WO2020135677A1 - 寡聚核酸分子及其应用 - Google Patents

寡聚核酸分子及其应用 Download PDF

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WO2020135677A1
WO2020135677A1 PCT/CN2019/129025 CN2019129025W WO2020135677A1 WO 2020135677 A1 WO2020135677 A1 WO 2020135677A1 CN 2019129025 W CN2019129025 W CN 2019129025W WO 2020135677 A1 WO2020135677 A1 WO 2020135677A1
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nucleic acid
acid molecule
small
strand
smn2
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PCT/CN2019/129025
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English (en)
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 EP19906151.6A priority Critical patent/EP3904517A4/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 US17/419,569 priority patent/US20220064642A1/en
Priority to MX2021007932A priority patent/MX2021007932A/es
Priority to JP2021538368A priority patent/JP2022515881A/ja
Priority to AU2019414608A priority patent/AU2019414608A1/en
Priority to KR1020217020377A priority patent/KR20210110310A/ko
Priority to CA3120534A priority patent/CA3120534A1/en
Priority to CN201980076095.6A priority patent/CN112996913B/zh
Priority to PE2021001108A priority patent/PE20212248A1/es
Priority to BR112021012422-5A priority patent/BR112021012422A2/pt
Priority to SG11202106327QA priority patent/SG11202106327QA/en
Publication of WO2020135677A1 publication Critical patent/WO2020135677A1/zh
Priority to IL284333A priority patent/IL284333A/en

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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C12N2320/30—Special therapeutic applications

Definitions

  • the present invention belongs to the technical field of nucleic acids, and in particular, relates to oligo nucleic acid molecules related to gene activation and uses thereof.
  • SMA Spinal muscular atrophy
  • SMA is an inherited neuromuscular disease, manifested by progressive skeletal and respiratory muscle weakness and atrophy, and is the first genetic disease that causes death in children under 2 years of age. According to clinical age and severity of the disease, it is divided into 4 types, from the heaviest SMA type I to the lightest SMA type IV (1). SMA is one of the most common childhood autosomal recessive genetic diseases, the incidence rate in live births is 1/11,000, and the frequency of adult carriers is as high as 1/67 to 1/40(2), China The population carrying rate is about 1/42 (3).
  • SMA is caused by mutations in the SMN1 (survival protein 1, motor neuron 1) gene, and patients with SMA carry highly homologous SMN2 genes with varying copy numbers (4; 5). Both SMN2 and SMN1 genes are located in the chromosome 5q13 region and encode the same protein called motor neuron survival protein (SMN). Compared with the SMN1 gene, the SMN2 gene has 11 nucleotide sites. Unlike SMN1, the other sequences are identical. Only one of the 11 nucleotides appears in the protein coding region of SMN2, that is, the C of nucleotide 6 of exon 7 is replaced by T (C6T), and this region is the splicing of the exon Enhancer region.
  • C6T the C6T
  • This substitution does not change the protein coding sequence, but it affects the effective splicing of exon 7 of SMN2, resulting in that most of the SMN2 precursor messenger RNA (pre-mRNA) misses exon 7 during splicing and translates into a large number Unstable mutant SMN protein (SMN ⁇ 7) and a small amount of normal functional full-length SMN protein (5; 6). Therefore, in SMA patients, the small amount of full-length SMN protein produced by SMN2 is not sufficient to compensate for SMN protein deletion caused by SMN1 mutations. Therefore, any method that can increase the expression of the full-length SMN protein from the SMN2 gene may become an effective method for the treatment of SMA.
  • pre-mRNA precursor messenger RNA
  • Small molecule compounds or oligonucleotides are used to alter the splicing of the SMN2 gene, thereby increasing the expression of full-length protein.
  • This splicing regulation strategy has been validated in animal SMA models and clinical trials (7-11).
  • the antisense oligonucleotide Spinraza was approved by the US FDA as the first small nucleic acid drug for the treatment of spinal muscular atrophy in children and adults in 2016.
  • regulating pre-mRNA splicing is an effective therapeutic strategy, it is limited by the amount of SMN2pre-mRNA available, that is, if the expression level of SMN2pre-mRNA itself is constant or low, the amount of SMN full-length protein produced under the influence of the drug is also limited.
  • Another strategy to increase SMN protein levels is to increase the transcription level of SMN2, which in turn increases SMN2 full-length protein expression.
  • small molecule histone deacetylase inhibitors histone deacetylase inhibitors, HDAC inhibitors
  • HDAC inhibitors histone deacetylase inhibitors
  • the present invention provides a small activation nucleic acid molecule based on the RNA activation process, such as a small activation RNA (saRNA) molecule, which activates/upregulates the transcription of SMN2 to increase the expression of full-length SMN protein to treat Diseases or conditions caused by SMN protein deficiency, such as spinal muscular atrophy.
  • saRNA small activation RNA
  • a small activation nucleic acid molecule such as a small activation RNA (saRNA) molecule, which activates or up-regulates the expression of SMN2 gene in a cell, one chain of the small activation nucleic acid molecule and the promoter of SMN2 gene
  • the fragment with a length of 16-35 nucleotides in the region has at least 75% homology or complementation.
  • the promoter region refers to 2000 nucleotides upstream of the start site of transcription, so as to achieve the expression of the gene Activate or increase.
  • one chain of the small activation nucleic acid molecule and the region from the transcription start site of -1639 to -1481 (SEQ ID NO: 476) and the region of -1090 to -1008 (SEQ ID NO) in the SMN2 gene promoter :477), -994 to -180 region (SEQ ID NO: 478) or -144 to -37 (SEQ ID NO: 479) consecutive fragments of 16-35 nucleotides in length have at least 75%, for example at least About 79%, about 80%, about 85%, about 90%, about 95%, or about 99% homology or complementarity.
  • one strand of the small activation nucleic acid molecule has at least 75%, such as at least about 79%, about 80%, about 85%, about any nucleotide sequence selected from SEQ ID NO:315-471 90%, about 95%, or about 99% homology or complementarity.
  • the small activation nucleic acid molecule includes a sense nucleic acid fragment and an antisense nucleic acid fragment, and the sense nucleic acid fragment and the antisense nucleic acid fragment contain complementary regions, and the complementary regions can form a double-stranded nucleic acid structure. Promote the expression of SMN2 gene in cells through RNA activation mechanism.
  • the sense nucleic acid fragment and the antisense nucleic acid fragment of the small activation nucleic acid may exist on two different nucleic acid strands, or may exist on the same nucleic acid strand.
  • the sense nucleic acid fragment and the antisense nucleic acid fragment are located on two strands respectively, at least one strand of the small activation nucleic acid has a 3'overhang of 0-6 nucleotides in length, preferably both strands have a length of 2 Or a 3'overhang of 3 nucleotides, the overhanging nucleotide is preferably dT.
  • the small activation nucleic acid molecule is a hairpin-type 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 length of the sense nucleic acid fragment and antisense nucleic acid fragment is 16-35 nucleotides, which can be 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 activation nucleic acid molecule of the present invention has at least 75%, such as at least about 79%, about 80%, about 85, with any nucleotide sequence selected from SEQ ID NO: 1-157 %, about 90%, about 95%, or about 99% homology, and its antisense strand has at least 75% of any nucleotide sequence selected from SEQ ID NO: 158-314, for example at least about 79 %, about 80%, about 85%, about 90%, about 95%, or about 99% homology.
  • the sense strand of the small activation nucleic acid molecule of the present invention includes any nucleotide sequence selected from SEQ ID NO: 1-157, or consists of any nucleotide sequence selected from SEQ ID NO: 1-157 , Or any nucleotide sequence selected from SEQ ID NO: 1-157;
  • the antisense strand of the small activation nucleic acid molecule of the present invention includes any nucleotide sequence selected from SEQ ID NO: 158-314, or It is composed of any nucleotide sequence selected from SEQ ID NO: 158-314, or is any nucleotide sequence selected from SEQ ID NO: 158-314.
  • nucleotides in the small activated nucleic acid molecule described herein may be natural or unchemically modified nucleotides, or at least one nucleotide may be a chemically modified nucleotide, the chemical modification is as follows One or a combination of modifications:
  • the chemical modification of the present invention is well known to those skilled in the art.
  • the modification of the phosphodiester bond refers to the modification of the oxygen in the phosphodiester bond, including phosphorothioate modification and boronated phosphate modification. Both modifications can stabilize the saRNA structure, maintaining the high specificity and high affinity of base pairing.
  • the ribose modification refers to the modification of 2'-OH in nucleotide pentose, that is, the introduction of certain substituents at the hydroxyl position of ribose, for example, 2'-fluoro modification, 2'-oxymethyl modification, 2'-oxyethylene methoxy modification, 2,4'-dinitrophenol modification, locked nucleic acid (LNA), 2'-amino modification, 2'-deoxy modification.
  • LNA locked nucleic acid
  • the base modification refers to the modification of the nucleotide base, for example, 5′-bromouracil modification, 5′-iodouracil modification, N-methyluracil modification, 2,6-diaminopurine Grooming.
  • lipophilic groups such as cholesterol can be introduced at the ends of the sense or antisense strands of small activated nucleic acid molecules to facilitate the passage of lipid bilayers
  • the formed cell membrane and nuclear membrane interact with the gene promoter region in the nucleus.
  • the small activation nucleic acid molecule provided by the present invention can effectively activate or up-regulate the expression of SMN2 gene in the cell after contact with the cell, and preferably the expression is up-regulated by at least 10%.
  • nucleic acid encoding the small activation nucleic acid molecule of the present invention.
  • the small nucleic acid molecule of the present invention is a small activation RNA (saRNA) molecule.
  • nucleic acid is a DNA molecule.
  • An aspect of the present invention provides a cell comprising the small activation nucleic acid molecule of the present invention or the nucleic acid encoding the small activation nucleic acid molecule of the present invention.
  • the cell is a mammalian cell, preferably a human cell.
  • the above-mentioned cells may be ex vivo, such as cell lines or cell lines, or may be present in mammals, such as humans.
  • compositions such as a pharmaceutical composition
  • a composition comprising the above-mentioned small activated nucleic acid molecule or a nucleic acid encoding the small activated nucleic acid molecule of the present invention and (optionally) a pharmaceutically acceptable Accepted carrier.
  • the pharmaceutically acceptable carrier includes an aqueous carrier, liposome, high molecular polymer or polypeptide.
  • the pharmaceutically acceptable carrier is selected from aqueous carriers, liposomes, high molecular polymers or polypeptides.
  • the aqueous carrier may be, for example, RNase-free water, or RNase-free buffer.
  • the composition may contain 1-150 nM, such as 1-100 nM, such as 1-50 nM, such as 1-20 nM, such as 10-100 nM, 10-50 nM, 20-50 nM, 20-100 nM, such as 50 nM of the above small activated nucleic acid molecules Or a nucleic acid encoding the small activation nucleic acid molecule of the present invention.
  • 1-150 nM such as 1-100 nM, such as 1-50 nM, such as 1-20 nM, such as 10-100 nM, 10-50 nM, 20-50 nM, 20-100 nM, such as 50 nM of the above small activated nucleic acid molecules Or a nucleic acid encoding the small activation nucleic acid molecule of the present invention.
  • 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, or the nucleic acid comprising the above small activation nucleic acid molecule or the nucleic acid encoding the small activation nucleic acid molecule described herein Use of the composition in preparing a preparation for activating/up-regulating expression of SMN2 gene in cells.
