# Patent

## Title
Development of universal cancer drugs and vaccines

## Abstract

This invention generally relates to a design and method for developing novel anti-tumor/cancer drugs, vaccines and therapies, using microRNA (miRNA) and its shRNA homologues/derivatives. More particularly, the present invention relates to the use of a nucleic acid composition capable of expressing mir-302-like gene silencing effectors upon delivery into human cells and then silencing mir-302-targeted cell cycle regulators and oncogenes, resulting in an inhibitory effect on tumor/cancer cell growth and metastasis. Mir-302 is the most predominant miRNA found in human embryonic stem (hES) and induced pluripotent stem (iPS) cells, yet its function is unclear. The present invention establishes that in humans mir-302 concurrently suppressed both cyclin-E-CDK2 and cyclin-D-CDK4/6 pathways and eventually blocked over 70% of the G1-S transition. Simultaneously, mir-302 also silences BMI-1, a cancer stem cell marker, and subsequently promotes the tumor suppressor functions of p16Ink4a and p14/p19Arf in inhibiting CDK4/6-mediated cell proliferation. Therefore, the present invention for the first time reveals the tumor suppressor function of mir-302 in humans. This novel finding advances the design and method for developing new cancer drugs, vaccines and therapies directed against multiple kinds of human tumors and cancers, in particular including, but not limited, malignant skin, prostate, breast and liver cancers as well as various tumors.

## Background
CLAIM OF THE PRIORITY
The present application claims priority to the U.S. Provisional Application Ser. No. 61/272,169 filed on Aug. 26, 2009, entitled “Mir-302 reprograms somatic cell stemness with high pluripotency in SCNT and the application of chemical reprogramming thereof”. The present application also claims priority to the U.S. Provisional Application Ser. No. 61/323,190 filed on Apr. 12, 2010, entitled “Mir-302 inhibits human iPS Cell tumorigenecity through co-suppression of CDK2 and CDK4/6 pathways”. The present application is a continuation-in-part application of the U.S. patent application Ser. No. 12/149,725 filed on May 7, 2008, entitled “Generation of Human Embryonic Stem-Like Cells Using Intronic RNA”, and the U.S. patent application Ser. No. 12/318,806 filed on Jan. 8, 2009, entitled “Generation of Tumor-Free Embryonic Stem-Like Pluripotent Cells Using Inducible Recombinant RNA Agents”, which are hereby incorporated by reference as if fully set forth herein.


