WO2019196883A1 - 一种激活p21基因表达的方法 - Google Patents

一种激活p21基因表达的方法 Download PDF

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WO2019196883A1
WO2019196883A1 PCT/CN2019/082126 CN2019082126W WO2019196883A1 WO 2019196883 A1 WO2019196883 A1 WO 2019196883A1 CN 2019082126 W CN2019082126 W CN 2019082126W WO 2019196883 A1 WO2019196883 A1 WO 2019196883A1
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nucleic acid
small activating
rag
gene
acid strand
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PCT/CN2019/082126
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French (fr)
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李龙承
姜武林
吴建成
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中美瑞康核酸技术(南通)研究院有限公司
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Priority to EP19785602.4A priority Critical patent/EP3778893A4/en
Priority to CN201980002538.7A priority patent/CN110678549B/zh
Priority to JP2020555223A priority patent/JP2021520220A/ja
Priority to US15/733,357 priority patent/US20210024915A1/en
Priority to KR1020207032370A priority patent/KR20200143428A/ko
Publication of WO2019196883A1 publication Critical patent/WO2019196883A1/zh

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Definitions

  • the present invention relates to the field of molecular biology, and in particular to the forward regulation of gene expression using a double-stranded small RNA targeting a gene promoter sequence.
  • Double-stranded small nucleic acid molecules including chemically synthesized oligoribonucleotides such as small activating RNA (saRNA) and naturally occurring oligoribonucleotides such as microribonucleotides (miRNAs) have been shown to target in a sequence-specific manner
  • the expression level of a gene encoding a gene, such as a promoter sequence is positively regulated at the transcriptional and epigenetic levels, and this phenomenon is called RNA activation (RNAa) (Li, Okino et al. (2006) Proc Natl Acad Sci U S A 103: 17337-17342; Janowski, Younger et al.
  • RNA activation is an evolutionarily conserved endogenous molecular mechanism from C. elegans to humans (Huang, Qin et al. (2010) PLoS One 5: e8848; Seth, Shirayama et al. (2013) Dev Cell 27: 656-663; Turner, Jiao et al. (2014) Cell Cycle 13: 772-781).
  • RNA activation has the advantage of being able to activate endogenous gene expression without changing the genome, and represents a new strategy for activating endogenous gene expression, which has great application value in disease treatment.
  • the p21 WAF1/CIP1 also known as CDKN1A, hereinafter referred to as p21
  • CDK cyclin-dependent kinase
  • p21 cyclin-dependent kinase
  • CDK cyclin-dependent kinase
  • the present invention provides a small activating RNA characterized in that one of the small activating RNAs is in the sequence shown in SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, and 12. Any contiguous fragment of 16-35 nucleotides in length having at least 75% homology or complementarity activates or up-regulates the expression of the p21 gene by targeting the human p21 gene promoter sequence. .
  • the target gene of the human p21 gene is selected from the group consisting of the sequences set forth in SEQ ID NOS: 5-12, wherein the human p21 gene promoter sequence is located at a distance from the transcription start position, respectively.
  • TSS Transcription start site upstream - 893 ⁇ - 801 bp (SEQ ID NO: 5), -717 ⁇ - 632 bp (SEQ ID NO: 6), -585 ⁇ - 551 bp (SEQ ID NO: 7), -554 ⁇ -504 bp (SEQ ID NO: 8), -514 to -485 bp (SEQ ID NO: 9), -442 to -405 bp (SEQ ID NO: 10), -352 to -313 bp (SEQ ID NO: 11), -325 to -260 bp (SEQ ID NO: 12).
  • the small activating RNA comprises a sense nucleic acid strand and an antisense nucleic acid strand, the sense nucleic acid strand and the antisense nucleic acid strand comprising a complementary region, wherein the complementary region is capable of forming a double stranded nucleic acid structure, wherein the sense nucleic acid strand or Any one of 16-35 nucleotides in length of the antisense nucleic acid strand and the human p21 gene promoter sequence has 75% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100%. Homology.
  • the sense nucleic acid strand and the antisense nucleic acid strand are present on two different nucleic acid strands as described above; the sense nucleic acid strand and the antisense nucleic acid strand are present on the same nucleic acid strand, A sandwich-type single-stranded nucleic acid molecule in which a complementary region of a sense nucleic acid strand and an antisense nucleic acid strand forms a double-stranded nucleic acid structure.
  • At least one of the small activating RNAs has a 3' overhang of 0 to 6 nucleotides in length; both of the small activating RNAs have a length of 2-3 3' overhang of nucleotides.
  • the sense nucleic acid strand or the antisense nucleic acid strand is 16-35 nucleotides in length, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.
  • the sense nucleic acid strand or the antisense nucleic acid strand is SEQ ID NOs: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108, 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249- 258, 261-262, 265-270, 275-280, 283-300, 303-308, 317-320, 323-324, 329-348, 351-352, 357-358, 361-366, 371-392,
  • the sequences shown at 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, 463-468 have at least 75%, such as 80%, 85%, 90. %, 95%, 99%, 100% homo
  • the sense nucleic acid strand or antisense nucleic acid strand sequence is SEQ ID NOs: 13-30, 35-46, 59-62, 67-74, 77-80, 85-96, 103-108 , 111-118, 121-132, 139-140, 147-180, 185-186, 189-190, 195-198, 201-202, 209-212, 215-218, 225-240, 243-246, 249 -258, 261-262, 265-270, 275-280, 283-300, 303-308, 317-320, 323-324, 329-348, 351-352, 357-358, 361-366, 371-392
  • the nucleotide sequences of any of 399-400, 405-412, 415-416, 419-424, 429-432, 439-442, 447-450, 453-458, and 463-468 are shown.
  • the invention also provides a method for preparing a small activating RNA according to any one of the preceding claims, which comprises the steps of: 1) selecting a target gene promoter sequence as a template and selecting 19 bases; The sequence serves as a target site; 2) synthesizes an RNA sequence having a homology of more than 75% of the sequence of the target site obtained in step 1) to obtain a sense oligonucleotide chain; 3) the sequence and the sense oligonucleoside obtained in step 2) The acid chain is complementary; 4) the sense oligonucleotide strand obtained in step 2) is mixed with the antisense oligonucleotide strand obtained in step 3) in the same molar number in the RNA annealing buffer, heated, and then naturally cooled to At room temperature, a double-stranded small activating RNA is obtained; wherein the human p21 gene promoter sequence is selected from the group consisting of the sequences set forth in SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, 12 .
  • At least one nucleotide of the small activating RNA is a chemically modified nucleotide, the chemical modification being at least one of the following modifications:
  • At least one nucleotide in the nucleotide sequence of the small activating nucleic acid molecule is a locked nucleic acid.
  • the activating or upregulating expression of the p21 gene is upregulated by at least 10%, such as 15%, 20%, 30%, 40%, 50%, 80%, 100%, 200% or more.
  • the invention also provides the use of a small activating RNA for the preparation of a preparation for activating or upregulating the expression of the p21 gene in a cell.
  • the small activating RNA is introduced directly into the cell or is produced in the cell after the nucleotide sequence encoding the small activating RNA is introduced into the cell.
  • the cell is a mammalian cell, preferably a human cell, more preferably a human tumor cell.
  • the human cell can be an isolated human cell line or can be present in a human body.
  • the human body is a patient having a tumor caused by insufficient expression of the p21 protein, and the small activating nucleic acid molecule is administered in an amount effective to achieve treatment of a tumor, preferably a bladder cancer, Prostate cancer, liver cancer or colorectal cancer.
  • the present invention provides an isolated p21 gene small activating RNA target site, wherein the target site is any consecutive 16-35 nucleosides selected from any one of SEQ ID Nos. 5-12. Acid sequence.
  • the invention features a method of activating or upregulating expression of a human p21 gene in a cell, wherein the method comprises administering to the subject or cell a small activating RNA as described in any one of the preceding.
  • the small activating RNA may be directly introduced into the cell, or may be produced in the cell after the nucleotide sequence encoding the small activating RNA is introduced into the cell.
  • the cell is a mammalian cell, preferably a human cell, more preferably a human tumor cell, and more preferably a human bladder cancer, prostate cancer, liver cancer or colorectal cancer cell.
  • the invention further discloses a composition
  • a composition comprising a small activating RNA as described above and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is a liposome, a high molecular polymer or a polypeptide.
  • the present invention also discloses the use of a small activating RNA as described above or a composition as described above for the preparation of a medicament for activating or upregulating the expression of the p21 gene, preferably in the preparation of an anti-tumor or benign proliferative lesion.
  • the tumor is bladder cancer, prostate cancer, liver cancer or colorectal cancer.
  • FIG. 1 shows the p21 gene promoter sequence from -1000 bp upstream of the transcription start site (TSS) to 3 bp downstream of the TSS.
  • the TSS is indicated by a curved arrow.
  • Figure 2 shows the screening of small activated RNA hotspot regions on the p21 gene promoter.
  • 439 double-stranded RNA molecules were designed and chemically synthesized for the p21 promoter sequence shown in Figure 1, and transfected into PC3 human prostate cancer cells, respectively.
  • the p21 gene mRNA level was analyzed after 72 hours using the QuantiGene 2.0 method.
  • Figure 3 shows the activation of the p21 gene by double-stranded RNA molecules of the hotspots 1 to 8.
  • Figure 4 shows the analysis of p21 gene mRNA levels by RT-qPCR method to verify the experimental results of QuantiGene 2.0.