  • the present invention also relates to a method of activating/up-regulating the expression of the SMN2 gene in a cell, the method comprising administering to the cell the small activation nucleic acid molecule described above, a nucleic acid encoding the small activation nucleic acid molecule of the invention, or comprising the above A small activation nucleic acid molecule or a composition of nucleic acids encoding the small activation nucleic acid molecule of the present invention.
  • the small activation nucleic acid molecule, the nucleic acid encoding the small activation nucleic acid molecule of the present invention, or the composition containing the small activation nucleic acid molecule or the nucleic acid encoding the small activation nucleic acid molecule of the present invention may be directly introduced into a cell, or may be
  • the nucleotide sequence encoding the small activation nucleic acid molecule is produced in a cell after being introduced into the cell; the cell is preferably a mammalian cell, and more preferably a human cell.
  • the above-mentioned cells may be ex vivo, such as cell lines or cell lines, or may be present in mammals, such as humans.
  • the human body is a patient suffering from a disease or symptom caused by decreased expression of SMN full-length protein, mutation or deletion of SMN1 gene or insufficient expression of full-length protein, and/or insufficient expression of SMN2 full-length protein, and the small activation nucleic acid molecule,
  • the nucleic acid encoding the small activation nucleic acid molecule of the present invention or the composition containing the above-mentioned small activation nucleic acid molecule or the nucleic acid encoding the small activation nucleic acid molecule of the present invention is administered in a sufficient amount to achieve treatment.
  • the symptoms caused by lack of SMN full-length protein amount, mutation or deletion of SMN1 gene or insufficient expression of full-length protein, and/or insufficient expression of SMN2 full-length protein include, for example, spinal muscular atrophy.
  • the disease caused by insufficient expression of SMN full-length protein or mutation or deletion of SMN1 gene or insufficient expression of full-length protein is spinal muscular atrophy.
  • the spinal muscular atrophy according to the present invention includes SMA type I, SMA type II, SMA type III, and SMA type IV.
  • Another aspect of the present invention provides an isolated SMN2 gene small activation nucleic acid molecule action site, the site having any contiguous 16-35 nucleotide sequences on the promoter region of the SMN2 gene, preferably, said The action site is any consecutive 16-35 nucleotide sequence on any sequence selected from SEQ ID NO:476-479. Specifically, the action site includes or is selected from the sequence shown in any nucleotide sequence of SEQ ID NO:315-471.
  • Another aspect of the present invention relates to a method for treating a disease caused by insufficient expression of SMN full-length protein, mutation or deletion of SMN1 gene, and/or insufficient expression of SMN2 full-length protein in an individual, comprising administering to the individual a therapeutically effective amount of
  • the individual may be a mammal, such as a human.
  • the disease caused by insufficient SMN full-length protein expression or a mutation in the SMN1 gene may include, for example, spinal muscular atrophy.
  • the disease caused by insufficient SMN full-length protein expression, SMN1 gene mutation or deletion, and/or insufficient SMN2 full-length protein expression is spinal muscular atrophy.
  • the spinal muscular atrophy according to the present invention includes SMA type I, SMA type II, SMA type III, and SMA type IV.
  • Another aspect of the present invention relates to a small activated nucleic acid molecule of the present invention, a nucleic acid encoding the small activated nucleic acid molecule of the present invention, or a nucleic acid comprising the small activated nucleic acid molecule of the present invention or a nucleic acid encoding the small activated nucleic acid molecule of the present invention
  • the individual may be a mammal, such as a human.
  • the disease caused by insufficient SMN full-length protein expression, SMN1 gene mutation or deletion or insufficient full-length protein expression, and/or insufficient SMN2 full-length protein expression may include, for example, spinal muscular atrophy.
  • the disease caused by insufficient SMN full-length protein expression, SMN1 gene mutation or deletion or insufficient full-length protein expression, and/or insufficient SMN2 full-length protein expression is spinal muscular atrophy.
  • the spinal muscular atrophy according to the present invention includes SMA type I, SMA type II, SMA type III, and SMA type IV.
  • the small activation nucleic acid molecule for activating/up-regulating SMN2 gene expression provided by the present invention, can efficiently and specifically up-regulate the expression of SMN2 gene and increase the expression amount of full-length SMN2mRNA, while having lower toxicity Side effects can be used to prepare a medicament for treating a disease or condition caused by a condition related to insufficient expression of SMN full-length protein, mutation or deletion of SMN1 gene or insufficient expression of full-length protein, and/or insufficient expression of full-length protein of SMN2.
  • saRNA small activation RNA
  • Figure 1 is a schematic diagram of the SMN2 gene structure and the promoter region and primer design site for designing a small activation nucleic acid molecule with a length of 2 kb.
  • A shows the SMN2 gene structure and the promoter region of 2 kb in length used for designing saRNA.
  • B Design of RT-PCR primers used to amplify SMN2 mRNA.
  • SMN F1+SMN R1 are RT-qPCR primers used for high-throughput screening;
  • SMN F2+SMN R2 are RT-qPCR primers used for verification;
  • SMN-exon6-F+SMN-exon8-R are used for conventional RT-PCR Primers.
  • Figure 2 shows SMN mRNA expression changes mediated by small activated nucleic acid molecules.
  • Human embryonic kidney cells HEK293T were transfected with 980 small activating nucleic acid molecules targeting the SMN2 gene promoter, and 72 hours later, SMN mRNA expression was analyzed by one-step RT-qPCR.
  • the figure shows the SMN expression changes caused by the treatment of 980 small activated nucleic acid molecules relative to the blank control (Mock) from highest to lowest.
  • Figure 3 shows the hot spot area of small activated nucleic acid molecules on the SMN2 promoter.
  • 980 small activated nucleic acid molecules targeting the SMN2 promoter were transfected into HEK293T cells, and 72 hours later, SMN mRNA expression was analyzed by one-step RT-qPCR.
  • the figure shows that the change in SMN expression caused by each small activated nucleic acid molecule treated with the blank control (Mock) is ranked from -1949 to -37 according to the target position of the small activated nucleic acid molecule on the SMN2 promoter.
  • the illustration also includes the distribution of 4 hotspot areas (H1 ⁇ H4, rectangular frame). The numbers above indicate the boundaries of the hotspot region (relative to SMN2 transcription start site).
  • Figure 4 is a quantitative analysis of the expression of SMN genes activated by 50 randomly selected 50 saRNAs in HEK293T cells.
  • the y-axis is the change value of SMN mRNA expression change caused by each saRNA-treated sample after being corrected with the internal reference gene relative to the blank control (Mock) treatment.
  • dsCon2 and siSMN2-1 are irrelevant sequence double-stranded RNA control and SMN2 small interfering RNA control, respectively.
  • Figure 5 is a schematic diagram of SMN gene mRNA expression and DdeI restriction enzyme digestion to identify SMN PCR products.
  • A Schematic diagram of the difference between SMN1 gene and SMN2 gene. Due to a G ⁇ A mutation in exon 8 of the SMN2 gene, a Ddel cleavage site was generated. CDNA was amplified with primers SMN-exon6-F and SMN-exon8-R to obtain (B) the full-length SMN product (507bp) and/or the exon 7 skip deletion (SMN2 ⁇ 7) product (453bp) (C). In order to identify the source of these two products, the PCR products were digested with DdeI enzyme and gel electrophoresed.
  • the product from full-length SMN1 cannot be digested, the product from full-length SMN2 is digested into 392 bp and 115 bp (B), and the product from SMN2 ⁇ 7 is digested into 338 bp and 115 bp products (C).
  • Figure 6 shows the electrophoresis results of 50 saRNAs randomly selected to increase the expression of full-length SMN2 mRNA in HEK293T cells.
  • Control treatments included blank control (Mock), dsCon2, siSMN2-1 and vector-mediated overexpression (SMN-vector) (bands 51, 52, 53 and 54 respectively).
  • RNA was extracted with Qiagen RNeasy kit. After reverse transcription, conventional RT-PCR amplification was performed and HPRT1 was amplified as an internal control.
  • the SMN gene amplification product was digested with DdeI and then subjected to gel electrophoresis, and the band brightness was quantified.
  • HPRT1 amplification products were directly subjected to gel electrophoresis.
  • (A) is the gel electrophoresis diagram of SMN gene amplification product after DdeI digestion;
  • (B) is the gel electrophoresis diagram of HPRT1 amplification product;
  • FL full-length amplification product; SMN2 ⁇ 7: exon 7 skip deletion product, SMN2 partial: SMN2-specific digested fragment.
  • the black arrow marks the saRNA that can increase the ratio of the SMN2 full-length product to the exon 7 skip deletion product.
  • Figure 7 shows that 50 randomly selected saRNAs specifically increase total SMN2mRNA expression and increase full-length SMN2 expression.
  • Quantitative analysis of the brightness of the electrophoretic bands in Figure 6 shows the change of the total expression of SMN2mRNA (A), and the change of the ratio of the expression of SMN2 full-length mRNA to the expression of SMN2 ⁇ 7 (B), the value is the internal reference gene After the band brightness of HPRT1 is corrected, it is further normalized with the value of the blank control (Mock).
  • Figure 8 shows the relationship between the effect of saRNA to activate SMN expression and increase full-length SMN2mRNA and protein expression.
  • Two SMN2saRNA (RAG6-281 and RAG6-550) were selected and transfected into HEK293T cells at the indicated concentrations (1nM, 10nM, 20nM, 50nM, 100nM) for 72 hours. Collect cells to extract total RNA and perform reverse transcription and free protein for Western blot analysis.
  • A The relative expression level of SMN total mRNA detected by RT-qPCR method. Simultaneously amplify TBP and HPRT1 and use the geometric mean of the two as internal reference.
  • the value below the electropherogram (SMN2FL/ ⁇ 7) represents the amount of change in the ratio of SMN2 full length to SMN2 ⁇ 7 relative to the ratio of the blank control (Mock) treatment.
  • M blank transfection control
  • C dsCon2 irrelevant sequence double-stranded RNA control
  • FL full-length amplification product
  • SMN2 ⁇ 7 exon 7 skip deletion product.
  • Fig. 9 is the PCR identification result of the neonatal neonatal rat genome Smn1.
  • the source is Smn1 +/- , SMN2 -/- and Smn1 -/- , SMN2 +/+ gene-deficient mice are mated to breed neonatal suckling rats.
  • Neonatal suckling mice identified Smn1 as homozygous deletion or heterozygosity by genomic PCR.
  • the genotype of Smn1 homozygous deficient mice (SMA type I mice) is Smn1 -/- , SMN2 +/- .
  • the genotypes of Smn1 heterozygous mice normal control group) are Smn1 +/- and SMN2 +/- .
  • the PCR bands of SMA type I mice are 160bp; the Smn1 hybrid mouse (Het) PCR bands are 160bp and 180bp.
  • Figure 10 shows the exercise ability of SMA type I mice after SMN2-saRNA administration.
  • the obtained newborn mice were divided into four groups, namely normal control rats (Het), SMA type I control rats (untreated), in vivo-jetPEI coated SMN2-saRNA RAG6-539 (DS06-0013B, 1mg /mL) group and HKP loaded SMN2-saRNA RAG6-538 (DS06-0002B, 2mg/mL) group.
  • Newborn mice were administered by lateral ventricle injection (ICV) on the first day after birth, and the exercise ability of SMA type I mice was tested by the flip reflex test (righting time test) on the 7th or 8th day after administration .
