformed cells were normally grown in Luria-Bertani (LB) broth supplemented with 10 mM MgS0 4 and 0.2 mM glucose at 37°C with frequent agitation at 170 rpm, whereas the transformed cells are cultivated in the above LB broth further supplemented with additional 100 ⁇ g/ml ampicillin.
  • Human liver cancer cell line HepG2 was obtained from ATCC and maintained according to manufacturer's suggestions. For transfection, 15 ⁇ g of pre-miR-302 was dissolved in 1 ml of fresh RPMI medium and mixed with 50 ⁇ of X-tremeGENE HP DNA transfection reagent (Roche, Indianapolis, IN). After 10 min incubation, the mixture is added into a 100-mm cell culture dish containing 50% ⁇ 60% confluency of HepG2. The medium was refreshed 12 to 18 hours later.
  • the medium was changed to a knockout DMEM/F-12 medium (Invitrogen) supplemented with 20% knockout serum, 1% MEM nonessential amino acids, 100 ⁇ ⁇ - mercaptoethanol, 1 mM GlutaMax, 1 mM sodium pyruvate, 10 ng/ml bFGF, 10 ng/ml FGF-4, 5 ng/ml LIF, 100 IU/ml penicillin/ 100 ⁇ streptomycin, 0.1 ⁇ A83-01, and 0.1 ⁇ valproic acid (Stemgent, San Diego, CA), and the cells were cultivated at 37°C under 5% CO 2 . The result is shown in FIG. 9.
  • DMEM/F-12 medium Invitrogen
  • MEM nonessential amino acids 100 ⁇ ⁇ -mercaptoethanol
  • 1 mM GlutaMax 1 mM sodium pyruvate
  • 10 ng/ml bFGF 10 ng/ml FGF-4
  • 5 ng/ml LIF
  • Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), electroblotted onto a nitrocellulose membrane and incubated in Odyssey blocking reagent (Li-Cor Biosciences, Lincoln, NB) for 2 hours at room temperature. Then, a primary antibody was applied to the reagent and incubated the mixture at 4°C. Primary antibodies included 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 goat anti-mouse IgG conjugated secondary antibody to Alexa Fluor 680 reactive dye (1 :2,000; Invitrogen-Molecular Probes), for 1 hour at the room temperature. After three additional TBS-T rinses, fluorescent scanning of the immunoblot and image analysis was conducted using Li-Cor Odyssey Infrared Imager and Odyssey Software v.10 (Li-Cor). The results are shown in FIG. 5.
  • RNAs (10 ⁇ g) were isolated with a mz ' rVanaTM miRNA isolation kit (Ambion, Austin, TX), fractionated by either 15% TBE-urea polyacrylamide gel or 3.5% low melting point agarose gel electrophoresis, and electroblotted onto a nylon membrane. Detection of miR-302s and the related pre-miR-302s was performed with a [LNAJ-DNA probe (5'- [TCACTGAAAC] ATGGAAGCAC TTA-3') (SEQ.ID.NO.10) probe.
  • the probe has been purified by high-performance liquid chromatography (HPLC) and tail-labeled with terminal transferase (20 units) for 20 min in the presence of [ 32 P]-dATP (> 3000 Ci/mM, Amersham International, Arlington Heights,IL). The results are shown in FIG. 6.
  • E. coli DHSalpha competent cells after transformation were cultivated in LB broth supplemented with 10 mM MgS0 4 and 0.2 mM glucose at 37°C with frequent agitation at 170 rpm.
  • MOPS metal-oxide-semiconductor
  • glycerin glycerin-semiconductor
  • ethanol aqueous saline
  • the amplified plasmid DNAs and expressed mRNAs/microRNAs in the transformed cells were isolated using a HiSpeed plasmid purification kit (Qiagen, Valencia, CA), following the manufacturer's protocol but with a minor modification that RNase A was not added into the PI buffer.
  • RNAs isolated from Example 5 were further extracted using a mz ' rVanaTM miRNA isolation kit (Ambion, Austin, TX), following the manufacturer's protocol. The final products so obtained were dissolved in DEPC-treated dd]3 ⁇ 40 and stored at -80°C before use. Because bacterial RNAs are naturally degraded very fast (within a few hours) whereas eukaryotic hairpin-like microRNA precursors (pre-miRNAs and pri-miRNAs) remain largely stable at 4°C (half- life up to 3-4 days), we can use this half-life difference to acquire relatively pure pri-/pre-miRNAs for other applications. For example, the pre-miR-302s so obtained can be used to reprogram somatic cells to hESC-like iPSCs, as shown in FIG. 9.
  • Embedding, sectioning and immunostaining tissue samples were performed as previously reported (Lin et al., 2008).
  • Primary antibodies include Oct4 (Santa Cruz) and RGFP (Clontech, Palo Alto, CA). Fluorescent dye-labeled goat anti -rabbit or horse anti- mouse antibody was used as the secondary antibody (Invitrogen-Molecular Probes, Carlsbad, CA). Positive results were examined and analyzed at lOOx or 200x magnification under a fluorescent 80i microscopic quantitation system with a Metamorph imaging program (Nikon). The result is shown in FIG. 7.
  • Genomic DNAs were isolated from -2,000,000 cells using a DNA isolation kit (Roche) and 1 ⁇ g of the isolated DNAs was further treated with bisulfite (CpGenome DNA modification kit, Chemicon, Temecula, CA), following the manufacturers' suggestion.
  • the bisulfite treatment converted all unmethylated cytosine to uracil, while methylated cytosine remained as cytosine.
  • the bisulfite- modified DNAs 50 ng were mixed with the primers (total 100 pmol) in lx PCR buffer, heated to 94°C for 2 min, and immediately cooled on ice.
  • 25 cycles of PCR were performed as follows: 94°C for 1 min and 70°C for 3 min, using an Expand High Fidelity PCR kit (Roche).
  • the PCR product with a correct size was further fractionized by 3% agarose gel electrophoresis, purified by a gel extraction filter (Qiagen), and then used in DNA sequencing. After that, a detailed profile of DNA methylation sites was generated by comparing the unchanged cytosine in the converted DNA sequence to the unconverted one, as shown in FIG. 8.
  • MicroRNA MicroRNA
  • RNAs from each cell culture were isolated, using the mz ' rVanaTM miRNA isolation kit (Ambion). The purity and quantity of the isolated small RNAs were assessed, using 1% formaldehyde-agarose gel electrophoresis and spectrophotometer measurement (Bio-Rad), and then immediately frozen in dry ice and submitted to LC Sciences (San Diego, CA) for miRNA microarray analyses. Each microarray chip was hybridized a single sample labeled with either Cy3 or Cy5 or a pair of samples labeled with Cy3 and Cy5, respectively. Background subtraction and normalization were performed as manufacturer's suggestions.
  • Xenografting human liver cancers into immunocompromised SCID-beige mice is a valid animal model for studying liver cancer metastasis and therapy.
  • HepG2 human hepatocarcinoma
  • matrix gel a cell that engrafts the mixture into each flank of the mouse hind limbs, respectively.
  • both sides of the mouse hind limbs were subjected to approximately the same amount of cancer cell engraftment. Cancers were observed about two weeks post-engraftment and sized about 15.6+8 mm 3 in average (starting cancer size before treatment).
  • pro-mir-302 To deliver pro-mir-302 into the targeted cancer regions in vivo, we contracted a professional formulation company, Latitude (San Diego, CA), to liposomally encapsulate pro- miR-302s into 160-200 nm-diameter nanoparticles. These pro-miR-302-containing nanoparticles have been tested to be almost 100% stable at room temperature for over two weeks and at 4°C for over one month, whereas other synthetic siRNA mimics (siRNA-302) were all quickly degraded over 50% within 3 to 5 days under the same conditions, indicating that pro-miRNA rather than siRNA is stable enough to be used as a drug for therapy.
  • siRNA-302 synthetic siRNA mimics
  • 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.

