WO2020221309A1 - 寡聚核酸分子及其在急性间歇性卟啉症治疗中的应用 - Google Patents

寡聚核酸分子及其在急性间歇性卟啉症治疗中的应用 Download PDF

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WO2020221309A1
WO2020221309A1 PCT/CN2020/087844 CN2020087844W WO2020221309A1 WO 2020221309 A1 WO2020221309 A1 WO 2020221309A1 CN 2020087844 W CN2020087844 W CN 2020087844W WO 2020221309 A1 WO2020221309 A1 WO 2020221309A1
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nucleic acid
acid molecule
strand
small
cell
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PCT/CN2020/087844
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French (fr)
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李龙承
姜武林
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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 EP20798178.8A priority Critical patent/EP3964582A4/en
Priority to CN202080006273.0A priority patent/CN113260702B/zh
Priority to BR112021021614A priority patent/BR112021021614A2/pt
Priority to CA3129976A priority patent/CA3129976A1/en
Priority to US17/594,740 priority patent/US20220211739A1/en
Priority to AU2020265062A priority patent/AU2020265062A1/en
Application filed by 中美瑞康核酸技术(南通)研究院有限公司 filed Critical 中美瑞康核酸技术(南通)研究院有限公司
Priority to MX2021013361A priority patent/MX2021013361A/es
Priority to JP2021555183A priority patent/JP2022530937A/ja
Priority to KR1020217033428A priority patent/KR20220002896A/ko
Publication of WO2020221309A1 publication Critical patent/WO2020221309A1/zh
Priority to IL287615A priority patent/IL287615A/en
Priority to CONC2021/0015542A priority patent/CO2021015542A2/es

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Definitions

  • the present invention belongs to the field of nucleic acid technology. Specifically, it relates to oligo nucleic acid molecules related to gene activation, such as small activating nucleic acid molecules, and small activating nucleic acid molecules in activating/up-regulating the expression of hydroxymethylbilin synthase (HMBS) genes.
  • HMBS hydroxymethylbilin synthase
  • Hereditary porphyria is a series of diseases caused by the lack of specific enzyme activity in the heme biosynthetic pathway (also known as the porphyrin pathway).
  • the lack of enzymes in the porphyrin pathway leads to insufficient heme production and accumulation of porphyrin precursors and porphyrins, while high tissue concentrations of porphyrin precursors and porphyrins cause tissue toxicity.
  • AIP acute intermittent porphyria
  • VP variant porphyria
  • VP hereditary fecal porphyria
  • Hereditary fecal porphyria chromaffin cells or HCP, such as autosomal dominant HCP
  • 5'aminolevulinic acid also known as delta-aminolevulinic acid or ALA
  • dehydratase-deficient porphyria is classified as acute hepatic porphyria, manifested as potentially life-threatening acute neurological symptoms involving autonomic, peripheral and central nervous systems, including severe abdominal pain, hypertension, and heartbeat Overspeed, constipation, exercise weakness, paralysis and seizures, etc.
  • cytochrome P450 drugs can increase the activity of 5'-aminolevulinic acid synthase 1 (ALAS1) in the liver to induce acute attacks of porphyria (Balwani and Desnick, Blood, 120 : 4496-4504, 2012).
  • omega-1 5'-aminolevulinic acid synthase 1
  • AIP also known as porphobinogen deaminase (PBGD) deficiency, or hydroxymethylbilane synthase (HMBS) deficiency
  • PBGD porphobinogen deaminase
  • HMBS hydroxymethylbilane synthase
  • PBGD porphobinogen deaminase
  • HMBS hydroxymethylbilane synthase
  • Intravenous injection of hemin is usually used for the treatment and prevention of acute attacks in AIP patients.
  • Chlormethrin provides exogenous hemoglobin to inhibit the negative feedback of ALAS1, thereby reducing the production of ALA and PBG.
  • patients usually respond well, the effect is slow, and it usually takes 2 to 4 days or more to normalize urine ALA and PBG concentrations.
  • intravenous heme is rapidly metabolized, it usually takes 3-4 infusions to effectively treat or prevent acute attacks. In addition, repeated infusions may cause iron overload and phlebitis.
  • the only curative treatment is liver transplantation, but liver transplantation is accompanied by significant complications and mortality, and the source of liver donors is limited.
  • the present invention provides the use of small activating RNA to specifically activate the expression level of HMBS gene in the body, and lastingly promote the production of endogenous porphobilinogen deaminase (also known as hydroxymethylbiliin synthase) in cells to restore the normal level in the cell so as to be effective Methods of treatment of AIP.
  • endogenous porphobilinogen deaminase also known as hydroxymethylbiliin synthase
  • the present invention provides a small activating nucleic acid molecule (saRNA) based on the RNA activation process, which activates/up-regulates the transcription of HMBS gene, thereby increasing the expression of HMBS protein to treat the underexpression or decreased activity of HMBS.
  • Diseases such as acute intermittent porphyria.
  • a small activating nucleic acid molecule that can activate or up-regulate the expression of the HMBS gene in a cell.
  • the length of a strand of the small activating nucleic acid molecule of the present invention and the promoter region of the HMBS gene is 16-35 nuclei.
  • the nucleotide fragments have at least 75% homology or complementarity, and the promoter region includes 400 nucleotide sequences upstream of the transcription start site of the HMBS gene, so as to activate or up-regulate the expression of the gene.
  • one strand of the small activating nucleic acid molecule of the present invention includes a nucleotide sequence having at least 75% homology or complementarity with any consecutive 16-35 nucleotides in the promoter region of the HMBS gene, wherein The promoter region refers to the 400 nucleotides upstream of the transcription start site of the HMBS gene.
  • one strand of the small activating nucleic acid molecule of the present invention includes or is selected from the region (region 1) from -395bp to -351bp upstream of the HMBS gene promoter (tagcctgggcaacatagtgaggccacctccccgctgtctctataa, SEQ ID NO:1) and- -1bp to 179bp region (the second region) (tgctgcctattttcaaggttgtagcaaagctaagtttgaacagagcaaaggaagcgccatagaagctgcactacttgctcatgtcacagctggggaatggggtggtcgaatggggaggtccactgtcgcaatgttccaattcccgcccagagggagggacctccccttcgagggagggcg, SEQ ID NO: 2) 16-
  • one strand of the small activation nucleic acid molecule of the present invention has at least 75 consecutive nucleotide sequences selected from SEQ ID NO: 1 and SEQ ID NO: 2. %, such as at least about 79%, about 80%, about 85%, about 90%, about 95%, or about 99% homology or complementarity.
  • a chain of the small activation nucleic acid molecule of the present invention includes or is selected from any one of SEQ ID NO: 1 and SEQ ID NO: 2 with 16-35 consecutive nucleotide sequences having A nucleotide sequence with at least 75%, for example at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% homology or complementarity.
  • one strand of the small activating nucleic acid molecule of the present invention has at least 75% of a continuous 16-35 nucleotide sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2. For example, at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% homology or complementarity of nucleotide sequences.
  • the small activating nucleic acid molecule of the present invention includes a double-stranded small activating nucleic acid molecule targeting the promoter region of the HMBS gene, comprising a first nucleic acid strand and a second nucleic acid strand, the first nucleic acid strand and the SEQ in the HMBS gene promoter Any one of the consecutive 16-35 nucleotides in ID No. 1 or SEQ ID No.
  • the first nucleic acid strand and the second nucleic acid strand can form a double-stranded nucleic acid structure through complementarity, and the double-stranded nucleic acid structure can activate the expression of HMBS gene in the cell.
  • the sense nucleic acid strand and the antisense nucleic acid strand of the small activating nucleic acid molecule of the present invention can exist on two different nucleic acid strands or on the same nucleic acid strand.
  • the sense nucleic acid strand and the antisense nucleic acid strand are located on two strands respectively, at least one strand of the small activation nucleic acid molecule can have protrusions or overhangs at either the 5'end and the 3'end, for example, the 3'end can have a length of Overhang of 0-6 nucleotides.
  • both strands of the small activation nucleic acid molecule of the present invention have protrusions, more preferably, the 3'ends of both strands of the small activation nucleic acid molecule can have protrusions of 0-6 nucleotides, most preferably , With an overhang of 2-3 nucleotides at the 3'end.
  • the overhanging nucleotide can be dT or U, or it can be a natural nucleotide overhang.
  • the natural nucleotide overhang in the present invention means that the end overhanging nucleotide of the sense nucleic acid fragment or the antisense nucleic acid fragment is identical or complementary to the nucleotide of the corresponding target sequence.
  • Small activating nucleic acid molecules can also include single-stranded RNA molecules that can form a double-stranded hairpin structure.
  • the small activating nucleic acid molecule of the present invention is a single-stranded RNA molecule targeted to the promoter region of the HMBS gene, wherein the small activating nucleic acid molecule can form a double-stranded hairpin structure.
  • the small activation nucleic acid molecule can be a hairpin single-stranded nucleic acid molecule, wherein the complementary regions of the sense nucleic acid fragment and the antisense nucleic acid fragment form a double strand Nucleic acid structure, the double-stranded nucleic acid structure can promote the expression of HMBS gene in cells through, for example, RNA activation mechanism.
  • the length of the sense nucleic acid fragment and the antisense nucleic acid fragment can be 16-35 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.
  • the sense strand of the small activating nucleic acid molecule of the present invention has at least 75%, such as at least about 79%, about 80%, about 75%, and any nucleotide sequence selected from SEQ ID NO: 30-48. 85%, about 90%, about 95%, about 99%, or about 100% identity or homology, and its antisense strand has any nucleotide sequence selected from SEQ ID NO: 49-67 At least 75%, such as at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% identity or homology.
  • the sense strand of the small activation nucleic acid molecule of the present invention includes or is selected from any nucleotide sequence selected from SEQ ID NO: 30-48 with at least 75%, such as at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% identity or homology sequence, and its antisense strand includes or is selected from SEQ ID NO: 49 Any nucleotide sequence of -67 has at least 75%, for example, at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% identity or identity Source sequence.
  • the sense strand of the small activation nucleic acid molecule of the present invention has at least 75%, such as at least about 79%, about 80%, and any nucleotide sequence selected from SEQ ID NO: 30-48. About 85%, about 90%, about 95%, about 99%, or about 100% identity or homology sequence is composed, and its antisense strand is composed of any sequence selected from SEQ ID NO: 49-67 A nucleotide sequence having at least 75%, for example at least about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% identity or homology sequence composition .
  • the sense strand of the small activation nucleic acid molecule of the present invention may be a sequence selected from any nucleotide sequence of SEQ ID NO: 30-48, and the antisense strand may be selected from SEQ ID NO: The sequence shown in any nucleotide sequence of 49-67.
  • the small activation nucleic acid molecules described herein can be synthesized, transcribed in vitro, or expressed in a vector.
  • nucleotides in the small activation nucleic acid molecules described herein can be natural non-chemically modified nucleotides, and can also include at least one modification.
  • the modification in the small activation nucleic acid molecule described herein may be a chemical modification, such as a chemical modification on at least one nucleotide.
  • the chemical modification used in the present invention may include or be selected from one or more of the following modifications or any combination thereof:
  • At least one nucleotide in the nucleotide sequence of the small activation nucleic acid molecule is a locked nucleic acid.
  • the chemical modification is well known to those skilled in the art, and the modification of the phosphodiester bond refers to the modification of the oxygen in the phosphodiester bond, including but not limited to phosphorothioate modification and boration phosphate modification. Both modifications can stabilize the structure of small activated nucleic acid molecules and maintain high specificity and affinity of base pairing.