  • IMV lateral ventricle injection
  • complementary refers to the ability of two oligonucleotide strands to form base pairs with each other. Base pairs are usually formed by hydrogen bonding between nucleotides in antiparallel oligonucleotide chains.
  • the complementary oligonucleotide strand can be base paired in a Watson-Crick manner (eg, AT, AU, CG), or in any other manner that allows the formation of duplexes (eg, Hoogsteen type or reverse Hoogsteen type base pairing) Base pairing.
  • Complementarity includes two situations of complete complementarity and incomplete complementarity.
  • Fully complementary or 100% complementary means that each nucleotide from the first oligonucleotide chain in the double-stranded region of the double-stranded oligonucleotide molecule can correspond to the nucleoside at the corresponding position of the second oligonucleotide chain
  • the acid forms hydrogen bonds without "mismatch”.
  • Incomplete complementarity refers to the situation where the nucleotide units of the two strands cannot all be hydrogen bonded to each other.
  • oligonucleotide strands with a double-stranded region of 20 nucleotides in length
  • the oligonucleotide strand exhibits a 10% Complementarity.
  • 18 base pairs on each strand can hydrogen bond to each other, the oligonucleotide strands exhibit 90% complementarity.
  • Substantial complementarity refers to at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, or 99% complementarity.
  • oligonucleotide refers to a polymer of nucleotides, and includes, but is not limited to, single-stranded or double-stranded molecules of DNA, RNA, or DNA/RNA hybrids, including alternating regularly and irregularly
  • the oligonucleotide used in the present invention to activate transcription of a target gene is a small activation nucleic acid molecule.
  • oligonucleotide strand and “oligonucleotide sequence” are interchangeable and refer to the general term for short-chain nucleotides of less than 35 bases (including DNA or RNA within ribonucleic acid) Nucleotides).
  • the length of the oligonucleotide chain may be any length of 16 to 35 nucleotides.
  • Gene refers to the entire nucleotide sequence required to encode a polypeptide chain or transcribe a functional RNA.
  • Gene may be a gene that is endogenous to the host cell or completely or partially recombined (for example, due to the introduction of an exogenous oligonucleotide encoding a promoter and a coding sequence or a heterologous promoter that will be adjacent to the endogenous coding sequence Into the host cell).
  • the term “gene” includes nucleic acid sequences that can consist of exons and introns.
  • a protein-encoding sequence is, for example, a sequence contained in an exon in an open reading frame between a start codon and a stop codon, as used herein, "gene” may refer to including, for example, a gene regulatory sequence such as a promoter , 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 a coding sequence or a non-coding sequence.
  • a “gene” may be used to describe a functional nucleic acid that contains regulatory sequences such as promoters or enhancers. The expression of the recombinant gene can be controlled by one or more heterologous regulatory sequences.
  • target gene may be a nucleic acid sequence, transgene, viral or bacterial sequence, chromosome or extrachromosomal and/or transient or stable transfection or incorporation into a cell and/or its chromatin that occurs naturally in an organism .
  • the target gene may be a protein-coding gene or a non-protein-coding gene (eg, microRNA gene, long-chain non-coding RNA gene).
  • the target gene usually contains a promoter sequence. Designing a small activation nucleic acid molecule with identity (also called homology) to the promoter sequence can achieve positive regulation of the target gene, which is manifested as an upregulation of the target gene expression.
  • Target gene promoter sequence refers to the non-coding sequence of the target gene, and in the present invention relates to "complementary to the target gene promoter sequence", the target gene promoter sequence refers to the coding strand of the sequence, also known as the non-template strand, That is, a nucleic acid sequence having the same sequence as the coding sequence of the gene.
  • target sequence refers to a sequence fragment to which the sense oligonucleotide chain or antisense oligonucleotide of the small activation nucleic acid molecule in the promoter sequence of the target gene is homologous or complementary.
  • sense strand and “sense oligonucleotide strand” are interchangeable, and the sense oligonucleotide strand of the small activation nucleic acid molecule refers to the promoter of the small activation nucleic acid molecule duplex containing the target gene
  • the coding strand of the sequence has the identity of the first nucleic acid strand.
  • antisense strand and “antisense oligonucleotide strand” are interchangeable, and the antisense oligonucleotide strand of the small activation nucleic acid molecule refers to the small activation nucleic acid molecule duplex and sense oligonucleotide A second nucleic acid strand complementary to the nucleotide chain.
  • coding strand refers to the DNA strand in the target gene that cannot be transcribed, and the nucleotide sequence of the strand is consistent with the sequence of the RNA produced by transcription (U is substituted for U in the RNA 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 coding strand of the target gene DNA.
  • template strand refers to the other strand of the double-stranded DNA of the target gene that is complementary to the coding strand, and that strand that can be transcribed into RNA as a template, which strand 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 RNA synthesis 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 exerts a regulatory effect on the transcription of a protein-coding or RNA-encoding nucleic acid sequence by its positional association.
  • eukaryotic gene promoters contain 100-5,000 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 may also be present in the exon and intron sequences.
  • transcription start site refers to a nucleotide that marks the start of transcription on the template strand of a gene.
  • the transcription start site may appear on the template chain of the promoter region.
  • a gene can have more than one transcription start site.
  • identity refers to a coding strand of a small activation RNA in which an oligonucleotide strand (sense strand or antisense strand) and a region of the promoter sequence of the target gene Or the similarity of the template chain.
  • the “identity” or “homology” may be at least about 75%, about 79%, about 80%, about 85%, about 90%, about 95%, or 99%.
  • overhang As used herein, the terms “overhang”, “overhang”, and “overhang” are interchangeable, and refer to the end of the oligonucleotide chain (5' or 3') non-base-paired nucleotide, which is extended beyond the double strand One of the strands within the oligonucleotide is produced by the other strand.
  • the single-stranded region extending beyond the 3'and/or 5'end of the duplex is called a protrusion.
  • gene activation or “activation gene” or “gene upregulation” or “upregulated gene” are interchangeable and refer to the measurement of gene transcription level, mRNA level, protein level, enzyme activity, methylation status, The chromatin state or configuration, level of translation, or its activity or state in a cell or biological system determines the increase in transcription, translation, or expression or activity of a nucleic acid. These activities or states can be measured directly or indirectly.
  • gene activation refers to an increase in activity related to nucleic acid sequences, regardless of the mechanism by which such activation occurs, for example, it plays a regulatory role as a regulatory sequence, It is transcribed into RNA, translated into protein and increases protein expression.
  • small activated RNA As used herein, the terms “small activated RNA”, “saRNA”, and “small activated nucleic acid molecule” are interchangeable, and refer to a nucleic acid molecule capable of promoting gene expression, and may be composed of a non-coding nucleic acid sequence (e.g., Sons, enhancers, etc.) a first nucleic acid fragment (antisense strand, also called antisense oligonucleotide strand) having a nucleotide sequence of sequence identity and a first nucleic acid fragment containing a nucleotide sequence complementary to the first nucleic acid fragment Two nucleic acid fragments (sense strand, also called sense strand or sense oligonucleotide strand), wherein the first nucleic acid fragment and the second nucleic acid fragment form a duplex.
  • a non-coding nucleic acid sequence e.g., Sons, enhancers, etc.
  • a first nucleic acid fragment antisense
  • the small activation nucleic acid molecule may also be composed of a single-stranded RNA molecule that is synthesized or expressed by a vector and can form a double-stranded region hairpin structure, where the first region contains a nucleotide sequence having 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 duplex region of the small activation nucleic acid molecule is generally about 10 to about 50 base pairs, about 12 to about 48 base pairs, about 14 to about 46 base pairs, about 16 to about 44 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 activation RNA” and "small activation 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 region of a gene promoter of at least 30 bp in length, in which a cluster of functional small activation nucleic acid molecule targets is exhibited, ie, small activation nucleic acid molecules targeting these hot spot regions are at least 30 % Can induce target gene mRNA expression to reach 1.2 times or more.
  • synthesis refers to the manner in which oligonucleotides are synthesized, including any means capable of synthesizing RNA, such as chemical synthesis, in vitro transcription, vector expression, and the like.
  • the expression of SMN2 gene is up-regulated by RNA activation, and related diseases are treated by increasing the expression of full-length SMN protein, especially spinal muscular atrophy.
  • the SMN2 gene is sometimes referred to as a target gene.
  • the preparation method of the small activated nucleic acid molecule provided by the present invention includes sequence design and sequence synthesis.
  • the synthesis of the small activated nucleic acid molecule sequence may use a chemical synthesis method, or entrust a biotechnology company specialized in nucleic acid synthesis.
  • the chemical synthesis method includes the following four processes: (1) synthesis of oligoribonucleotides; (2) deprotection; (3) purification and separation; (4) desalination and annealing.
  • the first cycle is connected to a base on the solid support, and then in the nth cycle (19 ⁇ n ⁇ 2), the One base is connected to the base connected in n-1 cycles, 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 with a concentration of 1 mol/ml, and then separated by high-pressure liquid chromatography reversed-phase C18 column to obtain purified saRNA single-stranded product.
  • the sense promoter sequence of the SMN2 gene from the transcription start site (TSS) to the upstream-2000 bp was obtained from the UCSC genome database (genome.ucsc.edu).
  • saRNA small activation RNA
  • Figure 1 a target of 19 bp in size was selected from -2000 bp upstream of the TSS.
  • a total of 1982 target sequences were obtained.
  • the target sequence is filtered, and the standard for retaining the target sequence is: 1) GC content is between 35% and 65%; 2) does not contain 5 or more than 5 consecutive identical nucleotides; 3) No more than 3 dinucleotide repeats; 4) No more than 3 trinucleotide repeats.
  • the remaining 980 target sequences are used as candidates to enter the screening process.
  • the corresponding double-stranded small activated RNA is chemically synthesized.
  • the length of the sense and antisense strands of the double-stranded small activated RNA used in this experiment are both 21 nucleotides, and 19 of the 5′ region of the first ribonucleic acid strand (sense strand) of the double-stranded saRNA
  • the nucleotide is 100% identical to the target sequence of the promoter, and its 3'end contains the dTdT sequence; 19 nucleotides in the 5'region of the second ribonucleic acid strand are complementary to the first ribonucleic acid strand sequence, its 3 The'end contains the dTdT sequence.
  • the two strands of the aforementioned double-stranded saRNA are mixed in the same amount of moles, and annealed to form a double-stranded s
  • HEK293T Human embryonic kidney cell line HEK293T ( CRL-3216 TM ) was cultured in DMEM medium (Gibco); all mediums contained 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco). The cells were cultured at 37°C under 5% CO2. HEK293T cells were seeded in 96-well plates at 5000 cells per well, using 0.3 ⁇ l RNAiMAX (Invitrogen, Carlsbad, CA) per well to reverse transfect a single saRNA into HEK293T cells at a final concentration of 10 nM (unless otherwise stated), The duration of transfection is 72 hours.
  • Control treatment includes blank control (Mock), non-specific oligonucleotide duplex (dsCon2, sense strand 5'-ACUACUGAGUGACAGUAGA[dT][dT]-3' (SEQ ID NO:472), antisense strand 5'- UCUACUGUCACUCAGUAGU[dT][dT]-3' (SEQ ID NO: 473)), SMN2 small interfering RNA (siMSN2-1, sense strand 5'-GGGAUGAUACAGCACUGAU[dT][dT]-3' (SEQ ID NO: 474) , Antisense strand 5'AUCAGUGCUGUAUCAUCCC[dT][dT]-3' (SEQ ID NO: 475)), where the blank control (Mock) treatment is a transfection treatment omitting nucleic acid.