  • A 439 double-stranded RNA molecules were divided into four regions (groups) according to their activity to induce p21 mRNA expression, and 5 double-stranded RNA molecules were randomly selected from each group and transfected into PC3 cells at a concentration of 10 nM, respectively. . After 72 hours, total cellular RNA was extracted, and p21 gene mRNA levels were analyzed by RT-qPCR after reverse transcription.
  • QuantiGene 2.0 (X-axis) and RT-qPCR (Y-axis) methods were used to detect the correlation of p21 gene-induced mRNA levels induced by double-stranded RNA molecules.
  • Figure 5 shows the effect of small activating RNA on the induction of p21 mRNA expression and inhibition of KU-7 cell proliferation.
  • the three small activating RNAs shown were transfected with KU-7 cells at 10 nM for 72 hours.
  • A RT-qPCR analysis of p21 gene mRNA expression levels.
  • B CCK-8 method to assess cell viability. The viability of the cells in the saRNA-treated group was expressed as a percentage of the cell viability relative to the control-treated group (Mock).
  • C Representative cell image at the end of transfection (100 x).
  • Figure 6 shows the effect of small activating RNA on the induction of p21 mRNA expression and inhibition of HCT116 cell proliferation.
  • the three small activating RNAs shown were transfected with HCT116 cells at 10 nM for 72 hours.
  • A RT-qPCR analysis of p21 gene mRNA expression levels.
  • B CCK-8 method was used to evaluate cell viability, and the viability of cells in the saRNA-treated group was expressed as a percentage of cell viability relative to the control-treated group (Mock).
  • C Representative cell image at the end of transfection (100 x).
  • Figure 7 shows the effect of small activating RNA on the induction of p21 mRNA expression and inhibition of HepG2 cell proliferation.
  • the three small activating RNAs shown were transfected with HepG2 cells at 10 nM for 72 hours.
  • A RT-qPCR analysis of p21 gene mRNA expression levels.
  • B CCK-8 method was used to evaluate cell viability, and the viability of cells in the saRNA-treated group was expressed as a percentage of cell viability relative to the control-treated group (Mock).
  • C Representative cell image at the end of transfection (100 x).
  • complementary refers to the ability of two oligonucleotide strands to form base pairs with each other.
  • Base pairs are typically formed by hydrogen bonds between nucleotide units in an antiparallel oligonucleotide chain.
  • the complementary oligonucleotide strand can be base paired in Watson-Crick mode (eg, AT, AU, CG), or in any other manner that allows for the formation of a duplex (eg, Hoogsteen type or reverse Hoogsteen type base pairing).
  • Base pairing “100% pairing" or “completely complementary” means having 100% complementarity, ie, the nucleotide units of both strands are all hydrogen bonded to each other.
  • Fully complementary or 100% paired means that each nucleotide unit from the first oligonucleotide strand in the double stranded region of the double stranded oligonucleotide molecule can form a hydrogen bond with the second oligonucleotide strand There is no "mismatch" situation. Incomplete complementarity refers to the case where the nucleotide units of both strands cannot all be hydrogen bonded to each other. For example, for an oligonucleotide strand of 20 nucleotides in length with two double stranded regions, if only two base pairs on each strand can hydrogen bond to each other, the oligonucleotide strand exhibits 10% Complementarity.
  • the oligonucleotide strand exhibits 90% complementarity.
  • Substantial complementarity refers to about 79%, about 80%, about 85%, about 90%, and about 95% complementarity.
  • oligonucleotide refers to a polymer of nucleotides and includes, but is not limited to, single or double stranded molecules of DNA, RNA, or DNA/RNA hybrids, including regularly and irregularly. Alternate deoxyribosyl moieties and oligonucleotide moieties, as well as modifications of these classes of oligonucleotides and to naturally occurring or non-naturally occurring backbones.
  • oligonucleotide refers to an oligonucleotide comprising two or more modified or unmodified ribonucleotides and/or analogs thereof.
  • oligonucleotide strand and "oligonucleotide sequence” as used herein are used interchangeably and refer to a generic term for short-chain nucleotides of less than 50 bases (nucleotides may be deoxyribonucleic acid DNA or RNA RNA).
  • the length of the oligonucleotide strand may be any length of 17 to 30 nucleotides.
  • gene refers to all nucleotide sequences required to encode a polypeptide chain or to transcribe a functional RNA.
  • a “gene” can be a gene that is endogenous or fully or partially recombined for a host cell (eg, due to the introduction of an exogenous oligonucleotide encoding a promoter and a coding sequence or a heterologous promoter that will be adjacent to an endogenous coding sequence). Introduced into the host cell).
  • the term “gene” includes nucleic acid sequences that can be composed of exons and introns.
  • the sequence encoding the protein is, for example, a sequence contained within an exon of an open reading frame between the initiation codon and the stop codon, and as used herein, "gene” may be taken to include, for example, a gene regulatory sequence such as a promoter.
  • a gene regulatory sequence such as a promoter.
  • a "gene” can be used to describe a functional nucleic acid comprising a regulatory sequence, such as a promoter or enhancer. Expression of the recombinant gene can be controlled by one or more heterologous regulatory sequences.
  • target gene may be a nucleic acid sequence, a transgene, a viral or bacterial sequence naturally occurring in an organism, a chromosomal or extrachromosomal and/or transient or stable transfection or incorporation into a cell and/or its chromatin.
  • the target gene may be a protein-coding gene or a non-protein-encoding gene (for example, a microRNA gene or a long-chain non-coding RNA gene).
  • the target gene usually contains a promoter sequence, and a small activating RNA designed to have identity (also referred to as homology) with the promoter sequence can achieve positive regulation of the target gene, which is manifested by up-regulation of target gene expression.
  • Target gene promoter sequence refers to a non-coding sequence of a target gene
  • a target gene promoter sequence in “complementary to a target gene promoter sequence” refers to a coding strand of the sequence, also referred to as a non-template strand, That is, a nucleic acid sequence which is the same sequence as the coding sequence of the gene.
  • a “target sequence” refers to a sequence fragment to which a sense oligonucleotide strand or an antisense oligonucleotide of a small activating RNA in a target gene promoter sequence is homologous or complementary.
  • sense strand As used herein, the terms “sense strand”, “sense oligonucleotide strand” are interchangeable, and a sense oligonucleotide strand of a small activating RNA refers to a promoter sequence containing a target gene in a small activating RNA duplex. A first ribonucleic acid strand having the identity of the coding strand.
  • antisense strand As used herein, the terms “antisense strand”, “antisense oligonucleotide strand” are interchangeable, and an antisense oligonucleotide strand of a small activating RNA refers to a small activating RNA duplex and a sense oligonucleotide. A second ribonucleic acid strand complementary to the strand.
  • coding strand refers to a DNA strand in a target gene that is not transcribed, and the nucleotide sequence of the strand is identical to the sequence of the RNA produced by transcription (in the RNA, the DNA is substituted with U). T).
  • the coding strand of the target gene promoter double-stranded DNA sequence described in the present invention refers to a promoter sequence on the same DNA strand as the target gene DNA coding strand.
  • template strand refers to another strand of a double-stranded DNA of a target gene that is complementary to the coding strand, which can be transcribed as a template for RNA, which is complementary to the transcribed RNA base (AU, GC).
  • RNA polymerase binds to the template strand and moves along the 3' ⁇ 5' direction of the template strand, catalyzing the synthesis of RNA in the 5' ⁇ 3' direction.
  • the template strand of the target gene promoter double-stranded DNA sequence described in the present invention refers to a promoter sequence on the same DNA strand as the target gene DNA template strand.
  • promoter refers to a nucleic acid sequence that does not encode a protein, which exerts a regulatory effect on their transcription by positional association with protein encoding or RNA encoding nucleic acid sequences.
  • a eukaryotic promoter comprises 100-5,000 base pairs, although this range of lengths is not meant to limit the term “promoter” as used herein.
  • the promoter sequence is typically located at the 5' end of the protein coding or RNA coding sequence, in some cases the promoter sequence is also present in the exon and intron sequences.
  • transcription initiation site refers to a nucleotide that marks the initiation of transcription on a template strand of a gene.
  • a transcription initiation site can occur on the template strand of the promoter region.
  • a gene can have more than one transcription initiation site.
  • identity refers to a coding strand of a region of a promoter sequence of a promoter gene of a small activation RNA (sense strand or antisense strand). Or the template chain has at least 75% similarity.
  • overhang refers to an oligonucleotide (5' or 3') non-base pairing nucleotide that is extended beyond the double strand. Another strand of one of the strands within the oligonucleotide is produced. A single-stranded region that extends beyond the 3' and/or 5' ends of the duplex is referred to as a protrusion.
  • gene activation or “activation gene” are used interchangeably to refer to measurement of gene transcription levels, mRNA levels, protein levels, enzymatic activity, methylation status, chromatin status or conformation, translation levels, Or its activity or state in a cell or biological system to determine the increase in transcription, translation or expression or activity of a nucleic acid. These activities or states can be determined directly or indirectly.
  • gene activation and activation gene refer to an increase in activity associated with a nucleic acid sequence, regardless of the mechanism by which such activation occurs, for example, it acts as a regulatory sequence, is transcribed into RNA, is translated into a protein and Increase protein expression.