Abstract

Cette invention concerne de manière générale une composition et une méthode permettant d'induire l'expansion et/ou la régénération d'une population de cellules souches adultes positives pour le CD34, qui est utilisable pour développer des médicaments, vaccins et/ou traitements contre diverses maladies dégénératives liées au vieillissement chez l'homme. En outre, la présente invention concerne les procédés de production et de purification nécessaires à la fabrication de grandes quantités de compositions de haute qualité de petits ARN en épingle à cheveux (ARNsh), tels que des précurseurs de microARN (pri-miARN et pré-miARN) et de petits ARN interférents (siARN), qui sont utilisables pour traiter des maladies liées au vieillissement, telles que, sans y être limitées, la maladie d'Alzheimer, la maladie de Parkinson, l'ostéoporose, le diabète et les cancers. Les ARNsh qui en résultent, de préférence des pré-miARN, sont utilisables pour développer des médicaments thérapeutiques contre des maladies dégénératives, en particulier par l'intermédiaire d'un mécanisme qui permet d'induire l'expansion et/ou la régénération de cellules souches positives pour le CD34.
PCT/US2016/049583 2015-12-02 2016-08-31 Utilisation de précurseurs de micro-arn comme médicaments permettant d'induire l'expansion de cellules souches adultes WO2017095489A1 (fr)

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JP2018528746A JP2018535684A (ja) 2015-12-02 2016-08-31 ＣＤ３４陽性成体幹細胞の増殖を誘導する薬物としてのｍｉｃｒｏＲＮＡ前駆体の使用
CN201680070742.9A CN108431227A (zh) 2015-12-02 2016-08-31 使用微核醣核酸前驱物作为诱导cd34阳性成体干细胞增殖的药物
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