  • dsCon2 non-specific oligonucleotide duplex
  • dsCon2 sense strand 5'-ACUACUGAGUGACAGUAGA
  • stage 1 reverse transcription reaction 42°C, 5 minutes; 95°C 10 seconds; stage 2 PCR reaction: 95°C 5 seconds, 60°C 20 seconds, and 45 cycles of amplification.
  • HPRT1 and TBP were used as internal reference genes.
  • the PCR primers used for SMN, HPRT1 and TBP are shown in Table 4 below, where SMN is amplified using SMN F1/R1 primer pairs.
  • 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 processed sample
  • CtR1m is the Ct value of the internal reference gene 1 from the blank control (Mock) processed sample
  • CtR1s is from the saRNA processing
  • 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.
  • HEK293T cells were transfected with the above 980 saRNAs at a transfection concentration of 10 nM. After 72 hours, the cells were lysed and subjected to one-step RT-qPCR analysis to obtain the relative SMN2 gene of each saRNA-treated sample ( Compared with the blank control (Mock treatment) expression value. As shown in Table 2, 157 (16.02%) and 416 (42.45%) saRNAs showed activation and inhibitory activity, respectively, and 407 (41.53%) saRNAs had no significant effect on SMN2 expression. The maximum activation amplitude is 1.82 times, and the maximum suppression amplitude is 0.33 times. These saRNAs with activation activity are called functional small activation nucleic acid molecules.
  • the active target sequence, sense sequence, antisense sequence and SMN relative expression data are shown in Table 3.
  • Figure 2 further shows the distribution of SMN2saRNA activity from highly activated to highly inhibited.
  • the activity of 980 saRNAs is arranged according to their position on the SMN2 promoter.
  • the distribution of functional saRNAs shows aggregation, that is, in some promoter regions, activated saRNAs gather in specific "hot spots ( hot spot” area ( Figure 3).
  • hot spot As shown in FIG. 3, in the promoter region of -1639 to -1481 (H1), region of -1090 to -1008 (H2), region of -994 to -180 (H3) and region of -144 to -37 (H4) Four hot spots appeared respectively, showing a high concentration of activating saRNA.
  • the analysis results show that the activated saRNA is not randomly distributed on the promoter, but there are specific hot spots.
  • Hotspot H1 -1639 to -1481) sequence (SEQ ID NO: 476):
  • Hotspot H2 (-1090 to -1008) sequence (SEQ ID NO: 477):
  • Hotspot H3 (-994 to -180) sequence (SEQ ID NO: 478):
  • Hotspot H4 (-144 to -37) sequence (SEQ ID NO: 479):
  • Example 3 Further screening and verification of functional saRNA capable of activating SMN gene
  • the applicant randomly selected 50 saRNAs from 157 active saRNAs to further verify that the saRNAs against SMN in HEK293T, HS27 and NHDF cells Activation effect of gene expression.
  • Transfect HEK293T cells, HS27 cells, and NHDF cells with saRNA (n 50, final concentration of 20 nM) shown in Table 5, cell culture as described in Example 2. After 72 hours, the cells were collected and RNA was extracted using Qiagen RNeasy kit.
  • FIG. 4 shows the activation effect of saRNA on SMN gene expression in HEK293T
  • Table 5 shows the activation effect of saRNA on SMN gene expression in HS27 and NHDF cells. From these results, it can be seen that the verified saRNA has different degrees of activation of SMN gene expression in different cells, and can be activated up to 19 times.
  • Table 5 50 saRNAs randomly selected for verification
  • RT-qPCR primers used above are not sufficient to distinguish the mRNA sequences of SMN2 and SMN1.
  • primer pairs SMN-exon6-F and SMN-exon8-R were used Amplify the cDNA from saRNA-treated cells, and then digest the PCR product with DdeI enzyme.
  • the full-length of the SMN2 gene and the mRNA with exon 7 deletion (SMN2 ⁇ 7) are judged by the brightness of the specific band.
  • the expression level The specific method is as follows: HEK293T cells are seeded in a 6-well plate at 2 to 3 ⁇ 10 5 cells/well, and the oligonucleotide duplex saRNA is transfected at a final concentration of 10 nM. Using the RNeasy Plus Mini kit (Qiagen), total cellular RNA was extracted according to its instructions.
  • RNA (1 ⁇ g) was reverse transcribed into cDNA using PrimeScript RT kit containing gDNA Eraser (Takara, Shlga, Japan), and the RT-PCR primers used were SMN-exon6-F and SMN-exon8-R.
  • the Takara (RR010A) reagent was used to perform ordinary PCR to amplify the resulting cDNA.
  • the reaction conditions were: 98°C for 10 seconds, 60°C for 15 seconds, and 72°C for 32 seconds.
  • the amplification cycle was 28 cycles. See Table 6 for details.
  • the PCR product is digested with DdeI to distinguish between SMN1 and SMN2, and then the digested product is separated by 2.5% agarose gel electrophoresis.
  • the intensity of each PCR product or digested digested band is determined by Image Lab ( BIO-RAD, Chemistry Doc tm MP imaging system), using the 500bp band of the Takara 100bp DNA ladder band (3407A) (5 ⁇ l loading contains ⁇ 150ng DNA) as a reference standard and then using a blank control (Mock) to perform the analysis See Table 7 for the enzyme digestion system and conditions. Taking HPRT1 as the internal reference gene, the primer sequences used are listed in Table 4.
  • SMN2 overexpression vector construction and transfection process used in this example are as follows:
  • Extract total cell RNA from HEK293T cells use reverse transcription of OligodT primers to obtain cDNA, use PCR to clone primers cSMN2-F2 (TAAGCA GGATCC ATG GGC ATG AGC AGC GGG GGG (SEQ ID NO:490)) and cSMN2-R2 (TAAGCA GAATTC TTA ATT TAA GGA ATG TGA GCA (SEQ ID NO: 491)) Amplify SMN2 full-length ORF to obtain PCR products.
  • the product was digested with BamHI and EcoRI enzymes.
  • the pcDNA3.1 plasmid (Invitrogen) was digested with the same enzyme.
  • the digested plasmid and PCR product were ligated with T4 ligase.
  • the ligation reaction product was used to transfect competent cells DH5 ⁇ , and the expanded cells were cultured overnight to extract plasmids using Qiagen Miniprep kit.
  • the resulting plasmid (1 ⁇ g) was transfected into HEK293T with Lipofectamine 3000 (Invitrogen). After 72 hours, cells were collected, total RNA was extracted, and RT-PCR and enzyme digestion experiments were performed.
  • Example 5 A dose-effect relationship study of saRNA activating SMN expression and increasing full-length SMN2 mRNA and protein expression
  • saRNAs (RAG6-281 and RAG6-550) were transfected into HEK293T cells at different concentrations (1nM, 10nM, 20nM, 50nM, 100nM), 72 hours later Cells were collected to extract RNA and protein, and RNA samples were obtained by reverse transcription to obtain cDNA, and then subjected to RT-qPCR and conventional RT-PCR amplification after DdeI digestion analysis; for protein samples, Western blot analysis was performed with SMN specific antibodies for detection SMN protein expression level.
  • the specific steps are as follows: collect the cells and lyse them with cell lysate (1 ⁇ RIPA buffer, Cell Signaling Technology (CST), Danvers, MA, USA, #9806). Protease inhibitor (Sigma, Lot#126M4015v) was added to the lysate. The protein samples were quantified by BCA method, and then separated by polyacrylamide gel electrophoresis. After the electrophoresis was completed, the protein samples were transferred to 0.45 ⁇ m PVDF membrane.
  • the blot was detected with mouse monoclonal anti-SMN (CST, #12976) or rabbit polyclonal anti- ⁇ / ⁇ -tubulin (tubulin) antibody (CST, #2148), and the secondary antibodies were anti-mouse IgG, HRP -Conjugated antibody (CST, #7076) or anti-rabbit IgG, HRP-conjugated antibody (CST, #7074). Scan the membrane with Image Lab to detect the signal.
  • RAG6-281 and RAG6-550 can significantly activate SMN mRNA expression by more than 1.5 times at a transfection concentration of 1 nM.
  • SMN expression was up-regulated respectively It reached 2.38 and 2.16 times, and when the concentration reached 100 nM, the expression of SMN did not increase further.
  • the applicant performed conventional PCR amplification on these cDNA samples and then digested with DdeI enzyme to perform gel electrophoresis on the digested products.
  • the transfection concentration of RAG6-281 at 1, 10, 20, 50 and 100 nM increased the ratio of SMN2 full-length mRNA to SMN2 ⁇ 7 mRNA by 1.9, 2.39, 2.41, 2.39 and 2.1-fold, respectively, while RAG6 -550 increased the ratio of SMN2 full-length mRNA to SMN2 ⁇ 7 mRNA at the same concentration by 1.52, 1.99, 1.91, 2.3, and 1.7 times, respectively.
  • the changes caused by both showed a dose-dependence in the transfection concentration range from 1 nM to 50 nM ( Figure 8B).
  • Example 6 SMA type I mice verify the improvement effect of saRNA on their exercise ability
  • Smn1 is Smn1 -/- , SMN2 +/- .
  • Smn1 heterozygous mice normal control group
  • SMN2-saRNA RAG6-539 (DS06-0013B) was dissolved in RNase-free water (Invitrogen, 2063810) to make a 5 mg/mL stock solution. Take 5 ⁇ L of DS06-0013B, 12.5 ⁇ L of 10% glucose solution (Polyplus-transfection, G181106) and 3.5 ⁇ L of RNase-free water and mix gently to prepare DS06-0013B working solution. The working solution was added to 4 ⁇ L of in vivo-jetPEI (Polyplus-transfection, 26031A1C) and mixed, incubated at room temperature for 15 minutes, and the final concentration was 1 mg/mL.
  • HKP preparation Dissolve SMN2-saRNA RAG6-538 (DS06-0002B) in RNase-free water to make a 4mg/mL stock solution. Take HKP (Suzhou Shengnuo Biomedical Technology Co., Ltd., AKF271/042-79-11) dissolved in RNase-free water to make a 16mg/mL stock solution. Quickly mix 7.5 ⁇ L of HKP stock solution with 7.5 ⁇ L of DS06-0002B stock solution, let stand at room temperature for 30 minutes, and the final concentration is 2 mg/mL.
  • mice The obtained newborn mice were divided into four groups, namely normal control group (Het), SMA type I control group (untreated), in vivo jetPEI-encapsulated SMN2-saRNA (DS06-0013B, 1 mg/mL) group and HKP contains SMN2-saRNA (DS06-0002B, 2mg/mL) group.
  • normal control group Het
  • SMA type I control group untreated
  • HKP contains SMN2-saRNA (DS06-0002B, 2mg/mL) group.
  • HKP contains SMN2-saRNA (DS06-0002B, 2mg/mL) group.
  • ICV lateral ventricle injection
  • the exercise ability of SMA type I mice was tested by flip reflex test.
  • the specific experiment is briefly described as follows: the mouse in the normal standing position is completely inverted, with its back touching the experimental table, with the limbs facing up, and then releasing the hand to start calculating the time for the mouse to fully return to the normal position, the time recording unit is seconds (s), this time is right-reflex time or righting time. If the mouse still can't flip to the normal posture within 60 seconds, the correction time is recorded as >60 seconds.
  • the centering time reflects the movement ability of the mouse. The shorter the centering time, the better the movement ability of the mouse.