  • small activating RNA As used herein, the terms “small activating RNA,” “saRNA,” “small activating nucleic acid molecule” are used interchangeably to refer to a ribonucleic acid molecule capable of promoting gene expression, and may be comprised of a non-coding nucleic acid sequence comprising a target gene (eg, Promoter, enhancer, etc.) a first ribonucleic acid strand of a ribonucleotide sequence having sequence identity (antisense strand, also referred to as an antisense oligonucleotide strand) and a nucleotide sequence complementary to the first strand A second ribonucleic acid chain (sense strand, also referred to as a sense strand or a sense oligonucleotide strand), wherein the first strand and the second strand form a duplex.
  • a target gene eg, Promoter, enhancer, etc.
  • antisense strand also referred to as an anti
  • Small activating RNAs can also be composed of synthetic or vector-expressed single-stranded RNA molecules that form a double-stranded region hairpin structure, wherein the first region comprises a nucleic acid sequence having sequence identity to the promoter sequence of the gene, and the second region comprises The nucleic acid sequence is complementary to the first region.
  • the duplex region of the small activating RNA molecule typically has a length of from about 10 to about 50 base pairs, from about 12 to about 48 base pairs, from about 14 to about 46 base pairs, from about 16 to about 44.
  • Base pairs about 18 to about 42 base pairs, about 20 and about 40 base pairs, about 22 and about 38 base pairs, about 24 and about 36 base pairs, about 26 and about 34 base pairs, about 28 and about 32 base pairs, typically about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50 base pairs.
  • saRNA and small activating RNA small activating nucleic acid molecule
  • nucleic acids other than ribonucleotide moieties, including but not limited to modified nucleotides or analogs.
  • hot spot refers to a gene promoter region of at least 30 bp in length, in which aggregation of functional small activation nucleic acid molecule targets, ie, small activation nucleic acid molecules targeting these hotspot regions, are at least 60 % can induce target gene mRNA expression to be 1.5 times or more.
  • p21 refers to the p21 WAF1/CIP1 gene, also known as the CDKN1A gene, is a cyclin-dependent kinase (CDK) inhibitor and an important tumor suppressor gene, sometimes referred to as a "target. gene".
  • CDK cyclin-dependent kinase
  • target. gene an important tumor suppressor gene
  • the term "synthetic” refers to the manner in which an oligonucleotide is synthesized, including any means by which RNA can be synthesized, such as chemical synthesis, in vitro transcription, vector expression, and the like.
  • the preparation methods of the small activating nucleic acid molecules provided by the present invention include sequence design and sequence synthesis.
  • the synthesis of the small activating nucleic acid molecule sequence can be carried out by chemical synthesis or by a biotechnology company specializing in nucleic acid synthesis.
  • the method of chemical synthesis includes the following four processes: (1) synthesis of oligoribonucleotides; (2) deprotection; (3) purification separation; (4) desalting and annealing.
  • the specific steps of chemical synthesis of the double-stranded RNA molecules of the present invention, such as saRNA are as follows:
  • RNA was synthesized on an automated DNA/RNA synthesizer (for example, Applied Biosystems EXPEDITE 8909) while setting the coupling time for each cycle to 10-15 minutes, and the starting material was 5' for solid phase attachment.
  • -O-p-dimethoxytrityl-thymidine support the first cycle is linked to a base on a solid support, and then in the nth (19 ⁇ n ⁇ 2) cycle, at One base is ligated to the base to which the n-1 cycle is ligated, and this cycle is repeated until the synthesis of all nucleic acid sequences is completed.
  • the obtained crude product of saRNA was dissolved in 2 ml of an aqueous solution of ammonium acetate having a concentration of 1 mol/ml, and then separated by a high pressure liquid chromatography reverse phase C18 column to obtain a purified saRNA single-chain product.
  • Cell lines RT4, KU-7, T24, J82, TCCSUP and HT-1197 were cultured in modified McCoy's 5A medium (Gibco); cell lines 5637, PC3 and Bel-7402 were cultured in RPMI 1640 medium (Gibco); The UM-UC-3 cell line was cultured in basal medium (Gibco). All media contained 10% calf serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Gibco). The cells were cultured at 5% CO 2 at 37 °C. The double-stranded RNA molecules designed in the experiments were transfected with RNAiMax (Invitrogen, Carlsbad, CA) at a concentration of 10 nM (unless otherwise stated) according to the manufacturer's instructions.
  • RNAiMax Invitrogen, Carlsbad, CA
  • RNA (1 ⁇ g) was reverse transcribed into cDNA using a PrimeScript RT kit containing gDNA Eraser (Takara, Shlga, Japan).
  • qPCR was performed using ABI 7500 Fast Real-time PCR System (Applied Biosystems) and SYBR Premix Ex Taq II (Takara, Shlga, Japan) reagents under the conditions of 95 ° C for 3 seconds, 60 ° C for 30 seconds, and amplification for 40 cycles. Take GAPDH as an internal reference. All primer sequences are listed in Table 1.
  • the cells were plated at 2-4 x 10 3 cells/well in 96-well plates, cultured overnight, and transfected with oligonucleotide duplexes. Three days after transfection, cell proliferation assay was performed using CCK8 (Dojindo) according to the instructions. The experimental procedure is briefly described as follows: 10 ⁇ L of CCK8 solution was added to each well, and incubated at 37 ° C for 1 hour, after which the absorbance at 450 nm was measured using a microplate reader.
  • the cells were plated in 96-well plates and transfected with oligonucleotide duplexes. After transfection for 72 hours, the mRNA level of the target gene was quantitatively detected using the QuantiGene 2.0 kit (AffyMetrix).
  • the QuantiGene 2.0 kit is a hybridization-based method that directly quantifies mRNA levels using gene-specific probes. The experimental procedure is briefly described as follows: The lysate was added to lyse the transfected cells, and the cell lysate was loaded into a capture well plate containing CDKN1A (p21) and HPRT1 (housekeeping gene) probes, and hybridized overnight at 55 °C.
  • hybridization was performed sequentially with 2.0 PreAMP, 2.0 AMP and 2.0 Lable Probe in 100 ⁇ L of the corresponding buffer (provided by the Quantigene 2.0 kit). All hybridizations were shaken for 1 hour at 50-55 °C. After the final wash, 2.0 Substrate was added and incubated for 5 minutes at room temperature. The optical signal was then detected using an Infinite 200PRO plate reader (Tecan, Switzerland).
  • Results are expressed as mean ⁇ standard deviation.
  • One-way analysis of variance was performed using GraphPad Prism software (GraphPad Software) followed by Tukey's t-test for statistical analysis. The criteria for statistical significance were set to *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001.
  • Example 1 Screening for functional small activator RNA (saRNA) targeting the promoter region of the p21 gene
  • a 1 kb promoter sequence of the p21 gene was obtained from the UCSC Genome database (Fig. 1).
  • a target of 19 bp in size was selected from the upstream of the transcription start site (TSS) at -1 kb, and moved to the TSS site by moving 1 bp each time to obtain a total of 982 target sequences.
  • the target sequence is subjected to a filtration treatment to exclude a GC sequence containing more than 65% or less than 35%, and a target sequence containing 5 or more than 5 consecutive identical nucleotides. After filtering, the remaining 439 target sequences entered the screening process as candidates.
  • the corresponding double-stranded double-stranded RNA molecules are chemically synthesized.
  • the length of the sense and antisense strands of the double-stranded RNA molecule used in the experiment are both 21 nucleotides, and the double-stranded RNA molecule, for example, the 5' of the first ribonucleic acid strand (sense strand) of the double-stranded saRNA
  • the 19 nucleotides of the region are 100% identical to the promoter target sequence, and the 3' end contains a dTdT overhang; the 19 nucleotide of the 5' region of the second ribonucleic acid strand is aligned with the first ribonucleic acid strand
  • the 19 nucleotides of the 3' region sequence are fully complementary and contain a dTdT overhang at the 3' end.
  • the aforementioned double-stranded RNA molecule was transfected into PC3 prostate cancer cells at a final concentration of 10 nM, and 72 hours later, p21 gene mRNA levels were detected using the QuantiGene 2.0 kit.
  • the fold change in p21 mRNA levels of each double-stranded RNA molecule relative to the blank control treatment was calculated and plotted in Figure 2.
  • the fold change in p21 gene mRNA caused by all double-stranded RNA molecules in this study ranged from 0.66 (inhibition) to 8.12 (induction) (Fig. 2B).
  • RNA molecules screened 132 double-stranded RNA molecules (30.1%) were able to induce p21 mRNA at least 2-fold; 229 (52.4%) double-stranded RNA molecules induced p21 mRNA at least 1.5-fold, and these induced p21 The mRNA expression increased by more than 10%.
  • the double-stranded RNA molecule is a saRNA, a functional saRNA. These functional saRNAs are dispersed throughout the p21 promoter region.
  • the hotspot region is defined as a region containing at least 10 corresponding small activating RNAs, wherein at least 60% of the small activating RNAs are capable of inducing p21 mRNA expression of 1.5-fold or more (Fig. 2A and Fig. 3).
  • the target sequences of the hotspots 1 to 8 and the corresponding small activating RNA sequences are listed in Tables 2 and 3, respectively.
  • hotspots include hotspot region 1, the corresponding target sequence is -893 bp to -801 bp of the p21 promoter sequence, and the sequence is as shown in SEQ ID NO: 93, and 44 functional saRNAs were found in this region (Table 3, Figure 3A).