  • Ribose modification refers to the modification of the 2'-OH in the nucleotide pentose, that is, the introduction of certain substituents at the hydroxyl position of the ribose sugar, for example, including but not limited to, 2'-fluoromodification, 2'-oxymethyl Group modification, 2'-oxyethylene methoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid (LNA), 2'-amino modification, 2'-deoxy modification, etc.
  • LNA locked nucleic acid
  • Base modification refers to the modification of nucleotide bases, for example, including but not limited to, 5'-bromouracil modification, 5'-iodouracil modification, N-methyluracil modification, 2,6- Diaminopurine modification, etc.
  • lipophilic groups such as cholesterol can be introduced at the end of the sense strand or antisense strand of the small activating nucleic acid molecule to facilitate passage through the lipid bilayer.
  • the formed cell membrane and nuclear membrane interact with the gene promoter region in the nucleus.
  • the small activating nucleic acid molecule provided by the present invention can effectively activate or up-regulate the expression of the HMBS gene in the cell after contacting the cell, and preferably the expression is up-regulated by at least 10%.
  • nucleic acids encoding the small activation nucleic acid molecules described herein.
  • the nucleic acid may be a DNA molecule.
  • the small activating nucleic acid molecule of the present invention may be a double-stranded small activating nucleic acid molecule targeting the promoter region of the HMBS gene, which includes a sense strand and an antisense strand.
  • the small activation nucleic acid molecule of the present invention may be a single-stranded nucleic acid molecule encoding a small activation nucleic acid molecule targeting the promoter region of the HMBS gene.
  • kits comprising the above-mentioned small activation nucleic acid molecule and the nucleic acid encoding the small activation nucleic acid molecule described herein.
  • Another aspect of the present invention relates to the application of the small activating nucleic acid molecule described above and the nucleic acid encoding the small activating nucleic acid molecule described herein in the preparation of a medicine for activating/up-regulating HMBS gene expression in cells.
  • Another aspect of the present invention relates to the small activation nucleic acid molecule described above, the nucleic acid encoding the small activation nucleic acid molecule described herein, and the cells of the present invention are caused by insufficient expression or decreased activity of HMBS in the preparation for use in the treatment of a subject.
  • diseases caused by insufficient expression or decreased activity of HMBS may include hereditary porphyria.
  • Hereditary porphyrias can include, for example, acute intermittent porphyrias.
  • Another aspect of the present invention relates to the small activating nucleic acid molecule described above, the nucleic acid encoding the small activating nucleic acid molecule described herein, and the cell of the present invention in the preparation of a medicament for the treatment of acute intermittent porphyria in a subject Applications.
  • Another aspect of the present invention also relates to a method for treating diseases caused by insufficient expression or decreased activity of HMBS in a subject, which comprises administering to the subject the small activating nucleic acid molecule described above, the nucleic acid encoding the small activating nucleic acid molecule described herein Or the cell of the present invention.
  • Another aspect of the present invention also relates to a method for treating acute intermittent porphyria in a subject, comprising administering to the subject the small activating nucleic acid molecule described above, the nucleic acid encoding the small activating nucleic acid molecule described herein, or the present invention The cell.
  • the present invention also relates to a method for activating/up-regulating the expression of the HMBS gene in a cell, which method comprises administering to the cell the small activation nucleic acid molecule described above or the nucleic acid encoding the small activation nucleic acid molecule described herein.
  • the small activating nucleic acid molecule of the present invention can be directly introduced into a cell, or a nucleic acid sequence encoding the small activating nucleic acid molecule can be introduced into the cell and then produced in the cell; the cell is preferably a mammalian cell, more preferably a human cell.
  • the above-mentioned cells may be in vitro, such as cell lines or cell lines, etc., or isolated from mammals, such as humans.
  • the human body may be a patient suffering from symptoms caused by decreased expression of HMBS protein.
  • the small activating nucleic acid molecules described herein can be administered in a sufficient amount to achieve the treatment of symptoms caused by the decrease in HMBS protein expression.
  • the symptoms caused by the lack of HMBS protein include or are selected from acute intermittent porphyria.
  • Another aspect of the present invention provides an isolated HMBS gene small activation nucleic acid molecule action site, which has any continuous 16-35 nucleotide sequence on the promoter region of the HMBS gene.
  • the The site of action is any continuous 16-35 nucleotide sequence on any sequence selected from SEQ ID NO: 1-2.
  • the action site is shown in any nucleotide sequence selected from SEQ ID NO: 11-29.
  • the small activating nucleic acid molecule capable of activating/up-regulating the expression of HMBS gene provided by the present invention can permanently activate the HMBS gene, thereby efficiently and specifically up-regulating or restoring the expression of HMBS gene and protein and enzyme activity, and at the same time has low toxicity Side effects, can be used to prepare drugs or preparations that activate/up-regulate the expression of HMBS genes and proteins in cells.
  • Figure 1 is a schematic diagram of the HMBS gene promoter. Region 1 and region 2 in the figure are the target sequence regions for designing small nucleic acid molecules. TSS is the transcription start site.
  • Figure 2 shows the change in HMBS mRNA expression mediated by small nucleic acid molecules.
  • 180 small nucleic acid molecules targeting the target region of the HMBS promoter were respectively transfected into human hepatocellular carcinoma Huh7 cells at a transfection concentration of 10 nM. After 72 hours, the cells were collected and analyzed for HMBS mRNA expression by one-step RT-PCR.
  • the graph shows the change in HMBS expression relative to the control treatment (mock), ranked from highest to lowest.
  • Figure 3 shows the change in HMBS mRNA expression mediated by small nucleic acid molecules.
  • 180 small nucleic acid molecules targeting the HMBS promoter were respectively transfected into Huh7 cells at a transfection concentration of 10 nM. After 72 hours, the cells were collected and analyzed for HMBS mRNA expression by one-step RT-PCR.
  • the graph shows the fold of the change in HMBS expression induced by each small nucleic acid molecule relative to the control treatment (mock). Small nucleic acid molecules are sorted according to their positions on the HMBS gene promoter from the most upstream to TSS.
  • Figure 4 shows that candidate saRNA (small activating RNA) activates HMBS gene mRNA expression in different liver cells.
  • dsCon2 sense chain 5'-ACUACUGAGUGACAGUAGATT-3' (SEQ ID NO: 68), antisense chain 5'-UCUACUGUCACUCAGUAGUTT-3' (SEQ ID NO: 69) and siHMBS (sense chain: 5'-CCUGUUUACCAAGGAGCUUTT-3 '(SEQ ID No: 3), antisense strand: 5'-AAGCUCCUUGGUAAACAGGTT-3' (SEQ ID No: 4)) are blank transfection, unrelated sequence double-stranded RNA transfection and small interfering RNA (siRNA) control transfection, respectively dye.
  • siRNA small interfering RNA
  • Figure 5 shows that candidate saRNA activates HMBS protein expression in liver cells.
  • the indicated saRNA final concentration of 20 nM
  • Mock, dsCon2 and siHMBS are blank transfection, double-stranded RNA transfection of unrelated sequence and small interfering RNA control transfection, respectively.
  • A Scanning image of western blot membrane.
  • B Use ImageJ software to quantitatively analyze the bands of A, and obtain the relative value of the intensity of HMBS bands of each treatment compared with Mock treatment.
  • Figure 6 shows the PPIX fluorescence intensity per unit (mg) of protein obtained from the ALA conversion analysis.
  • Mock, dsCon2 and siHMBS are blank transfection, double-stranded RNA transfection of unrelated sequence and small interfering RNA control transfection, respectively.
  • the Y-axis is the PPIX fluorescence intensity in units (mg) of protein.
  • Figure 7 shows the PPIX fluorescence intensity per unit (mg) protein obtained by the candidate saRNA activation of HMBS gene mRNA and protein expression in Li-7 cells and the ALA conversion analysis.
  • A Transfect human liver cancer cell Li-7 with the indicated saRNA and the indicated concentration. After 72 hours, the cells were collected and the RNA was extracted with Qiagen RNeasy kit. After reverse transcription, the HMBS gene was amplified by qPCR with the 7500FAST real-time PCR system. At the same time, HPRT1 gene was amplified as an internal control.
  • B The above picture shows the human liver cancer cell Li-7 transfected with the indicated saRNA and the indicated concentration.
  • Figure 8 shows that candidate saRNA activates HMBS gene mRNA and protein expression in GM01623 cells from patients with acute intermittent porphyria (AIP).
  • A GM01623 cells were transfected with the saRNA shown at a final concentration of 20nM. After 72 hours, the cells were collected and RNA was extracted with Qiagen RNeasy kit. After reverse transcription, the HMBS gene was amplified by qPCR using 7500FAST real-time PCR system. Amplify HPRT1 and TBP genes as internal controls.
  • B GM01623 cells were transfected with the saRNA shown at a final concentration of 20nM. After 72 hours, the cells were collected for Western blot analysis.
  • HMBS protein was detected with anti-human HMBS antibody, and Tubulin was also detected as the protein. Accuracy control of sample amount. Mock, dsCon2 and siHMBS are blank transfection, double-stranded RNA transfection of unrelated sequence and small interfering RNA control transfection, respectively.
  • Figure 9 shows that candidate saRNA activates HMBS gene mRNA and protein expression in GM01624 cells from patients with acute intermittent porphyria (AIP).
  • A GM01624 cells were transfected with the saRNA shown at a final concentration of 20nM. After 72 hours, the cells were collected and RNA was extracted with Qiagen RNeasy kit. After reverse transcription, the HMBS gene was amplified by qPCR using the 7500FAST real-time PCR system. Amplify HPRT1 and TBP genes as internal controls.
  • B GM01624 cells were transfected with the saRNA shown at a final concentration of 20 nM. After 72 hours, the cells were collected for Western blot analysis.
  • HMBS protein was detected with anti-human HMBS antibody, and Tubulin was also detected as the protein. Accuracy control of sample amount. Mock, dsCon2 and siHMBS are blank transfection, double-stranded RNA transfection of unrelated sequence and small interfering RNA control transfection, respectively.
  • Figure 10 shows that candidate saRNA activates HMBS gene mRNA and protein expression in GM01625 cells from patients with acute intermittent porphyria (AIP).
  • A GM01625 cells were transfected with the saRNA shown at a final concentration of 20nM. After 72 hours, the cells were collected and RNA was extracted with Qiagen RNeasy kit. After reverse transcription, the HMBS gene was amplified by qPCR using the 7500FAST real-time PCR system. Amplify HPRT1 and TBP genes as internal controls.
  • B GM01625 cells were transfected with the saRNA shown at a final concentration of 20 nM. After 72 hours, the cells were collected for Western blot analysis.
  • HMBS protein was detected with anti-human HMBS antibody, and Tubulin was also detected as the protein. Accuracy control of sample amount. Mock, dsCon2 and siHMBS are blank transfection, double-stranded RNA transfection of unrelated sequence and small interfering RNA control transfection, respectively.
  • Complementary refers to the ability of two oligonucleotide strands to form base pairs with each other. Base pairs are usually formed by hydrogen bonds between nucleotides in antiparallel oligonucleotide chains.
  • Complementary oligonucleotide strands can be base paired in a Watson-Crick manner (e.g., AT, AU, CG), or in any other manner that allows duplex formation (e.g., Hoogsteen type or reverse Hoogsteen type base pairing) performs base pairing.
  • Complementarity includes two situations: complete complementarity and incomplete complementarity.
  • Complete complementarity or 100% complementarity means that each nucleotide from the first oligonucleotide strand in the double-stranded region of the double-stranded oligonucleotide molecule can correspond to the nucleoside at the corresponding position of the second oligonucleotide strand.
  • the acid forms a hydrogen bond without "mismatch”.
  • Incomplete complementarity refers to the situation where all the nucleotide units of the two strands cannot be hydrogen bonded to each other.
  • oligonucleotide chain exhibits 10% Complementarity.