  • the specific mouse centering time is shown in Table 9.
  • Figure 10 shows the exercise ability of SMA type I mice after SMN2-saRNA administration.
  • the correction time of normal control rats Het
  • the correction time of SMA type I control group untreated
  • 2 complete Loss of exercise capacity centering time> 60 seconds.
  • the correction time of the two groups of mice treated with RAG6-538 DS06-0002B-H group
  • DS06-0013B DS06-0013B-J group
  • the applicant screened the saRNA targeting the SMN promoter through high-throughput and discovered a number of saRNAs that can significantly activate SMN gene expression. These saRNAs not only upregulated the expression of the SMN2 gene in a dose-dependent manner, but also significantly increased the ratio of full-length SMN2 protein to SMN2 ⁇ 7 protein in the cell. At the same time, in vivo experiments proved that the saRNA of the present invention can significantly improve the exercise ability of SMA type I mice. These results clearly suggest that using saRNA targeting the MSN2 promoter to activate SMN2 transcription at the transcriptional level to increase full-length SMN protein expression is a promising strategy for treating SMA.

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Abstract

提供了用于治疗脊髓性肌萎缩症的小激活核酸分子及其应用。该小激活核酸分子包含正义核酸链和反义核酸链，正义核酸链和反义核酸链分别为长度为16-35个核苷酸的寡核苷酸链，并且其中一条核苷酸链与选自靶基因SMN2启动子区的靶点存在至少75％的碱基同源性或互补性。也提供了包含小激活核酸分子和可选地药用载体的药物组合物，以及使用该小激活核酸分子或包含小激活核酸分子的药物组合物上调靶基因在细胞中表达及治疗由靶基因表达不足引起的疾病的方法。

Description

寡聚核酸分子及其应用 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及其用途。
背景技术
脊髓性肌萎缩症(spinal muscular atrophy，SMA)是一种遗传性神经肌肉疾病，表现为进行性的骨骼肌和呼吸肌无力、萎缩，是导致2岁以下儿童死亡的首位遗传疾病。临床上根据发病年龄及病症严重程度分为4种类型，从最重的SMA I型到最轻的SMA IV型(1)。SMA为最常见的儿童期常染色体隐性遗传病之一，在活产婴儿中的发病率为1/11,000，而成人携带者的频率更是高达1/67至1/40(2)，中国人群携带率约为1/42(3)。
SMA是由于SMN1(运动神经元生存蛋白1，survival of motor neuron 1)基因突变引起的，同时SMA患者携带拷贝数不等的高度同源的SMN2基因(4；5)。SMN2和SMN1基因均位于染色体5q13区域，编码同一个被称为运动神经元生存蛋白(survival of motor neuron)(SMN)的蛋白质，与SMN1基因相比，SMN2基因有11个核苷酸位点有别于SMN1，其他序列完全相同。该11个核苷酸中只有一个核苷酸出现于SMN2的蛋白编码区，即7号外显子的第6位核苷酸的C被T替代(C6T)，而该区域为外显子的剪接增强子区。这一替代并不改变蛋白编码序列，但影响SMN2第7号外显子的有效拼接，导致大部分的SMN2的前体信使RNA(pre-mRNA)在拼接时遗漏掉7号外显子，翻译成大量不稳定的突变SMN蛋白(SMNΔ7)和少量正常功能的全长SMN蛋白(5；6)。因此，在SMA病人中，由SMN2产生的少量全长SMN蛋白的功能不足以代偿SMN1突变导致的SMN蛋白缺失。因此，任何能够增加来自SMN2基因的SMN全长蛋白表达的方法将有可能成为治疗SMA的有效方法。
采用小分子化合物或寡核苷酸来改变SMN2基因的剪接，从而增加全长蛋白的表达量，这一剪接调节策略已在动物SMA模型以及临床试验中得到验证(7-11)。其中，反义寡核苷酸Spinraza已于2016年被美国FDA批准为首个用于治疗儿童和成人脊髓性肌萎缩症的小核酸药物。尽管调节pre-mRNA剪接是个有效的治疗策略，但它受到SMN2pre-mRNA可用量的限制，即如果SMN2pre-mRNA本身的表达量恒定或者低下，在该药影响下产生的SMN全长蛋白的量也是有限的。另一个提高SMN蛋白水平的策略是提高SMN2的转录水平，进而增加SMN2全长蛋白的表达。有数据表明，小分子组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitor，HDAC抑制剂)通过抑制组蛋白去乙酰化酶能够激活SMN2基因，提高SMN2转录水平，从而产生更多的SMN2pre-mRNA，在SMA动物模型中也表现出良好的药效(12；13)，但在SMA病人中并未获得临床疗效，可能的原因是，小分子HDAC抑制剂在抑制组蛋白去乙酰化酶后导致很多基因的表达上调而不具备针对SMN2启动子的高特异性和高 活性(14；15)。本发明独辟蹊径，提供了一种通过靶向SMN2启动子而高度特异性激活SMN2转录的小激活核酸分子。
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例如小激活RNA(saRNA)分子，其通过激活/上调SMN2转录，提高全长SMN蛋白的表达量来治疗由全长SMN蛋白缺乏而导致的疾病或状况，如脊髓性肌萎缩症。
在本发明的一个一方面，提供了小激活核酸分子例如小激活RNA(saRNA)分子，其激活或者上调细胞中SMN2基因的表达，所述的小激活核酸分子的一条链与SMN2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％以上的同源或互补，启动子区是指包括转录起始位点上游的2000个核苷酸，从而实现所述基因表达的激活或者上调。具体地，所述小激活核酸分子的一条链与SMN2基因启动子中距转录起始位点-1639至-1481的区域(SEQ ID NO:476)、-1090至-1008的区域(SEQ ID NO:477)、-994至-180区域(SEQ ID NO:478)或-144至-37(SEQ ID NO:479)中的连续16-35个核苷酸长度的片段具有至少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。更具体地，所述小激活核酸分子的一条链与选自SEQ ID NO:315-471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％，或约99％的同源性或互补性。
在本发明中，所述小激活核酸分子包含正义核酸片段和反义核酸片段，所述正义核酸片段和反义核酸片段含有互补区域，互补区域能形成双链核酸结构，该双链结构核酸分子通过RNA激活机制促进SMN2基因在细胞中的表达。小激活核酸的正义核酸片段和反义核酸片段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。当正义核酸片段和反义核酸片段分别位于两条链上时，小激活核酸的至少一条链具有长度为0-6个核苷酸的3’突出端，优选情况下两条链具有长度为2或3个核苷酸的3’突出端，突出端的核苷酸优选为dT。当正义核酸片段和反义核酸片段存在于同一条核酸链上时，优选情况下该小激活核酸分子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。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为16-35个核苷酸，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。
在一个实施方案中，本发明的小激活核酸分子的正义链与选自SEQ ID NO:1-157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％，或约99％的同源性，并且其反义链与选自SEQ ID NO:158-314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％，或约99％的同源性。具体地，本发明的小激活核酸分子的正义链包括选自SEQ ID NO:1-157的任一核苷酸序列，或者由选自SEQ ID NO:1-157的任一核苷酸序列组成，或者是选自SEQ ID NO:1-157的任一核苷酸序列；本发明的小激活核酸分子的反义链包括选自SEQ ID NO:158-314的任一核苷酸序列，或者由选自SEQ ID NO:158-314的任一核苷酸序列组成，或者是选自SEQ ID NO:158-314的任一核苷酸序列。
本文所述小激活核酸分子中所有的核苷酸都可以为天然的或未经化学修饰的核苷酸，也 可以至少有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一种或其组合：
(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。
本发明的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，包括硫代磷酸修饰和硼烷化磷酸盐修饰。两种修饰都能稳定saRNA结构，保持碱基配对的高特异性和高亲和力。
所述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，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。
所述碱基修饰是指对核苷酸的碱基进行修饰，例如，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。
这些修饰可以增加所述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胆固醇等亲脂性的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SMN2基因的表达，优选情况下表达至少上调10％。
本发明的一个方面提供了编码本发明所述的小激活核酸分子的核酸。在一个实施方式中，本发明的小核酸分子为小激活RNA(saRNA)分子。在一个实施方式中，所述核酸是DNA分子。
本发明的一个方面提供了包含本发明所述的小激活核酸分子或编码本发明所述的小激活核酸分子的核酸的细胞。在一个实施方式中，所述细胞是哺乳动物细胞，优选地是人类细胞。上述细胞可以是离体的，如细胞系或细胞株等，也可以存在于哺乳动物体中，如人体中。
本发明的另一方面提供了一种组合物(如药物组合物)，该组合物包含上述小激活核酸分子或编码本发明所述的小激活核酸分子的核酸以及(任选地)药学上可接受的载体。在一个实施方案中，所述药学上可接受的载体包括水性载体、脂质体、高分子聚合物或多肽。在一个实施方案中，所述药学上可接受的载体选自水性载体、脂质体、高分子聚合物或多肽。在一个实施方式中，所述水性载体可以是，例如，无RNA酶的水，或无RNA酶的缓冲液。所述组合物可以含有1-150nM，例如1-100nM，例如1-50nM，例如1-20nM，例如10-100nM、10-50nM、20-50nM、20-100nM，例如50nM的上述小激活核酸分子或编码本发明所述的小激活核酸分子的核酸。
本发明的另一方面涉及上文所述的小激活核酸分子、编码本发明所述的小激活核酸分子的核酸或包含上述小激活核酸分子或编码本发明所述的小激活核酸分子的核酸的组合物在制备用于激活/上调SMN2基因在细胞中表达的制剂中的应用。