  • the corresponding target sequence of the hot spot region 2 (Table 3, Figure 3B) is -71 to -632 bp of the p21 promoter sequence, and the sequence is as shown in SEQ ID NO: 94.
  • 31 functional saRNAs were found in this region, respectively, RAG -693, RAG-692, RAG-688, RAG-696, RAG-694, RAG-687, RAG-691, RAG-690, RAG-689, RAG-682, RAG-686, RAG-662, RAG-695 , RAG-654, RAG-658, RAG-685, RAG-704, RAG-714, RAG-705, RAG-661, RAG-656, RAG-698, RAG-697, RAG-657, RAG-715, RAG -652, RAG-651, RAG-650, RAG-716, RAG-717, RAG-711;
  • Hot spot 3 (Table 3, Figure 3C), the corresponding target sequence is -585 bp to -551 bp of the p21 promoter sequence, the sequence is shown as SEQ ID NO: 95, and it is found in this region that it contains 9 functional saRNAs, respectively RAG-580, RAG-577, RAG-569, RAG-576, RAG-570, RAG-574, RAG-585, RAG-579, RAG-584;
  • Hot spot 4 (Table 3, Figure 3D), the corresponding target sequence is -554 bp to -505 bp of the p21 promoter sequence, and the sequence is shown as SEQ ID NO: 96.
  • 17 functional saRNAs were found, respectively RAG-524, RAG-553, RAG-537, RAG-526, RAG-554, RAG-523, RAG-534, RAG-543, RAG-525, RAG-535, RAG-546, RAG-545, RAG- 542, RAG-531, RAG-522, RAG-529, RAG-552;
  • Hot spot 5 (Table 3, Figure 3E), the corresponding target sequence is -514 bp to -485 bp of the p21 promoter sequence, and the sequence is shown as SEQ ID NO: 97.
  • 9 functional saRNAs were found, respectively RAG-503, RAG-504, RAG-505, RAG-506, RAG-507, RAG-508, RAG-509, RAG-510, RAG-511, RAG-512, RAG-513, RAG-514;
  • Hot spot 6 (Table 3, Figure 3F), the corresponding target sequence is -442 bp to -405 bp of the p21 promoter sequence, and the sequence is shown as SEQ ID NO: 98. In this region, 12 functional saRNAs were found, respectively.
  • Hot spot 7 (Table 3, Figure 3G), the corresponding target sequence is -352 bp to -313 bp of the p21 promoter sequence, the sequence is shown as SEQ ID NO: 99, and 13 functional saRNAs were found in this region, respectively RAG-335, RAG-351, RAG-352, RAG-331, RAG-344, RAG-342, RAG-341, RAG-333, RAG-345, RAG-346, RAG-336, RAG-332, RAG- 343;
  • Hot spot 8 (Table 3, Figure 3H), the corresponding target sequence is -325 bp to -260 bp of the p21 promoter sequence, the sequence is shown as SEQ ID NO: 100, and 18 functional saRNAs were found in this region, respectively RAG-294, RAG-285, RAG-286, RAG-292, RAG-291, RAG-284, RAG-279, RAG-280, RAG-325, RAG-293, RAG-322, RAG-321, RAG- 281, RAG-289, RAG-278, RAG-283, RAG-282, RAG-295.
  • Example 2 saRNA induces p21 gene mRNA expression and inhibits cancer cell proliferation
  • the saRNA (RAG1-431, RAG1-553, RAG1-688) screened by QuantiGene 2.0 was transfected into the cancer cell line KU-7 (bladder cancer), HCT116 (colon cancer) and HepG2 (hepatocellular carcinoma). The results showed that in all of the above cell lines, saRNA induced at least twice the expression level of p21 gene mRNA and inhibited cell proliferation, revealing the efficacy of saRNA-mediated p21 induction.
  • RAG1-431, RAG1-553, and RAG1-688 were transfected into KU7 cells, respectively, to induce p21 mRNA expression of 14.0, 36.9, and 31.9 fold, respectively, and the survival rate relative to blank treatment was 71.7%, 60.7%, 67.4% (Figure 5).
  • RAG1-431, RAG1-553, and RAG1-688 were transfected into HCT116 cells, respectively, to induce p21 mRNA expression of 2.3, 3.5, and 2.4 fold, respectively, and the survival rates relative to blank treatment were 45.3%, 22.5%, and 38.5% ( Figure 6).
  • RAG1-431, RAG1-553, and RAG1-688 were transfected into HepG2 cells, respectively, to induce p21 mRNA expression of 2.2, 3.3, and 2.0 fold, respectively, and the survival rate of the corresponding blank treatment was 76.7%, 64.9%, and 79.9% (Fig. 7).

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Abstract

提供了一种由正义寡核苷酸链和反义寡核苷酸链组成的激活或上调人p21基因表达的小激活RNA，该正义核酸链或反义核酸链与人p21基因启动子靶序列中的任一长度为16-35个核苷酸的连续片段具有75％以上的同源性，其中人p21基因启动子靶序列选自由SEQ ID NO：5、6、7、8、9、10、11、12所示的序列组成的组。

Description

一种激活p21基因表达的方法 技术领域
本发明涉及分子生物学领域，具体涉及利用靶向基因启动子序列的双链小RNA正向调控基因表达。
背景技术
双链小核酸分子包括化学合成的寡核糖核苷酸如小激活RNA(saRNA)和天然存在的寡核糖核苷酸如微小核糖核苷酸(miRNA)已被证明能够以序列特异性的方式靶向蛋白编码基因的调控序列如启动子序列而在转录和表观遗传水平正向调控基因的表达水平，这种现象被称为RNA激活(RNAa)(Li，Okino et al.(2006)Proc Natl Acad Sci U S A 103：17337-17342；Janowski，Younger et al.(2007)Nat Chem Biol 3：166-173；Place，Li et al.(2008)Proc Natl Acad Sci U S A105：1608-1613；Huang，Place et al.(2012)Nucleic Acids Res 40：1695-1707；Li(2017)Adv Exp Med Biol 983：1-20)。研究表明RNA激活是从秀丽隐杆线虫到人的进化保守的内源性分子机制(Huang，Qin et al.(2010)PLoS One 5：e8848；Seth，Shirayama et al.(2013)Dev Cell 27：656-663；Turner，Jiao et al.(2014)Cell Cycle13：772-781)。