  • the oligonucleotide strand exhibits 90% complementarity.
  • Substantial complementarity refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% complementarity.
  • oligonucleotide or "small nucleic acid molecule” as used herein refers to a polymer of nucleotides, including but not limited to single-stranded or double-stranded molecules of DNA, RNA or DNA/RNA hybrids, including regularly And the oligonucleotide chains of deoxyribosyl moieties and ribosyl moieties that alternate irregularly, as well as modifications of these types of oligonucleotides and naturally occurring or non-naturally occurring backbones.
  • the oligonucleotides for activating transcription of target genes described in the present invention are small activating nucleic acid molecules.
  • oligonucleotide chain and “oligonucleotide sequence” are used interchangeably, and refer to the general term for short-chain nucleotides of less than 35 bases (including deoxyribonucleic acid DNA or ribonucleic acid Nucleotides within RNA).
  • the length of the oligonucleotide chain can be any length from 16 to 35 nucleotides.
  • first nucleic acid strand can be either the sense strand or the antisense strand.
  • the sense strand of the small activating RNA refers to the small activating RNA duplex that contains the coding strand of the promoter DNA sequence of the target gene.
  • the identical nucleic acid strand, the antisense strand refers to the nucleic acid strand complementary to the sense strand in the small activating RNA duplex.
  • the term "second nucleic acid strand" can also be a sense strand or an antisense strand.
  • the first oligonucleotide strand is the sense strand
  • the second oligonucleotide strand is the antisense strand
  • the first oligonucleotide strand is the antisense strand
  • the second oligonucleotide strand is the sense strand.
  • gene refers to the entire nucleotide sequence required to encode a polypeptide chain or transcribe a functional RNA.
  • a “gene” may be a gene endogenous to the host cell or fully or partially recombined (for example, due to the introduction of an exogenous oligonucleotide and coding sequence encoding a promoter or a heterologous promoter adjacent to an endogenous coding sequence Into host cells).
  • the term “gene” includes nucleic acid sequences that can be composed of exons and introns.
  • a sequence encoding a protein is, for example, a sequence contained within an exon in an open reading frame between a start codon and a stop codon.
  • gene may refer to including, for example, gene regulatory sequences such as promoters , Enhancers and all other sequences known in the art that control the transcription, expression or activity of another gene, regardless of whether the other gene contains coding or non-coding sequences.
  • gene regulatory sequences such as promoters , Enhancers and all other sequences known in the art that control the transcription, expression or activity of another gene, regardless of whether the other gene contains coding or non-coding sequences.
  • “gene” can be used to describe a functional nucleic acid containing regulatory sequences such as promoters or enhancers. The expression of recombinant genes 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, a chromosomal or extrachromosomal and/or transient or stable transfection or incorporation into a cell and/or its chromatin, naturally occurring in an organism .
  • the target gene may be a protein-coding gene or a non-protein-coding gene (for example, microRNA genes, long-chain non-coding RNA genes).
  • the target gene usually contains a promoter sequence, and the design of a small activating nucleic acid molecule that has the same identity (also called homology) with the promoter sequence can realize the positive regulation of the target gene, which is represented by the up-regulation of the target gene expression.
  • target gene promoter sequence refers to the non-coding sequence of the target gene.
  • the target gene promoter sequence refers to the coding strand of the sequence, also known as the non-template strand. It is a nucleic acid sequence that is the same sequence as the gene coding sequence.
  • Target sequence refers to a sequence fragment homologous or complementary to the sense oligonucleotide strand or antisense oligonucleotide of the small activating nucleic acid molecule in the promoter sequence of the target gene.
  • sense strand and “sense oligonucleotide strand” are used interchangeably.
  • the sense oligonucleotide strand of a small activating nucleic acid molecule refers to a small activating nucleic acid molecule duplex containing a target gene
  • the coding strand of the promoter sequence has the first nucleic acid strand of identity.
  • antisense strand and “antisense oligonucleotide strand” are used interchangeably.
  • the antisense oligonucleotide strand of a small activating nucleic acid molecule refers to the small activating nucleic acid molecule in the duplex
  • the second nucleic acid strand complementary to the oligonucleotide strand refers to the small activating nucleic acid molecule in the duplex
  • the second nucleic acid strand complementary to the oligonucleotide strand are used interchangeably.
  • coding strand refers to the DNA strand that cannot be transcribed in the target gene.
  • the nucleotide sequence of this strand is consistent with the sequence of the RNA generated by transcription (in RNA, U replaces the DNA in DNA). T).
  • the coding strand of the double-stranded DNA sequence of the target gene promoter in the present invention refers to the promoter sequence on the same DNA strand as the target gene DNA coding strand.
  • template strand refers to another strand of the double-stranded DNA of the target gene that is complementary to the coding strand, and that strand that can be used as a template to be transcribed into RNA, which strand is complementary to the base of the transcribed RNA (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 double-stranded DNA sequence of the target gene promoter in the present invention refers to the promoter sequence on the same DNA strand as the target gene DNA template strand.
  • promoter refers to a sequence that regulates the transcription of protein-coding or RNA-coding nucleic acid sequences by being positionally associated with them.
  • eukaryotic gene promoters contain 100 to 5,000 base pairs, although this length range is not meant to limit the term “promoter” as used herein.
  • the promoter sequence is generally located at the 5'end of the protein coding or RNA coding sequence, the promoter sequence can also be present in exon and intron sequences.
  • transcription start site refers to a nucleotide on the template strand of a gene that marks the start of transcription.
  • the transcription initiation site can appear on the template strand in the promoter region.
  • a gene can have more than one transcription start site.
  • identity refers to the coding strand between one of the oligonucleotide strands (sense strand or antisense strand) of the small activating RNA and a region of the promoter sequence of the target gene Or the similarity in the template chain.
  • the "identity” or “homology” may be at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100%. %.
  • overhang As used herein, the terms “overhang”, “overhang”, and “overhang” are used interchangeably and refer to non-base paired nucleotides at the end (5' or 3') of an oligonucleotide chain, which are extended beyond A double-stranded oligonucleotide is produced from one strand to the other strand.
  • the single-stranded region that extends beyond the 3'and/or 5'end of the duplex is called an overhang.
  • gene activation or “activation of genes” or “gene up-regulation” or “up-regulation of genes” are used interchangeably and refer to the measurement of gene transcription levels, mRNA levels, protein levels, enzyme activity, methylation State, chromatin state or configuration, translation level, or its activity or state in a cell or biological system are used to determine the increase in transcription, translation, or expression or activity of a certain nucleic acid. These activities or states can be measured directly or indirectly.
  • gene activation refers to the increase in activity related to nucleic acid sequences, regardless of the mechanism by which such activation occurs, for example, it acts as a regulatory sequence, It is transcribed into RNA, is translated into protein and increases protein expression.
  • the small activating RNA molecule provided by the present invention can up-regulate gene or protein expression or increase activity by at least 10%.
  • small activating RNA As used herein, the terms “small activating RNA”, “saRNA”, and “small activating nucleic acid molecule” are used interchangeably, and refer to nucleic acid molecules that can promote gene expression, and can be composed of non-coding nucleic acid sequences (for example, a promoter, an enhancer, etc.) a first nucleic acid fragment (antisense strand, also called an antisense oligonucleotide strand) of a nucleotide sequence with sequence homology or identity, and a first nucleic acid fragment that is complementary to the first nucleic acid fragment
  • the second nucleic acid fragment (sense strand, also called sense strand or sense oligonucleotide strand) of the nucleotide sequence is composed, wherein the first nucleic acid fragment and the second nucleic acid fragment form a duplex.
  • the small activation nucleic acid molecule can also be composed of a synthetic or vector-expressed single-stranded RNA molecule that can form a double-stranded region hairpin structure, where the first region contains a nucleotide sequence that has sequence identity with the target sequence of the promoter of the gene, The nucleotide sequence contained in the second region is complementary to the first region.
  • the length of the duplex region of a small activation nucleic acid molecule is usually about 10 to about 50 base pairs, about 12 to about 48 base pairs, about 14 to about 46 base pairs, and about 16 to about 44 base pairs.
  • Base pairs about 18 to about 42 base pairs, about 20 to about 40 base pairs, about 22 to about 38 base pairs, about 24 to about 36 base pairs, about 26 to about 34 base pairs, about 28 to about 32 base pairs, usually about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50 Base pairs.
  • the terms "saRNA”, “small activating RNA” and “small activating nucleic acid molecule” also contain nucleic acids other than ribonucleotide moieties, including but not limited to modified nucleotides or the like.
  • hot spot refers to a gene promoter region with a length of at least 30 bp. In these regions, there is an aggregation of functional small activating nucleic acid molecule targets, that is, the small activating nucleic acid molecules targeting these hot spots are at least 30 bp. % Can induce target gene mRNA expression to 1.2 times or more.
  • synthetic refers to the synthesis method of oligonucleotides, including any method capable of synthesizing RNA, such as chemical synthesis, in vitro transcription, vector expression, and the like.
  • the present invention up-regulates the expression of HMBS gene by means of RNA activation, and promotes the production of heme by increasing the expression of HMBS protein.
  • the HMBS gene is sometimes referred to as a target gene.
  • the preparation method of the small activation nucleic acid molecule provided by the present invention includes sequence design and sequence synthesis.
  • the synthesis of small activating nucleic acid molecule sequences can be done by chemical synthesis, or by entrusting biotechnology companies specializing in nucleic acid synthesis.
  • the method of chemical synthesis includes the following four processes: (1) synthesis of oligoribonucleotides; (2) deprotection; (3) purification and separation; (4) desalting and annealing.
  • oligo nucleic acid molecules of the present invention are as follows:
  • the first cycle connects a base to the solid support, and then in the nth cycle (19 ⁇ n ⁇ 2), in the first cycle One base is connected to the connected base in n-1 cycles, and this cycle is repeated until the synthesis of all nucleic acid sequences is completed.
  • the obtained crude saRNA product was dissolved in 2 ml of ammonium acetate aqueous solution with a concentration of 1 mol/ml, and then separated by a high-pressure liquid chromatography reverse phase C18 column to obtain a purified single-stranded oligo nucleic acid molecule.
  • filter the target sequence and the criteria for retaining the target sequence are: 1) The GC content is between 35% and 75%; 2) Does not contain 5 or more than 5 consecutive identical nucleotides; 3) Does not contain more than 3 dinucleotide repeats; 4) Does not contain more than 3 trinucleotide repeats.
  • the remaining 180 target sequences will enter the screening process as candidates. Based on these candidate sequences, the corresponding double-stranded oligo nucleic acid molecules are chemically synthesized.
  • the length of the sense and antisense strands of the double-stranded oligonucleotide molecule used in this experiment are both 21 nucleotides, and the first ribonucleic acid strand (sense strand) of the double-stranded oligonucleotide molecule is 5'
  • the 19 nucleotides of the region have 100% identity with the promoter target sequence, and its 3'end contains the TT sequence;
  • the 19 nucleotides of the 5'region of the second ribonucleic acid chain are the same as the first ribonucleic acid chain sequence Complementary, its 3'end contains TT sequence.
  • the two strands of the aforementioned oligo-nucleic acid molecule are mixed in the same amount of moles, and annealed to form a double-stranded oligo-nucleic acid molecule.
  • Human liver cancer cell lines Huh7 and HepG2 were cultured in DMEM medium (Gibco).
  • Human embryonic liver cells CCC-HEL-1 and human liver cancer cells Li-7 were cultured in RPMI-1640 medium (Gibco). All media contained 10% calf serum (Sigma-Aldrich) and 1% penicillin/streptomycin ( Gibco). The cells were cultured under 5% CO 2 and 37°C. According to the manufacturer's instructions, RNAiMax (Invitrogen, Carlsbad, CA) was used to transfect double-stranded oligo nucleic acid molecules at a concentration of 10 nM (unless otherwise specified) in a reverse transfection manner.