FIELD OF THE INVENTION
This invention generally relates to a design and utilization of DNA/RNA-based therapeutical drugs and/or vaccines for cancer therapy. More particularly, the present invention relates to the design and method of using a nucleic acid composition capable of generating and expressing small RNA-based gene silencing effectors upon delivery into human cells and then silencing mir-302-targeted cell cycle regulators and oncogenes, resulting in an inhibitory effect on tumor/cancer cell growth and metastasis. Preferably, the small RNA-based gene silencing effectors include microRNA (miRNA) such as mir-302a, mir-302b, mir-302c, mir-302d, mir-302e, and their precursors (pre-miRNA) as well as manually re-designed shRNA/siRNA homologues/derivatives, and a combination thereof. The human cells of interest include isolated somatic or tumor/cancer cells in vitro, ex vivo and/or in vivo.
BACKGROUND OF THE INVENTION
Mir-302 is the most predominant microRNA (miRNA) found in human embryonic stem (hES) and induced pluripotent stem (iPS) cells, yet its function is unclear. Previous studies have shown that ectopic expression of mir-302 is able to reprogram human cancer cells to hES-like pluripotent cells with a distinct slow cell cycle rate and dormant cell-like morphology (Lin et al., 2008). Relative quiescence is a defined characteristic of these mir-302-reprogrammed pluripotent stem (mirPS) cells, while other three/four factors (i.e. Oct4-Sox2-Klf4-c-Myc or Oct4-Sox2-Nanog-Lin28)-induced pluripotent stem (iPS) cells have dramatic proliferative ability and inexorable tumorigenetic tendency (Takahashi et al., 2006; Yu et al., 2007; Wernig et al., 2007). Despite the mechanism underlying this anti-proliferative characteristic of mirPS cells is largely unknown, we have identified the possible involvement of two mir-302-targeted G1-checkpoint regulators, cyclin-dependent kinase 2 (CDK2) and cyclin D (Lin et al., 2008). Progression in the eukaryotic cell cycle is driven by activities of cyclin-dependent kinases (CDKs), which forms functional complexes with positive regulatory subunits, cyclins, as well as by negative regulators, CDK inhibitors (CKIs, such as p14/p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1/Waf1, and p27Kip1). In mammalian cells, different cyclin-CDK complexes are involved in regulating different cell cycle transitions, such as cyclin-D-CDK4/6 for G1 progression, cyclin-E-CDK2 for G1-S transition, cyclin-A-CDK2 for S-phase progression, and cyclinA/B-CDC2 (cyclin-A/B-CDK1) for entry into M-phase. Thus, it is conceivable that the anti-proliferative function of mir-302 may result from the co-suppression of CDK2 and cyclin D during G1-S transition.
However, studies of the mir-291/294/295 family, an analog to human mir-302 in mouse, revealed a totally different result from the mir-302 function in human mirPS cells. In mouse embryonic stem (mES) cells, ectopic expression of mir-291/294/295 promoted fast cell proliferation and G1-S transition through direct silencing of p21Cip1 (also named CDKN1A) and serine/threonine-protein kinase Lats2 (Wang et al., 2008). This tumor-prone result was presumed due to the tumor suppressor nature of p21Cip1 and Lats2. Transgenic mice lacking p21Cip1/Waf1 were shown to display normal development with a defect in the G1 checkpoint control (Deng et al., 1995). Yet, the role of Lats2 remains to be determined because of its function in recruitment of amma-tubulin and spindle formation at the onset of mitosis. Loss of Lats2 in mouse embryos was found to cause severe mitotic defects and lethality, indicating that silencing of Lats2 may hinder rather than facilitate cell division (Yabuta et al., 2007). Taken together, silencing of p21Cip1 seems to be the key mechanism underlying such mir-291/294/295-induced tumorigenecity. Nevertheless, our recent effort to screen the mir-302 target site in human p21Cip1 gene shows negative. The same negative result was also predicted by online computing programs TARGETSCAN and PICTAR-VERT. Therefore, mir-302 and its analog mir-291/294/295 likely have different functions in hES and mES cells, lending different characteristics to human and mouse iPS cells. This finding suggests that the role of mir-291/294/295 in mES cells cannot serve as an equivalent model for evaluating mir-302 function in hES and iPS cells.
MiRNA is a cytoplasmic inhibitor and often functions to suppress the translation of it targeted gene transcripts (mRNAs) with high complementarity. The binding stringency between miRNA and its target genes determines the real function of a miRNA. Depending on the cellular condition, miRNA may present different preferences in gene targeting. However, there is no report related to either the concentration effect of mir-302 or the stringency of mir-302-target gene interaction. To resolve this problem, our present invention provides insight into these important details and for the first time reveals that mir-302 functions very differently in human and mouse cells. In humans, mir-302 strongly targets CDK2, cyclins D1/D2 and BMI-1, but interestingly, not p21Cip1. Unlike mouse p21Cip1, human p21Cip1 does not contain any target site for mir-302. This different gene targeting leads to a significant schism between respective cell cycle regulations. In mES cells, mir-302 silences p21Cip1 and promotes tumor-like cell proliferation (Wang et al., 2008; Judson, 2009), whereas p21Cip1 expression is preserved in human mirPS cells and may cause slower cell proliferation and lower tumorigenecity. Additionally, mouse BMI-1 is not a target gene for mir-302 either due to lack of a proper target site. We have found that silencing of human BMI-1 in human mirPS cells stimulates p16Ink4a/p14ARF expression to attenuate cell proliferation, whereas mir-302 cannot silence mouse BMI-1 to raise the same effect in mouse cells. Since p16Ink4a/p14ARF are elevated while p21Cip1 is not affected in mirPS cells, the anti-proliferative function of mir-302 in human cells most likely goes through p16Ink4a-Rb and/or p14/19ARF-p53 pathways in addition to the co-suppression of cyclin-E-CDK2 and cyclin-D-CDK4/6 pathways. These distinct targeting preferences of mir-302 to human and mouse genes imply that the mechanisms underlying their cell cycle regulations are fundamentally different in human and mouse cells.
In sum, prior arts overlooked the stringency of miRNA-target gene interaction and thus misled human mir-302 function into a wrong assumption. To clarify this misunderstanding, our present invention adopted an inducible mir-302 expression system to reveal a novel function of mir-302 in inhibition of human tumor/cancer cell growth, of which our new finding is useful for developing universal anti-tumor/cancer drugs and/or vaccines for cancer therapy as well as prevention. Therefore, there remains a need for effective and safe designs and methods for utilizing mir-302 and its precursors as well as homologues/derivatives in drug/vaccine development and cancer therapy.
SUMMARY OF THE INVENTION
The present invention is a design and utilization of DNA/RNA-based therapeutical drugs and/or vaccines for cancer therapy. More particularly, the present invention uses a recombinant nucleic acid composition capable of expressing small RNA gene silencing effectors upon delivery into human cells to inhibit the activities of mir-302-targeted cell cycle regulators and oncogenes, resulting in a tumor suppressor effect directed against human tumor/cancer cell growth and metastasis. Preferably, the small RNA gene silencing effectors include microRNA (miRNA) and miRNA-like agents, such as mir-302a, mir-302b, mir-302c, mir-302d, and their hairpin-like microRNA precursors (pre-miRNAs) and manually re-designed small hairpin RNA (shRNA) homologues/derivatives as well as a combination thereof. The designs of shRNA homologues/derivatives include mismatched and perfectly matched nucleic acid compositions of small hairpin RNA (shRNA) and small interfering RNA (siRNA) constructs in a single separate unit or a multiple unit cluster, all of which may improve the target specificity and reduce the copy number of mir-302 required for delivery and therapy.
Native microRNA (miRNA) is sized approximately 18-27 nucleotides (nt) in length and capable of either directly degrading its targeted messenger RNA (mRNA) or suppressing the translation of its targeted mRNA, depending on their mutual complementarity between miRNA and the targets. The mir-302 family (mir-302s) is a group of highly homologous miRNAs conserved in many mammals. Mir-302s consists of four members which are transcribed together as a non-coding RNA cluster containing mir-302b, mir-302c mir-302a, mir-302d and mir-367 in a 5′ to 3′ direction (Suh et al., 2004). Recently, the fifth mir-302 member was also found outside the familial cluster, namely mir-302e. Although mir-367 and mir-302s are co-expressed but their functions are actually different from each other, we prefer to re-design the mir-302 cluster for expressing only mir-302s. In addition, we also prefer to use manually re-designed hairpin loops, such as 5′-GCTAAGCCAG GC-3′ (SEQ.ID.NO.1) and 5′-GCCTGGCTTA GC-3′ (SEQ.ID.NO.2), to replace the original mir-302 precursor (pre-mir-302) loops for making a more compact cluster for easy delivery and expression. Normally, mir-302 is only expressed abundantly in mammalian embryonic stem (ES) cells, except mouse ES cells, and quickly decreased after cell differentiation and/or proliferation (Tang et al., 2007; Suh et al., 2004). Given that miRNA is characterized as small inhibitory RNAs capable of silencing its target genes with high complementarity (Bartel, D. P., 2004), mir-302s is likely a key inhibitor responsible for preventing errant and premature growth of ES cells during early embryogenesis, which may also prevent tumor formation from the stem cells. In fact, ES cells before the morula stage (32-64 cell stage) often present a very slow cell cycle rate. These findings suggest that mir-302 plays an important role in regulating normal stem cell maintenance and renewal, which may also help to inhibit tumor/cancer formation.
All mir-302 members share a totally identical sequence in their first 5′-seventeen (17) nucleotides, including the entire seed motif 5′-UAAGUGCUUC CAUGUUU-3′ (SEQ.ID.NO.3), and contain over 85% homology in their complete 23-nucleotide mature miRNA sequences. Based on the predicted results of on-line computing programs TARGETSCAN and PICTAR-VERT, these members currently and concurrently target against almost the same cellular genes, including over 607 human genes. In addition, mir-302 also shares many overlapping target genes with mir-93, mir-367, mir-371, mir-372, mir-373, and mir-520 familial members, which may provide certain similarity in functionality. Most of these target genes are developmental signals and transcriptional factors involved in initiation and/or establishment of lineage-specific cell differentiation during early embryogenesis (Lin et al., 2008). Many of these target genes are also well-known oncogenes. Therefore, the function of mir-302s is more likely to suppress the global production of developmental signals and differentiation-related transcription factors rather than to directly create transcriptional stimulation on certain embryonic signaling pathways like what the previous iPS methods did. Furthermore, since many of these targeted developmental signals and differentiation-related transcription factors are oncogenes, mir-302s likely functions as a tumor suppressor to prevent the deviation of normal hES cell growth into tumor/cancer formation.
In one preferred embodiment, the inventors have designed and developed an inducible pTet-On-tTS-miR302s expression vector (FIG. 1A) in conjunction with either viral infection, electroporation or liposomal/polysomal transfection methods to deliver mir-302 into normal and/or cancerous human cells. The redesigned mir-302 construct is consisting of four small non-coding RNA members: mir-302a, b, c and d in one cluster (mir-302s; FIG. 1B). The expression of this mir-302s construct is driven by a tetracycline-responsive-element (TRE)-controlled cytomegaloviral (CMV) promoter in response to doxycycline (Dox) stimulation. After infection/transfection, the expression of mir-302 followed the natural miRNA biogenesis pathway, in which the mir-302s construct is co-transcribed with a reporter gene such as red-shifted fluorescent protein (RGFP), and then further processed into individual mir-302 members by spliceosomal components and/or cytoplasmic RNaseIII Dicers (FIG. 2A) (Lin et al., 2003). As a result of this strategy, miRNA microarray analysis (Example 3) shows that all sense mir-302 members were efficiently expressed in transfected cells after Dox stimulation (FIG. 1C). The procedure for transducing the mir-302 expression vector into human cells is summarized in FIGS. 2B-C.
By mimicking the natural intronic miRNA biogenesis pathway (FIG. 2A), the inventors have devised an intronic miRNA expression system to transcribe a recombinant RGFP gene, namely SpRNAi-RGFP, which contains a man-made/artificial splicing-competent intron (SpRNAi) capable of producing intronic miRNA and/or shRNA-like gene silencing effectors (Lin et al., 2003; Lin et al., (2006) Methods Mol. Biol. 342: 295-312). The SpRNAi is co-transcribed within the pre-mRNA of the SpRNAi-RGFP gene by Pol-II RNA polymerases and cleaved out by RNA splicing components. Subsequently, the spliced SpRNAi is further processed into mature gene silencing effectors, such as native miRNAs and man-made shRNAs, so as to trigger specific RNA interference (RNAi) effects on target genes. Meanwhile, after intron splicing, the exons of the SpRNAi-RGFP gene transcript are linked together to form a mature mRNA for translation of a RGFP reporter protein useful for identifying the miRNA/shRNA expression. For quantification measurement, one fold RGFP concentration equaled to four folds the mir-302 concentration. Alternatively, some functional protein exons may be used in place of RGFP to provide additional gene functions, such as hES gene markers for somatic cell reprogramming. Given that there are currently over 1000 native miRNA species found in vertebrates without clear function and many more new miRNAs continue to be identified, our intronic miRNA expression system may also serve as a powerful tool for testing these miRNA functions in vitro and in vivo.
The SpRNAi intron contains several consensus nucleotide components, consisting of a 5′-splice site, a branch-point (BrP) motif, a poly-pyrimidine tract, and a 3′-splice site. In addition, a hairpin miRNA or shRNA precursor is inserted in between the 5′-splice site and the BrP motif. This portion of intron usually forms a lariat structure during RNA splicing and processing. Moreover, the 3′-end of SpRNAi contains a multiple translational stop codon region (T codon) to increase the accuracy of intronic RNA splicing and processing. When presented in a cytoplasmic mRNA, this T codon signals the activation of intracellular nonsense-mediated decay (NMD) system to degrade any unstructured RNA accumulated in the cell for preventing cytotoxicity. However, the highly structured shRNA and precursor miRNA (pre-miRNA) will be preserved for further Dicer cleavage to form mature siRNA and miRNA, respectively. For intronic miRNA/shRNA expression, we manually incorporate the SpRNAi in the DraII restriction site of a RGFP gene (Lin et al., 2006 and 2008). This forms a recombinant SpRNAi-RGFP gene. The cleavage of RGFP with DraII generates an AG-GN nucleotide break with three recessing nucleotides in each end, which will form 5′- and 3′-splice sites, respectively, after SpRNAi insertion. Because this intronic insertion disrupts the integrity of RGFP protein, which can be recovered by intron splicing, we are able to determine the expression of mature miRNA/shRNA through the appearance of red RGFP in the transfected cells. The RGFP gene also contains multiple exonic splicing enhancers (ESEs) to increase RNA splicing accuracy and efficiency.
In details, the SpRNAi intron contains a 5′-splice site homologous to either 5′-GTAAGAGK-3′ (SEQ.ID.NO.4) or GU(A/G)AGU motifs (SEQ.ID.NO.35) (i.e. 5′-GTAAGAGGAT-3′ (SEQ.ID.NO.36), 5′-GTAAGAGT-3′ (SEQ.ID.NO.37), 5′-GTAGAGT-3′ (SEQ.ID.NO.38) and 5′-GTAAGT-3′ (SEQ.ID.NO.39)), while its 3′-end is a 3′-splice site that is homologous to either GWKSCYRCAG (SEQ.ID.NO.5) or CT(A/G)A(C/T)NG (SEQ.ID.NO.40) motifs (i.e. 5′-GATATCCTGCAG-3′ (SEQ.ID.NO.41), 5′-GGCTGCAG-3′ (SEQ.ID.NO.42) and 5′-CCACAG-3′ (SEQ.ID.NO.43)). Moreover, a branch point sequence is located between the 5′- and 3′-splice sites, containing high homology to 5′-TACTWAY-3′ (SEQ.ID.NO.6) motifs, such as 5′-TACTAAC-3′ (SEQ.ID.NO.44) and 5′-TACTTAT-3′ (SEQ.ID.NO.45). The adenosine “A” nucleotide of the branch-point sequence can form a part of (2′-5′)-linked lariat intron RNA by cellular (2′-5′)-oligoadenylate synthetases and spliceosomes in almost all spliceosomal introns. Furthermore, a poly-pyrimidine tract is closely located between the branch-point and 3′-splice site, containing a high T or C content sequence homologous to either 5′-(TY)m(C/−)(T)nS(C/−)-3′ (SEQ.ID.NO.7) or 5′-(TC)nNCTAG(G/−)-3′ (SEQ.ID.NO.8) motifs. The symbols of “m” and “n” indicate multiple repeats ≧1; most preferably, the m number is equal to 1 ̃3 and the n number is equal to 7 ̃12. The symbol “−” refers a nucleotide that can be skipped in the sequence. There are also some linker nucleotide sequences for the connection of all these synthetic intron components. Based on the guideline of 37 CFR 1.822 for symbols and format to be used for nucleotide and/or amino acid sequence data, the symbol W refers to an adenine (A) or thymine (T)/uracil (U), the symbol K refers to a guanine (G) or thymine (T)/uracil (U), the symbol S refers to a cytosine (C) or guanine (G), the symbol Y refers to a cytosine (C) or thymine (T)/uracil (U), the symbol R refers to an adenine (A) or guanine (G), and the symbol N refers to an adenine (A), cytosine (C), guanine (G) or thymine (T)/uracil (U).”
In another preferred embodiment, the present invention is a direct (exonic) mir-302 miRNA/shRNA expression system, which can be used for generating mir-302-like gene silencing effectors directly from the expression system without going through intracellular RNA splicing and/or NMD mechanisms. However, the drawback of this method is that the expression of mir-302-like gene silencing effectors is not regulated by any intracellular surveillance system, such as NMD, and may therefore over-saturate the natural miRNA biogenesis pathway to cause cytotoxicity (Grimm et al., 2006). The expression system used for this method can be a linear or circular nucleic acid composition selected from the group of plasmid, viral vector, lentiviral vector, transposon, retrotransposon, jumping gene, protein-coding gene, non-coding gene, artificially recombinant transgene, and a combination thereof. The mir-302-like gene silencing effectors, including miRNA, shRNA, siRNA and their precursors as well as homologues/derivatives, are expressed under the control of a tissue-specific or non-specific RNA promoter selected from the group consisting of type-II RNA polymerase (Pol-II), viral polymerase, type-III RNA polymerase (Pol-III), type-I RNA polymerase (Pol-I), and tetracycline responsive element-controlled RNA polymerase (TRE) promoters. The viral promoters are Pol-II-like RNA promoters isolated but not limited from cytomegalovirus (CMV), retrovirus long-terminal region (LTR), hepatitis B virus (HBV), adenovirus (AMV), and adeno-associated virus (AAV). For example, a lentiviral LTR promoter is sufficient to produce up to 5×105 copies of pre-mRNA transcripts per cell. It is also feasible to insert a drug-sensitive repressor (i.e. tTS) in front of the RNA polymerase promoter in order to control the transcription rate of the gene silencing effectors. The repressor can be inhibited by a chemical drug or antibiotics selected from the group of G418, neomycin, tetracycline, doxycycline, ampicillin, kanamycin, puromycin, and their derivatives, etc.
In another aspect, multiple transgenes and/or vectors expressing various intronic gene silencing effectors may be used to achieve gene silencing on the mir-302-targeted genes. Alternatively, multiple gene silencing effectors may be generated from one intronic insert. For example, it has been reported that the ectopic expression of one anti-EGFP pre-miRNA-containing intron in zebrafish generates two different size miRNAs, namely miR-EGFP (282/300) and miR-EGFP (280-302), indicating that one insert of the SpRNAi may generate multiple gene-silencing effectors (Lin et al. (2005) Gene 356: 32-38). In certain cases, intronic gene-silencing effectors can hybridize with a target gene transcript (i.e. mRNA) to form double-stranded siRNAs for triggering secondary RNA interference (RNAi) effects. Because these gene-silencing effectors are constantly produced from the transgene vector, it will alleviate the concerns of fast RNA degradation in vivo. The advantage of this strategy is in its stable delivery through the vector-based transgene transfection or viral infection, providing a reliable long-term gene silencing efficacy.