如何安全和选择性增强人体细胞内源性基因或者蛋白质表达仍然是基因治疗领域的一个巨大挑战。传统的基于病毒基因治疗系统具有其固有的缺陷，包括有可能改变宿主基因组及引起免疫反应等副作用。而RNA激活具有能够激活内源基因表达而不改变基因组的优点，代表了一种激活内源性基因表达的新策略，在疾病治疗方面具有巨大的应用价值。
p21 WAF1/CIP1(也称CDKN1A，下称p21)基因为细胞周期蛋白依赖性激酶(CDK)抑制剂，是一个重要的肿瘤抑制基因(Harper，Adami et al.(1993)Cell75：805-816；Fang，Igarashi et al.(1999)Oncogene 18：2789-2797)。研究表明，异位载体过表达p21或者通过激活内源性p21的转录可以有效抑制肿瘤细胞和在体肿瘤的生长(Harper，Adami et al.(1993)Cell 75：805-816；Eastham，Hall et al.(1995)Cancer Res 55：5151-5155；Wu，Bellas et al.(1998)J Exp Med 187：1671-1679；Harrington，Spitzweg et al.(2001)J Urol 166：1220-1233)。因此，靶向激活p21基因的方法有可能在诸多疾病例如癌症的治疗上有广泛应用价值。
发明内容
本发明提供了一种一种小激活RNA，其特征在于，所述小激活RNA中的一条链与SEQ ID NO：5、6、7、8、9、10、11、12所示的序列中的任一长度为16-35个核苷酸的连续片段具有至少75％的同源性或互补性，所述小激活RNA通过靶向人p21基因启动子序列激活或上调p21基因的表达。。
在本发明的实施例中，所述人p21基因的靶基因选自由SEQ ID NO：5-12所示的序列组成的组，其中，所述人p21基因启动子序列分别位于距离转录起始位点(transcription start site，TSS)上游-893～-801bp(SEQ ID NO：5)、-717～-632bp(SEQ ID NO：6)、-585～-551bp(SEQ ID NO：7)、-554～-504bp(SEQ ID NO：8)、-514～-485bp(SEQ ID NO：9)、-442～-405bp(SEQ ID NO：10)、-352～-313bp(SEQ ID NO：11)、-325～-260bp(SEQ ID NO：12)。
在一个实施例中，所述小激活RNA包含正义核酸链和反义核酸链，所述正义核酸链和反义核酸链含有互补区域，互补区域能形成双链核酸结构，其中的正义核酸链或反义核酸链与人p21基因启动子序列中的任一长度为16-35个核苷酸的连续片段具有75％以上、80％以上、90％以上、95％以上、99％以上或100％的同源性。
在一个实施例中，如前所述所述正义核酸链和反义核酸链存在于两条不同的核酸链上；所述正义核酸链和反义核酸链存在于同一条核酸链上，为发夹型单链核酸分子，其中正义核酸链和反义核酸链的互补区域形成双链核酸结构。
在一个具体实施例中，所述小激活RNA中的至少一条链具有长度为0至6个核苷酸的3’突出端；所述小激活RNA中的两条链都具有长度为2-3个核苷酸的3’突出端。
在一个实施例中，所述正义核酸链或反义核酸链的长度为16-35个核苷酸，例如16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、或35个核苷酸。
在一个具体实施例中，所述正义核酸链或反义核酸链与SEQ ID NO：13-30、35-46、59-62、67-74、77-80、85-96、103-108、111-118、121-132、139-140、147-180、185-186、189-190、195-198、201-202、209-212、215-218、225-240、243-246、249-258、261-262、265-270、275-280、283-300、303-308、317-320、323-324、329-348、351-352、357-358、361-366、371-392、399-400、405-412、 415-416、419-424、429-432、439-442、447-450、453-458、463-468所示序列具有至少75％例如80％、85％、90％、95％、99％、100％的同源性。
在一个具体实施例中，所述正义核酸链或反义核酸链序列如SEQ ID NO：13-30、35-46、59-62、67-74、77-80、85-96、103-108、111-118、121-132、139-140、147-180、185-186、189-190、195-198、201-202、209-212、215-218、225-240、243-246、249-258、261-262、265-270、275-280、283-300、303-308、317-320、323-324、329-348、351-352、357-358、361-366、371-392、399-400、405-412、415-416、419-424、429-432、439-442、447-450、453-458、463-468任一核苷酸序列所示。
本发明还提供了一种如前任一项所述的小激活RNA的制备方法，其特征在于，所述方法包括以下步骤：1)以靶基因启动子序列为模板，选取包含19个碱基的序列作为靶位点；2)合成与步骤1)所得靶位点序列75％以上的同源性的RNA序列，得到正义寡核苷酸链；3)序列与步骤2)得到的正义寡核苷酸链互补；4)将步骤2)得到的正义寡核苷酸链与步骤3)得到的反义寡核苷酸链以相同的摩尔数在RNA退火缓冲液中混合、加热，然后自然冷却至室温，即得到双链的小激活RNA；其中人p21基因启动子序列选自由SEQ ID NO：5、6、7、8、9、、10、11、12所示的序列组成的组。
在一个实施例中，所述小激活RNA中的至少一个核苷酸为化学修饰的核苷酸，所述化学修饰为如下修饰中的至少一种：
(1)对所述小激活RNA的核苷酸序列中连接核苷酸的磷酸二酯键的修饰；
(2)对所述小激活RNA的核苷酸序列中的核糖的2’-OH的修饰；
(3)对所述小激活RNA的核苷酸序列中的碱基的修饰；
(4)所述小激活核酸分子的核苷酸序列中的至少一个核苷酸为锁核酸。
在一个实施例中，所述激活或上调p21基因的表达为上调至少10％，例如15％、20％、30％、40％、50％、80％、100％、200％以上。
本发明还提供了小激活RNA在制备用于激活或上调p21基因在细胞中表达的制剂中的应用。所述的小激活RNA被直接导入所述细胞中，或者是在编码该小激活RNA的核苷酸序列导入细胞后在细胞中产生的。所述细胞是哺乳动物细胞，优选为人类细胞，更优选为人类肿瘤细胞。该人类细胞可以是分离的人细胞系，也可以存在于人体中。
在一个实施例中，所述的人体是患有由p21蛋白表达不足引发的肿瘤患者，并且所述小激活核酸分子被施用以有效量以实现对肿瘤的治疗，所述肿瘤优选为膀胱癌、前列腺癌、肝癌或结直肠癌。
又一方面，本发明还提供一种分离的p21基因小激活RNA靶位点，其中所述靶位点为选自SEQ ID No 5-12的任一条序列上的任意连续16-35个核苷酸序列。
再一方面，本发明公开了一种激活或上调人p21基因在细胞中表达的方法，其中所述方法包括向受试者或细胞施予如前任一项所述的小激活RNA。所述小激活RNA可以被直接导入细胞中，也可以是在编码该小激活RNA的核苷酸序列导入细胞后在细胞中产生的。所述的细胞是哺乳动物细胞，优选为人类细胞，更优选为人类肿瘤细胞，再优选为人膀胱癌、前列腺癌、肝癌或结直肠癌细胞。
本发明进一步公开了一种包含如前所述的小激活RNA和药学上可接受的载体的组合物。其中，药学上可接受的载体为脂质体、高分子聚合物或多肽。
本发明还公开了如前所述的小激活RNA或如前所述的组合物在制备用于激活或上调p21基因表达的药物中的应用，优选为在制备抗肿瘤或良性增殖性病变中的药物中的应用，更优选地，所述肿瘤为膀胱癌、前列腺癌、肝癌或结直肠癌。
附图说明
图1示出p21基因启动子序列，从转录起始位点(TSS)上游-1000bp至TSS下游3bp。TSS由弯曲的箭头表示。
图2示出筛选p21基因启动子上的小激活RNA热点区域。针对图1所示p21启动子序列设计并化学合成439个双链RNA分子，分别转染至PC3人前列腺癌细胞。72小时后用QuantiGene 2.0方法分析p21基因mRNA水平。(A)439个双链RNA分子(X轴)分别所致的p21 mRNA水平相对于对照处理的倍数变化(Y轴)。X轴上的双链RNA分子按照它们相对于p21基因的TSS的位置(从最上游的RAG-898到最下游的RAG-177)进行排序。图示中标出了8个热点(hotspot)区域(浅灰色长方形框)。(B)对(A)的数据按照每个双链RNA分子诱导p21 mRNA表达改变的倍数大小从低到高进行排序。(A)和(B)中的虚线表示2倍诱导。
图3示出热点区1至8的双链RNA分子对p21基因的激活作用。
图4示出采用RT-qPCR方法分析p21基因mRNA水平，以验证QuantiGene 2.0的实 验结果。(A)将439个双链RNA分子按照它们诱导p21 mRNA表达的活性大小分成四个区域(组)，从每组中随机挑选5个双链RNA分子，分别以10nM的浓度转染至PC3细胞。72小时后，提取细胞总RNA、在反转录后采用RT-qPCR方法分析p21基因mRNA水平。(B)QuantiGene 2.0(X轴)和RT-qPCR(Y轴)方法检测双链RNA分子诱导的p21基因相对mRNA水平的相关性。
图5示出小激活RNA诱导p21 mRNA表达和抑制KU-7细胞增殖的作用。所示3个小激活RNA分别以10nM转染KU-7细胞72小时。(A)RT-qPCR分析p21基因mRNA表达水平。(B)CCK-8方法评估细胞活力。saRNA处理组细胞的活力表示为相对于对照处理组(Mock)细胞活力的百分比。(C)转染结束时具代表性的细胞图像(100×)。