  • stage 1 reverse transcription reaction: 42°C for 5 minutes; 95°C for 10 seconds; stage 2, PCR reaction: 95°C for 5 seconds, 60°C for 20 seconds, and 45 cycles of amplification.
  • HPRT1 and TBP were used as internal reference genes.
  • the PCR primers used for HMBS, HPRT1 and TBP are shown in Table 1. Among them, HMBS is amplified by HMBS F1/R1 primer pair.
  • CtTm is the Ct value of the target gene from the Mock sample
  • CtTs is the Ct value of the target gene from the saRNA-treated sample
  • CtR1m is the Ct value of the internal reference gene 1 from the Mock-treated sample
  • CtR1s is the internal reference gene from the saRNA-treated sample
  • the Ct value of 1 is the Ct value of 1,
  • CtR2m is the Ct value of the internal reference gene 2 from the Mock-treated sample
  • CtR2s is the Ct value of the internal reference gene 2 from the saRNA-treated sample.
  • Huh7 cells were transfected with the above 180 double-stranded oligonucleic acid molecules at a transfection concentration of 10 nM. After 72 hours, the cells were lysed and subjected to one-step RT- in the same manner as described above. The qPCR analysis obtained the relative (compared with Mock treatment) expression value of the HMBS gene of each saRNA-treated sample. The results showed that 19 saRNAs showed activation activity. These double-stranded oligo nucleic acid molecules with activating activity are called activating saRNA.
  • Figures 2 and 3 further show the activity distribution of HMBS saRNA and the changes in the expression of HMBS mRNA mediated by saRNA.
  • Example 3 saRNA promotes HMBS gene expression in different cell lines
  • 72 hours after transfection Collect the cells and extract RNA with Qiagen RNeasy kit.
  • After reverse transcription use 7500FAST real-time PCR system to amplify HMBS gene by qPCR, and amplify HPRT1 and TBP genes as internal controls.
  • Mock, dsCon2 and siHMBS are blank transfection, double-stranded RNA transfection of unrelated sequence and small interfering RNA control transfection, respectively.
  • the PCR results were analyzed according to the method described in Example 2. As shown in Figure 4, the candidate saRNA can promote the mRNA expression level of the HMBS gene in different cell lines.
  • Example 4 saRNA promotes HMBS protein expression
  • Human hepatocellular carcinoma HepG2 was reversely transfected with the saRNA shown in Figure 5 (final concentration 20 nM). After 72 hours, the cells were collected and lysed with an appropriate amount of cell lysate (1 ⁇ RIPA buffer, Cell Signaling Technology) containing protease inhibitors. BCA method for protein quantification, followed by polyacrylamide gel electrophoresis separation and transfer to 0.45 ⁇ m PVDF membrane.
  • the primary antibody used was: rabbit monoclonal anti-HMBS (Abcam, ab129092), ⁇ / ⁇ -tubulin antibody (Cell Signaling Technology, 2148s) to detect the blot; the secondary antibody was anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology).
  • the signal was detected by scanning the membrane with Image Lab (BIO-RAD, Chemistry Doc tm MP Imaging System).
  • Image Lab BIO-RAD, Chemistry Doc tm MP Imaging System.
  • the candidate saRNA increased the expression of HMBS protein in HepG2 cells by nearly 2-fold, and the expression of some proteins was increased to 2.5-fold.
  • Activating saRNA has a significant activating effect on HMBS protein expression in liver cells.
  • Example 5 saRNA promotes HMBS enzyme activity
  • AIP is the third enzyme in the heme synthesis pathway. Defects or insufficient activity of hydroxymethylbiliin synthase (HMBS) leads to the accumulation of ⁇ -aminolevulinic acid (ALA) and PBG (porphobilinogen) in the body and the synthesis of heme A disease caused by insufficient.
  • ALA is a simple endogenous 5-carbon chemical substance that participates in the biosynthesis of heme in the body.
  • ALA generates protoporphyrin IX (Proto-porphyrin IX, abbreviated as PPIX) with strong photosensitivity in the mitochondria under the action of a series of enzymes such as ALA dehydratase.
  • PPIX combines with Fe ions Heme is produced, which is the final intermediate of heme biosynthesis.
  • the heme biosynthesis pathway is regulated by the body's negative feedback, that is, the synthesis of ALA is regulated by the heme content in the cell, so there will not be too much ALA accumulation in the body.
  • the heme synthesis pathway of the cell can convert ALA into PPIX.
  • the content of PPIX can be detected by fluorescence method.
  • the fluorescence intensity of PPIX can be used to indirectly reflect the activity of HMBS and the biosynthesis of heme (Sassa et al., J Exp Med 1975; 142: 722-731, Divaris et al., Am J Pathol 1990; 136: 891-897 , Kennedy et al., J Photochem Photobiol 1992; 14: 275-292).
  • This detection method is called ALA transition analysis.
  • RNAiMax Invitrogen, Carlsbad, CA
  • saRNA shown in Figure 6 into the cells according to the reverse method provided by the manufacturer’s instructions
  • a final concentration of 20 nM cells were treated with 1 mM ALA for 0 hours (baseline) and 24 hours at 48 hours after transfection.
  • Example 6 saRNA dose-dependently promotes the expression of HMBS gene mRNA and protein and increases HMBS enzyme activity
  • the cell culture was as described in Example 2.
  • Human liver cancer cells (Li-7) were seeded in a 6-well plate at 2 ⁇ 10 5 cells/well and transformed using RNAiMax (Invitrogen, Carlsbad, CA) according to the reverse method provided by the manufacturer’s instructions
  • the saRNA (RAG5-386) shown in Figure 7 was transfected into the cells, and the final transfection concentrations were 1, 10, 20, 50, and 100 nM, respectively.
  • Three sets of the same saRNA (RAG5-386) were transfected for different experiments. One group of cells was transfected for 24 hours, and 1 mM ALA was added to treat the cells for 48 hours.
  • the cells were collected and analyzed for the fluorescence intensity of PPIX, and the cells were lysed to determine the protein concentration. The other two groups of cells were transfected and cultured for 72 hours. Cells were collected to detect the expression of HMBS gene mRNA and protein. The methods for cell mRNA extraction and protein lysis quantification are as described in Example 3 and Example 4.
  • saRNA (RAG5-386) As shown in Figure 7, as the dose of saRNA (RAG5-386) increased, the mRNA and protein expression of HMBS gene increased to varying degrees.
  • (A) is the mRNA expression after saRNA (RAG5-386) treatment: Compared with the control treatment, the mRNA expression of the siHMBS group was reduced by more than 90%, indicating the effectiveness of the transfection system in the experiment, saRNA (RAG5-386) group It has a 2-fold activation effect, especially when treated with 20nM saRNA (RAG5-386).
  • (B) is the protein expression level after saRNA (RAG5-386) treatment: Compared with the control treatment, the HMBS protein expression level gradually increases with the increase of the saRNA (RAG5-386) dose, and both are more than 1.5 times The activation effect showed a significant dose-dependent expression.
  • (C) is the change of PPIX fluorescence intensity after saRNA (RAG5-386) treatment.
  • the saRNA (RAG5-386) group all have different degrees of increase. The above results indicate that saRNA (RAG5-386) promotes HMBS gene activation, improves the activity of HMBS enzyme and ultimately promotes the synthesis of heme.
  • Example 7 saRNA promotes the expression of HMBS gene mRNA and protein in AIP patient cells GM01623
  • GM01623, GM01624 and GM01625 cells were cultured in MEM medium (Gibco). All mediums contained 15% calf serum (Sigma-Aldrich), 1% NEAA (non-essential Amino acids, purchased from Thermo Fisher; article number: 11140050) and 1% penicillin/streptomycin (Gibco). The cells were cultured under 5% CO 2 and 37°C. GM01623 cells (1 ⁇ 10 5 cells/well) were seeded in a 6-well plate and transfected with RNAiMax. The final concentration of transfection was 20 nM. The cells were collected 72 hours after transfection. The methods for cell mRNA extraction and protein lysis quantification are as described in Example 3 and Example 4.
  • the candidate saRNA promotes the expression of HMBS gene mRNA and protein in AIP patient cells GM01623.
  • (A) is the mRNA expression after the candidate saRNA treatment: Compared with the control treatment, siHMBS is knocked down by more than 90%, which has obvious effect as a small interfering RNA, indicating the effectiveness of the transfection system in the experiment. The relative expression of mRNA after saRNA treatment was higher than that of the control treatment group.
  • the expression values of the five groups of RAG5-126, RAG5-373, RAG5-179, RAG5-386 and RAG5-374 increased by 56%, 14%, and 45%, respectively , 96% and 25%, indicating that the candidate saRNA provided by the present invention can activate the expression of HMBS gene mRNA.
  • (B) is the protein expression after the candidate saRNA treatment: Compared with the control treatment, the protein amount of the RAG5-126, RAG5-373, RAG5-179 and RAG5-386 groups all increased to varying degrees after saRNA treatment, in which RAG5 The -126 group increased by 50% and had obvious activation effect, indicating that all the candidate saRNAs provided by the present invention can activate the expression of HMBS protein.
  • Example 8 saRNA promotes the expression of HMBS gene mRNA and protein in AIP patient cells GM01624
  • the GM01624 cell culture and transfection conditions are as described in Example 7, and the cell mRNA extraction and protein lysis quantification methods are as described in Example 3 and Example 4.
  • the candidate saRNA can promote the expression of HMBS gene mRNA and protein in AIP patient cells GM01624.
  • (A) is the mRNA expression after the candidate saRNA treatment: Compared with the control treatment, siHMBS is knocked down by 90%, which has a significant effect as a small interfering RNA, indicating the effectiveness of the transfection system in the experiment. The relative expression of mRNA after saRNA treatment was higher than that of the control treatment group.
  • the expression values of the three groups of RAG5-126, RAG5-179 and RAG5-386 were increased by more than 50% respectively, with significant activation effects, indicating that the candidates provided by the present invention saRNA can activate the expression of HMBS gene mRNA.
  • (B) is the protein expression after the candidate saRNA treatment: Compared with the control treatment, the protein amount of the RAG5-126, RAG5-373, RAG5-179 and RAG5-386 groups all increased to varying degrees after saRNA treatment, indicating that this The candidate saRNA provided by the invention can activate the expression of HMBS protein to varying degrees.
  • Example 9 saRNA promotes the expression of HMBS gene mRNA and protein in AIP patient cells GM01625
  • the candidate saRNA can promote the expression of HMBS gene mRNA and protein in AIP patient cells GM01625.
  • A is the mRNA expression after the candidate saRNA treatment: Compared with the control treatment, siHMBS knocks down the HMBS mRNA expression by more than 90%, which has a significant effect as a small interfering RNA, indicating the effectiveness of the transfection system in the experiment. The relative expression of mRNA after saRNA treatment was higher than that of the control treatment group.
  • RAG5-126, RAG5-373, RAG5-179, RAG5-386 and RAG5-374 groups increased by 4%, 26%, 60%, respectively. 123% and 14%, RAG5-386 has extremely significant activation effect, indicating that the candidate saRNA provided by the present invention can activate the expression of HMBS gene mRNA and has extremely significant activation effect.
  • B is the protein expression after the candidate saRNA treatment: Compared with the control treatment, the protein expression of the RAG5-126, RAG5-373, RAG5-179 and RAG5-374 groups increased after saRNA treatment, indicating that the present invention provides The candidate saRNA can activate the expression of HMBS protein.