本发明还涉及激活/上调SMN2基因在细胞中的表达的方法，该方法包括给所述细胞施用上文所述的小激活核酸分子、编码本发明所述的小激活核酸分子的核酸或包含上述小激活核酸分子或编码本发明所述的小激活核酸分子的核酸的组合物。
上述小激活核酸分子、编码本发明所述的小激活核酸分子的核酸或包含上述小激活核酸分子或编码本发明所述的小激活核酸分子的核酸的组合物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苷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，也可以存在于哺乳动物体中，如人体中。该人体是患有由SMN全长蛋白表达减少、SMN1基因突变或缺失或全长蛋白表达不足、和/或SMN2全长蛋白表达不足引发的疾病或症状的患者，并且所述小激活核酸分子、编码本发明所述的小激活核酸分子的核酸或包含上述小激活核酸分子或编码本发明所述的小激活核酸分子的核酸的组合物被施以足够的量以实现对所述疾病或症状的治疗。具体情况下，所述由SMN全长蛋白量的缺乏、SMN1基因突变或缺失或全长蛋白表达不足、和/或SMN2全长蛋白表达不足引发的症状包括例如脊髓性肌萎缩症。在一个实施方案中，所述由SMN全长蛋白表达不足或SMN1基因突变或缺失或全长蛋白表达不足引发的疾病是脊髓性肌萎缩症。在一个实施方案中，本发明所述的脊髓性肌萎缩症包括SMA I型、SMA II型、SMA III型、和SMA IV型。
本发明另一方面提供了一种分离的SMN2基因小激活核酸分子作用位点，该位点具有SMN2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:476-479的任一条序列上任意连续16-35个核苷酸序列。具体地，所述作用位点包括或选自SEQ ID NO:315-471的任一核苷酸序列所示的序列。
本发明的另一方面涉及治疗个体中由SMN全长蛋白表达不足、SMN1基因突变或缺失、和/或SMN2全长蛋白表达不足引发的疾病的方法，包括给所述个体施用治疗有效量的上文所述的小激活核酸分子、编码本发明所述的小激活核酸分子的核酸或包含本发明的小激活核酸分子或编码本发明所述的小激活核酸分子的核酸的组合物。所述个体可以是哺乳动物，例如人。在一个实施方案中，所述由SMN全长蛋白表达不足或SMN1基因突变引发的疾病可以包括例如脊髓性肌萎缩症。在一个实施方案中，所述由SMN全长蛋白表达不足、SMN1基因突变或缺失、和/或SMN2全长蛋白表达不足引发的疾病是脊髓性肌萎缩症。在一个实施方案中，本发明所述的脊髓性肌萎缩症包括SMA I型、SMA II型、SMA III型、和SMA IV型。
本发明的另一方面涉及本发明的小激活核酸分子、编码本发明所述的小激活核酸分子的核酸或包含本发明的小激活核酸分子或编码本发明所述的小激活核酸分子的核酸的组合物在制备用于治疗由SMN全长蛋白表达不足、SMN1基因突变或缺失或全长蛋白表达不足、和/或SMN2全长蛋白表达不足引发的疾病或状况的药物中的应用。所述个体可以是哺乳动物，例如人。在一个实施方案中，所述由SMN全长蛋白表达不足、SMN1基因突变或缺失或全长蛋白表达不足、和/或SMN2全长蛋白表达不足引发的疾病可以包括例如脊髓性肌萎缩症。在一个实施方案中，所述由SMN全长蛋白表达不足、SMN1基因突变或缺失或全长蛋白表达不足、和/或SMN2全长蛋白表达不足引发的疾病是脊髓性肌萎缩症。在一个实施方案中，本发明所述的脊髓性肌萎缩症包括SMA I型、SMA II型、SMA III型、和SMA IV型。
本发明的有益效果
本发明提供的激活/上调SMN2基因表达的小激活核酸分子例如小激活RNA(saRNA)分子，其能够高效、特异地上调SMN2基因的表达并且增加全长SMN2mRNA的表达量，同时具有较低的毒副作用，可用于制备治疗与SMN全长蛋白表达不足相关的病症、SMN1基因突变或缺失或全长蛋白表达不足、和/或SMN2全长蛋白表达不足引发的疾病或状况的药物。
附图说明
图1为SMN2基因结构及用于设计小激活核酸分子的长度为2kb的启动子区域和引物设计位点示意图。(A)所示为SMN2基因结构及用于设计saRNA的长度为2kb的启动子区域。(B)为用于扩增SMN2mRNA的RT-PCR引物设计。SMN F1+SMN R1为高通量筛选所用RT-qPCR引物；SMN F2+SMN R2为用于验证的RT-qPCR引物；SMN-exon6-F+SMN-exon8-R为用于常规RT-PCR的引物。
图2为小激活核酸分子介导的SMN mRNA表达改变。用靶向SMN2基因启动子的980个小激活核酸分子分别转染人胚胎肾细胞HEK293T，72小时后用一步法RT-qPCR分析SMN的mRNA表达。图示为相对于空白对照(Mock)处理980个小激活核酸分子引起的SMN表达改变从最高到最低排序。
图3为小激活核酸分子在SMN2启动子上的热点区域。靶向SMN2启动子的980个小激活核酸分子分别转染HEK293T细胞，72小时后用一步法RT-qPCR分析SMN mRNA表达。图示为相对于空白对照(Mock)处理的每个小激活核酸分子所致的SMN表达改变按照该小激活核酸分子在SMN2启动子上的靶点位置从-1949到-37排序。图示还包括4个热点区域(H1～H4，长方形框)的分布。上方的数字表示热点区域的界限(相对于SMN2转录起始位点)。
图4为随机选择的50个saRNA在HEK293T细胞中激活SMN基因表达的定量分析。用所示saRNA(n＝50,终浓度为20nM)转染HEK293T细胞。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SMN进行qPCR扩增，同时扩增HPRT1和TBP基因并以其几何平均值作为内参。y轴为每个saRNA处理样本所致的SMN mRNA表达改变在用内参基因矫正后相对于空白对照(Mock)处理的改变值。dsCon2和siSMN2-1分别为无关序列双链RNA对照及SMN2小干扰RNA对照。
图5为SMN基因mRNA表达和DdeI限制性内切酶消化鉴定SMN PCR产物示意图。(A)SMN1基因与SMN2基因差异示意图。由于SMN2基因第8号外显子存在一个G→A的变异，而产生了一个Ddel的酶切位点。用引物SMN-exon6-F和SMN-exon8-R扩增cDNA得到(B)全长SMN产物(507bp)和/或外显子7跳跃缺失(SMN2Δ7)的产物(453bp)(C)。为了鉴别该两种产物的来源，将PCR产物用DdeI酶消化后凝胶电泳。来自全长SMN1产物不能被消化，来自全长SMN2的产物被消化成392bp和115bp(B)，而SMN2Δ7产物则会被消化成338bp和115bp的产物(C)。
图6为随机选择的50个saRNA在HEK293T细胞中增加全长SMN2mRNA表达的电泳结果。用所示saRNA(n＝50,终浓度为20nM)转染HEK293T细胞。对照处理包括空白对照 (Mock)、dsCon2、siSMN2-1及载体介导的过表达(SMN-vector)(分别为条带51、52、53和54)。72小时后收集细胞用Qiagen RNeasy试剂盒提取RNA，反转录后进行常规RT-PCR扩增，同时扩增HPRT1作为内参。SMN基因扩增产物用Dde I消化后进行凝胶电泳，并对条带亮度进行定量。HPRT1扩增产物直接进行凝胶电泳。(A)为SMN基因扩增产物在DdeI消化后的凝胶电泳图；(B)为HPRT1扩增产物的凝胶电泳图；(C)中列出了(A)和(B)中条带的样本名称。FL：全长扩增产物；SMN2Δ7：外显子7跳跃缺失产物，SMN2部分(partial)：SMN2特异消化片段。黑色箭头标记为能够增加SMN2全长产物与外显子7跳跃缺失产物比值的saRNA。
图7为随机选择的50个saRNA特异性增加总SMN2mRNA表达及增加全长SMN2表达。对图6中的电泳条带进行亮度定量分析，得出SMN2mRNA的总表达量的改变(A)，及SMN2全长mRNA表达量与SMN2Δ7表达量的比值的改变(B)，数值为用内参基因HPRT1的条带亮度矫正后进一步用空白对照(Mock)处理的值进行归一化处理。
图8为saRNA激活SMN表达及增加全长SMN2mRNA与蛋白表达的剂效关系。选取2个SMN2saRNA(RAG6-281和RAG6-550)用所示浓度(1nM，10nM，20nM,50nM,100nM)分别转染HEK293T细胞72小时。收集细胞提取总RNA后进行逆转录以及游离蛋白质进行Western印迹分析。(A)用RT-qPCR方法检测的SMN总mRNA相对表达水平。同时扩增TBP与HPRT1并用二者的几何平均值作为内参。(B)常规RT-PCR扩增MSN mRNA，对扩增产物用DdeI消化后进行凝胶电泳。同时扩增HPRT1作为内参基因，扩增产物直接进行凝胶电泳。电泳图下方的数值(SMN2FL/Δ7)代表SMN2全长与SMN2Δ7比值相对于空白对照(Mock)处理的比值的改变量。(C)免疫印迹分析检测SMN蛋白表达，同时检测α/β微管蛋白(tubulin)作为内参蛋白。M：空白转染对照；C：dsCon2无关序列双链RNA对照；FL：全长扩增产物；SMN2Δ7：外显子7跳跃缺失产物。
图9为新生乳鼠基因组Smn1的PCR鉴定结果。来源为Smn1 +/-，SMN2 -/-与Smn1 -/-，SMN2 +/+的基因缺陷小鼠交配繁殖新生乳鼠，新生乳鼠通过基因组PCR鉴定Smn1为纯合缺失或杂合。Smn1纯合缺陷小鼠(SMA I型小鼠)的基因型为Smn1 -/-，SMN2 +/-。Smn1杂合小鼠(正常对照组)的基因型为Smn1 +/-，SMN2 +/-。SMA I型小鼠PCR条带为160bp；Smn1杂合小鼠(Het)PCR条带为160bp和180bp。
图10显示了SMN2-saRNA给药后SMA I型小鼠的运动能力。将获得的新生小鼠分为四组，分别为正常对照组鼠(Het)、SMA I型对照组鼠(未治疗)、in vivo-jetPEI包载SMN2-saRNA RAG6-539(DS06-0013B，1mg/mL)组和HKP包载SMN2-saRNA RAG6-538(DS06-0002B，2mg/mL)组。新生小鼠出生后第1天通过侧脑室注射(ICV)给药，在给药后的第7或8天通过翻转反射实验(扶正时间(righting time)测试)检测SMA I型小鼠的运动能力。
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％或99％的互补。
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，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，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，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％或99％。
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”或“基因上调”或“上调基因”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”、“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。
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SMN2基因的表达，通过增加全长SMN蛋白的表达量来治疗相关疾病，尤其是脊髓型肌萎缩症。本发明中SMN2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。
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。
本研究发现，将上述saRNA导入细胞后，能够有效提高全长SMN2mRNA和蛋白的表达。
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。
实施例一 靶向SMN2启动子的小激活核酸分子设计与合成
从UCSC基因组数据库(genome.ucsc.edu)获得SMN2基因从转录起始位点(TSS)到上游-2000bp的正义启动子序列。
为了筛选能够激活SMN2基因表达的功能性小激活RNA(saRNA)，以长度为2000bp的SMN2的启动子序列为模板(图1)，从TSS上游-2000bp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1982个靶点序列。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％～65％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980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小激活RNA。其中，该实验中使用的双链小激活RNA的正义和反义链的长度均为21个核苷酸，所述双链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dTdT序列。将前述双链saRNA的两条链以同等量的摩尔数混合，退火后形成双链saRNA。