Because the stem-loop structures of some native pre-miRNAs are too large and/or complicated to fit in a miRNA expression system/vector, the inventors often use a manually re-designed tRNAmet loop (i.e. 5′-(A/U)UCCAAGGGGG-3′ (SEQ.ID.NO.46)), to replace the native pre-miRNA loops. The tRNAmet loop has been shown to efficiently facilitate the export of manually re-designed miRNAs from nucleus to cytoplasm through the same Ran-GTP and Exportin-5 transporting mechanisms as native miRNAs do (Lin et al., 2005). Advantageously, the present invention now uses a pair of manually improved pre-mir-302 loops, including 5′-GCTAAGCCAG GC-3′ (SEQ.ID.NO.1) and 5′-GCCTGGCTTA GC-3′ (SEQ.ID.NO.2), which provide the same nuclear export efficiency as the native pre-miRNAs but not interfere with the tRNA exportation. Also, this improvement enhances the formation of mir-302a-mir-302a* and mir-302c-mir-302c* duplexes, which may increase the overall function and stability of mir-302s. The design of these new pre-miRNA loops is modified by the combination of the tRNAmet loop and the short stem-loops of mir-302b/mir-302a, which are highly expressed in human ES cells but not in other differentiated tissue cells. Thus, the use of these recombinant/man-made/artificial hairpin loops in the structure of mir-302 will not interfere with the native miRNA pathway in human body, resulting in a much less cytotoxicity and more safety.
The cluster of familial mir-302 pre-miRNAs is formed by hybridization and linkage/ligation of synthetic mir-302 homologues, consists of four parts: mir-302a, mir-302b, mir-302c and mir-302d pre-miRNAs in a 5′ to 3′ direction (FIG. 1B). All of these manually re-designed mir-302 miRNA/shRNA molecules possess an identical 5′-end in their first 17 nucleotides [e.g. 5′-UAAGUGCUUC CAUGUUU-3′ (SEQ.ID.NO.3)]. Synthetic oligonucleotides used for DNA recombination of the mir-302 pre-miRNA cluster are listed: including mir-302a-sense, 5′-GTCACGCGTT CCCACCACTT AAACGTGGAT GTACTTGCTT TGAAACTAAA GAAGTAAGTG CTTCCATGTT TTGGTGATGG ATAGATCTCT C-3′ (SEQ.ID.NO.9); mir-302a-antisense, 5′-GAGAGATCTA TCCATCACCA AAACATGGAA GCACTTACTT CTTTAGTTTC AAAGCAAGTA CATCCACGTT TAAGTGGTGG GAACGCGTGA C-3′ (SEQ.ID.NO.10); mir-302b-sense, 5′-ATAGATCTCT CGCTCCCTTC AACTTTAACA TGGAAGTGCT TTCTGTGACT TTGAAAGTAA GTGCTTCCAT GTTTTAGTAG GAGTCGCTCA TATGA-3′ (SEQ.ID.NO.11); mir-302b-antisense, 5′-TCATATGAGC GACTCCTACT AAAACATGGA AGCACTTACT TTCAAAGTCA CAGAAAGCAC TTCCATGTTA AAGTTGAAGG GAGCGAGAGA TCTAT-3′ (SEQ.ID.NO.12); mir-302c-sense, 5′-CCATATGGCT ACCTTTGCTT TAACATGGAG GTACCTGCTG TGTGAAACAG AAGTAAGTGC TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3′ (SEQ.ID.NO.13); mir-302c-antisense, 5′-ATGTCTAGAC GCCTCCACTG AAACATGGAA GCACTTACTT CTGTTTCACA CAGCAGGTAC CTCCATGTTA AAGCAAAGGT AGCCATATGG-3′ (SEQ.ID.NO.14); mir-302d-sense, 5′-CGTCTAGACA TAACACTCAA ACATGGAAGC ACTTAGCTAA GCCAGGCTAA GTGCTTCCAT GTTTGAGTGT TCGCGATCGC AT-3′ (SEQ.ID.NO.15); and mir-302d-antisense, 5′-ATGCGATCGC GAACACTCAA ACATGGAAGC ACTTAGCCTG GCTTAGCTAA GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3′ (SEQ.ID.NO.16). Alternatively, we may use the manually re-designed shRNA formed by the hybrid of synthetic miR-302s-sense, 5′-GCAGATCTCG AGGTACCGAC GCGTCCTCTT TACTTTAACA TGGAAATTAA GTGCTTCCAT GTTTGAGTGG TGTGGCGCGA TCGATATCTC TAGAGGATCC ACATC-3′ (SEQ.ID.NO.17) and mir-302s-antisense, 5′-GATGTGGATC CTCTAGAGAT ATCGATCGCG CCACACCACT CAAACATGGA AGCACTTAAT TTCCATGTTA AAGTAAAGAG GACGCGTCGG TACCTCGAGA TCTGC-3′ (SEQ.ID.NO.18), in place of the mir-302 pre-miRNA cluster for easy intronic insertion. The mir-302 shRNA shares over 85% homology to all native mir-302 members and targets the same cellular genes in human. In design of mir-302 homologues, thymine (T) can be used in place of uracil (U) or vice versa.
For intronic insertion of the mir-302 pre-miRNA/shRNA, given that the insertion site of the recombinant SpRNAi-RGFP transgene is flanked with a PvuI and an MluI restriction/cloning site at its 5′- and 3′-ends, respectively, the primary insert can be easily removed and replaced by various pre-miRNA/shRNA inserts (e.g. mir-302 pre-miRNA/shRNA), which possess matched cohesive ends to the PvuI and an MluI restriction sites. By changing the intronic inserts directed against various gene transcripts, the present invention of the intronic mir-302s expression system can be used as a powerful tool for inducing targeted gene silencing in vitro, ex vivo and in vivo. After intronic insertion, the mir-302-inserted SpRNAi-RGFP transgene is further inserted into the restriction/cloning site (i.e. a XhoI-HindIII site) of a Dox-inducible pSingle-tTS-shRNA vector to form a pTet-On-tTS-mir302s expression vector for intracellular expression (FIG. 1A).
Delivery of the mir-302-expressing nucleic acid composition into human cells can be accomplished using a non-transgenic or transgenic method selected from the group of liposomal/polysomal/chemical transfection, DNA recombination, electroporation, gene gun penetration, transposon/retrotransposon insertion, jumping gene integration, micro-injection, viral infection, retroviral/lentiviral infection, and a combination thereof. To prevent the risks of random transgene insertion and cell mutation, the inventors preferably use liposomal or polysomal transfection to deliver the pTet-On-tTS-mir302s vector into the targeted human cells (i.e. tumor/cancer cells). The expression of mir-302s is dependent on the activation of the TRE-regulated CMV promoter of the pTet-On-tTS-mir302s vector, in the presence of various Dox concentrations. Therefore, the present invention provides an inducible mechanism by a defined drug (i.e. Dox) to control the expression of mir-302s in vitro, ex vivo and/or in vivo, which serves as a second safeguard in addition to the intracellular NMD system. As a result of such Dox-mediated control, we did not observe any cytotoxicity of RNA accumulation or over-saturation in the treated cells. Alternatively, the present invention is a constitutive mir-302 expression system capable of consistently expressing the mir-302-like gene silencing effectors for a certain period of time. Preferably, the expression of mir-302-like gene silencing effectors is driven by a CMV promoter, which is often silenced after about one-month activation in human cells due to DNA methylation. Such a one-month activation mechanism is beneficial for cancer therapy to prevent RNA accumulation or over-saturation in the treated cells.
In sum, the present invention has adopted a novel design and strategy for either inducible or constitutive expression of mir-302-like gene silencing effectors in the transfected cells. Mir-302-like gene silencing effectors include mir-302a, mir-302b, mir-302c, mir-302d, and their hairpin-like microRNA precursors (pre-miRNAs) as well as manually re-designed small hairpin RNA (shRNA) homologues/derivatives, and a combination thereof. In one preferred embodiment, the present invention provides a design and method for using a recombinant nucleic acid composition capable of being delivered, transcribed and processed into mir-302-like gene silencing effectors in targeted human cells and thus inducing specific gene silencing effects on mir-302-targeted cell cycle regulators and oncogenes in the cells, comprising the steps of: a) providing a recombinant nucleic acid composition capable of being delivered, transcribed and processed into at least a gene silencing effector interfering a plurality of cellular genes targeted by mir-302, and b) treating a cell substrate with said recombinant nucleic acid composition. The transcription of mir-302-like gene silencing effectors is driven either by a constitutive (i.e. CMV) or drug-inducible (i.e. TRE-CMV) promoter. Preferably, the drug-inducible recombinant nucleic acid composition is a Tet-On vector containing a recombinant transgene inserted with either a recombinant mir-302 family cluster (mir-302s; hybrid of SEQ.ID.NOs.9-16) or a manually re-designed mir-302 shRNA homologue (i.e. hybrid of SEQ.ID.NOs.17 and 18). The cell substrate may express the mir-302 target genes either in vitro, ex vivo or in vivo. By silencing the mir-302-targeted cell cycle regulators and oncogenes, the present invention is able to suppress cell tumorigenecity and reprogram the treated cells into non-tumor/cancer cells.
Using the present invention, the inventors have gathered evidence for the success of mir-302-mediated tumor/cancer therapy in six areas: First, transfection of mir-302 in normal human cells (mirPS-hHFC) causes cell cycle attenuation but not apoptosis nor cell death (FIGS. 1D-F and FIGS. 3A-C). Second, transfection of mir-302 in human normal cells is able to reprogram the cells into a stem cell-like state, which is beneficial for healing damaged tissues (FIGS. 4A-B and FIGS. 5A-D). Third, transfection of mir-302 in human tumor/cancer cells (mirPS-MCF7, mirPS-HepG2 and mirPS-NTera2) strongly inhibits tumor/cancer cell tumorigenecity and causes >98% cell death or apoptosis (FIGS. 6A-E). Fourth, mir-302 can inhibit tumor/cancer cell tumorigenecity not only through co-suppression of multiple cell cycle regulators, such as CDK2, cyclin D1/D2 and BMI-1, but also activation of tumor suppressors, p16INK4a and p14/p19Arf (FIGS. 7A-D). Fifth, In vivo delivery of mir-302 into tumors can inhibit >90% tumor cell growth (FIGS. 8A-C). Last, mir-302 does not cause cell senescence through telomere shortening (FIGS. 9A-C). Furthermore, the inventors have successfully used polysome/liposome-based transfection to deliver the mir-302-like gene silencing effectors into targeted tumor cells in vivo, preventing the risks of retroviral infection and transgenic mutation (FIGS. 8A-C). These findings provide strong evidence for using mir-302-like gene silencing effectors as therapeutic drugs and/or vaccines for tumor/cancer therapy. Given that mir-302 also functions to rejuvenate human cell stemness (Lin et al., 2008), the present invention may provide beneficial applications in both stem cell and cancer therapies.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the USPTO upon request and payment of the necessary fee.
Referring particularly to the drawings for the purpose of illustration only and not limitation, there is illustrated:
FIGS. 1A-F show inducible mir-302 expression and its effect on normal human hair follicle cell (hHFC) proliferation. (A) Construct of the Dox-inducible pTet-On-tTS-miR302s vector. (B) Structure of the mir-302 familial cluster (mir-302s). (C) MiRNA microarray analysis of induced mir-302 expression at 6 hours after 10 μM Dox treatment (n=3, p<0.01). (D) Northern and western blot analyses of the dose-dependent mir-302 effect on the expression patterns of core reprogramming factors Oct3/4-Sox2-Nanog and melanocytic marker genes TRP1 and cytokeratin 16 (n=5, p<0.01). (E) Bar charts of flow cytometry analyses showing the dose-dependent mir-302 effect on the changes of mitotic (M phase) and dormant (G0/G1 phase) mirPS-hHFC populations. (F) Mir-302-induced apoptotic DNA laddering effects after treatment of 10 μM Dox in various mirPS cell lines.
FIGS. 2A-C depict the biogenesis of mir-302s and generation of mirPS cells. (A) Mechanism of intronic mir-302 biogenesis. The mir-302 familial cluster was transcribed with a gene encoded for red fluorescent protein (RGFP) and then further spliced into individual mir-302 members by spliceosomal components and cytoplasmic RNaseIII Dicers, while the RGFP served as a indicator for mir-302 production. One fold RGFP concentration equaled to four folds the mir-302 concentration. (B)-(C) Schematic procedure for mir-302 transfection with liposome/polysome/electroporation. The inducible mir-302-expressing pTet-On-tTS-miR302s vector (FIG. 1A) was transduced into adult hHFC by electroporation at 300-400 volts for 150 μsec in a hypo-osmolar PH buffer (200 μl; Eppendorf). In each test, 10 μg of the pTet-On-tTS-miR302s vector was used to transfect 200,000 cultured hHFC derived from as few as two human hair follicles (dermal papillae). After doxycycline (Dox)-induced expression, the biogenesis of mir-302 relied on the natural intronic miRNA pathway.
FIGS. 3A-C show the changes of mirPS-hHFC cell properties by Dox-induced mir-302 expression (Dox=5 or 10 μM). (A)-(B) Changes of cell morphology and cell cycle rate before and after Dox-induced reprogramming. Each cell DNA content respective to cell cycle stages was shown by a chart of flow cytometry analysis above the cell morphology (n=3, p<0.01). The first (left) and second (right) peaks of the charts represented the ratios of resting G0/G1 and mitotic M phase cells in the entire tested cell population, respectively. Scale bars=100 μm. (C) Time-course formation of embryoid body (EB) from a single mirPS-hHFC cell after limiting dilution. The cell cycle was estimated to be approximately 20-24 hours at start but gradually accelerated after 72 hours. Scale bars=100 μm.
FIGS. 4A-B show analysis of pluripotent marker expression and in vivo pluripotent differentiation/assimilation. (A) Northern blot analysis of hES marker gene expression patterns induced by a high mir-302 concentration in mirPS cells compared to those in hES WA01-H1 (H1) and WA09-H9 (H9) cells (n=5, p<0.01). After treatment of 7.5 μM Dox, mir-302 concentration was raised to over 30% higher than that of H1 and H9 cells (>30 fold higher than untreated hHFCs) and began inducing co-expression of the major pluripotent markers Oct3/4, Sox2, Nanog, Lin28 and undifferentiated embryonic cell transcription factor 1 (UTF1). (B) Assimilation of mirPS cell-differentiated tissues into the surrounding tissues around the injection sites of immunocompromised SCID-beige mice one week after the implantation. White arrows indicated the direction of injection. Intercalated disks were marked by yellow triangles. The mirPS-hHFC-derived tissue cysts were not grown in mouse organs/tissues other than uterus and peritoneal cavity. In view of this, we further dissected and examined the surrounding tissue formation around the injection sites one week after the implantation. One day prior to dissection, the mice were treated with 10 μg Dox by tail vein injection. We observed that the RGFP-positive mirPS cells differentiated into the same cell types as the surrounding tissues in the implanted sites, including gut epithelium after intraperitoneal injection, cardiac muscle after heart puncture, and skeleton muscle by dorsal flank injection. Not only that, the assimilated mirPS cells also expressed the same tissue-specific makers as the surrounding tissues, such as MUC2 for gut epithelium, troponin T type 2 (cTnT) for cardiac muscle, and myosin heavy chain (MHC) for skeleton muscle.
FIGS. 5A-D show gain of hES-like properties in Dox-induced mirPS-hHFCs (Dox=10 μM). (A) Analysis of global gene expression patterns before and after mir-302-induced somatic cell reprogramming (SCR), using human genome GeneChip U133 plus 2.0 arrays (Affymetrix; n=3, p<0.01-0.05). (B) HpaII cleavage showing the vast loss of global CpG methylation, identified by increased presence of smaller DNA fragments, at a genome-wide scale in mirPS cells treated with 10 μM but not 5 μM Dox. (C) Bisulfite DNA sequencing in the promoter regions of Oct3/4 and Nanog, showing the detailed methylation maps. Black and white circles indicate the methylated and unmethylated cytosine sites, respectively. (D) Pluripotent differentiation of mirPS-hHFCs into teratoma-like tissue cysts, containing various tissues derived from all three embryonic germ layers.
FIGS. 6A-E show in vitro tumorigenecity assays of various tumor/cancer-derived mirPS cells in response to mir-302 expression induced by 10 μM Dox. (A)-(B) Changes of cell morphology and cell cycle rate before and after Dox-induced mir-302 expression. Each cell DNA content respective to cell cycle stages was shown by a chart of flow cytometry analysis above the cell morphology (n=3, p<0.01). (C) Bar charts of flow cytometry analyses showing the dose-dependent mir-302 effect on the changes of mitotic (M phase) and dormant (G0/G1 phase) cell populations of various tumor/cancer-derived mirPS cells. (D) Functional analysis of mir-302—suppressed tumor invasion in Matrigel chambers (n=4, p<0.05). (E) Comparison of cell adhesion to the hBMEC monolayer before and after Dox-induced mir-302 expression (n=4, p<0.05).
FIGS. 7A-D show luciferase 3′-UTR reporter assays of mir-302-induced gene silencing effects on targeted G1-checkpoint regulators. (A) Constructs of the luciferase 3′-UTR reporter genes, which carry either two normal (T1+T2) or two mutant (M1+M2), or a mixture of both (T1+M2 or M1+T2), mir-302 target sites in the 3′-UTR. The mutant sites contained a mismatched TCC motif in place of the uniform 3′-CTT end of the normal target sites. (B) Effects of Dox-induced mir-302 on the luciferase expression (n=5, p<0.01). Dox=5 or 10 μM. CCND1 and CCND2 refer to cyclin D1 and D2, respectively. (C) and (D) Western blot analyses showing the changes of major mir-302-targeted G1-checkpoint regulator induced by high (10 μM Dox) and low (5 μM Dox) mir-302 concentrations in mirPS cells compared to those found in hES H1 and H9 cells (n=4, p<0.01).
FIGS. 8A-C show In vivo tumorigenecity assays of mirPS-NTera2 cells in response to either constitutive mir-302s (NTera2+mir-302s) or mir-302d* (NTera2+mir-302d*) expression (n=3, p<0.05). Mir-302s and mir-302d* were transcribed from the pCMV-miR302s and pCMV-miR302d* vectors in the transfected neoplastic Tera-2 (NTera2) cells, respectively. (A) Morphological evaluation of average tumor sizes three weeks after the in-situ injection (post-is). All tumors were localized in the original implant sites (black arrows). No signs of cachexia or tumor metastasis were observed in all tested mice. (B) Northern and western blot analyses and (C) Immunohistochemical staining analyses of the in vivo mir-302 effect on the expression patterns of core reprogramming factors Oct3/4-Sox2-Nanog and mir-302-targeted G1-checkpoint regulators CDK2, cyclins D1/D2 and BMI-1 as well as p16Ink4a and p14Arf.
FIGS. 9A-C show analyses of telomerase activity in mirPS-hHFC and various tumor/cancer-derived mirPS cell lines in response to the mir-302 expression induced by 10 μM Dox. (A) Telomerase activities shown by TRAP assays (n=5, p<0.01). Telomerase activity was sensitive to RNase treatment (hHFC+RNase). (B) Western blotting confirming the consistent increase of hTERT and decrease of AOF2 and HDAC2 expression in various mirPS cell lines (n=5, p<0.01). (C) Telomerase activities measured by telomerase PCR ELISA assays (OD470-OD680; n=3, p<0.01).
FIGS. 10A-D show analyses of mir-302-induced silencing effects on its targeted epigenetic genes. (A) Constructs of the luciferase 3′-UTR reporter genes, which carry either two normal (T1+T2) or two mutant (M1+M2), or a mixture of both (T1+M2 or M1+T2), mir-302 target sites in the 3′-UTR. The mutant sites contained a mismatched TCC motif in place of the uniform 3′-CTT end of the normal target sites. (B) Effects of Dox-induced mir-302 on the luciferase expression (n=5, p<0.01). (C) and (D) Western blot analyses showing the changes of major mir-302-targeted epigenetic gene expression induced by high (10 μM Dox) and low (5 μM Dox) mir-302 concentrations in mirPS cells compared to those found in hES H1 and H9 cells (n=4, p<0.01).
FIG. 11 depicts the proposed mechanism of mir-302-mediated SCR and cell cycle regulation. Based on our previous and current studies, two collateral events were discovered. First, reprogramming is initiated by strong silencing of multiple epigenetic regulators AOF1/2, MECP1/2 and HDAC2, leading to global genomic DNA demethylation, whereby re-activating hES cell marker genes essential for SCR induction (marked in gray). Second, cell cycle attenuation is caused by co-suppression of G1-checkpoint regulators CDK2, cyclins D1/D2 and BMI-1 as well as activation of p16Ink4a and p14/p19Arf to quench all cellular activities ready for SCR (marked in black). Quiescence at this dormant G0/G1 state also prevents the possible random growth and/or tumor-like transformation of the reprogrammed pluripotent stem cells. Collectively, the synergistic effect of these two events results in a more accurate and safe reprogramming process, by which pre-mature cell differentiation and tumorigenecity are both inhibited.