  • saRNA targeting the promoter of HMBS gene Based on the above results, the inventors through high-throughput screening of saRNA targeting the promoter of HMBS gene, and found multiple saRNAs that can significantly activate the expression of HMBS gene. These saRNAs promote heme production by up-regulating the mRNA and protein expression of the HMBS gene. These results clearly indicate that the use of saRNA targeting the HMBS gene promoter will be a promising strategy for the treatment of AIP.

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Abstract

提供提高HMBS基因表达的小激活核酸分子及其应用。该小激活核酸分子是靶向HMBS基因启动子区的双链或单链RNA分子，包含各自含有能形成双链核酸结构的互补区域的第一和第二核酸链。该双链核酸结构可以通过例如RNA激活机制促进HMBS基因在细胞中的表达。第一或第二核酸链长度分别为16-35个核苷酸，且其中第一条链与选自靶基因启动子区的靶点存在至少75%的同一性或互补性，另一条链与第一条链存在至少75%的互补性，两条寡核苷酸链的3'端可以具有0-6个核苷酸的突出。使用该小激活核酸分子能够上调HMBS基因的mRNA和蛋白在细胞中的表达并促进其酶活性。

Description

寡聚核酸分子及其在急性间歇性卟啉症治疗中的应用 技术领域
本发明属于核酸技术领域，具体来讲，涉及基因激活相关的寡聚核酸分子，例如小激活核酸分子，和小激活核酸分子在激活/上调羟甲基胆素合酶(HMBS)基因表达中的应用，以及其在由HMBS表达不足或活性降低而导致的疾病，如急性间歇性卟啉症治疗中的应用。
背景技术
遗传性卟啉病是由血红素生物合成途径(亦称卟啉途径)中特定酶的活性缺乏引起的一系列疾病。卟啉途径的酶缺乏导致血红素产生不足以及卟啉前体和卟啉的积累，而高组织浓度的卟啉前体和卟啉导致组织毒性。
在遗传性卟啉症中，急性间歇性卟啉症(acute intermittent porphyria，AIP，例如，常染色体显性遗传性AIP)，变异性卟啉症(VP，例如，常染色体显性遗传性VP)，遗传性粪便性卟啉症(嗜铬细胞或HCP，例如常染色体显性HCP)和5’氨基乙酰丙酸(也被称为δ-氨基乙酰丙酸或ALA)脱水酶缺乏性卟啉症(ADP，例如常染色体隐性ADP)被归类为急性肝性卟啉症，表现为可能危及生命的涉及自主神经、外周神经和中枢神经的急性神经系统症状，包括剧烈腹痛、高血压、心动过速、便秘、运动无力、瘫痪和癫痫发作等。如果治疗不当，可能会出现四肢瘫痪，呼吸障碍和甚至死亡。很多能够诱导细胞色素P450的药物、节食和激素水平变化的因素可以通过增加肝脏5’-氨基乙酰丙酸合成酶1(ALAS1)的活性诱发卟啉症的急性发作(Balwani和Desnick，Blood，120：4496-4504，2012)。
AIP，也称为胆色素原脱氨酶(porphobinogen deaminase，PBGD)缺乏症，或羟甲基胆素合酶(hydroxymethylbilane synthase，HMBS)缺乏症，为最常见的急性肝性卟啉症，是由HMBS基因突变引起的常染色体显性遗传病，发病率为每10万人口中有5～10人，其中约有5-10％的患者有症状。在AIP病人，HMBS基因的一个等位发生突变，引起羟甲基胆素合酶的蛋白表达量减半(单倍剂量不足)而致其酶活性低下，进而导致ALA和PBG(胆色素原)在体内积累及血红素合成不足。
静脉注射氯高铁血红素(hemin)通常用于AIP患者急性发作期间的治疗和预防。氯高铁血红素提供外源血红素以抑制ALAS1的负反馈，从而减少ALA和PBG的产生。虽然患者通常反应良好，但其效果缓慢，通常需要2至4天或更长时间才能使尿ALA和PBG浓度正常化。 由于静脉注射的氯高铁血红素迅速代谢，通常需要3-4次输注才能有效治疗或预防急性发作。此外，重复输注可能导致铁超负荷和静脉炎。目前唯一治愈性的疗法是肝脏移植，但肝移植伴随有显著的并发症和和死亡率，并且肝脏供体来源有限。
鉴于目前治疗方法的缺陷，需要一种更有效、更持久、更快速和安全的替代治疗方法。
本发明提供了利用小激活RNA特异地激活体内HMBS基因表达水平，持久促进细胞产生内源性胆色素原脱氨酶(亦称羟甲基胆素合酶)以恢复其细胞内正常水平从而有效治疗AIP的方法。
发明内容
为解决上述问题，本发明提供了一种基于RNA激活过程的小激活核酸分子(saRNA)，通过激活/上调HMBS基因转录，从而提高HMBS蛋白的表达量来治疗由HMBS表达不足或活性降低而导致的疾病，如急性间歇性卟啉症。
在本发明的一个方面，提供了可激活或者上调细胞中HMBS基因的表达的小激活核酸分子，本发明的小激活核酸分子的一条链与HMBS基因的启动子区的长度为16-35个核苷酸的片段具有至少75％以上的同源性或互补性，启动子区是指包括HMBS基因转录起始位点上游的400个核苷酸序列，从而实现所述基因表达的激活或者上调。具体地，本发明的小激活核酸分子的一条链包括与HMBS基因启动子区中的任意连续16-35个核苷酸具有至少75％以上的同源性或互补性的核苷酸序列，其中启动子区是指包括HMBS基因转录起始位点上游的400个核苷酸。在一个具体的实施方式中，本发明的小激活核酸分子的一条链包括或选自与HMBS基因启动子上游-395bp至-351bp的区域(区域1)(tagcctgggcaacatagtgaggccacctccccgctgtctctataa，SEQ ID NO：1)和-179bp至-1bp的区域(区域2)(tgctgcctatttcaaggttgtagcaaagctaagtttgaacagagcaaaggaagcgccatagaagctgcactacttgctcatgtcacagctggggaatggggtggtcgaatggggaggtccactgtcgcaatgttccaattcccgcccagagggagggacctccccttcgagggagggcg，SEQ ID NO：2)中的连续16-35个核苷酸具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或100％的同源性或互补性的核苷酸序列。更具体地，在一个实施方式中，本发明的小激活核酸分子的一条链与选自SEQ ID NO：1和SEQ ID NO：2中的任一连续16-35个核苷酸序列具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、或约99％的同源性或互补性。在一个具体的实施方式中，本发明的小激活核酸分子的一条链包括或选自与选自SEQ ID NO：1和SEQ ID NO：2中的任一连续16-35个核苷酸序列具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或100％的同源性或互补性的核苷酸序列。在更具体的实施方式中，本发明的小激活核酸分子的一条链由与选自SEQ ID NO：1和SEQ ID NO：2中的任一连续16-35个核苷酸序列具有至少 75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或100％的同源性或互补性的核苷酸序列组成。
在本发明中，本发明的小激活核酸分子包括靶向HMBS基因启动子区的双链小激活核酸分子，包含第一核酸链和第二核酸链，第一核酸链与HMBS基因启动子中SEQ ID No.1或SEQ ID No.2中的任一连续16-35个核苷酸具有至少75％的同源性或互补性，第二核酸链与第一核酸链具有至少75％的互补性，第一核酸链和第二核酸链能通过互补形成双链核酸结构，双链核酸结构能够激活HMBS基因在细胞中的表达。
本发明的小激活核酸分子的正义核酸链和反义核酸链可以存在于两条不同的核酸链上，也可以存在于同一条核酸链上。当正义核酸链和反义核酸链分别位于两条链上时，小激活核酸分子的至少一条链可以在5’端和3’端的任一端具有突出或悬垂，例如在3’端可以具有长度为0-6个核苷酸的突出。优选地，本发明的小激活核酸分子的两条链都具有突出，更优选地，小激活核酸分子的两条链的3’端均可以具有0-6个核苷酸的突出，最优选地，具有2-3个核苷酸的3’端的突出。优选地，突出的核苷酸可以是dT或U，也可以为天然核苷酸突出。本发明中所述的天然核苷酸突出，是指正义核酸片段或反义核酸片段的末端突出的核苷酸与对应的靶点序列的核苷酸相同或互补。
小激活核酸分子也可以包括可形成双链区发夹结构的单链RNA分子。在一个实施方式中，本发明的小激活核酸分子是靶向HMBS基因启动子区的单链RNA分子，其中所述小激活核酸分子可形成双链区发夹结构。当正义核酸链和反义核酸链存在于同一条核酸链上时，优选地，小激活核酸分子可以为发夹型单链核酸分子，其中正义核酸片段和反义核酸片段的互补区域形成双链核酸结构，该双链核酸结构可以通过例如RNA激活机制促进HMBS基因在细胞中的表达。
上述小激活核酸分子中，正义核酸片段和反义核酸片段的长度可以分别为16-35个核苷酸，例如，可以为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。
在一个实施方式中，本发明的小激活核酸分子的正义链与选自SEQ ID NO：30-48中的任一核苷酸序列具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同一性或同源性，并且其反义链与选自SEQ ID NO：49-67的任一核苷酸序列具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同一性或同源性。在一个实施方式中，本发明的小激活核酸分子的正义链包括或者选自与选自SEQ ID NO：30-48中的任一核苷酸序列具有至少75％，例如至少约79％、约80％、约85％、 约90％、约95％、约99％、或约100％的同一性或同源性的序列，并且其反义链包括或者选自与选自SEQ ID NO：49-67中的任一核苷酸序列具有至少75％，例如至少约79％，约80％，约85％，约90％，约95％，约99％、或约100％的同一性或同源性的序列。在一个实施方式中，本发明的小激活核酸分子的正义链由与选自SEQ ID NO：30-48中的任一核苷酸序列具有至少75％，例如至少约79％、约80％、约85％、约90％、约95％、约99％、或约100％的同一性或同源性的序列组成，并且其反义链由与选自SEQ ID NO：49-67中的任一核苷酸序列具有至少75％，例如至少约79％，约80％，约85％，约90％，约95％，约99％、或约100％的同一性或同源性的序列组成。在具体的实施方式中，本发明的小激活核酸分子的正义链可以是选自SEQ ID NO：30-48的任一核苷酸序列所示的序列，并且其反义链可以是如选自SEQ ID NO：49-67中的任一核苷酸序列所示的序列。
在一个实施方式中，本文所述的小激活核酸分子可以是合成的、体外转录的或者载体表达的。
本文所述的小激活核酸分子中所有的核苷酸都可以为天然的未经化学修饰的核苷酸，也可以包括至少一种修饰。在一个实施方式中，本文所述的小激活核酸分子中的修饰可以为化学修饰，如在至少一个核苷酸上具有化学修饰。本发明使用的化学修饰可以包括或选自如下修饰中的一种或多种或其任意组合：
(1)对所述小激活核酸分子的核苷酸序列中核苷酸的磷酸二酯键的修饰；
(2)对所述小激活核酸分子的核苷酸序列中的核糖的2’-OH的修饰；
(3)对所述小激活核酸分子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。
所述化学修饰为本领域技术人员所公知，所述磷酸二酯键的修饰是指对磷酸二酯键中的氧进行修饰，包括但不限于，硫代磷酸修饰和硼烷化磷酸盐修饰。两种修饰都能稳定小激活核酸分子结构，保持碱基配对的高特异性和高亲和力。