图6示出小激活RNA诱导p21 mRNA表达和抑制HCT116细胞增殖的作用。所示3个小激活RNA分别以10nM转染HCT116细胞72小时。(A)RT-qPCR分析p21基因mRNA表达水平。(B)CCK-8方法评估细胞活力，saRNA处理组细胞的活力表示为相对于对照处理组(Mock)细胞活力的百分比。(C)转染结束时具代表性的细胞图像(100×)。
图7示出小激活RNA诱导p21 mRNA表达和抑制HepG2细胞增殖的作用。所示3个小激活RNA分别以10nM转染HepG2细胞72小时。(A)RT-qPCR分析p21基因mRNA表达水平。(B)CCK-8方法评估细胞活力，saRNA处理组细胞的活力表示为相对于对照处理组(Mock)细胞活力的百分比。(C)转染结束时具代表性的细胞图像(100×)。
具体实施方式
下面将通过具体描述，对本发明作进一步的说明。
除非另有限定，本文中所使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解相同的含义。
本申请中，单数形式“一个”、“该”包括复数对象，除非上下文另外清楚规定。
定义
如本文所用的术语“互补”是指两条寡核苷酸链彼此形成碱基对的能力。碱基对通常由反向平行的寡核苷酸链中的核苷酸单元之间的氢键形成。互补寡核苷酸链可以Watson-Crick方式碱基配对(例如，A-T，A-U，C-G)，或以允 许形成双链体的任何其他方式(例如Hoogsteen型或者反向Hoogsteen型碱基配对)进行碱基配对。“100％配对”或“完全互补”是指具有100％的互补性，即两条链的核苷酸单元全部互相氢键结合。
完全互补或100％配对是指双链寡核苷酸分子的双链区中来自第一条寡核苷酸链的每个核苷酸单元可以与第二条寡核苷酸链形成氢键而没有“错配”的情况。不完全互补是指两条链的核苷酸单元不能全部互相氢键结合的情况。例如，对于两条双链区为20个核苷酸长度的寡核苷酸链，如果每条链上只有两个碱基对可以彼此氢键结合，则寡核苷酸链展现出10％的互补性。在同一实例中，如果每条链上的18个碱基对可以彼此氢键结合，则寡核苷酸链展现出90％的互补性。基本互补性是指约79％，约80％，约85％，约90％，约95％以上的互补性。
如本文所用的术语“寡核苷酸”是指核苷酸的聚合物，并且包括但不限于DNA、RNA、或DNA/RNA杂交体的单链或双链分子，包括规则地和不规则地交替的脱氧核糖基部分和核糖基部分的寡核苷酸链，以及这些种类的寡核苷酸的修饰和以天然存在的或非天然存在的骨架。
如本文所用的术语“寡核糖核苷酸”是指包含两个或更多个经修饰或未经修饰的核糖核苷酸和/或其类似物的寡核苷酸。
如本文所用的术语“寡核苷酸链”和“寡核苷酸序列”可互换，是指50个以下碱基的短链核苷酸的总称(核苷酸可以为脱氧核糖核酸DNA或核糖核酸RNA)。在本发明中，寡核苷酸链的长度可以是17至30个核苷酸的任一长度。
如本文所用的术语“基因”是指编码一条多肽链或转录一条功能RNA所需的全部核苷酸序列。“基因”可以是对于宿主细胞而言内源的或完全或部分重组的基因(例如，由于引入编码启动子的外源寡核苷酸和编码序列或将邻近内源编码序列的异源启动子导入宿主细胞)。例如，术语“基因”包括可以由外显子和内含子组成的核酸序列。编码蛋白质的序列是，例如，包含在起始密码子和终止密码子之间的开放阅读框中的外显子内的序列，如本文所用，“基因”可以指包括例如基因调控序列例如启动子，增强子和本领域已知的控制另一基因的转录，表达或活性的所有其他序列，无论另一基因是否包含编码序列或非编码序列。在一种情况下，例如，“基因”可以用于描述包含调控序列例如启动子或增强子的功能性核酸。重组基因的表达可以通过一种或多种异源调节序 列来控制。
如本文所用的术语“靶基因”可以是天然存在于生物体中的核酸序列、转基因、病毒或细菌序列、染色体或染色体外和/或瞬时或稳定转染或掺入细胞和/或其染色质。靶基因可以为蛋白质编码基因，也可为非蛋白编码基因(例如微小RNA基因、长链非编码RNA基因)。靶基因通常含有启动子序列，设计与启动子序列具有同一性(也称同源性)的小激活RNA可以实现对靶基因的正性调控，表现为靶基因表达的上调。“靶基因启动子序列”是指靶基因的非编码序列，在本发明中涉及“与靶基因启动子序列互补”中靶基因启动子序列是指该序列的编码链，亦称非模板链，即为与该基因编码序列为同一序列的核酸序列。“靶点序列”是指靶基因启动子序列中小激活RNA的正义寡核苷酸链或反义寡核苷酸与之同源或互补的序列片段。
如本文所用，术语“正义链”、“正义寡核苷酸链”可互换，小激活RNA的正义寡核苷酸链是指小激活RNA双链体中含与靶基因的启动子序列的编码链具有同一性的第一核糖核酸链。
如本文所用，术语“反义链”、“反义寡核苷酸链”可互换，小激活RNA的反义寡核苷酸链是指小激活RNA双链体中与正义寡核苷酸链互补的第二核糖核酸链。
如本文所用的术语“编码链”是指靶基因中不能进行转录的那一条DNA链，该链的核苷酸序列与转录生成的RNA的序列一致(在RNA中是以U取代了DNA中的T)。本发明中所述的靶基因启动子双链DNA序列的编码链是指与靶基因DNA编码链在同一条DNA链上的启动子序列。
如本文所用的术语“模板链”是指靶基因的双链DNA中与编码链互补的另一条链，可作为模板转录为RNA的那条链，该链与转录的RNA碱基互补(A-U，G-C)。在转录过程中，RNA聚合酶与模板链结合，并沿着模板链的3′→5′方向移动，按照5′→3′方向催化RNA的合成。本发明中所述的靶基因启动子双链DNA序列的模板链是指与靶基因DNA模板链在同一条DNA链上的启动子序列。
如本文所用的术语“启动子”是指不编码蛋白质的核酸序列，通过与蛋白质编码或RNA编码核酸序列在位置上关联而对它们的转录发挥调控作用。通常，真核启动子包含100-5,000个碱基对，尽管此长度范围并不意味着限制本文所用的术语“启动子”。虽然启动子序列一般位于蛋白质编码或者RNA编码序列的 5′端，但有些情况下启动子序列也存在于外显子及内含子序列中。
如本文所用的术语“转录起始位点”是指在基因的模板链上标志转录起始的核苷酸。转录起始位点可出现于启动子区的模板链上。一个基因可以有多于一个的转录起始位点。
如本文所用的术语“同一性”或“同源性”是指小激活RNA的其中一条寡核苷酸链(正义链或者反义链)与靶基因的启动子序列的某一区域的编码链或者模板链存在至少75％的相似性。
如本文所用的术语“突出”、“overhang”、“悬垂”可互换，是指寡核苷酸链末端(5′或3′)非碱基配对核苷酸，其是由延伸超出双链寡核苷酸内的其中一条链的另一条链产生的。延伸超出双链体3′和/或5′端的单链区域被称为突出。
如本文所用，术语“基因激活”或“激活基因”可互换，是指通过测量基因转录水平、mRNA水平、蛋白水平、酶活性、甲基化状态、染色质状态或构型、翻译水平、或其在细胞或生物系统中的活性或状态来测定某一核酸转录、翻译或表达或活性的增加。这些活动或状态可以直接或间接的测定。此外，“基因激活”、“激活基因”是指与核酸序列相关的活性增加，而不管发生这种激活的机制如何，例如其作为调节序列发挥调控作用、被转录成RNA，被翻译为蛋白质并增加蛋白质的表达。
如本文所用，术语“小激活RNA”、“saRNA”、“小激活核酸分子”可互换，是指能够促进基因表达的核糖核酸分子，并且可以由包含与靶基因的非编码核酸序列(例如启动子、增强子等)具有序列同一性的核糖核苷酸序列的第一核糖核酸链(反义链，也称反义寡核苷酸链)和包含与第一链互补的核苷酸序列的第二核糖核酸链(正义链，也称有义链或正义寡核苷酸链)组成，其中所述第一链和第二链形成双链体。小激活RNA也可以由合成的或者载体表达的可形成双链区发卡结构的单链RNA分子组成，其中第一区域包含与基因的启动子靶序列具有序列同一性的核酸序列，第二区域包含的核酸序列与第一区域互补。小激活RNA分子的双链体区域长度通常为约10至约50个碱基对、约12个至约48个碱基对、约14个至约46个碱基对、约16个至约44个碱基对、约18个至约42个碱基对、约20个和约40个碱基对、约22个和约38个碱基对、约24个和约36个碱基对、约26个和约34个碱基对、约28个和约32个碱基对、通常约10个、 约15个、约20、约25、约30、约35、约40、约45、约50个碱基对。此外，术语“saRNA”和“小激活RNA”、“小激活核酸分子”还含有除核糖核苷酸部分之外的核酸，包括但不限于修饰的核苷酸或类似物。
如本文所用，术语“热点”是指长度至少为30bp的基因启动子区域，在这些区域，呈现出功能性小激活核酸分子靶点的聚集，即靶向这些热点区域的小激活核酸分子至少60％能够诱导靶基因mRNA表达达到1.5倍或以上。
如本文所用，术语“p21”是指p21 WAF1/CIP1基因，也称CDKN1A基因，是一种细胞周期蛋白依赖性激酶(CDK)抑制剂，也是一个重要的肿瘤抑制基因，有时也称为“靶基因”。过表达p21或者通过激活内源性p21的转录可以有效抑制肿瘤细胞和在体肿瘤的生长。
如本文所用，术语“合成”是指寡核苷酸的合成方式，包括任何能够合成RNA的方式，例如化学合成、体外转录、载体表达等。本发明提供的小激活核酸分子的制备方法包括序列设计和序列合成。所述小激活核酸分子序列的合成可以采用化学合成的方法，或者委托专门从事核酸合成的生物技术公司合成。一般来说，所述化学合成的方法包括以下四个过程：(1)寡聚核糖核苷酸的合成；(2)脱保护；(3)纯化分离；(4)脱盐及退火。例如，本发明所述双链RNA分子例如saRNA化学合成的具体步骤如下：
(1)寡聚核糖核苷酸的合成
在自动DNA/RNA合成仪(例如，Applied Biosystems EXPEDITE8909)上设定合成1微摩尔的RNA，同时设定每个循环的偶联时间为10-15分钟，起始物为固相连接的5’-O-对二甲氧基三苯甲基-胸苷支持物，第一个循环在固相支持物上连接一个碱基，然后在第n次(19≥n≥2)循环中，在第n-1次循环所连接的碱基上连接一个碱基，重复此循环直至完成全部核酸序列的合成。
(2)脱保护
将连接有saRNA的固相支持物加入到试管中，并在此试管中加入1毫升的乙醇/氨水溶液(体积比为1∶3)，然后密封，置于25-70℃温箱中，孵育2-30小时，过滤含有saRNA的固相支持物的溶液并收集滤液，用双蒸水淋洗固相支持物2次(每次1毫升)并收集滤液，合并收集洗脱液，在真空条件下干燥1-12小时。然后，加入1毫升四丁基氟化铵的四氢呋喃溶液(1M)，室温放置4-12小时，再加入2毫升正丁醇，高速离心收集沉淀即得到saRNA单链的粗产物。
(3)纯化分离