DETAILED DESCRIPTION OF THE INVENTION
Although specific embodiments of the present invention will now be described with reference to the drawings, it should be understood that such embodiments are by way of example only and merely illustrative of but a small number of the many possible specific embodiments which can represent applications of the principles of the present invention. Various changes and modifications obvious to one skilled in the art to which the present invention pertains are deemed to be within the spirit, scope and contemplation of the present invention as further defined in the appended claims.
The present invention provides a novel nucleic acid composition and method for inhibiting the proliferation and tumorigenecity of human tumor/cancer cells, using recombinant mir-302-like gene silencing effectors. Unlike previous shRNA designs, the presently invented shRNAs may contain a mismatched stem-arm similar to the precursors of native mir-302 (pre-mir-302). Further, the presently invented shRNAs may also contain an improved pre-mir-302 stem-loop, such as 5′-GCTAAGCCAG GC-3′ (SEQ.ID.NO.1) and 5′-GCCTGGCTTA GC-3′ (SEQ.ID.NO.2), which can provide the same nuclear export efficiency as native pre-miRNAs but not interfere with the tRNA exportation. Without being bound by any particular theory, such an anti-proliferative and anti-tumorigenetic effect of the present invention is directed to a newly discovered mir-302-mediated gene silencing mechanism, triggered by transfection of a recombinant nucleic acid composition capable of expressing either a mir-302 family cluster (mir-302s) or a mir-302-homologous shRNA. All of the manually re-designed mir-302 miRNA/shRNA molecules possess an identical 5′-end in their first 17 nucleotides, 5′-UAAGUGCUUC CAUGUUU-3′ (SEQ.ID.NO.3). The protocols for constructing the mir-302-like gene silencing effectors and the nucleic acid composition expressing mir-302 are described in Examples 2 and 3. In design of sequences homologous to mir-302, thymine (T) can be used in place of uracil (U).
To address the mechanistic role of mir-302 in human cell cycle, we designed an inducible pTet-On-tTS-miR302s expression vector (FIG. 1A; Example 2) to transfect normal and cancerous human cells. Mir-302 is a hES-specific microRNA (miRNA) family that consists of four small non-coding RNA members, mir-302b, c, a, and d, in one cluster (mir-302s; FIG. 1B) (Suh et al., 2004). In our design, the expression of mir-302s was driven by a tetracycline-response-element (TRE)-controlled cytomegaloviral (CMV) promoter in response to doxycycline (Dox) stimulation. After transfection, the biogenesis of mir-302 followed the natural intronic miRNA pathway, in which mir-302s was transcribed with a reporter gene encoded for red fluorescent protein (RGFP) and then further spliced into individual mir-302 members by spliceosomal components and cytoplasmic RNaseIII Dicers (FIG. 2A) (Lin et al., 2008). For quantification measurement, one fold RGFP concentration equaled to four folds the mir-302 concentration. MiRNA microarray analysis confirmed that all mir-302 members except mir-302b* were efficiently expressed in transfected cells after Dox stimulation (FIG. 1C; Example 3). The procedure for transfecting cells with the pTet-On-tTS-miR302s expression vector is summarized in FIGS. 2B-C.
Moreover, the mir-302-expressing nucleic acid composition, such as pTet-On-tTS-miR302s, may contain a Kozak consensus translation initiation site to increase translation efficiency in eukaryotic cells, multiple SV40 polyadenylation signals downstream of the mir-302-expressing construct, a pUC origin of replication for propagation in prokaryotic cells, at least two restriction sites for incorporation of the mir-302-expressing construct (i.e. SpRNAi-RGFP) into the nucleic acid composition, an optional SV40 origin for replication in mammalian cells that express the SV40 T antigen, and an optional SV40 early promoter for expressing an antibiotic resistance gene in replication-competent prokaryotic cells. The expression of antibiotic resistance genes is used to serve as a selective marker for isolating positive clones with the transgene expression. The antibiotics are selected from the group consisted of G418, neomycin, puromycin, penicillin G, ampicillin, kanamycin, streptomycin, erythromycin, spectromycin, phophomycin, tetracycline, doxycycline, rifapicin, amphotericin B, gentamycin, chloramphenicol, cephalothin, tylosin, and a combination thereof.
Mir-302 Attenuates the Normal Cell Cycle Rate without Causing Apoptosis
Our previous studies have shown that increasing mir-302 expression in human melanoma Colo-829 and prostate cancer PC3 cells reprogrammed these malignant cancer cells into a hES-like pluripotent state (Lin et al., 2008). During this somatic cell reprogramming (SCR) process, mir-302 caused apoptosis in >98% of the cancer cell population and greatly reduced the proliferation rate of the remaining (<2%) reprogrammed cells. Although this feature may benefit cancer therapy, it is uncertain how mir-302 functions in normal human cells. To evaluate this effect, we introduced the inducible pTet-On-tTS-miR302s expression vector into normal human hair follicle cells (hHFCs). hHFCs were chosen due to their abundance, accessibility and fast growth. Following an increase of Dox concentration up to 10 μM, we observed that the core reprogramming factors Oct4-Sox2-Nanog were concurrently stimulated by a threshold of Dox>7.5 μM (FIG. 1D; Example 5) and meanwhile the proliferative cell population was reduced by 70% from 37%±2% to 11%±2% (FIG. 1E, M phase; Example 7). Accordingly, the dormant cell population was increased by 41% from 56%±3% to 79%±5% (FIG. 1E, G0/G1 phase; Example 7), reflecting a strong anti-proliferative effect similar to our previous observation in mir-302-reprogrammed pluripotent stem cells (mirPS cells; Lin et al., 2008). However, the mir-302-reprogrammed hHFC cells (mirPS-hHFC) did not display any detectable sign of apoptotic DNA laddering or cell death (FIG. 1F; Example 6), indicating that normal cells are more tolerable than tumor/cancer cells to the anti-proliferative effect of mir-302. It is conceivable that tumor/cancer cells are very difficult to survive in such a dormant state due to their high metabolism and rapid growth expansion.
Most notably, when treated with Dox>7.5 μM, mirPS-hHFC morphology was changed from a spindle to a sphere shape, resembling a dormant cell (FIGS. 3A-B, red RGFP-positive cells). The cellular mir-302 concentration stimulated by 7.5 μM Dox was approximately 1.3 folds the level in the hES H1 and H9 cells (FIG. 4A; Example 4). At this higher level, mirPS-hHFCs strongly expressed Oct3/4, Sox2, Nanog, and other standard hES cell markers (FIG. 4A). Microarray analysis of global gene expression further showed that approximately half of the transcriptome changed from a somatic hHFC mode to a uniform hES-like expression pattern sharing an average of >93% similarity to that of H1/H9 cells (FIG. 5A; Example 8). Global genomic DNA demethylation, the first sign of SCR initiation, was also clearly detected in these mirPS-hHFCs identical to those demethylation patterns in H1/H9 cells (FIGS. 5B and 5C; Example 9). Moreover, each individual mirPS-hHFC cell could grow into a single embryoid body-like colony and the cell division rate was 20-24 hours per cycle consistent with the anti-proliferative effect of mir-302 (FIG. 3C). We particularly noted that these mirPS-hHFCs were pluripotent but not tumorigenetic because they formed teratoma-like tissue cysts only in the uteruses and peritoneal cavities of pseudopregnant immunocompromised SCID-beige mice. These teratoma-like cysts contained various tissues derived from all three embryonic germ layers, ectoderm, mesoderm and definitive endoderm (FIG. 5D; Example 10). Alternatively when xenografted into normal male mice, these mirPS-hHFCs were assimilated by the surrounding tissues and presented the same tissue makers, demonstrating a possible use for healing damaged tissues (FIG. 4B). Taken together, these findings suggest that mir-302 can reprogram somatic hHFCs to hES-like iPS cells. Given that Oct3/4 and Sox2 are crucial transcription factors for mir-302 expression (Marson et al., 2008; Card et al., 2008), mir-302 may be used in place of Oct3/4-Sox2 for inducing SCR.
Mir-302-induced SCR and cell cycle attenuation are two collateral events, depending on the mir-302 concentration. We noted that both events occur almost simultaneously at a mir-302 concentration over 1.3 folds the level in the hES H1/H9 cells, indicating that this specific concentration is the minimal threshold for initiating both events. Previous studies using a single mir-302 member or at a lower concentration equal to the level in H1/H9 cells failed to elicit these events. Also, we have shown that the mir-302 concentration induced by a lower 5 μM Dox cannot silence either the target sites of the reporter gene or the targeted G1-checkpoint regulators. Compared to the natural development, early embryonic cells before the morula stage (32-64 cell stage) often present a very slow cell cycle rate similar to that of mirPS cells. However, such cell cycle regulation is not found in blastocyst-derived hES cells. It is presumable that a lower mir-302 concentration in hES cytoplasm may fail to silence the targeted G1-checkpoint regulators and oncogenes. This may also explain why blastocyst-derived hES cells have dramatic proliferative ability and tend to form tumors. Thus, the present invention may be applied to reduce the tumorigenecity of hES cells for stem cell therapy.
Mir-302 Inhibits Tumorigenecity and Causes Apoptosis in Various Tumor/Cancer Cells
In view of mir-302 function in causing cancer cell apoptosis and cell cycle attenuation, we then investigated the possibility of using mir-302 as a universal drug to treat human tumor/cancer cells. As our previous studies have shown the feasibility of this approach in melanoma and prostate cancer cells (Lin et al., 2008), we further tested human breast cancer MCF7, hepatocellular carcinoma Hep G2, and embryonal teratocarcinoma Tera-2 (NTera-2) cells in the present invention. As shown in FIGS. 6A-B, all three kinds of tumor/cancer cells were reprogrammed to dormant mirPS cells and formed embryoid body-like colonies after transfected with the pTet-On-tTS-miR302s vector stimulated by 10 μM Dox. Mir-302 at this level also induced significant apoptosis (>95%) in all three tumor/cancer cell types (FIG. 1F; Example 6). Flow cytometry analysis comparing DNA content to cell cycle stages, further showed a significant reduction in all of the mirPS mitotic cell populations (FIG. 6C; Example 7). The mitotic cell population (M phase) was decreased by 78% from 49%±3% to 11%±2% in mirPS-MCF7, by 63% from 46%±4% to 17%±2% in mirPS-HepG2, and by 62% from 50%±6% to 19%±4% in mirPS-NTera2 cells, whereas the resting/dormant cell population (G0/G1 phase) was increased by 80% from 41%±4% to 74%±5% in mirPS-MCF7, by 65% from 43%±3% to 71%±4% in mirPS-HepG2, and by 72% from 40%±7% to 69%±8% in mirPS-NTera2 cells, respectively. These results indicate that mir-302 can effectively attenuate the fast cell cycle rates and cause significant apoptosis in these tumor/cancer cells.
In vitro tumorigenecity assays, using Matrigel chambers (cell invasion assay) and cell adhesion to the human bone marrow endothelial cell (hBMEC) monolayer (cell adhesion assay), revealed two more anti-tumorigenetic effects of mir-302 in addition to its anti-proliferative feature. Cell invasion assay showed that all three dormant mirPS-tumor/cancer cells lost their ability to migrate (reduced to <1%) while the original tumor/cancer cells aggressively invaded into the chambered areas supplemented with higher nutrients, representing over 9%±3% of MCF7, 16%±4% of Hep G2 and 3%±2% of NTera-2 cell populations (FIG. 6D; Example 11). Consistently, cell adhesion assay also showed that none of these mirPS-tumor/cancer cells could adhere to hBMECs whereas a significant population of original MCF7 (7%±3%) and Hep G2 (20%±2%) cells quickly metastasize into the hBMEC monolayer after 50 min incubation (FIG. 6E; Example 12). In sum, all of the findings thus far strongly and repeatedly suggest that mir-302 is a human tumor suppressor capable of attenuating fast cell growth, causing tumor/cancer cell apoptosis, and inhibiting tumor/cancer cell invasion as well as metastasis. Most importantly, this novel mir-302 function may offer a universal treatment against multiple kinds of human cancers/tumors, including but not limited in malignant skin (Colo-829), prostate (PC-3), breast (MCF7) and liver (HepG2) cancers as well as various tumors in view of the variety of different tissue types in teratomas (NTera-2).
Mir-302-Mediated Anti-Proliferation Functions Through Co-Suppression of CDK2, Cyclins-D1/D2 and BMI-1
To validate the physical interactions between mir-302 and its targeted G1-checkpoint regulators, we used a luciferase 3′-untranslated region (3′-UTR) reporter assay (FIG. 7A; Example 15), which showed that treatments with various mir-302 concentrations resulted in very different inhibitory effects on the targeted G1-checkpoint regulators, including CDK2, cyclins-D1/D2 and BMI1 polycomb ring finger oncogene (BMI-1). In the presence of 10 μM Dox, mir-302 effectively bound to the target sites of CDK2, cyclins D1/D2 and BMI-1 transcripts and successfully silenced >80% of the reporter luciferase expression in all targets (FIG. 7B; Example 15). Suppression of the real target genes in mirPS cells was also confirmed by western blot analyses consistent with the results of the luciferase 3′-UTR reporter assay (FIG. 7C; Example 5). In contrast, a lower mir-302 expression induced by 5 μM Dox failed to trigger any significant silencing effect on either the target sites of the reporter gene or the targeted G1-checkpoint regulators, except cyclin D2 (FIGS. 7B and 7D), indicating that mir-302 fine-tunes the cell cycle rate in a dose-dependent manner. Given that the G1-S transition of mammalian cell cycle is normally controlled by two compensatory cyclin-CDK complexes, cyclin-D-CDK4/6 and cyclin-E-CDK2 (Berthet et al., 2006), we found that high concentrated mir-302 inactivated both complexes through simultaneous suppression of CDK2 and cyclins D1/D2, thus blocking both G1-S transition pathways and attenuating the cell cycle rate of the reprogrammed mirPS cells. In hHFCs and mirPS cells, cyclin D3 was expressed at a limited level insufficient to compensate the loss of cyclins D1/D2 in the mirPS cells.
Accompanying BMI-1 silencing, we further detected a mild increase of p16Ink4a and p14Arf expression (gain 63%±17% and 57%±13% of the levels in hHFCs, respectively), whereas no change was found in p21Cip1 expression (FIG. 7C). Deficiency of BMI-1, an oncogenic cancer stem cell marker, has been shown to inhibit G1-S transition through enhancement of p16Ink4a and p14Arf tumor suppressor activities (Jacobs et al., 1999). In this scenario, 16Ink4a directly inhibits cyclin-D-dependent CDK4/6 activity in the phosphorylation of retinoblastoma protein Rb and thus prevents Rb from releasing E2F-dependent transcription required for S phase entry (Parry et al., 1995; Quelle et al., 1995). In addition, p14Arf prevents HDM2 from binding to p53 and permits the p53-dependent transcription responsible for G1 arrest or apoptosis (Kamijo et al., 1997). However, because embryonic stem cells are known to exempt from cyclin-D-dependent CDK regulation (Burdon et al., 2002; Jirmanova et al., 2002; Stead et al., 2002), current understanding of cell cycle regulation in hES cells has implicated CDK2 as the main determinant of the G1-S transition. As a result, the silencing of CDK2 likely contributes most of the G1-arrest efficacy in mirPS cells, while the co-suppression of cyclin-D and BMI-1 as well as co-activation of p16Ink4a and p14Arf may provide additional inhibition against tumorigenetic signal-induced cell proliferation. Furthermore, the loss of cyclin-D and activation of p16Ink4a may also explain the deficiency of cyclin-D-dependent CDK activity in embryonic stem cells.
Therefore, the stringency of miRNA-target gene interaction determines the real function of the miRNA. Depending on the cellular condition, miRNA may present different preferences in gene targeting. To this, the present invention provides insight into these important details and for the first time reveals that mir-302 functions very differently in human and mouse cells. In humans, mir-302 strongly targets CDK2, cyclins D1/D2 and BMI-1, but interestingly, not p21Cip1. Unlike mouse p21Cip1, human p21Cip1 does not contain any target site for mir-302. This different gene targeting leads to a significant schism between respective cell cycle regulations. In mES cells, mir-302 silences p21Cip1 and promotes tumor-like cell proliferation (Wang et al., 2008; Judson, 2009), whereas p21Cip1 expression is preserved in human mirPS cells and may cause slower cell proliferation and lower tumorigenecity. Additionally, mouse BMI-1 is not a target gene for mir-302 either due to lack of a proper target site. We have shown that silencing of human BMI-1 in mirPS cells slightly stimulates p16Ink4a/p14ARF expression to attenuate cell proliferation, whereas mir-302 cannot silence mouse BMI-1 to raise the same effect. Since p16Ink4a/p14ARF were elevated while p21Cip1 was not affected in mirPS cells, the anti-proliferative and anti-tumorigenetic effects of mir-302 in human cells most likely goes through p16Ink4a-Rb and/or p14/19ARF-p53 pathways in addition to the co-suppression of cyclin-E-CDK2 and cyclin-D-CDK4/6 pathways. These distinct targeting preferences of mir-302 to human and mouse genes imply that the mechanisms underlying their cell cycle regulations are fundamentally different.