核糖修饰是指对核苷酸戊糖中2’-OH的修饰，即，在核糖的羟基位置引入某些取代基，例如，包括但不限于，2’-氟代修饰、2’-氧甲基修饰、2’-氧亚乙基甲氧基修饰、2，4’-二硝基苯酚修饰、锁核酸(LNA)、2’-氨基修饰、2’-脱氧修饰等。
碱基修饰是指对核苷酸的碱基进行修饰，例如，包括但不限于，5′-溴尿嘧啶修饰、5′-碘尿嘧啶修饰、N-甲基脲嘧啶修饰、2，6-二氨基嘌呤修饰等。
这些修饰可以增加小激活核酸分子的生物可利用性，提高与靶序列的亲和性，增强在细胞内抵抗核酸酶水解的能力。
此外，为了促进小激活核酸分子进入细胞，可以在以上修饰的基础上，在小激活核酸分子的正义链或反义链的末端引入例如胆固醇等亲脂性基团以利于通过由脂质双分子层构成的细胞膜及核膜与细胞核内的基因启动子区发生作用。
本发明提供的小激活核酸分子在与细胞接触后可有效激活或上调细胞中HMBS基因的表达，优选情况下表达至少上调10％。
本发明的另一方面还涉及编码本文所述的小激活核酸分子的核酸。在一个实施方式中，所述核酸可以是DNA分子。
在本发明的另一方面，提供了包含上文所述的小激活核酸分子或编码本文所述的小激活核酸分子的核酸的细胞。在一个实施方式中，本发明的小激活核酸分子可以是靶向HMBS基因启动子区的双链小激活核酸分子，其包括正义链和反义链。在另一实施方式中，本发明的小激活核酸分子可以是编码靶向HMBS基因启动子区的小激活核酸分子的单链核酸分子。
在本发明的另一方面，提供了包含上文所述的小激活核酸分子、编码本文所述的小激活核酸分子的核酸的试剂盒。
本发明的另一方面涉及上文所述的小激活核酸分子、编码本文所述的小激活核酸分子的核酸在制备用于在细胞中激活/上调HMBS基因表达的药物中的应用。
本发明的另一方面涉及上文所述的小激活核酸分子、编码本文所述的小激活核酸分子的核酸、本发明的细胞在制备用于治疗对象中由HMBS表达不足或活性降低而导致的疾病的药物中的应用。在一个实施方式中，由HMBS表达不足或活性降低而导致的疾病可以包括遗传性卟啉症。遗传性卟啉症可以包括例如急性间歇性卟啉症。
本发明的另一方面涉及上文所述的小激活核酸分子、编码本文所述的小激活核酸分子的核酸、本发明的细胞在制备用于治疗对象中的急性间歇性卟啉症的药物中的应用。
本发明的另一方面还涉及治疗对象中由HMBS表达不足或活性降低而导致的疾病的方法，包括给对象施用上文所述的小激活核酸分子、编码本文所述的小激活核酸分子的核酸或本发明所述的细胞。
本发明的另一方面还涉及治疗对象中的急性间歇性卟啉症的方法，包括给对象施用上文所述的小激活核酸分子、编码本文所述的小激活核酸分子的核酸、或本发明所述的细胞。
本发明还涉及在细胞中激活/上调HMBS基因的表达的方法，该方法包括给所述细胞施用上文所述的小激活核酸分子或编码本文所述的小激活核酸分子的核酸。
本发明的小激活核酸分子可以被直接导入细胞中，也可以是将编码该小激活核酸分子的核酸序列导入细胞后在细胞内产生；所述细胞优选为哺乳动物细胞，更优选为人类细胞。上 述细胞可以是离体的，如细胞系或细胞株等，也可以分离自哺乳动物体内，如人体中。该人体可以是患有由HMBS蛋白表达减少引发的症状的患者。本文所述的小激活核酸分子可以被施以足够的量以实现对由HMBS蛋白表达减少引发的症状的治疗。具体情况下，所述由HMBS蛋白量的缺乏引发的症状包括或选自急性间歇性卟啉症。
本发明另一方面提供了一种分离的HMBS基因小激活核酸分子作用位点，该位点具有HMBS基因的启动子区上任意连续的16-35个核苷酸序列，优选情况下，所述作用位点为选自SEQ ID NO：1-2的任一条序列上任意连续16-35个核苷酸序列。具体地，所述作用位点如选自SEQ ID NO：11-29的任一核苷酸序列所示。
本发明的有益效果
本发明提供的能够激活/上调HMBS基因表达的小激活核酸分子，能够持久地激活HMBS基因，因而高效、特异地上调或恢复HMBS基因和蛋白的表达和酶的活性，并同时具有较低的毒副作用，可用于制备激活/上调细胞中HMBS基因和蛋白表达的药物或制剂。
附图说明
图1为HMBS基因启动子示意图。图中区域1和区域2为设计小核酸分子使用的靶序列区域。TSS为转录起始位点。
图2为小核酸分子介导的HMBS mRNA表达改变。靶向HMBS启动子靶点区的180个小核酸分子分别转染人肝细胞癌Huh7细胞，转染浓度为10nM，72小时后收集细胞用一步法RT-PCR分析HMBS mRNA表达。图示为相对于对照处理(mock)的HMBS表达改变从最高到最低排序。
图3为小核酸分子介导的HMBS mRNA表达改变。靶向HMBS启动子的180个小核酸分子分别转染Huh7细胞，转染浓度为10nM，72小时后收集细胞用一步法RT-PCR分析HMBS mRNA表达。图示为相对于对照处理(mock)的各个小核酸分子诱导HMBS表达改变的倍数。小核酸分子按照其在HMBS基因启动子上的位置从最上游到TSS排序。
图4为候选saRNA(small activating RNA)在不同肝脏细胞中激活HMBS基因mRNA表达。用所示saRNA(n＝13，终浓度为20nM)分别转染人肝细胞癌Huh7、HepG2和人胚胎肝细胞CCC-HEL-1。72小时后收集细胞用Qiagen RNeasy试剂盒提取RNA，反转录后用7500FAST实时PCR系统对HMBS基因进行qPCR扩增，同时扩增HPRT1和TBP基因作为内参。Mock、dsCon2(正义链5’-ACUACUGAGUGACAGUAGATT-3’(SEQ ID NO：68)，反义链5’-UCUACUGUCACUCAGUAGUTT-3’(SEQ ID NO：69)和siHMBS(正义链：5’-CCUGUUUACCAAGGAGCUUTT-3’(SEQ ID No：3)，反义链：5’-AAGCUCCUUGGUAAACAGGTT-3’(SEQ ID No：4))分别为空白转染、无关序列双链RNA转染及小干扰RNA(siRNA)对照转染。
图5为候选saRNA在肝脏细胞中激活HMBS蛋白表达。用所示saRNA(终浓度为20nM)转染人肝细胞癌HepG2细胞。72小时后收集细胞进行蛋白印迹分析，用抗人HMBS抗体检测HMBS蛋白表达量，同时检测微管蛋白(Tubulin)作为蛋白上样量准确性控制。Mock、dsCon2和siHMBS分别为空白转染、无关序列双链RNA转染及小干扰RNA对照转染。(A)蛋白印迹膜的扫描图。(B)用ImageJ软件对A图的条带进行定量分析，得出每种处理的HMBS条带强度与Mock处理比较的相对值。
图6为ALA转化分析得出的单位(mg)蛋白的PPIX荧光强度。用所示的saRNA转染人胚胎肝细胞CCC-HEL-1，48小时后向细胞加入ALA底物，在加入前(0h)和加入后24小时(24h)收集细胞分析PPIX的荧光强度，同时裂解细胞测定蛋白浓度。Mock、dsCon2和siHMBS分别为空白转染、无关序列双链RNA转染及小干扰RNA对照转染。Y轴为单位(mg)蛋白的PPIX荧光强度。
图7为候选saRNA在Li-7细胞中激活HMBS基因mRNA和蛋白表达以及ALA转化分析得出的单位(mg)蛋白的PPIX荧光强度。(A)为用所示的saRNA和所示浓度转染人肝癌细胞Li-7，72小时后收集细胞用Qiagen RNeasy试剂盒提取RNA，反转录后用7500FAST实时PCR系统对HMBS基因进行qPCR扩增，同时扩增HPRT1基因作为内参。(B)上图为用所示的saRNA和所示浓度转染人肝癌细胞Li-7，72小时后收集细胞进行蛋白印迹分析，用抗人HMBS抗体检测HMBS蛋白表达量，同时检测微管蛋白(Tubulin)作为蛋白上样量准确性控制。下图为用ImageJ软件对A图的条带进行定量分析，得出每种处理的HMBS条带强度与Mock处理比较的相对值。M：Mock处理，C：dsCon2处理。(C)为用所示的saRNA和所示浓度转染人肝癌细胞Li-7，24小时后向细胞加入ALA底物，在加入后48小时(48h)收集细胞分析PPIX的荧光强度，同时裂解细胞测定蛋白浓度。Mock、dsCon2和siHMBS分别为空白转染、无关序列双链RNA转染及小干扰RNA对照转染。
图8为候选saRNA在急性间歇性卟啉症(AIP)病人细胞GM01623细胞中激活HMBS基因mRNA和蛋白表达。(A)为用所示的saRNA转染GM01623细胞，终浓度为20nM，72小时后收集细胞用Qiagen RNeasy试剂盒提取RNA，反转录后用7500FAST实时PCR系统对HMBS基因进行qPCR扩增，同时扩增HPRT1和TBP基因作为内参。(B)为用所示的saRNA转染GM01623细胞，终浓度为20nM，72小时后收集细胞进行蛋白印迹分析，用抗人HMBS抗体检测HMBS蛋白表达量，同时检测微管蛋白(Tubulin)作为蛋白上样量准确性控制。Mock、dsCon2和siHMBS分别为空白转染、无关序列双链RNA转染及小干扰RNA对照转染。
图9为候选saRNA在急性间歇性卟啉症(AIP)病人细胞GM01624细胞中激活HMBS基因 mRNA和蛋白表达。(A)为用所示的saRNA转染GM01624细胞，终浓度为20nM，72小时后收集细胞用Qiagen RNeasy试剂盒提取RNA，反转录后用7500FAST实时PCR系统对HMBS基因进行qPCR扩增，同时扩增HPRT1和TBP基因作为内参。(B)为用所示的saRNA转染GM01624细胞，终浓度为20nM，72小时后收集细胞进行蛋白印迹分析，用抗人HMBS抗体检测HMBS蛋白表达量，同时检测微管蛋白(Tubulin)作为蛋白上样量准确性控制。Mock、dsCon2和siHMBS分别为空白转染、无关序列双链RNA转染及小干扰RNA对照转染。
图10示候选saRNA在急性间歇性卟啉症(AIP)病人细胞GM01625细胞中激活HMBS基因mRNA和蛋白表达。(A)为用所示的saRNA转染GM01625细胞，终浓度为20nM，72小时后收集细胞用Qiagen RNeasy试剂盒提取RNA，反转录后用7500FAST实时PCR系统对HMBS基因进行qPCR扩增，同时扩增HPRT1和TBP基因作为内参。(B)为用所示的saRNA转染GM01625细胞，终浓度为20nM，72小时后收集细胞进行蛋白印迹分析，用抗人HMBS抗体检测HMBS蛋白表达量，同时检测微管蛋白(Tubulin)作为蛋白上样量准确性控制。Mock、dsCon2和siHMBS分别为空白转染、无关序列双链RNA转染及小干扰RNA对照转染。
具体实施方式
在本发明中，相关术语采用如下定义：
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之间通过氢键形成。互补寡核苷酸链可以沃森-克里克(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。
互补包括完全互补和不完全互补两种情况。完全互补或100％互补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可以与第二条寡核苷酸链相应位置的核苷酸形成氢键而没有“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。实质互补是指至少约75％、约79％、约80％、约85％、约90％、约95％、约99％、或约100％的互补。
如本文所用的术语“寡核苷酸”或“小核酸分子”是指核苷酸的聚合物，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及天然存在的或非天然存在的骨架。本发明中所述的用于激活靶基因转录的寡核苷酸为小激活核酸分子。
如本文所用的术语“寡核苷酸链”和“寡核苷酸序列”可互换地使用，是指35个以下碱基的短链核苷酸的总称(包括脱氧核糖核酸DNA或核糖核酸RNA内的核苷酸)。在本发明中，寡核苷酸链的长度可以是16至35个核苷酸的任一长度。
如本文所用，术语“第一核酸链”可以是正义链也可以是反义链，小激活RNA的正义链是指小激活RNA双链体中含与靶基因的启动子DNA序列的编码链具有同一性的核酸链，反义链是指小激活RNA双链体中与正义链互补的核酸链。
如本文所用，术语“第二核酸链”也可以是正义链或者反义链。当第一寡核酸链为正义链时，第二寡核酸链为反义链，当第一寡核酸链为反义链时，第二寡核酸链为正义链。
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)的小激活核酸分子可以实现对靶基因的正向调控，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核酸分子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”可互换地使用，小激活核酸分子的正义寡核苷酸链是指小激活核酸分子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酸链。