将得到的saRNA的粗产物溶解于2毫升浓度为1摩尔/毫升的乙酸铵水溶液中，然后通过高压液相色谱反相C18柱进行分离，得到纯化的saRNA单链产物。
(4)脱盐及退火
用体积排阻凝胶过滤法去除盐份，将正义链和反义链的寡聚核糖核酸单链按相同摩尔比混合在1-2毫升的缓冲液中(10mM Tris，pH＝7.5-8.0，50mM NaCl)，将此溶液加热至95℃，然后缓缓将此溶液冷却至室温，得到含有saRNA的溶液。
材料和方法
细胞培养和转染
细胞系RT4、KU-7、T24、J82、TCCSUP和HT-1197培养在改良的McCoy′s5A培养基(Gibco)中；细胞系5637、PC3和Bel-7402培养在RPMI1640培养基(Gibco)中；UM-UC-3细胞系培养在基础培养基(Gibco)中。所有培养基含有10％小牛血清(Sigma-Aldrich)和1％青霉素/链霉素(Gibco)。细胞在5％CO 2，37℃条件下培养。依照制造商的说明，使用RNAiMax(Invitrogen，Carlsbad，CA)以10nM(除非另有说明)浓度转染实验中设计的双链RNA分子。
RNA分离和反转录聚合酶链式反应
细胞以2-3×10 5个细胞/孔接种在6孔板中，反向转染寡核苷酸双链体。使用RNeasy Plus Mini试剂盒(Qiagen)，按照其说明书提取细胞总RNA。使用含有gDNA Eraser(Takara，Shlga，Japan)的PrimeScript RT试剂盒将RNA(1μg)反转录为cDNA。qPCR采用ABI 7500Fast Real-time PCR System(Applied Biosystems)和SYBR Premix Ex Taq II(Takara，Shlga，Japan)试剂，反应条件为：95℃3秒，60℃30秒，扩增40个循环。以GAPDH为内参。所有引物序列列于表1中。
表1.qRT-PCR分析的引物序列
Figure PCTCN2019082126-appb-000001
细胞增殖测定
将细胞以2-4×10 3个细胞/孔铺板于96孔板中，培养过夜，转染寡核苷酸双链体。转染三天后，使用CCK8(Dojindo)，依照其说明书进行细胞增殖检测。实验步骤简述如下：将10μL CCK8溶液加入到每孔中，37℃孵育1小时，后使用酶标仪测定450nm处吸光值。
QuantiGene 2.0分析
将细胞铺板于96孔板中并转染寡核苷酸双链体，转染72小时后，使用QuantiGene 2.0试剂盒(AffyMetrix)定量检测目的基因mRNA水平。QuantiGene 2.0试剂盒是基于杂交技术的方法，使用基因特异性探针直接定量mRNA水平。实验步骤简述如下：添加裂解液以裂解转染后的细胞，细胞裂解物加样至包有CDKN1A(p21)和HPRT1(管家基因)探针的捕获孔板中，55℃杂交过夜。为了增强杂交信号，在100μL相应的缓冲液(Quantigene 2.0试剂盒提供)中顺次与2.0PreAMP，2.0AMP和2.0Lable Probe杂交。所有杂交均在50-55℃震荡1小时。最后一步洗涤后，加入2.0Substrate并在室温下孵育5分钟。然后使用Infinite 200PRO读板仪(Tecan，瑞士)检测光信号。
统计分析
结果表示为平均值±标准偏差。使用GraphPad Prism软件(GraphPad Software)进行单因素方差分析，后进行Tukey′s t检验以进行统计分析。统计学显著性的标准设定为*p＜0.05，**p＜0.01和***p＜0.001。
实施例
通过以下实施例进一步说明本发明。提供实施例仅用于说明目的，并且不应被解释为以任何方式限制本发明的范围或内容。
实施例1：筛选靶向p21基因启动子区的功能性小激活RNA(saRNA)
为了筛选能够激活p21基因表达的功能性小激活RNA，从UCSC Genome数据库中检索获得p21基因的1kb启动子序列(图1)。从转录起始位点(TSS)上游-1kb处开始选定大小为19bp的靶点，通过每次移动1bp的方式，向TSS位点移动，获得总共982个靶点序列。对靶点序列进行过滤处理，以排除GC含量高于65％或低于35％，和含有5个或者多于5个的连续同一核苷酸的靶点序列。过滤后，剩余439个靶点序列作为候选进入筛选过程。基于这些候选序列，化学合 成相应的双链双链RNA分子。其中，该实验中使用的双链RNA分子的正义和反义链的长度均为21个核苷酸，所述双链RNA分子例如双链saRNA的第一核糖核酸链(正义链)的5’区域的19个核苷酸与启动子靶点序列具有100％的同一性，其3’末端含有dTdT突出；第二核糖核酸链的5’区域的19个核苷酸与第一核糖核酸链的3’区域序列的19个核苷酸完全互补，其3’末端含有dTdT突出。
将前述双链RNA分子以10nM的终浓度转染至PC3前列腺癌细胞，72小时后，使用QuantiGene 2.0试剂盒检测p21基因mRNA水平。计算每个双链RNA分子相对于空白对照处理的p21 mRNA水平的倍数变化并绘制在图2中。该研究中所有双链RNA分子所引起的p21基因mRNA的倍数变化涵盖从0.66(抑制)至8.12(诱导)的范围(图2B)。有361个(82.2％)双链RNA分子诱导p21表达1.01至8.12倍；74个(16.9％)双链RNA分子表现出抑制作用(0.99至0.66倍)；4个双链RNA分子(0.9％)对p21基因mRNA水平无影响(1.0倍)。
在被筛选的439个双链RNA分子中，132个双链RNA分子(30.1％)能够诱导p21 mRNA至少2倍；229个(52.4％)双链RNA分子诱导p21 mRNA至少1.5倍，这些诱导p21 mRNA表达上升10％以上双链RNA分子为saRNA，即具有功能的saRNA。这些具有功能的saRNA分散在整个p21启动子区域。然而，在8个游离的区域呈现功能性小激活RNA的聚集，这些区域被称为“热点”。热点区域的定义为一个区域内含有至少10个对应的小激活RNA，其中，至少60％的小激活RNA能够诱导p21 mRNA表达达到1.5倍或以上(图2A和图3)。热点1至8的靶序列及对应的小激活RNA序列分别在表2和表3中列出。
表2 p21启动子热点区域靶序列
Figure PCTCN2019082126-appb-000002
Figure PCTCN2019082126-appb-000003
表3位于p21启动子热点区域的经设计的双链RNA分子
Figure PCTCN2019082126-appb-000004
Figure PCTCN2019082126-appb-000005
Figure PCTCN2019082126-appb-000006
Figure PCTCN2019082126-appb-000007
Figure PCTCN2019082126-appb-000008
Figure PCTCN2019082126-appb-000009
Figure PCTCN2019082126-appb-000010
Figure PCTCN2019082126-appb-000011
Figure PCTCN2019082126-appb-000012
这些热点包括热点区域1，相应的靶序列为p21启动子序列的-893bp至-801bp，序列如SEQ ID NO：93所示，在这个区域发现有44个具有功能的saRNA(表3、图3A)，分别为RAG-834、RAG-845、RAG-892、RAG-846、RAG-821、RAG-884、RAG-864、RAG-843、RAG-854、RAG-844、RAG-887、RAG-838、RAG-858、RAG-835、RAG-876、RAG-870、RAG-853、RAG-881、RAG-828、RAG-872、RAG-841、RAG-831、RAG-829、RAG-820、RAG-822、RAG-868、RAG-849、RAG-862、RAG-865、RAG-893、RAG-848、RAG-824、RAG-866、RAG-840、RAG-875，、RAG-880、RAG-871、RAG-888、RAG-885、RAG-894、RAG-833、RAG-825、RAG-889、RAG-823；
热点区域2(表3、图3B)相应的靶序列为p21启动子序列的-717至-632bp，序列如SEQ ID NO：94所示，在这个区域发现31个具有功能的saRNA，分别为RAG-693、RAG-692、RAG-688、RAG-696、RAG-694、RAG-687、RAG-691、 RAG-690、RAG-689、RAG-682、RAG-686、RAG-662、RAG-695、RAG-654、RAG-658、RAG-685、RAG-704、RAG-714、RAG-705、RAG-661、RAG-656、RAG-698、RAG-697、RAG-657、RAG-715、RAG-652、RAG-651、RAG-650、RAG-716、RAG-717、RAG-711；
热点3(表3、图3C)，相应的靶序列为p21启动子序列的-585bp至-551bp，序列如SEQ ID NO：95所示，在这个区域发现其包含9个具有功能的saRNA，分别为RAG-580、RAG-577、RAG-569、RAG-576、RAG-570、RAG-574、RAG-585、RAG-579、RAG-584；
热点4(表3、图3D)，相应的靶序列为p21启动子序列的-554bp至-505bp，序列如SEQ ID NO：96所示，在这个区域发现包含17个具有功能的saRNA，分别为RAG-524、RAG-553、RAG-537、RAG-526、RAG-554、RAG-523、RAG-534、RAG-543、RAG-525、RAG-535、RAG-546、RAG-545、RAG-542、RAG-531、RAG-522、RAG-529、RAG-552；
热点5(表3、图3E)，相应的靶序列为p21启动子序列的-514bp至-485bp，序列如SEQ ID NO：97所示，在这个区域发现包含9个具有功能的saRNA，分别是RAG-503、RAG-504、RAG-505、RAG-506、RAG-507、RAG-508、RAG-509、RAG-510、RAG-511、RAG-512、RAG-513、RAG-514；