实施例二 靶向SMN2启动子的saRNA的高通量筛选
(1)细胞培养和转染
人胚胎肾细胞系HEK293T(
Figure PCTCN2019129025-appb-000001
CRL-3216 TM)培养在DMEM培养基(Gibco)中；所有培养基均含有10％胎牛血清(Gibco)和1％青霉素/链霉素(Gibco)。细胞在5％CO2，37℃条件下培养。HEK293T细胞以每孔5000个细胞接种在96孔板中，每孔使用0.3μl RNAiMAX(Invitrogen,Carlsbad,CA)以10nM的终浓度(除非另有说明)反向转染单个saRNA到HEK293T细胞中，转染持续时间为72小时。对照处理包括空白对照(Mock)、非特异性寡核苷酸双链体(dsCon2，正义链5’-ACUACUGAGUGACAGUAGA[dT][dT]-3’(SEQ ID NO:472)，反义链5’-UCUACUGUCACUCAGUAGU[dT][dT]-3’(SEQ ID NO:473))、SMN2小干扰RNA (siMSN2-1,正义链5’-GGGAUGAUACAGCACUGAU[dT][dT]-3’(SEQ ID NO:474)，反义链5’AUCAGUGCUGUAUCAUCCC[dT][dT]-3’(SEQ ID NO:475)),其中，空白对照(Mock)处理为省略核酸的转染处理。
(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100μl细胞裂解液(Power
Figure PCTCN2019129025-appb-000002
Green Cells-to-Ct TMKit,Life Technologies)，室温孵育5分钟。每孔取0.5μl细胞裂解液使用One Step TB Green TM PrimeScrip TM RT-PCR kit II(Takara,RR086A)以及Bravo Automated Liquid Handling Platform(Agilent)在Roche Lightcycler 480进行qPCR分析，每个样本重复3个复孔扩增，PCR反应条件见表1。
表1.PCR反应制备
Figure PCTCN2019129025-appb-000003
反应条件为阶段1反转录反应：42℃，5分钟；95℃10秒；阶段2PCR反应：95℃5秒，60℃20秒，扩增45个循环。以HPRT1及TBP为内参基因。SMN、HPRT1及TBP所用PCR引物见下表4，其中SMN用SMN F1/R1引物对扩增。
为了计算某个saRNA转染样本的SMN2(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为来自空白对照(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SMN2转录的saRNA，用上述980个saRNA分别转染HEK293T细胞，转染浓度为10nM，72小时后裂解细胞进行一步法RT-qPCR分析得到每个saRNA处理样本的SMN2基因的相对(与空白对照(Mock)处理比较)表达值。如表2所示，分别有157个(16.02％)和416个(42.45％)saRNA显示出激活和抑制活性，407个(41.53％)saRNA对SMN2的表达不产生明显影响。激活的最大幅度为1.82倍，最大抑制幅度为0.33倍。这些具有激活活性的saRNA被称为功能性小激活核酸分子。其活性靶点序列、正义序列、反义序 列以及SMN相对表达数据显示于表3。
表2.SMN2saRNA的高通量筛选结果统计
Figure PCTCN2019129025-appb-000004
Figure PCTCN2019129025-appb-000005
Figure PCTCN2019129025-appb-000006
Figure PCTCN2019129025-appb-000007
Figure PCTCN2019129025-appb-000008
Figure PCTCN2019129025-appb-000009
Figure PCTCN2019129025-appb-000010
Figure PCTCN2019129025-appb-000011
Figure PCTCN2019129025-appb-000012
Figure PCTCN2019129025-appb-000013
Figure PCTCN2019129025-appb-000014
表4.RT-qPCR分析的引物序列
Figure PCTCN2019129025-appb-000015
图2进一步展示了SMN2saRNA从高度激活到高度抑制的活性分布。同时，对980个saRNA的活性按照其在SMN2启动子上的位置排列，很明显地看到功能性saRNA的分布呈现聚集现象，即在有些启动子区域，激活性saRNA聚集在特定的“热点(hot spot)”区域(图3)。如图3所示，在启动子的-1639至-1481的区域(H1)、-1090至-1008的区域(H2)、-994至-180的区域(H3)和-144至-37的区域(H4)分别出现4个热点区域，表现为激活性saRNA的高度聚集。该分析结果表明激活性saRNA在启动子上并非随机分布，而是存在特定的热点区域。
热点H1(-1639至-1481)序列(SEQ ID NO:476)：
Figure PCTCN2019129025-appb-000016
热点H2(-1090至-1008)序列(SEQ ID NO:477)：
Figure PCTCN2019129025-appb-000017
热点H3(-994至-180)序列(SEQ ID NO:478)：
Figure PCTCN2019129025-appb-000018
Figure PCTCN2019129025-appb-000019
热点H4(-144至-37)序列(SEQ ID NO:479)：
Figure PCTCN2019129025-appb-000020
实施例三 能够激活SMN基因的功能性saRNA的进一步筛选和验证
为了进一步筛选和验证能够激活SMN基因的功能性saRNA，基于高通量筛选结果，申请人从157个具有激活活性的saRNA中随机选择50个saRNA进一步验证saRNA在HEK293T、HS27和NHDF细胞中对SMN基因表达的激活效果。用表5所示saRNA(n＝50,终浓度为20nM)分别转染HEK293T细胞、HS27细胞和NHDF细胞，细胞培养如实施例二所述。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SMN进行qPCR扩增，同时扩增HPRT1和TBP基因并以其几何平均值作为内参。图4显示了saRNA在HEK293T中对SMN基因表达的激活效果，表5中显示了saRNA在HS27和NHDF细胞中对SMN基因表达的激活效果。从这些结果可以看出，所验证的saRNA在不同细胞中对SMN基因表达均具有不同程度的激活作用，最高可激活19倍。
表5随机选择用于验证的50个saRNA
Figure PCTCN2019129025-appb-000021
Figure PCTCN2019129025-appb-000022
实施例四 RT-PCR及酶切法检测SMN2的表达
由于SMN2基因与SMN1基因的高度相似性，上述所用RT-qPCR引物不足以区分SMN2与SMN1的mRNA序列。为了特异性检测SMN2mRNA在saRNA处理后的表达改变，以及SMN2第7号外显子在mRNA前体拼接成成熟mRNA的过程中是否被剪切，用引物对SMN-exon6-F和SMN-exon8-R扩增来自saRNA处理细胞的cDNA，然后用DdeI酶消化PCR产物，对消化产物进行凝胶电泳后通过特定条带的亮度来判断SMN2基因的全长及有外显子7缺失(SMN2Δ7)的mRNA表达水平。具体方法如下：HEK293T细胞以2～3×10 5细胞/孔接种在6孔板中，以10nM的终浓度反向转染寡核苷酸双链体saRNA。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，日本)的PrimeScript RT试剂盒将RNA(1μg)反转录为cDNA，所用RT-PCR引物为SMN-exon6-F和SMN-exon8-R。采用Takara(RR010A)试剂进行普通PCR扩增所得cDNA，反应条件为：98℃ 10秒，60℃ 15秒，72℃ 32秒扩增28个循环具体见表6。PCR完成后将PCR产物用DdeI进行酶切用于区分SMN1和SMN2，随后将酶切产物用2.5％的琼脂糖凝 胶电泳进行分离，每个PCR产物或者酶切消化条带强度用Image Lab(BIO-RAD,Chemistry Doc tm MP成像系统)进行分析，以Takara 100bp DNA梯状条带(3407A)的500bp条带(5μl加载量含有～150ng DNA)为参考标准然后用空白对照(Mock)进行归一化，酶切体系及条件见表7。以HPRT1为内参基因，所用引物序列列于表4。
本实施例中所用的SMN2过表达载体构建及转染过程如下：
从HEK293T细胞提取细胞总RNA，用OligodT引物进行逆转录获得cDNA，用PCR克隆引物cSMN2-F2(TAAGCA GGATCC ATG GCG ATG AGC AGC GGC GGC(SEQ ID NO:490))及cSMN2-R2(TAAGCA GAATTC TTA ATT TAA GGA ATG TGA GCA(SEQ ID NO:491))扩增SMN2全长ORF获得PCR产物。产物用BamHI和EcoRI酶消化。用同样的酶消化pcDNA3.1质粒(Invitrogen)。用T4连接酶连接消化的质粒与PCR产物。用连接反应产物转染感受态细胞DH5α，过夜培养扩增细胞后用Qiagen Miniprep试剂盒提取质粒。所得质粒(1μg)用Lipofectamine 3000(Invitrogen)转染进入HEK293T，72小时后收集细胞，提取总RNA，进行RT-PCR及酶切实验。
表6 RT-PCR体系及条件
Figure PCTCN2019129025-appb-000023
表7 DdeI酶切反应体系及反应条件
内切体系组分(NEB,R0175L) 体积(μl)
限制性内切酶DdeI 1
cDNA 8
10×NEB缓冲液 1
总反应体积 10
孵育温度 37℃
孵育时间 1h
根据图6和图7A所示，与空白对照(Mock)和对照寡核苷酸双链体(dsCON2)处理(条带51-52)相比，所有saRNA处理的细胞均展现出SMN2基因的总mRNA表达量明显增加，最高达2.49倍。同时，大部分(28个，56％)saRNA导致了SMN2基因的全长mRNA与外显子7缺失的mRNA表达之比值增加(图6黑色箭头所示，图7B)。
实施例五 saRNA激活SMN表达及增加全长SMN2 mRNA和蛋白表达的量效关系研究
为了确定saRNA处理与SMN激活的剂量-效应关系，选取2个saRNA(RAG6-281和RAG6-550)以不同的浓度(1nM，10nM,20nM，50nM，100nM)分别转染HEK293T细胞，72小时后收集细胞提取RNA和蛋白，RNA样本通过反转录获得cDNA，然后分别进行RT-qPCR及常规RT-PCR扩增后DdeI酶切分析；对于蛋白质样本，用SMN特异性抗体进行蛋白质印迹分析，检测SMN蛋白表达水平。具体步骤如下：收集细胞，用细胞裂解液(1×RIPA缓冲液,Cell Signaling Technology(CST)，Danvers,MA，USA，#9806)裂解。裂解液里加蛋白酶抑制剂(Sigma,Lot#126M4015v)。BCA法定量蛋白质样品，随后进行聚丙烯酰胺凝胶电泳分离,电泳完成后将蛋白样品转移到0.45μm的PVDF膜。用小鼠单克隆抗SMN(CST，#12976)或兔多克隆抗α/β-tubulin(微管蛋白)抗体(CST，#2148)对印迹进行检测，二抗分别用抗小鼠IgG,HRP-连接的抗体(CST，#7076)或抗兔IgG,HRP-连接的抗体(CST,#7074)。用Image Lab扫膜检测信号。