如本文所用，术语“反义链”、“反义寡核苷酸链”可互换地使用，小激活核酸分子的反 义寡核苷酸链是指小激活核酸分子双链体中与正义寡核苷酸链互补的第二核酸链。
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的序列。通常，真核基因启动子包含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，但启动子序列也可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的相似性。在本文中，所述“同一性”或“同源性”可以是至少约75％、约79％、约80％、约85％、约90％、约95％、约99％、或约100％。
如本文所用的术语“突出”、“overhang”、“悬垂”可互换地使用，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”或“基因上调”或“上调基因”可互换地使用，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”、“基因上调”、“上调基因”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。优选情况下本发明提供的小激活RNA分子能够上调基因或蛋白质表达或活性增加至 少10％。
如本文所用，术语“小激活RNA”、“saRNA”、“小激活核酸分子”可互换地使用，是指能够促进基因表达的核酸分子，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)具有序列同源性或同一性的核苷酸序列的第一核酸片段(反义链，也称反义寡核苷酸链)和包含与第一核酸片段互补的核苷酸序列的第二核酸片段(正义链，也称有义链或正义寡核苷酸链)组成，其中所述第一核酸片段和第二核酸片段形成双链体。小激活核酸分子也可以由合成的或者载体表达的可形成双链区发夹结构的单链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苷酸序列，第二区域包含的核苷酸序列与第一区域互补。小激活核酸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个至约40个碱基对、约22个至约38个碱基对、约24个至约36个碱基对、约26个至约34个碱基对、约28个至约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”、“小激活RNA”和“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30％能够诱导靶基因mRNA表达达到1.2倍或以上。
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。
本发明通过RNA激活方式上调HMBS基因的表达，通过增加HMBS蛋白的表达量来促进血红素的生成。本发明中HMBS基因有时也称为靶基因。
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。
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，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，本发明所述寡聚核酸分子化学合成的具体步骤如下：
(1)寡聚核酸分子的合成
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
将连接有寡聚核酸分子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，过滤含有寡聚核酸分子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，得到纯化的寡聚核酸分子单链产物。
(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，得到含有寡聚核酸分子的溶液。
下面结合具体实施例及附图，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1 靶向HMBS启动子的小激活核酸分子的设计与合成
从UCSC基因组数据库(genome.ucsc.edu)获得HMBS基因从转录起始位点(TSS)到上游-395bp的长度为395bp的正义启动子序列，其中一段重复序列(-350～-180)被排除在靶点之外，以余下的两段序列(长度为45bp的区域1：-395～-351和长度为179bp的区域2：-179～-1)为模板，从最上游处向下游移动，选定大小为19bp的靶点，区域1共获得27个靶点，区域2共获得161个靶点(图1)。然后对靶点序列进行过滤处理，保留靶点序列的标准为：1)GC含量在35％至75％之间；2)不含有5个或者多于5个的连续同一核苷酸；3)不含多于3个的二核苷酸重复序列；4)不含多于3个的三核苷酸重复序列。过滤后，剩余180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寡聚核酸 分子。其中，该实验中使用的双链寡聚核酸分子的正义和反义链的长度均为21个核苷酸，所述双链寡聚核酸分子的第一核糖核酸链(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TT序列；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序列互补，其3’末端含有TT序列。将前述寡聚核酸分子的两条链以同等量的摩尔数混合，退火后形成双链寡聚核酸分子。
实施例2 靶向HMBS启动子的saRNA的筛选
(1)细胞培养和转染
人肝癌细胞系Huh7、HepG2培养在DMEM培养基(Gibco)中。人胚胎肝细胞CCC-HEL-1和人肝癌细胞Li-7培养在RPMI-1640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)浓度以逆转染的方式转染双链寡聚核酸分子。
(2)一步法RT-qPCR
转染结束后，弃掉培养基，每孔加入150μl PBS清洗一次，弃掉PBS，每孔加入50μl细胞裂解液(裂解液的成分或来源？)，室温孵育5分钟。每孔取1μl细胞裂解物使用一步法TB Green TM PrimeScrip TM RT-PCR试剂盒II(Takara，RR086A)在
Figure PCTCN2020087844-appb-000001
480系统(Roche)上进行qPCR分析，每个样本重复3个复孔扩增，PCR反应条件见下表1。
表1.PCR反应制备
Figure PCTCN2020087844-appb-000002
反应条件为：阶段1，反转录反应：42℃，5分钟；95℃ 10秒；阶段2，PCR反应：95℃ 5秒，60℃ 20秒，扩增45个循环。以HPRT1及TBP为内参基因。HMBS、HPRT1及TBP所用PCR引物见表1，其中HMBS用HMBS F1/R1引物对扩增。
表2.qRT-PCR分析的引物序列
Figure PCTCN2020087844-appb-000003
为了计算某个saRNA转染样本的HMBS(目的基因)的相对于对照处理(Mock)的表达值(E rel)，用公式1代入目的基因及2个内参基因的Ct值计算。
E rel＝2 (CtTm-CtTs)/((2 (CtR1m-CtR1s)*2 (CtR2m-CtR2s)) (1/2))      (公式1)
其中，CtTm为来自Mock样本的目的基因的Ct值，CtTs为来自saRNA处理样本的目的基因的Ct值，CtR1m为来自Mock处理样本的内参基因1的Ct值，CtR1s为来自saRNA处理样本的内参基因1的Ct值，CtR2m为来自Mock处理样本的内参基因2的Ct值，CtR2s为来自saRNA处理样本的内参基因2的Ct值。
(3)功能性saRNA筛选
为了获得能够激活HMBS转录的saRNA，用上述180个双链寡聚核酸分子分别转染Huh7细胞，转染浓度为10nM，72小时后以如上所述相同的方法，裂解细胞并进行一步法RT-qPCR分析得到每个saRNA处理样本的HMBS基因的相对(与Mock处理比较)表达值。结果表明，有19个saRNA显示出激活活性。这些具有激活活性的双链寡聚核酸分子被称为激活性saRNA。
图2和图3进一步显示了HMBS saRNA的活性分布和saRNA介导的HMBS mRNA表达改变情况。
Figure PCTCN2020087844-appb-000004
Figure PCTCN2020087844-appb-000005
Figure PCTCN2020087844-appb-000006
实施例3 saRNA在不同细胞系中促进HMBS基因表达
用表4所示saRNA(n＝13，终浓度为20nM)按照实施例2中描述的方法分别转染人肝细胞癌Huh7、HepG2和人胚胎肝细胞CCC-HEL-1，转染72小时后，收集细胞用Qiagen RNeasy试剂盒提取RNA，反转录后用7500FAST实时PCR系统对HMBS基因进行qPCR扩增，同时扩增HPRT1和TBP基因作为内参。Mock、dsCon2和siHMBS分别为空白转染、无关序列双链RNA转染及小干扰RNA对照转染。按照实施例2中描述的方法对PCR结果进行分析，结果如图4所示，候选saRNA在不同细胞系中均能促进HMBS基因的mRNA表达水平。
实施例4 saRNA促进HMBS蛋白表达
用图5所示saRNA(终浓度为20nM)逆向转染人肝细胞癌HepG2，72小时后收集细胞，使用适量含有蛋白酶抑制剂的细胞裂解液(1×RIPA缓冲液，Cell Signaling Technology)裂解。BCA法进行蛋白质定量，随后进行聚丙烯酰胺凝胶电泳分离并转至0.45μm的PVDF膜。所用一抗为：兔单克隆抗HMBS(Abcam，ab129092)，α/β-微管蛋白抗体(Cell Signaling Technology，2148s)对印迹进行检测；二抗用抗兔IgG，HRP-连接的抗体(Cell Signaling Technology)。用Image Lab(BIO-RAD，Chemistry Doc tmMP Imaging System)扫膜检测信号。如图5所示，候选saRNA增加HepG2细胞中HMBS蛋白表达量接近2倍，部分蛋白表达量被提高至2.5倍。激活性saRNA对肝脏细胞中HMBS蛋白表达的激活效应效果显著。
实施例5 saRNA促进HMBS酶活性
AIP是血红素合成途径上第3个酶羟甲基胆素合成酶(HMBS)缺陷或是活性不足导致δ-氨基乙酰丙酸(ALA)和PBG(胆色素原)在体内积累而血红素合成不足引发的一种疾病。ALA是一种简单的内源性5碳化学物质，参与体内血红素的生物合成。作为血红素的前体物，ALA在ALA脱水酶等一系列酶的作用下，在线粒体内生成具有强光敏作用的原卟啉IX(Proto-porphyrin IX，缩写为PPIX)，PPIX与Fe离子结合生成血红素，为血红素生物合成的最后一步中间体。正常情况下，血红素生物合成途径受到机体负反馈调节，即ALA的合成受细胞内血红素含量调控，所以体内不会有过多的ALA蓄积。在细胞中加入外源性ALA时，细胞的血红素合成途径可以将ALA转化成PPIX。PPIX的含量可以通过荧光方法检测。因此可以通过检测PPIX的荧光强度间接反映HMBS的活性强弱以及血红素的生物合成量(Sassa等，J Exp Med 1975；142：722-731，Divaris等，Am J Pathol 1990；136：891-897，Kennedy等，J Photochem Photobiol 1992；14：275-292)。这种检测方法称为ALA转变分析。