Treatment of Mir-302 Eliminates >90% of Tumor Cell Growth In Vivo without Changing Stem Cell Pluripotency
After identifying the tumor suppressor function of mir-302 and its different effects between normal and tumor/cancer cells, we tested the possible use of mir-302 as a drug for treating NTera2-derived teratomas in eight-week-old male athymic mice (BALB/c nu/nu strain) (Example 13). The neoplastic Tera-2 (NTera-2) cell line is a pluripotent human embryonal teratocarcinoma cell line that can differentiate into a variety of primitive somatic tissues in vivo, in particular primitive glandular and neural tissues (Andrews et al., 1984). Due to its pluripotency, NTera2-derived teratoma may serve as a model for treating various tumor types in vivo. For drug delivery, we adopted in situ injection of polyethylenimine (PEI)-formulated pCMV-miR302s expression vector in close proximity to the tumor site. The pCMV-miR302s vector was formed by changing the TRE-controlled CMV promoter to a regular CMV promoter (Example 2), of which the expression duration was approximately one month in human cells due to DNA methylation. By injecting up to 10 μg of the pCMV-miR302s vector per g mouse weight (the maximal amount of one shot injection in a mouse), we observed no signs of sickness or cachexia in the mice, indicating the safety of this approach. Histological examination also showed no detectable tissue lesions in brain, heart, lung, liver, kidney and spleen.
We detected a significant inhibitory effect on teratoma growth after five treatments (three-day intervals for each treatment) of 2 μg pCMV-miR302s vector (total 10 μg) per g mouse weight. As shown in FIG. 8A (Example 13), when treated with the pCMV-miR302s vector, the average size of NTera2-derived teratomas decreased by >89% (11±5 mm3, n=6) compared to that of non-treated ones (104±23 mm3, n=4). In contrast, treating the same amount of PEI-formulated antisense-mir-302d expression vector (pCMV-miR302e) increased the teratoma sizes by 140% (250±73 mm3, n=3). Based on that, NTera-2 cells were found to express a moderate level of mir-302 (FIG. 8B). Northern blotting also showed that mir-302 expression levels in these differently treated teratoma cells negatively correlated to the tumor sizes (FIG. 8B), suggesting that modulating mir-302 expression can effectively control the teratoma growth in vivo. To validate the previous findings in vitro, we performed western blotting to confirm the co-suppression of G1-checkpoint regulators CDK2-cyclins-D1/D2-BMI-1 and the co-activation of core reprogramming factors Oct3/4-Sox2-Nanog in the mir-302-treated teratomas (FIG. 8B). The same results were also confirmed by immunohistochemical (IHC) staining of these proteins in teratoma tissue sections (FIG. 8C; Example 14). Most noteworthily, we found that mir-302 inhibited teratoma cell growth without affecting its nature in pluripotent differentiation, indicating that high concentrated mir-302 plays a dual role as a tumor suppressor and a reprogramming factor. Based on this dual function of mir-302 and the consistent data in vitro and in vivo, we conclude that the same anti-proliferative mechanism of mir-302 observed in vitro can be applied to inhibit teratoma growth in vivo, which may serve as a potential treatment for a variety of tumors.
Mir-302 May Cause Cell Senescence Through p16Ink4a/p14ARF Activation Rather than Telomere Shortening
Human iPS cells have been reported to exhibit problems of early senescence and limited expansion (Banito et al., 2009; Feng et al., 2010). Normal adult cells also undergo a limited number of divisions and finally reach a quiescence state called replicative senescence. Cells that escape from replicative senescence often become immortal cells such as tumor/cancer cells; thus, replicative senescence is a normal defense mechanism against tumor/cancer cell formation. In this study, we have found that mir-302 can directly silence BMI-1 to induce p16Ink4a/p14ARF-associated cell cycle regulation. Other studies have further implicated that BMI-1 can also activate human telomerase reverse transcriptase (hTERT) transcription and increase telomerase activity to bypass replicative senescence and increase the cell life span (Dimri et al., 2002). Thus, it is conceivable that mir-302 overexpression may cause hTERT-associated senescence in mirPS cells. To clarify this point, we performed telomeric repeat amplification protocol (TRAP) assay (Example 16) to measure the telomerase activity. Surprisingly, as shown in FIG. 9A, all mirPS cells treated with 10 μM Dox exhibit a strong telomerase activity similar to that of their original tumor/cancer cells and hES H1/H9 cells. Moreover, western blotting also showed that hTERT expression was increased rather than decreased in these mirPS cells (FIG. 9B). The increase of relative telomerase activity was also confirmed by telomerase PCR ELISA assay (FIG. 9C). In addition, we further detected the silencing of lysine-specific histone demethylase AOF2 (also known as KDM1/LSD1) and histone deacetylase HDAC2 in these mirPS cells (FIG. 9B). Previous studies have reported that AOF2 is required for the transcriptional suppression of hTERT and deficiency of both AOF2 and HDAC2 induces hTERT overexpression (Won et al., 2002; Zhu et al., 2008). Our recent study has also found that both AOF2 and HDAC2 are strong targets of mir-302 and are both silenced in the mirPS cells (FIG. 10). Therefore, mir-302 actually increases telomerase activity rather than causes hTERT-associated senescence in mirPS cells. However, the effect of this increased hTERT activity may be counteracted by the mir-302-induced BMI-1 suppression and p16Ink4a/p14ARF activation, resulting in a balance for preventing tumor/cancer cell formation in mirPS cells.
In sum, our present invention utilizes a novel tumor suppressor function of mir-302 for cancer therapy. We found that mir-302-mediated cell cycle regulation involves a highly coordinate mechanism between co-suppression of G1-checkpoint regulators and activation of CDK inhibitors. All these genetic events must occur simultaneously to prevent any loophole for G1-S progression. Quiescence at G0/G1 phase cell cycle is important for SCR initiation. In this dormant state, somatic cell genomes can be largely demethylated and over 91% of the cellular transcriptome are reprogrammed to a hES-like gene expression pattern. Through deciphering the interactions between mir-302 and its target genes, we learned the intricate mechanism for mir-302-associated cell cycle regulation during SCR as shown in FIG. 11. Our previous studies have demonstrated that mir-302 silences its targeted epigenetic regulators to activate Oct3/4-Sox2-Nanog co-expression, and in turn these reprogramming factors function to induce SCR (Lin et al., 2008). In advance, this invention further reveals that mir-302 concurrently silences CDK2, cyclins D1/D2 and BMI-1 to attenuate cell division during SCR. Proper control of the cell cycle rate is of critical biological importance in preventing the tumorigenecity of oncogenes that are often activated during SCR. To this, mir-302 silences CDK2 and cyclins D1/D2 to hinder the G1-S transition at this critical moment. Meanwhile, inhibition of BMI-1 further enhances the tumor suppressor activities of p16Ink4a and p19Arf. Through these synergistic cell cycle regulation pathways, mir-302 is able to initiate SCR while not aggravating cell tumorigenecity.
Advantageously, there are at least five breakthroughs in the present invention. First, one mir-302-like gene effector can replace all four reprogramming transcription factors Oct4-Sox2-Klf4-c-Myc and Oct4-Sox2-Nanog-Lin28 for reprogramming human cells to hES-like stem cells. These reprogrammed cells are useful for stem cell therapy. Second, because of the small size (about 23 ribonucleotides) of a mir-302-like gene silencing effector, the vector expressing such a small sized RNA can be designed to be very compact and highly efficient for in vivo transfection. Third, the RNA-related cytotoxicity is prevented by intracellular NMD system and inducible expression. Fourth, mir-302-induced apoptosis only occurs in tumor/cancer cells rather than normal human cells. Last, the present invention has used polysomal, liposomal and electroporation-based transfection in place of retroviral/lentiviral infection to deliver the mir-302-expressing nucleic acid composition into tumor/cancer cells, confirming the safety and therapeutic use of the mir-302-like gene silencing effectors in vitro and in vivo. Taken together, these advantages have shown the feasibility of using the mir-302-like gene silencing effector and its expression composition for tumor/cancer therapy, providing a completely novel design for the development of universal cancer drugs and/or vaccines.
A. DEFINITIONS
To facilitate understanding of the invention, a number of terms are defined below:
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 (1′ 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.
Oligonucleotide: a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. 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, reverse transcription, or a combination thereof.
Nucleic Acid: a polymer of nucleotides, either single or double stranded.
Nucleotide Analog: a purine or pyrimidine nucleotide that differs structurally from A, T, G, C, or U, but is sufficiently similar to substitute for the normal nucleotide in a nucleic acid molecule.
Nucleic Acid Composition: a nucleic acid composition refers to polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in either single-stranded or double-stranded molecular structures.
Gene: a nucleic acid whose nucleotide sequence codes for an RNA and/or a polypeptide (protein). A gene can be either RNA or DNA.
Base Pair (bp): a partnership of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule. In RNA, uracil (U) is substituted for thymine. Generally the partnership is achieved through hydrogen bonding.
Precursor messenger RNA (pre-mRNA): primary ribonucleotide transcripts of a gene, which are produced by type-II RNA polymerase (Pol-II) machineries in eukaryotes through an intracellular mechanism termed transcription. A pre-mRNA sequence contains a 5′-end untranslated region, a 3′-end untranslated region, exons and introns.
Intron: a part or parts of a gene transcript sequence encoding non-protein-reading frames, such as in-frame intron, 5′-untranslated region (5′-UTR) and 3′-UTR.
Exon: a part or parts of a gene transcript sequence encoding protein-reading frames (cDNA), such as cDNA for cellular gene, mammalian gene, embryonic stem cell marker gene, fluorescent protein marker gene, luciferase gene, lac-Z reporter gene, viral gene, jumping gene, transposon, and a combination thereof.
Messenger RNA (mRNA): assembly of pre-mRNA exons, which is formed after intron removal by intranuclear spliceosomal machineries and served as a protein-coding RNA for protein synthesis.
cDNA: a single stranded DNA that is complementary to an mRNA sequence and does not contain any intronic sequences.
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 “−” symbol or with an “a” or “antisense” in front of the DNA or RNA, e.g., “aDNA” or “aRNA”.
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.
Conserved: a nucleotide sequence is conserved with respect to a pre-selected (referenced) sequence if it non-randomly hybridizes to an exact complement of the pre-selected sequence.
Complementary or Complementarity or Complementation: used in reference to polynucleotides (i.e. a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “A-G-T” is complementary to the sequence “T-C-A,” and also to “T-C-U.” 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 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.
Homologous or Homology: refers to a polynucleotide sequence having similarities with 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 also be expressed as a percentage determined by the number of similar nucleotides over the total number of nucleotides.
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. Where a primer (or splice template) “hybridizes” with target (template), such complexes (or hybrids) are sufficiently stable to serve the priming function required by a DNA polymerase to initiate DNA synthesis. There is a specific, i.e. non-random, interaction between two complementary polynucleotides that can be competitively inhibited.
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 transgenes or small inhibitory RNAs.
RNA Interference (RNAi): 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.
Non-coding RNA: an RNA transcript that cannot be used to synthesize peptides or proteins through intracellular translation machineries.
MicroRNA (miRNA): single-stranded RNAs capable of binding to targeted gene transcripts that have partial complementarity to the miRNA. MiRNA is usually about 17-27 oligonucleotides in length and is able to either directly degrade its intracellular mRNA target(s) or suppress the protein translation of its targeted mRNA, depending on the complementarity between the miRNA and its target mRNA. Natural miRNAs are found in almost all eukaryotes, functioning as a defense against viral infections and allowing regulation of gene expression during development of plants and animals.
Pre-miRNA: hairpin-like single-stranded RNAs containing stem-arm and stem-loop regions for interacting with intracellular RNaseIII endoribonucleases to produce one or multiple microRNAs (miRNAs) capable of silencing a targeted gene or genes complementary to the microRNA sequence(s). The stem-arm of a pre-miRNA can form either a perfectly (100%) or a partially (mis-matched) hybrid duplexes, while the stem-loop connects one end of the stem-arm duplex to form a circle or hairpin-loop conformation.
Small interfering RNA (siRNA): short double-stranded RNAs sized about 18-25 perfectly base-paired ribonucleotide duplexes and capable of degrading target gene transcripts with almost perfect complementarity.
Small or short hairpin RNA (shRNA): single-stranded RNAs that contain 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”. Particularly important vectors allow cloning of cDNA from mRNAs produced using a reverse transcriptase. A vector may contain components consisting of a viral or a type-II RNA polymerase (Pol-II) 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.
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.
Promoter: a nucleic acid to which a polymerase molecule recognizes, perhaps binds to, and initiates synthesis. For the purposes of the instant invention, a promoter can be a known polymerase binding site, an enhancer and the like, any sequence that can initiate synthesis by a desired polymerase.
Antibody: a peptide or protein molecule having a pre-selected conserved domain structure coding for a receptor capable of binding a pre-selected ligand.
Primary RNA Transcript: an ribonucleotide sequence selected from the group consisting of mRNA, hnRNA, rRNA, tRNA, snoRNA, snRNA, pre-microRNA, viral RNA and their RNA precursors as well as derivatives.
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.
Donor Splice Site: a nucleic acid sequence either containing or homologous to the SEQ.ID.NO.4 sequence or 5′-GTAAG-3′ (SEQ.ID.NO.47).
Acceptor Splice Site: a nucleic acid sequence either containing or homologous to the SEQ.ID.NO.5 sequence or 5′-CTGCAG-3′ (SEQ.ID.NO.48).
Branch Point: an adenosine (A) nucleotide located within a nucleic acid sequence containing or homologous to the SEQ.ID.NO.6 sequence or 5′-TACTAAC-3′ (SEQ.ID.NO.44).
Poly-Pyrimidine Tract: a high T or C content nucleic acid sequence containing or homologous to the SEQ.ID.NO.7 or SEQ.ID.NO.8 sequence.
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, ampicillin, neomycin, G418, paromycin, kanamycin, streptomycin, erythromycin, spectromycin, phophomycin, tetracycline, rifapicin, amphotericin B, gentamycin, chloramphenicol, cephalothin, tylosin, and a combination thereof.
Type-II RNA Polymerase Equivalent: a transcription machinery selected from the group consisting of type-II (Pol-II), type-III (Pol-III), type-I (Pol-I), and viral RNA polymerases.
Restriction/Cloning Site: a DNA motif for restriction enzyme cleavage including but not limited 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, PmII, Ppu10I, PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI, XmaI cleavage site.
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 incorporation, transposon insertion, jumping gene integration, retroviral infection, and a combination thereof.
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, p14/p19Arf, p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1/Waf1, and p27Kip1, and a combination thereof.
Tumor Suppression: a cellular anti-tumor and anti-cancer mechanism consisting but not limited of cell cycle attenuation, G0/G1-checkpoint arrest, tumor suppression, anti-tumorigenecity, cancer cell apoptosis, and a combination thereof.
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.
B. COMPOSITIONS AND METHODS
A design and method for using a recombinant nucleic acid composition capable of being delivered, transcribed and processed into mir-302-like gene silencing effectors in targeted human cells and thus inducing specific gene silencing effects on mir-302-targeted cell cycle regulators and oncogenes in the cells, comprising the steps of:

    a) providing a recombinant nucleic acid composition capable of being delivered, transcribed and processed into at least a gene silencing effector interfering a plurality of cellular genes targeted by mir-302; and
    b) treating a cell substrate with said recombinant nucleic acid composition.


The above recombinant nucleic acid composition, further comprises:

    a) A plurality of exons, wherein said exons can be linked to form a gene transcript possessing a desired function; and
    b) At least an intron, wherein said intron contains a recombinant mir-302 homologue and can be cleaved out of the exons through intracellular RNA splicing and processing mechanisms.


The intron of the above recombinant nucleic acid composition, further comprises:

    a) A 5′-donor splice site for spliceosomal binding;
    b) A gene-silencing effector insert homologous to members of the mir-302 family;
    c) A branch point motif for spliceosomal recognition;
    d) A poly-pyrimidine tract for spliceosomal interaction;
    e) A 3′-acceptor splice site for spliceosomal binding; and
    f) A plurality of linkers for connecting each of the above components in a 5′ to 3′ direction.


Preferably, the present invention has adopted a novel design and strategy for either inducible or constitutive expression of mir-302-like gene silencing effectors in the transfected cells. Mir-302-like gene silencing effectors include mir-302a, mir-302b, mir-302c, mir-302d, and their hairpin-like microRNA precursors (pre-miRNAs) as well as manually re-designed small hairpin RNA (shRNA) homologues/derivatives, and a combination thereof. The transcription of mir-302-like gene silencing effectors is driven either by a constitutive (i.e. CMV) or drug-inducible (i.e. TRE-CMV) promoter. Preferably, the drug-inducible recombinant nucleic acid composition is a Tet-On vector containing a recombinant transgene inserted with either a recombinant mir-302 family cluster (mir-302s; hybrid of SEQ.ID.NOs.9-16) or a manually re-designed mir-302 shRNA homologue (i.e. hybrid of SEQ.ID.NOs.17 and 18). The cell substrate may express the mir-302 target genes either in vitro, ex vivo or in vivo. By silencing the mir-302-targeted cell cycle regulators and oncogenes, the present invention is able to suppress cell tumorigenecity and reprogram the treated cells into non-tumor/cancer cells.
EXAMPLES
The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations apply: M (molar); mM (millimolar); μm (micromolar); mol (moles); pmol (picomolar); gm (grams); mg (milligrams); μg (micrograms); ng (nanograms); L (liters); ml (milliliters); μl (microliters); ° C. (degrees Centigrade); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double-stranded DNA); dNTP (deoxyribonucleotide triphosphate); RNA (ribonucleic acid); PBS (phosphate buffered saline); NaCl (sodium chloride); HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid); HBS (HEPES buffered saline); SDS (sodium dodecylsulfate); Tris-HCl (tris-hydroxymethylaminomethane-hydrochloride); ATCC (American Type Culture Collection, Rockville, Md.); hES (human embryonic stem cells); iPS (induced pluripotent stem cells); and SCR (somatic cell reprogramming).
Example 1
Cell Culture and Transfection
Human cancer NTera-2, HepG2, MCF7, PC3 and Colo829 cell lines were acquired from ATCC, while human hair follicle cells (hHFCs) were isolated and dissociated from a minimum of two hair dermal papillae by 4 mg/ml collagenase I digestion for 45 min in fresh RPMI 1640 medium supplemented with 20% FBS. For culturing melanocytes, the isolated cells were cultivated in Medium 254 with the addition of human melanocytes growth supplement-2 (HMGS-2, Invitrogen, Carlsbad, Calif.) in the absence of antibiotics at 37° C. under 5% CO2. Cultures were passaged at 70%-80% confluency by exposing cells to trypsin/EDTA solution for 1 min and rinsing once with phenol red-free DMEM medium (Invitrogen), and the detached cells were replated at 1:10 dilution in fresh Medium 254 with HMGS-2 supplements. For electroporation, a mixture of pTet-On-tTS-mir302s (10 μg) and pTet-On-Adv-Neo(−) (50 μg) was added with the isolated cells (20,000-50,000) in a hypoosmolar buffer (200 μl; Eppendorf, Westbury, N.Y.) and electroporation was performed using Eppendorf Multiporator at 300-400 volts for 150 μsec. The electroporated cells were first grown in phenol red-free DMEM medium (Invitrogen) supplemented with 20% knockout serum, 1% MEM nonessential amino acids, 10 ng/ml bFGF, 1 mM GlutaMax, and 1 mM sodium pyruvate, for 24 hours at 37° C. under 5% CO2. Then, 850 μg/ml G418 and >3.75 μg/ml doxycycline (Dox) were added and refreshed daily for 3-5 days till the cells expressed strong red fluorescent RGFP. Next, the individual red fluorescent cell (mirPS) was monitored under a TE2000 inverted microscopic system (Nikon, Melville, N.Y.) and separately collected into a 96-well, using MO-188NE 3D micromanipulators (Nikon). In the absence of Dox, the mirPS cells were grown and passaged in knockout DMEM/F-12 medium (Invitrogen) supplemented with 20% knockout serum, 1% MEM nonessential amino acids, 100 μM β-mercaptoethanol, 1 mM GlutaMax, 1 mM sodium pyruvate, 10 ng/ml bFGF, 100 IU/ml penicillin/100 μg/ml streptomycin/250 μg/ml G418, 0.1 μM A83-01, and 0.1 μM valproic acid (Stemgent, San Diego, Calif.), at 37° C. under 5% CO2. Alternatively, in the presence of Dox (3.75-5 μg/ml; Sigma-Aldrich, St. Louis, Mo.), the mirPS cells were cultivated and passaged in the same feeder-free cultural condition with addition of 0.05 μM GSK inhibitor SB216763 (Stemgent). Addition of GSK inhibitor could facilitate mirPS cell proliferation but with a slight tendency to cause neural differentiation. For neural cell induction, the mirPS cells were grown in the above feeder-free cultural condition with 0.05 μM SB216763 but no Dox.
Example 2
Construction of Recombinant Vectors Expressing mir-302s
The mir-302 familial cluster (mir-302s) was generated as reported (Lin et al., 2008). The mir-302s cluster consists of four parts, including precursor miRNAs (pre-miRNAs) of mir-302a, b, c, and d. Synthetic oligonucleotides (Sigma-Genosys, St. Louis, Mo.) used for constructing the mir-302s cluster were listed below. For expression, we mixed an equal amount (1:1) of the mir-302s cluster and a pre-made SpRNAi-RGFP recombinant gene (Lin et al., 2006 and 2008), and then digested the mixture with MluI/PvuI restriction enzymes at 37° C. for 4 hours. The digested mixture was collected with a gel extraction filter (Qiagen, CA) in 30 μl of ddH2O and ligated together using T4 DNA ligase at 8° C. for 16 hours. This formed a recombinant mir-302-expressing SpRNAi-RGFP gene, which was further cleaved with XhoI/HindIII restriction enzymes and inserted into a Dox-inducible pSingle-tTS-shRNA vector (Clontech, Palo Alto, Calif.). This formed an inducible pTet-On-tTS-mir302s expression vector. Then, we further modified the pTet-On-tTS-mir302s vector by replacing its U6 promoter with a TRE-CMV promoter isolated from a pTRE-Tight plasmid (Clontech). For generating a non-inducible, constitutive pCMV-miR302s expression vector, we cleaved the modified pTet-On-tTS-mir302s vector with EcoR1 restriction enzyme, removed the upstream tTS-TRE sequence (1.5 kb) by gel electrophoresis, and recovered the cleaved vector from the gel for further DNA ligation to complete the formation of the non-inducible pCMV-miR302s vector.
Synthetic oligonucleotides for DNA recombination of the mir-302 familial pre-miRNA cluster were listed as follows: mir-302a-sense, 5′-GTCACGCGTT CCCACCACTT AAACGTGGAT GTACTTGCTT TGAAACTAAA GAAGTAAGTG CTTCCATGTT TTGGTGATGG ATAGATCTCT C-3′ (SEQ.ID.NO.9); mir-302a-antisense, 5′-GAGAGATCTA TCCATCACCA AAACATGGAA GCACTTACTT CTTTAGTTTC AAAGCAAGTA CATCCACGTT TAAGTGGTGG GAACGCGTGA C-3′ (SEQ.ID.NO.10); mir-302b-sense, 5′-ATAGATCTCT CGCTCCCTTC AACTTTAACA TGGAAGTGCT TTCTGTGACT TTGAAAGTAA GTGCTTCCAT GTTTTAGTAG GAGTCGCTCA TATGA-3′ (SEQ.ID.NO.11); mir-302b-antisense, 5′-TCATATGAGC GACTCCTACT AAAACATGGA AGCACTTACT TTCAAAGTCA CAGAAAGCAC TTCCATGTTA AAGTTGAAGG GAGCGAGAGA TCTAT-3′ (SEQ.ID.NO.12); mir-302c-sense, 5′-CCATATGGCT ACCTTTGCTT TAACATGGAG GTACCTGCTG TGTGAAACAG AAGTAAGTGC TTCCATGTTT CAGTGGAGGC GTCTAGACAT-3′ (SEQ.ID.NO.13); mir-302c-antisense, 5′-ATGTCTAGAC GCCTCCACTG AAACATGGAA GCACTTACTT CTGTTTCACA CAGCAGGTAC CTCCATGTTA AAGCAAAGGT AGCCATATGG-3′ (SEQ.ID.NO.14); mir-302d-sense, 5′-CGTCTAGACA TAACACTCAA ACATGGAAGC ACTTAGCTAA GCCAGGCTAA GTGCTTCCAT GTTTGAGTGT TCGCGATCGC AT-3′ (SEQ.ID.NO.15); and mir-302d-antisense, 5′-ATGCGATCGC GAACACTCAA ACATGGAAGC ACTTAGCCTG GCTTAGCTAA GTGCTTCCAT GTTTGAGTGT TATGTCTAGA CG-3′ (SEQ.ID.NO.16). Alternatively, we used a manually re-designed shRNA formed by the hybrid of synthetic miR-302s-sense, 5′-GCAGATCTCG AGGTACCGAC GCGTCCTCTT TACTTTAACA TGGAAATTAA GTGCTTCCAT GTTTGAGTGG TGTGGCGCGA TCGATATCTC TAGAGGATCC ACATC-3′ (SEQ.ID.NO.17) and mir-302s-antisense, 5′-GATGTGGATC CTCTAGAGAT ATCGATCGCG CCACACCACT CAAACATGGA AGCACTTAAT TTCCATGTTA AAGTAAAGAG GACGCGTCGG TACCTCGAGA TCTGC-3′ (SEQ.ID.NO.18), in place of the mir-302 pre-miRNA cluster for easy intronic insertion. In design of mir-302 homologues, thymine (T) can be used in place of uracil (U) or vice versa. All these synthetic sequences were purified with PAGE gel extraction before ligation.
The recombinant mir-302 familial pre-miRNA cluster (mir-302s) was formed by linkage of four mir-302a-d hybrids, including mir-302a-sense and mir-302a-antisense, mir-302b-sense and mir-302b-antisense, mir-302c-sense and mir-302c-antisense, and mir-302d-sense and mir-302d-antisense. The hybrids of mir-302a, mir-302b, mir-302c, and mir-302d were digested by PvuI/XhoI, XhoI/NheI, NheI/XbaI, and XbaI/MluI restriction enzymes, respectively, and collected together by a gel extraction filter column in 35 μl autoclaved ddH2O (Qiagen, CA). Immediately after that, the mixed hybrids were ligated to form a cluster with T4 DNA ligase (Roche, 20 U) and further inserted into the PvuI/MluI-linearized SpRNAi-RGFP recombinant gene. Alternatively, the mir-302 shRNA made by hybridizing SEQ.ID.NO.17 and SEQ.ID.NO.18 was cleaved with PvuI/MluI restriction enzymes and inserted into the PvuI/MluI-linearized SpRNAi-RGFP.
The pTet-On-tTS-mir302s and CMV-mir302s vectors were propagated in E. coli DH5α LB culture containing 100 μg/ml ampicillin (Sigma Chemical, St. Louis, Mo.). The propagated pTet-On-tTS-mir302s and CMV-mir302s vectors were isolated and purified using an Endo-Free Maxi-Prep Plasmid Extraction Kit (Qiagen, CA).
Example 3
MicroRNA (miRNA) Microarray Analysis
At 70% confluency, small RNAs from each cell culture were isolated, using the mirVanaTM 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, Calif.) for miRNA microarray analysis. 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. For a dual sample assay, a p-value calculation was performed and a list of differentially expressed transcripts more than 3-fold was produced. The result was shown in FIG. 1C.
Example 4
Northern Blot Analysis
Total RNAs (10 μg) were isolated with a mirVanaTM miRNA Isolation Kit (Ambion, Austin, Tex.), fractionated by either 15% TBE-urea polyacrylamide gel or 3.5% low melting point agarose gel electrophoresis, and electroblotted onto a nylon membrane. Detection of mir-302 was performed with a [LNA]-DNA probe (5′-[TCACTGAAAC] ATGGAAGCAC TTA-3′) (SEQ.ID.NO.19), while probes for other gene detection were synthesized and listed in Table 1. All probes were purified by high-performance liquid chromatography (HPLC) and tail-labeled with terminal transferase (20 units) for 20 min in the presence of [32P]-dATP (>3000 Ci/mM, Amersham International, Arlington Heights, Ill.). Hybridization was carried out in the mixture of 50% freshly deionized formamide (pH 7.0), 5×Denhardt's solution, 0.5% SDS, 4×SSPE and 250 mg/mL denatured salmon sperm DNA fragments (18 hr, 42° C.). Membranes were sequentially washed twice in 2×SSC, 0.1% SDS (15 min, 25° C.), and once in 0.2×SSC, 0.1% SDS (45 min, 37° C.) before autoradiography. The results were shown in FIGS. 1D, 4A and 8B.
Example 5
Western Blot Analysis
Cells (106) were lysed with a CelLytic-M lysis/extraction reagent (Sigma) supplemented with protease inhibitors, Leupeptin, TLCK, TAME and PMSF, following the manufacturer's suggestion. Lysates were centrifuged at 12,000 rpm for 20 min at 4° C. and the supernatant was recovered. Protein concentrations were measured using an improved SOFTmax protein assay package on an E-max microplate reader (Molecular Devices, Calif.). Each 30 μg of cell lysate was added to SDS-PAGE sample buffer under reducing (+50 mM DTT) and non-reducing (no DTT) conditions, and boiled for 3 min before loading onto a 6 ̃8% polyacylamide gel. 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, Calif.), Sox2 (Santa Cruz), Nanog (Santa Cruz), Lin28 (Abcam Inc., Cambridge, Mass.), UTF1 (Abcam), Klf4 (Santa Cruz), TRP1 (Santa Cruz), keratin 16 (Abcam), CDK2 (Santa Cruz), cyclin D1 (Santa Cruz), cyclin D2 (Abcam), BMI-1 (Santa Cruz), AOF2 (Sigma), HDAC2 (Abcam), hTERT (Santa Cruz), 8-actin (Chemicon, Temecula, Calif.), 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 were conducted using Li-Cor Odyssey Infrared Imager and Odyssey Software v.10 (Li-Cor). The results were shown in FIGS. 1D, 4B, 7C, 7D, 8B and 9B.
Example 6
Apoptotic DNA Laddering Assay