如图8A所示，RAG6-281和RAG6-550在转染浓度为1nM即能显著激活SMN mRNA表达达到1.5倍以上，在浓度为50nM时，它们对SMN的激活效果达到顶峰，分别上调SMN表达达到2.38和2.16倍，而浓度达到100nM时，SMN的表达并没有进一步增加。同时，申请人对这些cDNA样本进行常规PCR扩增后用DdeI酶消化，对消化产物进行凝胶电泳。结果提示RAG6-281及RAG6-550均上调了SMN1及SMN2的mRNA表达，与RT-qPCR的结果一致。通过定量分析SMN2全长条带与SMN2Δ7条带的比率，发现RAG6-281及RAG6-550在所有浓度均显著增加了SMN2全长mRNA的表达(图8B)。与空白对照(Mock)处理比较，RAG6-281在1，10，20，50和100nM的转染浓度分别增加SMN2全长mRNA与SMN2Δ7mRNA的比率为1.9，2.39，2.41，2.39和2.1倍；而RAG6-550在同样浓度增加SMN2全长mRNA与SMN2Δ7mRNA的比率分别为1.52，1.99，1.91，2.3和1.7倍。二者引起的改变在转染浓度从1nM到50nM范围内均表现出剂量依赖性(图8B)。进一步蛋白质印迹分析SMN蛋白表达的改变与SMN RT-qPCR的结果高度一致，表现为RAG6-281和RAG6-550均以浓度依赖的方式，显著上调SMN全长蛋白的表达(图8C)。但未能够检测到第7号外显子缺乏的SMN蛋白质(SMN2Δ7)条带，原因可能是SMN2Δ7蛋白质高度不稳定而不能被蛋白质印迹法检出，与以前文献报道的结果一致(Hua et al,PLoS Biol 2007；5(4)e73)。
实施例六.SMA I型小鼠体内验证saRNA对其运动能力的改善效果
(1)SMA I型小鼠模型的繁殖及鉴定
通过来源为Smn1 +/-，SMN2 -/-与Smn1 -/-，SMN2 +/+的基因缺陷小鼠交配以繁殖新生乳鼠(由北京瑞希罕见病基因治疗技术研究所提供)，新生乳鼠通过基因组PCR鉴定Smn1为纯合缺失或杂合，具体鉴定结果见图9。Smn1纯合缺陷小鼠(SMA I型小鼠)的基因型为Smn1 -/-，SMN2 +/-。Smn1杂合小鼠(正常对照组)的基因型为Smn1 +/-，SMN2 +/-。
(2)制备体内jetPEI和HKP包载的寡核苷酸
制备体内jetPEI制剂：将SMN2-saRNA RAG6-539(DS06-0013B)溶解于无RNase水(Invitrogen，2063810)中制成5mg/mL的储备液。取5μL DS06-0013B与12.5μL 10％葡萄糖溶液(Polyplus-transfection，G181106)和3.5μL无RNase水温和混匀，制备成DS06-0013B工作液。将该工作液加入到4μL体内-jetPEI(Polyplus-transfection，26031A1C)混匀，室温孵育15分钟，终浓度为1mg/mL。
制备HKP制剂：将SMN2-saRNA RAG6-538(DS06-0002B)溶解于无RNase水中制成4mg/mL的储备液。取HKP(苏州圣诺生物医药技术有限公司，AKF271/042-79-11)于无RNase水中溶解，制成16mg/mL的储备液。将7.5μL HKP储备液与7.5μL DS06-0002B储备液快速混合，室温放置30分钟，终浓度为2mg/mL。
(3)SMA I型小鼠侧脑室注射体内jetPEI和HKP包载的寡核苷酸
将获得的新生小鼠分为四组，分别为正常对照组鼠(Het)、SMA I型对照组鼠(未治疗)、体内jetPEI包载SMN2-saRNA(DS06-0013B，1mg/mL)组和HKP包载SMN2-saRNA(DS06-0002B，2mg/mL)组。在新生小鼠出生的第1天通过侧脑室注射(ICV)给药，注射体积为5μL，具体的给药途径、注射体积及给药时间点见表8。
表8.saRNA给药及对照组设置
Figure PCTCN2019129025-appb-000024
(4)检测SMA I型小鼠的运动能力
在给药后的第7或8天通过翻转反射实验检测SMA I型小鼠的运动能力。具体实验简述如下：将保持正常站立姿势的小鼠完全反转放置，使其背部接触实验台面，四肢朝上，然后松开手开始计算小鼠完全恢复正常姿势的时间，时间记录单位为秒(s)，这个时间即为翻转反射时间(Righting-reflex time)或扶正时间。小鼠在60秒内仍然不能翻转至正常姿势则记录扶正时间为>60秒。扶正时间反应了小鼠的运动能力，扶正时间越短，小鼠的运动能力越好，具体的小鼠扶正时间见表9。
表9.扶正时间测试
Figure PCTCN2019129025-appb-000025
Figure PCTCN2019129025-appb-000026
图10所示为SMN2-saRNA给药后SMA I型小鼠的运动能力。如表9中小鼠的扶正时间所示，正常对照组鼠(Het)的扶正时间均至2秒之内，而SMA I型对照组鼠(未治疗)扶正时间均大于12秒，有2只完全失去运动能力(扶正时间>60秒)。经RAG6-538(DS06-0002B-H组)和DS06-0013B(DS06-0013B-J组)治疗的两组小鼠的扶正时间接近正常鼠，尤其是DS06-0002B-H组。与SMA I型对照组鼠比较，DS06-0002B-H组小鼠的扶正时间缩短了近10倍，DS06-0013B-J组小鼠的扶正时间缩短了2.5倍(表9，图10)。上述结果显示，在SMN2-saRNA给药后，SMA I型小鼠的运动能力可明显改善。这提示saRNA治疗能够延缓疾病发生时间。
综合上述结果，申请人通过高通量筛选靶向SMN启动子的saRNA，发现了多个能够显著激活SMN基因表达的saRNA。这些saRNA不仅以剂量依赖方式上调SMN2基因的表达，而且显著增加细胞内全长SMN2蛋白与SMN2Δ7蛋白的比例。同时体内实验证明，本发明的saRNA可明显改善SMA I型小鼠的运动能力。这些结果明确提示用靶向MSN2启动子的saRNA在转录水平激活SMN2转录来增加全长SMN蛋白的表达是具有很好前景的治疗SMA的策略。
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Claims (41)

  1. 一种小激活核酸分子，所述小激活核酸分子的一条链与SMN2基因启动子中距转录起始位点-1639至-1481的区域(SEQ ID NO:476)、-1090至-1008的区域(SEQ ID NO:477)、-994至-180区域(SEQ ID NO:478)或-144至-37(SEQ ID NO:479)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。
  2. 根据权利要求1所述的小激活核酸分子，其特征在于包含正义核酸链和反义核酸链，所述正义核酸链和所述反义核酸链含有互补区域，互补区域能形成双链核酸结构，该双链核酸能够激活SMN2基因在细胞中的表达。
  3. 根据权利要求2所述的小激活核酸分子，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. 根据权利要求2所述的小激活核酸分子，其中所述正义核酸片段和反义核酸片段存在于同一条核酸链上，优选地是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。
  5. 根据权利要求3所述的小激活核酸分子，其特征在于至少一条链具有长度为0至6个核苷酸的3’突出端。
  6. 根据权利要求5所述的小激活核酸分子，其特征在于两条链都具有长度为2-3个核苷酸的3’突出端。
  7. 根据权利要求2-6任一项所述的小激活核酸分子，其特征在于正义核酸链和反义核酸链的长度分别为16至35个核苷酸。
  8. 根据权利要求1所述的小激活核酸分子，其特征在于所述小激活核酸分子的一条链与选自SEQ ID NO:315-471的任一核苷酸序列具有至少75％的同源性或互补性。
  9. 根据权利要求8所述的小激活核酸分子，其特征在于其正义链与选自SEQ ID NO:1-157的任一核苷酸序列具有至少75％的同源性，并且其反义链与选自SEQ ID NO:158-314的任一核苷酸序列具有至少75％的同源性。
  10. 权利要求9所述的小激活核酸分子，其特征在于其正义链包括选自SEQ ID NO:1-157的任一核苷酸序列，或选自SEQ ID NO:1-157的任一核苷酸序列，并且其反义链包括选自SEQ ID NO:158-314的任一核苷酸序列，或选自SEQ ID NO:158-314的任一核苷酸。
  11. 根据权利要求1-10任一项所述的小激活核酸分子，其中至少一个核苷酸为化学修饰的核苷酸。
  12. 根据权利要求11所述的小激活核酸分子，其中所述化学修饰为如下修饰中的至少一种：
    (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。
  13. 权利要求1-12任一项所述的小激活核酸分子，其激活/上调SMN2基因表达至少10％。
  14. 编码权利要求1-10任一项所述的小激活核酸分子的核酸。
  15. 权利要求14所述的核酸，其特征在于所述核酸是DNA分子。
  16. 包含权利要求1-13任一项所述的小激活核酸分子或权利要求14或15所述的核酸的细胞。
  17. 包含权利要求1-13任一项所述的小激活核酸分子或权利要求14或15所述的核酸，和任选地药学上可接受的载体的组合物。
  18. 权利要求17所述的组合物，其特征在于所述药学上可接受的载体包括或选自水性载体、脂质体、高分子聚合物或多肽。
  19. 权利要求17或18所述的组合物，其中所述组合物含有1-150nM的所述小激活核酸分子。
  20. 治疗个体中由SMN蛋白表达不足、SMN1基因突变或缺失或全长蛋白表达不足、和/或SMN2全长蛋白表达不足引发的疾病或状况的方法，包括给所述个体施用治疗有效量的权利要求1-13任一项所述的小激活核酸分子、权利要求14-15中任一项所述的核酸、或权利要求17-19中任一项所述的组合物。
  21. 权利要求20的方法，其中所述疾病或状况包括遗传性神经肌肉疾病，优选地是脊髓性肌萎缩症。
  22. 权利要求20或21所述的方法，其中所述个体是哺乳动物，优选地是人。
  23. 权利要求1-13任一项所述的小激活核酸分子、权利要求14-15中任一项所述的核酸、或权利要求17-19中任一项所述的组合物在制备用于治疗个体中由SMN蛋白表达不足、SMN1基因突变或缺失或全长蛋白表达不足、和/或SMN2全长蛋白表达不足引发的疾病或状况的药物中的应用。
  24. 权利要求23的应用，其中所述疾病或状况包括遗传性神经肌肉疾病，优选地是脊髓性肌萎缩症。
  25. 权利要求23或24所述的应用，其中所述个体是哺乳动物，优选地是人。
  26. 权利要求1-13的任一项的小激活核酸分子、权利要求14-15中任一项所述的核酸、或权利要求17-19中任一项所述的组合物在制备用于激活/上调SMN2基因在细胞中表达的制剂 中的应用。
  27. 根据权利要求26所述的应用，其中所述的小激活核酸分子、权利要求14-15中任一项所述的核酸、或权利要求17-19中任一项所述的组合物被直接导入所述细胞中。
  28. 根据权利要求26所述的应用，其中所述的小激活核酸分子是在编码该小激活核酸分子的核苷酸序列导入所述细胞后在细胞内产生的。
  29. 根据权利要求26-28的任一项所述的应用，其中所述的细胞是哺乳动物细胞，优选地，所述的细胞是人类细胞。
  30. 根据权利要求29所述的应用，其中所述的细胞存在于人体中。
  31. 根据权利要求30所述的应用，其中所述的人体是患有由SMN蛋白表达不足、SMN1基因突变或缺失或全长蛋白表达不足、和/或SMN2全长蛋白表达不足引发的症状的患者，并且所述小激活核酸分子、所述核酸、或所述组合物被施用足够的量以实现对所述症状的治疗。
  32. 根据权利要求31所述的应用，其中所述由SMN全长蛋白表达缺乏引发的症状包括遗传性神经肌肉疾病，优选地是脊髓性肌萎缩症。
  33. 一种分离的SMN2基因小激活核酸分子靶位点，其中所述靶位点为选自SEQ ID NO:476-479的任一条序列上的任意连续16-35个核苷酸序列。
  34. 根据权利要求33所述的小激活核酸分子靶位点，其中所述靶位点如选自SEQ ID NO:315-471的任一核苷酸序列所示。
  35. 激活/上调SMN2基因在细胞中的表达的方法，该方法包括给所述细胞施用权利要求1-13中任一项所述的小激活核酸分子、权利要求14-15中任一项所述的核酸、或权利要求17-19中任一项所述的组合物。
  36. 根据权利要求35所述的方法，其中所述的小激活核酸分子、权利要求14-15中任一项所述的核酸、或权利要求17-19中任一项所述的组合物被直接导入所述细胞中。
  37. 根据权利要求35所述的方法，其中所述的小激活核酸分子是在编码该小激活核酸分子的核苷酸序列导入所述细胞后在细胞内产生的。
  38. 根据权利要求35-37的任一项所述的方法，其中所述的细胞是哺乳动物细胞，优选是人类细胞。
  39. 根据权利要求38所述的方法，其中所述的细胞存在于人体中。
  40. 根据权利要求39所述的方法，其中所述的人体是患有由SMN全长蛋白表达不足、SMN1基因突变或缺失或全长蛋白表达不足、和/或SMN2全长蛋白表达不足引发的症状的 患者，并且所述小激活核酸分子、所述核酸、或所述组合物被施用足够的量以实现对所述症状的治疗。
  41. 根据权利要求40所述的方法，其中所述由SMN全长蛋白表达缺乏引发的症状包括遗传性神经肌肉疾病，优选地是脊髓性肌萎缩症。
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