在6孔板中接种人胚胎肝细胞CCC-HEL-1(2×10 5细胞/孔)使用RNAiMax(Invitrogen，Carlsbad，CA)按照生产商说明书提供的逆转法转染图6所示saRNA至细胞，终浓度为20nM， 转染后第48小时用1mM的ALA处理细胞0小时(基线)和24小时。转染后第三天收集细胞于1.5ml的离心管中，取出样品的一部分用300μl 1N 1∶1MeOH-PCA在冰上裂解细胞10分钟，10000g 4℃离心10分钟后取100μl上清液加入到黑色96孔板，使用多功能酶标仪(TECAN Infintie M200PRO)在400nM激发光和660nM发射光检测荧光强度。样品的另一部分用1×RIPA裂解液(Cell Signaling Technology)裂解细胞随后用BCA法检测蛋白浓度。最后用荧光强度/蛋白浓度对数据进行归一化处理。
根据图6所示，与Mock和dsCon2对照组相比，转染激活性saRNA的细胞中单位蛋白浓度对应的荧光强度明显增加，说明saRNA导致的HMBS基因激活提高了HMBS酶的活性并最终促进了血红素的合成。
实施例6 saRNA剂量依赖性地促进HMBS基因mRNA和蛋白的表达以及增加HMBS酶活性
细胞培养如实施例2所述，人肝癌细胞(Li-7)以2×10 5细胞/孔接种于6孔板中，使用RNAiMax(Invitrogen，Carlsbad，CA)按照生产商说明书提供的逆转法转染图7所示saRNA(RAG5-386)至细胞，转染的终浓度分别为1、10、20、50和100nM，转染三组相同的saRNA(RAG5-386)分别用于不同的实验。其中一组细胞转染24小时后，加入1mM的ALA处理细胞48小时后收集细胞分析PPIX的荧光强度，同时裂解细胞测定蛋白浓度。另外两组细胞转染培养72小时后收集细胞分别用于检测HMBS基因mRNA和蛋白的表达。细胞mRNA提取和蛋白裂解定量方法如实施例3和实施例4所述。
如图7所示，随着saRNA(RAG5-386)剂量的增加，HMBS基因的mRNA和蛋白表达都有不同程度的增加。(A)为经saRNA(RAG5-386)处理后mRNA的表达：与对照处理相比，siHMBS组mRNA表达降低了90％以上，说明实验中转染系统的有效性，saRNA(RAG5-386)组具有2倍左右的激活效果，尤其在20nM的saRNA(RAG5-386)处理下达到高峰。(B)为经saRNA(RAG5-386)处理后蛋白的表达量：与对照处理相比，HMBS蛋白的表达量随着saRNA(RAG5-386)剂量的增加而逐渐上调，均有1.5倍以上的激活效果，展示了明显的剂量依赖性表达。(C)为经saRNA(RAG5-386)处理后PPIX荧光强度的变化，saRNA(RAG5-386)组均有不同程度的增加。上述结果说明saRNA(RAG5-386)促进HMBS基因激活提高了HMBS酶的活性并最终促进了血红素的合成。
实施例7 saRNA促进HMBS基因mRNA和蛋白在AIP病人细胞GM01623中的表达
GM01623、GM01624和GM01625细胞(购自Coriell Institute，Camden，NJ，USA)培养在MEM培养基(Gibco)中，所有培养基含有15％小牛血清(Sigma-Aldrich)、1％NEAA(non-essential amino acids，购自Thermo Fisher；货号：11140050)和1％青霉素/链霉素(Gibco)。细胞在5％CO 2，37℃条件下培养。在6孔板中接种GM01623细胞(1×10 5细胞/ 孔)使用RNAiMax进行转染，转染的终浓度为20nM，转染72小时后收集细胞。细胞mRNA提取和蛋白裂解定量方法如实施例3和实施例4所述。
如图8所示，候选saRNA促进HMBS基因mRNA和蛋白在AIP病人细胞GM01623中的表达。(A)为经候选saRNA处理后mRNA的表达：与对照处理相比，siHMBS敲低了90％以上，作为小干扰RNA有明显的效果，说明实验中转染系统的有效性。saRNA处理后mRNA的相对表达量均高于对照处理组，RAG5-126、RAG5-373、RAG5-179、RAG5-386和RAG5-374五组的表达值分别增加了56％、14％、45％、96％和25％，说明本发明提供的候选saRNA均能激活HMBS基因mRNA的表达。(B)为经候选saRNA处理后蛋白的表达：与对照处理相比，saRNA处理后RAG5-126、RAG5-373、RAG5-179和RAG5-386组的蛋白量均有不同程度的增加，其中RAG5-126组上升了50％，有明显的激活效果，说明本发明提供的候选saRNA均能激活HMBS蛋白的表达。
实施例8 saRNA促进HMBS基因mRNA和蛋白在AIP患者细胞GM01624中的表达
GM01624细胞培养及转染条件如实施例7所述，细胞mRNA提取和蛋白裂解定量方法如实施例3和实施例4所述。如图9所示，候选saRNA能够促进HMBS基因mRNA和蛋白在AIP患者细胞GM01624中的表达。(A)为经候选saRNA处理后mRNA的表达：与对照处理相比，siHMBS敲低了90％，作为小干扰RNA有显著的效果，说明实验中转染系统的有效性。saRNA处理后mRNA的相对表达量均高于对照处理组，RAG5-126、RAG5-179和RAG5-386三组的表达值分别增加了50％以上，有显著的激活效果，说明本发明提供的候选saRNA能激活HMBS基因mRNA的表达。(B)为经候选saRNA处理后蛋白的表达：与对照处理相比，saRNA处理后RAG5-126、RAG5-373、RAG5-179和RAG5-386组的蛋白量均有不同程度的增加，说明本发明提供的候选saRNA能不同程度的激活HMBS蛋白的表达。
实施例9 saRNA促进HMBS基因mRNA和蛋白在AIP病人细胞GM01625中的表达
GM01625细胞培养及转染条件如实施例7所述，细胞mRNA提取和蛋白裂解定量方法如实施例3和实施例4所述。如图10所示，候选saRNA能够促进HMBS基因mRNA和蛋白在AIP病人细胞GM01625中的表达。(A)为经候选saRNA处理后mRNA的表达：与对照处理相比，siHMBS敲低了HMBS mRNA表达90％以上，作为小干扰RNA有显著的效果，说明实验中转染系统的有效性。saRNA处理后mRNA的相对表达量均高于对照处理组，RAG5-126、RAG5-373、RAG5-179、RAG5-386和RAG5-374组的表达值分别增加了4％、26％、60％、123％和14％，RAG5-386有极为显著的激活效果，说明本发明提供的候选saRNA能激活HMBS基因mRNA的表达，并具有极为显著的激活效果。(B)为经候选saRNA处理后蛋白的表达：与对照处理相比，saRNA处理后RAG5-126、RAG5-373、RAG5-179和RAG5-374组的蛋白表达量均有增加，说明本发明提 供的候选saRNA能激活HMBS蛋白的表达。
综合上述结果，发明人通过高通量筛选靶向HMBS基因启动子的saRNA，发现了多个能够显著激活HMBS基因表达的saRNA。这些saRNA通过上调HMBS基因的mRNA和蛋白表达，促进血红素生成。这些结果明确提示用靶向HMBS基因启动子的saRNA将是一种很有前景的治疗AIP的策略。

Claims (33)

  1. 一种小激活核酸分子，包含第一核酸链和第二核酸链，其中第一核酸链与HMBS基因启动子中SEQ ID No.1或SEQ ID No.2中的任一连续16-35个核苷酸具有至少75％的同源性或互补性，第二核酸链与第一核酸链具有至少75％的互补性，第一核酸链和第二核酸链能通过互补形成双链核酸结构，双链核酸结构能够激活HMBS基因在细胞中的表达。
  2. 根据权利要求1所述的小激活核酸分子，其中所述第一核酸链和所述第二核酸链存在于两条不同的核酸链上。
  3. 根据权利要求1所述的小激活核酸分子，其中所述第一核酸链和第二核酸链存在于同一条核酸链上，优选地，为发夹型单链核酸分子，其中所述第一核酸链和所述第二核酸链的互补区域形成双链核酸结构。
  4. 根据权利要求2所述的小激活核酸分子，其中所述小激活核酸分子的至少一条链在3’端具有0至6个核苷酸的突出。
  5. 根据权利要求4所述的小激活核酸分子，其中，所述小激活核酸分子的两条链在3’端具有0至6个核苷酸的突出，优选地具有2-3个核苷酸的突出。
  6. 根据权利要求1-5任一项所述的小激活核酸分子，其中所述第一核酸链和第二核酸链的长度分别为16至35个核苷酸。
  7. 根据权利要求1-6所述的小激活核酸分子，其中，所述小激活核酸分子的一条链包括，或者由与选自SEQ ID NO：11-SEQ ID NO：29的任一核苷酸序列具有至少75％的同源性或互补性的序列组成。
  8. 根据权利要求1-7所述的小激活核酸分子，其中，所述第一核酸链与选自SEQ ID NO：30-48中的任一核苷酸序列具有至少75％的同源性，并且所述第二核酸链与选自SEQ ID NO：49-67的任一核苷酸序列具有至少75％的同源性。
  9. 权利要求1-8所述的小激活核酸分子，其中，所述第一核酸链包括或选自SEQ ID NO：30-48的任一核苷酸序列所示的序列，并且所述第二核酸链包括或选自SEQ ID NO：49-67的任一核苷酸序列所示的序列。
  10. 根据权利要求1-9任一项所述的小激活核酸分子，其中所述小激活核酸分子包括至少一个修饰，优选地，所述修饰为化学修饰。
  11. 根据权利要求10所述的小激活核酸分子，其中所述化学修饰包括或选自如下修饰中的一种或多种：
    (1)对所述小激活核酸分子的核苷酸序列中连接核苷酸的磷酸二酯键的修饰；
    (2)对所述小激活核酸分子的核苷酸序列中的核糖的2’-OH的修饰；
    (3)对所述小激活核酸分子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。
  12. 根据权利要求10所述的小激活核酸分子，其中所述化学修饰包括或选自如下修饰中的一种或多种：2’-氟代修饰、2’-氧甲基修饰、2’-氧亚乙基甲氧基修饰、2，4’-二硝基苯酚修饰、锁核酸(LNA)、2’-氨基修饰、2’-脱氧修饰、5′-溴尿嘧啶修饰、5′-碘尿嘧啶修饰、N-甲基脲嘧啶修饰、2，6-二氨基嘌呤修饰、硫代磷酸修饰和硼烷化磷酸盐修饰。
  13. 编码权利要求1-9任一项所述的小激活核酸分子的核酸。
  14. 权利要求13所述的核酸，其中所述核酸是DNA分子。
  15. 包含权利要求1-12任一项所述的小激活核酸分子或权利要求13或14所述的核酸的细胞。
  16. 试剂盒，其包含权利要求1-12任一项所述的小激活核酸分子、权利要求13或14所述的核酸、或权利要求15所述的细胞。
  17. 权利要求1-12任一项所述的小激活核酸分子，其激活/上调HMBS基因表达至少10％。
  18. 权利要求1-12任一项所述的小激活核酸分子或权利要求13或14所述的核酸在制备用于在细胞中激活/上调HMBS基因表达的制剂中的应用。
  19. 根据权利要求18所述的应用，其中所述的小激活核酸分子被直接导入所述细胞中。
  20. 根据权利要求18所述的应用，其中所述小激活核酸分子是在权利要求13或14所述的核酸导入所述细胞后在细胞内产生的。
  21. 根据权利要求18-20任一项所述的应用，其中所述细胞是哺乳动物细胞。
  22. 根据权利要求21所述的应用，其中所述细胞是人类细胞。
  23. 根据权利要求22所述的应用，其中所述细胞存在于人体中。
  24. 一种分离的HMBS基因小激活核酸分子靶位点，其中所述靶位点包括或由选自SEQ ID NO：1-2的任一条序列上的任意连续16-35个核苷酸序列组成。
  25. 根据权利要求24所述的小激活核酸分子靶位点，其中所述靶位点包括或选自SEQ ID NO：11-29的任一核苷酸序列所示的序列。
  26. 在细胞中激活/上调HMBS基因的表达的方法，包括给所述细胞施用权利要求1-12中任一项所述的小激活核酸分子或权利要求13或14所述的核酸。
  27. 根据权利要求26所述的方法，其中所述的小激活核酸分子被直接导入所述细胞中。
  28. 根据权利要求27所述的方法，其中所述的小激活核酸分子是在权利要求13或14所述的核酸导入所述细胞后在细胞内产生的。
  29. 根据权利要求26-28任一项所述的方法，其中所述的细胞是哺乳动物细胞。
  30. 根据权利要求29所述的方法，其中所述的细胞是人类细胞。
  31. 根据权利要求30所述的方法，其中所述的细胞存在于人体中。
  32. 根据权利要求26所述的方法，其中给所述细胞施用终浓度为1-150nM的小激活核酸分子。
  33. 包含权利要求1-12任一项所述的小激活核酸分子、权利要求13或14所述的核酸或权利要求15所述的细胞在制备用于治疗对象中的急性间歇性卟啉症的药物中的应用。
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