Genomic DNAs were isolated from about two million cells using an Apoptotic DNA Ladder Kit (Roche Biochemicals, Indianapolis, Iowa) and 2 μg of the isolated DNAs were further assessed by 2% agarose gel electrophoresis, according to the manufacturers' suggestion. The result was shown in FIG. 1F.
Example 7
DNA-Density Flow Cytometry
Cells were trypsinized, pelleted and fixed by re-suspension in 1 ml of pre-chilled 70% methanol in PBS for 1 hour at −20° C. The cells were pelleted and washed once with 1 ml of PBS. The cells were pelleted again and resuspended in 1 ml of 1 mg/ml propidium iodide, 0.5 μg/ml RNase in PBS for 30 min at 37° C. Approximately 15,000 cells were then analyzed on a BD FACSCalibur (San Jose, Calif.). Cell doublets were excluded by plotting pulse width versus pulse area and gating on the single cells. The collected data were analyzed using the software package Flowjo using the “Watson Pragmatic” algorithm. The result was shown in FIGS. 3A-B, 6A-B and 6C.
Example 8
Genome-Wide Microarray Analysis
Human genome GeneChip U133 plus 2.0 arrays (Affymetrix, Santa Clara, Calif.) were used to detect the alterations of over 47,000 human gene expression patterns in tested cells. Total RNAs from each tested sample were isolated using a mirVanaTM miRNA Isolation Kit (Ambion), following the manufacturer's suggestion. The purity and quantity of isolated RNAs were assessed using 1% formaldehyde-agarose gel electrophoresis and spectrophotometer measurement (Bio-Rad). The sample signals were normalized using the total average difference between perfectly matched probes and mismatched probes. Alterations of genome-wide gene expression patterns were analyzed using Affymetrix Microarray Suite version 5.0, Expression ConsoleTM version 1.1.1 (Affymetrix) and Genesprings (Silicon Genetics) softwares. Changes in gene expression rates more than 1-fold were considered as positive differential genes. For gene clustering, a plug-in program Genetrix (Epicenter Software) was used in conjunction with the Affymetrix softwares. Signals of the sample were normalized with the internal house-keeping control average in each microarray. The result of scatter plot analysis was shown in FIG. 5A.
Example 9
DNA Demethylation Assays
Genomic DNAs were isolated from about two million cells using a DNA Isolation Kit (Roche) and 1 μg of the isolated DNAs were further treated with bisulfite (CpGenome DNA modification kit, Chemicon, Temecula, Calif.), according to the manufacturers' suggestions. Meanwhile, 2 μg of untreated DNAs were digested with a CCGG-cutting restriction enzyme HpaII and then analyzed by 1% agarose gel electrophoresis to determine genome-wide demethylation (FIG. 5B). The treatment with bisulfite converted all unmethylated cytosine to uracil, while methylated cytosine remained as cytosine. For bisulfite DNA sequencing analyses, we amplified the promoter regions of Oct3/4 and Nanog with PCR. Primers included 5′-GAGGCTGGAG CAGAAGGATT GCTTTGG-3′ (SEQ.ID.NO.20) and 5′-CCCTCCTGAC CCATCACCTC CACCACC-3′ (SEQ.ID.NO.21) for Oct3/4, and 5′-TGGTTAGGTT GGTTTTAAAT TTTTG-3′ (SEQ.ID.NO.22) and 5′-AACCCACCCT TATAAATTCT CAATTA-3′ (SEQ.ID.NO.23) for Nanog. The bisulfite-modified DNAs (50 ng) were first mixed with the primers (total 100 pmole) in 1×PCR buffer, heated to 94° C. for 2 min, and immediately cooled on ice. Next, 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 amplified DNA product with a correct size was further fractionized by 3% agarose gel electrophoresis, purified with a gel extraction filter (Qiagen), and then used in DNA sequencing. A detailed profile of the DNA methylation sites was generated by comparing the unchanged cytosine in the converted DNA sequence to the unconverted one. The result was shown in FIG. 5C.
Example 10
Implantation and Teratoma Formation
Approximately 5-10 mirPS cell-derived embryoid bodies (4- to 8-cell-stage) were suspended in 50 μl of a mixture of DMEM and Matrigel (2:1), followed by implantation into the uterus of a 6-week-old female pseudopregnant immunocompromised SCID-beige mouse. The pseudopregnant mice were prepared by intraperitoneal injection of 1IU human menopausal gonadotrophin (HMG) for two days and then human chorionic gonadotrophin (hCG) for one more day. The cells and mice were not treated with Dox before or after implantation. The mice were anesthetized with 2.5% Avertin solution, 0.4 ml per mouse during implantation. Xenografted masses were monitored 3-4 weeks after the implantation or when the sizes were grown to over 100 mm3. Cysts/teratomas were dissected and the volumes were calculated using the formula (length×width2)/2. Cyst/teratoma lesions were counted, weighed and subjected to further histological analysis. Formation of teratoma-like tissue cysts was usually observed at approximately 2.5-week post-implantation. The result was shown in FIG. 5D.
Example 11
Cell Invasion Assay
Chamber inserts (12-μm pore size, Chemicon) were coated with 200 μg/ml of Matrigel alone or supplemented with 20% FBS in phenol red-free-DMEM with 1% L-glutamine and dried overnight under sterile conditions. Cells were harvested, washed, and resuspended in phenol red-free-DMEM to give a final cell density of 1×105 cells/ml. Five hundred microliters of the resulting cell suspension was then dispensed into the top chamber whereas DMEM conditioned medium (1.5 ml) was added to the bottom chamber to create a chemotactic gradient. Invasion was measured after overnight incubation at 37° C. for 16 hour. Top chambers were wiped with cotton wool, and invading cells on the underside of the membrane were fixed in 100% methanol for 10 min, air dried, stained in cresyl violet for 20 min, and gently rinsed in water. When dry, the cresyl violet stain on membranes was eluted using a 100% ethanol/0.2 M NaCitrate (1:1) wash for 20 min and absorbance read at 570 nm using a Precision Microplate Reader (Molecular Dynamics). The percentage of invading cells was calculated by comparison of absorbance in test samples against absorbance determined on membrane inserts that were not wiped (total cells). The result was shown in FIG. 6D.
Example 12
Cell Adhesion Assay
Cell Adhesion assay was performed as reported (Lin et al., 2007). Human bone marrow endothelial cells (hBMECs) were seeded at a density of 1×105 cells/ml in 96-well plates and washed with adhesion medium [RPMI 1640/0.1% BSA/20 mM HEPES (pH7.4)] before assays. Tested cells were trypsinized (tumor/cancer cells) or collagenase-digested (mirPS cells), washed in sterile saline, and resuspended at 1×106 cells/ml in PBS with 10 μM fura-4 acetoxymethyl ester (fluorescent probe, Sigma) for 1 hour at 37° C. in the dark. The cells were then pelleted, washed in serum-free medium containing 1% (v/v) of probenecid (100 mM) and incubated for 20 min in adhesion medium at 37° C. in the dark to activate the intracellular fluorescent probe. After that, 105 cells (in 300-0 cell suspension/well) were added to the confluent hBMEC endothelial monolayer and incubated for 50 min at 37° C. Non-adherent cells were removed using 2×250 μl washes of adhesion medium. Plates were read in a fluorescent plate reader (Molecular Dynamics) at 37° C. using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The result was shown in FIG. 6E.
Example 13
In Vivo Tumorigenecity Assay
The inventors xenografted NTera-2 cells (2×106 cells in a total volume of 100 μl Matrigel-PBS) into the flanks (e.g. right hind limb) of eight-week-old male mice (BALB/c nu/nu strain). Tumors were monitored weekly and in situ injection of pCMV-miR302s vector or pCMV-miR302d* was conducted one week after the NTera-2 xenograft. Five treatments (three-day intervals for each treatment) of 2 μg PEI-formulated pCMV-miR302s or pCMV-miR302d* vector (total 10 μg) per g mouse weight were performed. In vivo-jetPEI Delivery Reagent (Polyplus-transfection Inc., New York, N.Y.) was used as the manufacturer's suggestion. Samples were collected either three weeks post injection or when untreated tumors grew to an average size of approximately 100 mm3. Major organs, such as the blood, brain, heart lung, liver, kidney and spleen, and the xenografts were removed for histological evaluation of tumor lesions and immunoreactive cytotoxicity. Tumor formation was monitored by palpation and tumor volume was calculated using the formula (length×width2)/2. Tumor lesions were counted, dissected, weighed, and subjected to histological examination using H&E and immunostaining assays. Histological examination showed no detectable tissue lesions in brain, heart, lung, liver, kidney and spleen. The result was shown in FIG. 8A.
Example 14
Immunostaining Assay
Tissue samples were fixed in 4% paraformaldehyde overnight at 4° C. The samples were washed sequentially with 1×PBS, methanol, isopropanol and tetrahydronaphthalene before embedded in paraffin wax. The embedded samples were then cut on a microtome at 7-10 μm thickness and mounted on clean TESPA-coated slides. Then, the slides were dewaxed with xylene and mounted under coverslips using mounting media (Richard Allan Scientific, Kalamazoo, Mich.) and stained by hematoxylin and eosin (H&E, Sigma) for morphological observation. Immunohistochemical (IHC) Staining Kits were purchased from Imgenex (San Diego, Calif.). Processes for antibody dilution and immunostaining were performed according to the manufacturers' suggestions. Primary antibodies included Oct3/4 (Santa Cruz), Sox2 (Santa Cruz), Nanog (Santa Cruz), CDK2 (Santa Cruz), cyclin D1 (Santa Cruz), cyclin D2 (Abcam), BMI-1 (Santa Cruz), and RGFP (Clontech). Secondary antibodies used were biotinylated goat anti-rabbit or biotinylated horse anti-mouse antibodies (Chemicon, Temecula, Calif.). Streptavidin-HRP was added as the tertiary antibody. After the slides were washed twice with PBT, the bound antibody was detected using DAB substrates. Positive results were observed under a 100× microscope with whole field scanning and measured at 200× magnification for quantitative analysis using a Metamorph Imaging program (Nikon 80i microscopic quantitation system). The result of scatter plot analysis was shown in FIG. 8C.
Example 15
Luciferase 3′-UTR Reporter Assay
Luciferase assays were performed using a modified pMir-Report miRNA Expression Reporter Vector System (Ambion). The mir-302 target sites (normal and/or mutant) were inserted in the 3′-UTR cloning site of the pMir-Report Luciferase Reporter vector. The two target sites were synthesized and separated by twelve -CAGT-repeats. Another pMir-Report β-gal Control vector was used as a no reporter control. We transfected 200 ng of the reporter vector into fifty thousand mirPS cells in the absence or presence of Dox treatment, using a FuGene HD reagent (Roche) following the manufacturer's suggestion. Cell lysates were harvested 48 hours after transfection, and the knockdown levels of luciferase were normalized and shown by ratios of relative luciferase activity (RFA), which was calculated by the level of luciferase activity in Dox-treated (Dox-on) mirPS cells divided by that of untreated (Dox-off) mirPS cells. Mir-434-expressing cells generated by electroporating hHFCs with pTet-On-tTS-miR434-5p were served as a negative control. The result was shown in FIG. 7B.
Example 16
TRAP Assay
Cells (106) were lysed with a CelLytic-M lysis/extraction reagent (Sigma) supplemented with protease inhibitors, Leupeptin, TLCK, TAME and PMSF, following the manufacturer's suggestion. Lysates were centrifuged at 12,000 rpm for 20 min at 4° C. and the supernatant was recovered. Protein concentrations were measured using an improved SOFTmax protein assay package on an E-max microplate reader (Molecular Devices, Calif.). Oligonucleotides 5′-AATCCGTCGAGCAGAGTT-3′ (SEQ.ID.NO.24) labeled with infrared Alexa Fluor 680 dye (TS Primer; Sigma-Genosys) and 5′-GTGTAACCCTAACCCTAACCC-3′ (CX primer; 30 μM) (SEQ.ID.NO.25) were used for detecting the PCR products. Telomerase inhibitors were directly added to the master mix. The optimal results for all tested cell lines were achieved using 50 ng proteins per reaction. After a 30-min incubation at 30° C., the samples were placed in a thermal cycler for 2 min at 94° C., followed by 35 PCR cycles of denaturation at 94° C. for 30 sec and synthesis at 57° C. for 30 sec as well as a single postsynthesis step at 57° C. for 30 sec. The PCR products were separated by electrophoresis on a 6% nondenaturing polyacrylamide gel and detected using Li-Cor Odyssey Infrared Imager and Odyssey Software v.10 (Li-Cor). The result was shown in FIG. 9A.
Example 17
Statistic Analysis
Any change over 75% of signal intensity in the analyses of immunostaining, western blotting and northern blotting was considered as a positive result, which in turn was analyzed and presented as mean±SE. Statistical analysis of data was performed by one-way ANOVA. When main effects were significant, the Dunnett's post-hoc test was used to identify the groups that differed significantly from the controls. For pairwise comparison between two treatment groups, the two-tailed student t test was used. For experiments involving more than two treatment groups, ANOVA was performed followed by a post-hoc multiple range test. Probability values of p<0.05 was considered significant. All p values were determined from two-tailed tests.
REFERENCES
The following references are hereby incorporated by reference as if fully set forth herein:

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It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art, and are to be included within the spirit and purview of the invention as set forth in the appended claims. All publications and patents cited herein are incorporated herein by reference in their entirety for all purposes.








TABLE 1







related to Example 4.









Probe
Sequence












Oct3/4
5′-GCAGTGTGGG TTTCGGGCAC TGCAGGAACA AATTCTCCAG GTTGCCTCTC




ACTCGGTTCT CGATACTGGT TCGCTTTCTC TTTCGGGCCT GCACGAGGGT



TTCTGCTTTG-3′



(SEQ.ID.NO. 26)


 


Sox2
5′-TGCTGTAGGT GGGCGAGCCG TTCATGTAGG TCTGCGAGCT GGTCATGGAG



TTGTACTGCA GGGCGCTCAC GTCGTAGCGG TGCATGGGCT GCATCTGCGC



TGCGCCGTGC-3′



(SEQ.ID.NO. 27)


 


Nanog
5′-CGTGTGAGGC ATCTCAGCAG AAGACATTTG CAAGGATGGA TAGTTTTCTT



CAGGCCCACA AATCACAGGC ATAGGTGAAG ATTCTTTACA GTCGGATGCT



TCAAAGCAAG-3′



(SEQ.ID.NO. 28)


 


Lin28
5′-AGGTCCGGTG ACACGGATGG ATTCCAGACC CTTGGCTGAC TTCTTAAAGG



TGAACTCCAC TGCCTCACCC TCCTTCAAGC TCCGGAACCC TTCCATGTGC



AGCTTACTCT-3′



(SEQ.ID.NO. 29)


 


UTF1
5′-CTGCTGGGCC AGCGCGGCCG ACACGCGGCG GTAGGTGGGC



AGGGCCTGGC GGCGGTCCAG GAGCAGCGCG CGCCACACGG CCGGTTGCAG



CAGCGTCCCC AGCAGCAGCT-3′



(SEQ.ID.NO. 30)


 


Klf4
5′-CTGCTCGACG GCGACGACGA AGAGGAGGCT GACGCTGACG



AGGACACGGT GGCGGCCACT GACTCCGGAG GATGGGTCAG CGAATTGGAG



AGAATAAAGT CCAGGTCCAG-3′



(SEQ.ID.NO. 31)


 


FUT3
5′-GAGCCCTAGG GGATCCAGTG GCATCGTCTC GGGACACACG CAGGTAGGAG



AAGAAACACA CAGCCACCAG CAGCTGAAAT AGCAGTGCGG CCAGACAGCG



GCGCCATGGC-3′



(SEQ.ID.NO. 32)


 


RGFP
5′-CGAAGGGGTT GCCGTCGCCC TCGCCCTCGC ACTTGAAGTA GTGGCCGTTC



ACGGTGCCCT CCATGTACAT CTTGATGCGC ATACTCTCCT TCAGCAGGCC



GCTCACCATA-3′



(SEQ.ID.NO. 33)


 


B-actin
5′-AATGTCACGC ACGATTTCCC GCTCGGCCGT GGTGGTGAAG CTGTAGCCGC



GCTCGGTGAG GATCTTCATG AGGTAGTCAG TCAGGTCCCG GCCAGCCAGG



TCCAGAGCGA-3′



(SEQ.ID.NO. 34)

## Claims

1. A method for using a recombinant nucleic acid composition to induce tumor suppression effects in at least a targeted cancer or tumor cell, comprising the steps of:
(a) providing the recombinant nucleic acid composition capable of being delivered and processed to provide at least a gene silencing effector capable of concurrently suppressing the expression of CDK2, cyclin D and BMI-1 oncogene, wherein said gene silencing effector is a hairpin RNA having a sequence consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and wherein said gene silencing effector can be further processed to provide mir-302a, mir-302b, mir-302c and mir-302d in the targeted cancer or tumor cell; and
(b) treating a cell substrate containing the targeted cancer or tumor cell with said recombinant nucleic acid composition under a condition that the concentration of said gene silencing effector is at least 1.3 fold higher than the natural expression level of mir-302 found in human embryonic stem cell lines H1 or H9 and is sufficient to suppress said CDK2, cyclin D and BMI-1 oncogene; wherein the cancer or tumor cell is identified as a cell type derived from human neoplastic Tera-2-derived teratocarcinomas.

2. The method as defined in claim 1, wherein said targeted cancer or tumor cell includes a human cell categorized as one of tumor types found in teratoma.

3. The method as defined in claim 1, wherein said recombinant nucleic acid composition includes an expression-competent vector.

4. The method as defined in claim 1, wherein said recombinant nucleic acid composition includes a type-II RNA polymerase-driven promoter.

5. The method as defined in claim 4, wherein said type-II RNA polymerase-driven promoter is inducible.

6. The method as defined in claim 1, wherein said cell substrate expresses mir-302-targeted genes selected from CDK2, cyclin D or BMI-1.

7. The method as defined in claim 1, wherein said recombinant nucleic acid composition further includes a 5′-donor splice site, an intronic insert site, a branch point motif, a poly-pyrimidine tract, and a 3′-acceptor splice site.

8. The method as defined in claim 7, wherein said intronic insert site includes at least one of said gene silencing effector.

9. The method as defined in claim 1, wherein said recombinant nucleic acid composition further includes a plurality of exons.

10. The method as defined in claim 1, wherein said recombinant nucleic acid composition is delivered into said cell substrate by a gene delivery method.

11. The method as defined in claim 1, wherein said tumor suppression effects include at least one of cell cycle attenuation or cancer cell apoptosis.

12. The method as defined in claim 1, wherein said tumor suppression effects result from RNA interference.

13. The method as defined in claim 1, wherein said recombinant nucleic acid composition is made by a genetic engineering method.

14. The method as defined in claim 1, wherein said gene silencing effector contains at least a loop structural sequence of SEQ ID NO: 2.

15. A method for using a recombinant nucleic acid composition to induce tumor suppression effects in at least a targeted cancer or tumor cell, comprising the steps of:
(a) providing the recombinant nucleic acid composition capable of being delivered and processed to provide at least a gene silencing effector capable of concurrently suppressing the expression of CDK2, cyclin D and BMI-1 oncogene, wherein said gene silencing effector contains at least a hairpin RNA having a sequence of SEQ ID NO: 3 and consisting of pre-miRNAs of mir-302a, mir-302b, mir-302c and mir-302d, wherein said hairpin RNA is further processed to provide mir-302a, mir-302b, mir-302c and mir-302d in the targeted cancer or tumor cell; and
(b) treating a cell substrate containing the targeted cancer or tumor cell with said recombinant nucleic acid composition under a condition that the concentration of said gene silencing effector is at least 1.3 fold higher than the concentration of mir-302 in human embryonic stem cells H1 or H9 and is sufficient to suppress said CDK2, cyclin D and BMI-1 oncogene; wherein the cancer or tumor cell is identified as a cell type derived from human neoplastic Tera-2-derived teratocarcinomas.

16. The method as defined in claim 15, wherein said recombinant nucleic acid composition includes an expression-competent vector.

17. The method as defined in claim 15, wherein said recombinant nucleic acid composition includes a type-II RNA polymerase-driven promoter.

18. The method as defined in claim 17, wherein said type-II RNA polymerase-driven promoter is inducible.

19. The method as defined in claim 15, wherein said recombinant nucleic acid composition further includes a 5′-donor splice site, an intronic insert site, a branch point motif, a poly-pyrimidine tract, and a 3′-acceptor splice site.

20. The method as defined in claim 15, wherein said recombinant nucleic acid composition further includes a plurality of exons.

21. The method as defined in claim 15, wherein said recombinant nucleic acid composition is delivered into said cell substrate by a gene delivery method.

22. The method as defined in claim 15, wherein said tumor suppression effects include at least one of cell cycle attenuation or cancer cell apoptosis.

23. The method as defined in claim 15, wherein said tumor suppression effects result from RNA interference.

24. The method as defined in claim 15, wherein said recombinant nucleic acid composition is made by a genetic engineering method.

25. The method as defined in claim 15, wherein said gene silencing effector contains at least a loop structural sequence of SEQ ID NO: 2.

26. The method as defined in claim 15, wherein said gene silencing effector consists of sequences of pre-miRNAs of mir-302a, mir-302b, mir-302c, and mir-302d.

27. A method for using a recombinant nucleic acid composition to induce tumor suppression effects in at least a targeted cancer or tumor cell, comprising the steps of:
(a) providing the recombinant nucleic acid composition capable of being delivered and processed to provide at least a gene silencing effector capable of concurrently suppressing the expression of CDK2, cyclin D and BMI-1 oncogene, wherein said gene silencing effector is a hairpin RNA consisting of sequences of pre-miRNAs of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and is further processed to provide mir-302a, mir-302b, mir-302c and mir-302d in the targeted cancer or tumor cell; and
(b) treating a cell substrate containing the targeted cancer or tumor cell with said recombinant nucleic acid composition under a condition that the concentration of said gene silencing effector is at least 1.3 fold higher than the concentration of mir-302 in human embryonic stem cells H1 or H9 and is sufficient to suppress said CDK2, cyclin D and BMI-1 oncogene; wherein the cancer or tumor cell is identified as a cell type derived from human neoplastic Tera-2-derived teratocarcinomas.