热点6(表3、图3F)，相应的靶序列为p21启动子序列的-442bp至-405bp，序列如SEQ ID NO：98所示，在这个区域发现包含12个具有功能的saRNA，分别是RAG-427、RAG-430、RAG-431、RAG-423、RAG-425、RAG-433、RAG-435、RAG-434、RAG-439、RAG-426、RAG-428、RAG-442；
热点7(表3、图3G)，相应的靶序列为p21启动子序列的-352bp至-313bp，序列如SEQ ID NO：99所示，在这个区域发现包含13个具有功能的saRNA，分别是RAG-335、RAG-351、RAG-352、RAG-331、RAG-344、RAG-342、RAG-341、RAG-333、RAG-345、RAG-346、RAG-336、RAG-332、RAG-343；
热点8(表3、图3H)，相应的靶序列为p21启动子序列的-325bp至-260bp，序列如SEQ ID NO：100所示，在这个区域发现包含18个具有功能的saRNA，分别是RAG-294、RAG-285、RAG-286、RAG-292、RAG-291、RAG-284、RAG-279、RAG-280、RAG-325、RAG-293、RAG-322、RAG-321、RAG-281、RAG-289、RAG-278、RAG-283、RAG-282、RAG-295。
为验证QuantiGene 2.0检测结果，将439个双链RNA分子按照激活p21mRNA表达的活性大小分成四个组(bin)，从每组中随机挑选5个双链RNA分子，分别以10nM的浓度转染至PC3细胞。72小时后，提取细胞总RNA、反转录后采用RT-qPCR方法分析p21基因mRNA水平。两种方法所揭示的p21基因mRNA表达水平显示出显著的相关性(R 2＝0.82)(图4)。QuantiGene 2.0方法所得的所有功能性saRNA均可通过RT-qPCR方法验证为真实的功能性小激活RNA，其中一些功能性saRNA在RT-qPCR分析中显示出更强的p21 mRNA表达诱导(表4)。
表4.对QuantiGene 2.0方法的验证
Figure PCTCN2019082126-appb-000013
综上，上述数据表明，p21启动子区的许多特定位点可以用作saRNA靶点序列以诱导p21表达，其中一些区域更具有敏感性，其对应的saRNA有更大的可能性激活p21表达。
实施例2：saRNA诱导p21基因mRNA表达并抑制癌细胞增殖
为了进一步评估p21saRNA诱导p21基因mRNA表达和抑制癌细胞增殖的作用，通过QuantiGene 2.0筛选的saRNA(RAG1-431、RAG1-553、RAG1-688)转染到癌细胞系KU-7(膀胱癌)、HCT116(结肠癌)和HepG2(肝细胞癌)。 结果显示，在上述所有细胞系中，saRNA均可诱导至少两倍的p21基因mRNA表达水平，并抑制细胞增殖，揭示了saRNA介导的p21诱导所产生的功效。具体而言，将RAG1-431、RAG1-553、RAG1-688分别转染KU7细胞，分别诱导p21 mRNA表达14.0、36.9和31.9倍，并产生相对于空白处理的存活率为71.7％、60.7％、67.4％(图5)。将RAG1-431、RAG1-553、RAG1-688分别转染HCT116细胞，分别诱导p21 mRNA表达2.3、3.5、和2.4倍，并产生相对于空白处理的存活率为45.3％、22.5％、38.5％(图6)。将RAG1-431、RAG1-553、RAG1-688分别转染HepG2细胞，分别诱导p21 mRNA表达2.2、3.3、和2.0倍，并产生相对空白处理的存活率为76.7％、64.9％、79.9％(图7)。
通过引用并入
本文引用的每个专利文献和科学文献的全部公开内容通过引用并入本文用于所有目的。
等效
本发明可以在不脱离其基本特征的情况下以其他具体形式实施。因此，前述实施例被认为是说明性的，而不是对本文所述的本发明的限制。本发明的范围由所附权利要求书而不是由前述说明书表示，并且意在将落入权利要求书的等同物的含义和范围内的所有改变包括在其中。

Claims (23)

  1. 一种小激活RNA，其特征在于，所述小激活RNA中的一条链与SEQ ID NO：5、6、7、8、9、10、11、12所示的序列中的任一长度为16-35个核苷酸的连续片段具有至少75％的同源性或互补性，所述小激活RNA通过靶向人p21基因启动子序列激活或上调p21基因的表达。
  2. 如权利要求1所述的小激活RNA，其中所述小激活RNA包含正义核酸链和反义核酸链，所述正义核酸链和反义核酸链含有互补区域，互补区域能形成双链核酸结构，其中的正义核酸链或反义核酸链与人p21基因启动子序列中的任一长度为16-35个核苷酸的连续片段具有75％以上、80％以上、90％以上、95％以上、99％以上或100％的同源性。
  3. 如权利要求2所述的小激活RNA，其中所述正义核酸链和反义核酸链存在于两条不同的核酸链上。
  4. 如权利要求2所述的小激活RNA，其中所述正义核酸链和反义核酸链存在于同一条核酸链上，为发夹型单链核酸分子，其中正义核酸链和反义核酸链的互补区域形成双链核酸结构。
  5. 如权利要求3所述的小激活RNA，其中所述小激活RNA中的至少一条链具有长度为0至6个核苷酸的3’突出端。
  6. 如权利要求5所述的小激活RNA，其中所述小激活RNA中的两条链都具有长度为2-3个核苷酸的3’突出端。
  7. 如权利要求2至6任一项所述的小激活RNA，其中所述正义核酸链或反义核酸链的长度为16-35个核苷酸。
  8. 如权利要求1所述的小激活RNA，其中所述正义核酸链或反义核酸链与SEQ ID NO：13-30、35-46、59-62、67-74、77-80、85-96、103-108、111-118、121-132、139-140、147-180、185-186、189-190、195-198、201-202、209-212、215-218、225-240、243-246、249-258、261-262、265-270、275-280、283-300、303-308、317-320、323-324、329-348、351-352、357-358、361-366、371-392、399-400、405-412、415-416、419-424、429-432、439-442、447-450、453-458、463-468所示序列具有至少75％的同源性。
  9. 如权利要求8所述的小激活RNA，其中所述正义核酸链或反义核酸链序列如SEQ ID NO：13-30、35-46、59-62、67-74、77-80、85-96、103-108、111-118、121-132、 139-140、147-180、185-186、189-190、195-198、201-202、209-212、215-218、225-240、243-246、249-258、261-262、265-270、275-280、283-300、303-308、317-320、323-324、329-348、351-352、357-358、361-366、371-392、399-400、405-412、415-416、419-424、429-432、439-442、447-450、453-458、463-468任一核苷酸序列所示。
  10. 如权利要求1至9任一项所述的小激活RNA，其中所述小激活RNA中的至少一个核苷酸为化学修饰的核苷酸，所述化学修饰为如下修饰中的至少一种：
    (1)对所述小激活RNA的核苷酸序列中连接核苷酸的磷酸二酯键的修饰；
    (2)对所述小激活RNA的核苷酸序列中的核糖的2’-OH的修饰；
    (3)对所述小激活RNA的核苷酸序列中的碱基的修饰；
    (4)所述小激活核酸分子的核苷酸序列中的至少一个核苷酸为锁核酸。
  11. 如权利要求1至10任一项所述的小激活RNA，所述激活或上调p21基因的表达为上调至少10％。
  12. 如权利要求1至11任一项所述的小激活RNA在制备用于激活或上调p21基因在细胞中表达的制剂中的应用。
  13. 如权利要求12所述的应用，其中所述的小激活RNA被直接导入所述细胞中。
  14. 如权利要求13所述的应用，其中所述的细胞是哺乳动物细胞，优选为人类细胞，更优选为人类肿瘤细胞。
  15. 如权利要求14所述的应用，其中所述的细胞存在于人体中。
  16. 如权利要求15所述的应用，其中所述的人体是患有由p21蛋白表达不足引发的肿瘤患者，并且所述小激活核酸分子被施用以有效量以实现对肿瘤的治疗，所述肿瘤优选为膀胱癌、前列腺癌、肝癌或结直肠癌。
  17. 一种分离的p21基因小激活RNA靶位点，其中所述靶位点为选自SEQ ID No5-12的任一条序列上的任意连续16-35个核苷酸序列。
  18. 一种激活或上调p21基因在细胞中表达的方法，其中所述方法包括向所述细胞施用如权利要求1至11任一项所述的小激活RNA。
  19. 如权利要求18所述的方法，其中所述的小激活RNA被直接导入所述细胞中。
  20. 如权利要求18或19所述的方法，其中所述的细胞是哺乳动物细胞，优选为人类细胞，更优选为人类肿瘤细胞，再优选为人膀胱癌、前列腺癌、肝癌或结直肠癌细胞。
  21. 一种包含如权利要求1至11任一项所述的小激活RNA和药学上可接受的载 体的组合物。
  22. 如权利要求21所述的组合物，其特征在用于药学上可接受的载体为脂质体、高分子聚合物或多肽。
  23. 如权利要求1至11任一项小激活RNA或如权利要求21或22所述的组合物在制备用于激活或上调p21基因表达的药物中的应用，优选为在制备抗肿瘤或良性增殖性病变中的药物中的应用，更优选地，所述肿瘤为膀胱癌、前列腺癌、肝癌或结直肠癌。
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JP2020555223A JP2021520220A (ja) 2018-04-10 2019-04-10 ｐ２１遺伝子の発現を活性化させる方法
US15/733,357 US20210024915A1 (en) 2018-04-10 2019-04-10 Method for activating p21 gene expression
KR1020207032370A KR20200143428A (ko) 2018-04-10 2019-04-10 P21 유전자 발현을 활성화하는 방법

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