WO2024060205A1 - 包含基于小分子药物的可变剪接调节元件的核酸分子 - Google Patents
包含基于小分子药物的可变剪接调节元件的核酸分子 Download PDFInfo
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Definitions
- the present invention relates generally to the fields of molecular biology and medicine, and specifically to a nucleic acid molecule containing an alternative splicing regulatory element based on a small molecule drug.
- Gene therapy is the transfer of exogenous genetic material into target cells through various methods. For example, it can be achieved by introducing normal genes to replace missing or abnormally mutated genes, or inhibiting the function of abnormal endogenous genes. Intervene in cellular physiological functions at a high level and ultimately be used to treat specific diseases (Liu Jian, Research Progress of Nucleic Acid Drugs and Non-Viral Delivery Vectors in Gene Therapy, Basic Medicine and Clinical Medicine, Vol. 42, Issue 1, No. 41 -50 pages, January 2022).
- the research focus of gene therapy in the past 20 years has mainly focused on gene delivery systems.
- the adeno-associated virus (AAV) capsid has been engineered to improve its targeting (Ravindra Kumar et al., Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types.Nat Methods 17,541–550(2020)), there is little attention and research on the delivered therapeutic genes themselves, mainly focusing on promoter engineering, riboswitches, and limiting expression of specific cell types 3' adjustment element etc.
- the expression level of the therapeutic gene When the therapeutic gene is delivered to the target tissue or cell through the delivery system, the expression level of the therapeutic gene will become an important factor affecting the effect of gene therapy. If the expression level of the therapeutic gene is insufficient or not expressed, the expected therapeutic effect cannot be obtained; on the other hand, if the expression level of the therapeutic gene is too high, that is, overexpression, it is likely to cause unexpected toxic side effects to the patient. How to regulate the expression level of the therapeutic gene delivered to the body is an aspect that must be considered in the gene therapy program. In 2021, Monteys, AM and other researchers reported a method of regulating gene therapy through drug-induced splicing, which finely controls protein translation by constructing a drug-inducible switch X on system.
- This system controls the variable splicing of RNA and can achieve the inclusion or exclusion of specific exons in mature mRNA.
- the X on system does not require the use of exogenous regulatory elements, but instead uses the drug LMI070, which is bioavailable to the human body after oral administration, to induce the splicing of exons containing start codons into mature mRNAs, thereby finely regulating gene expression (Monteys, AM et al., Regulated control of gene therapies by drug-induced splicing. Nature 596, 291–295 (2021)).
- WO2020033473A1 discloses alternative splicing regulation and treatment methods of gene expression, which uses a chimeric transactivator minigene, in which the alternative splicing of the minigene determines whether the transactivator is expressed, and the expression of the transactivator can cause the promoter Transcription of the target gene under sequence control, or providing a chimeric target gene minigene, in which alternative splicing of the minigene directly determines whether the target gene is expressed.
- WO2021163556A1 provides a chimeric minigene, in which the alternative splicing of the minigene determines whether the encoding gene is expressed, and the minigene is alternatively spliced in response to a splicing-modulating drug, such that the encoding gene is only expressed in the presence of the splicing-regulating drug.
- WO2021014428A1 discloses a regulatable expression system, and specifically provides a composition including a minigene with a splicing regulator binding sequence, a composition that can be used for regulatable gene expression, and a system and use method thereof.
- the alternative splicing regulatory elements reported in the existing literature are derived from cells cultured in vitro, rather than organs in the body, and lack adaptation and optimization to the physiological and pathological environment in the body; (3) The application of existing elements lacks organ specificity and is not conducive to target The targeted nature of gene therapy when cells or tissues are present in specific organs, and the potential for toxic side effects to occur in undesirable organs.
- expanding the selection of alternative splicing-modulating small molecule drugs is also beneficial in terms of treatment selection.
- a nucleic acid molecule comprising an alternative splicing regulatory element and a target gene located at the 3' end of the alternative splicing regulatory element; wherein the alternative splicing regulatory element is from 5
- the 'end to the 3' end includes the first exon, the first intron, the pseudo exon, the second intron, and the second exon in sequence;
- the sequence of the pseudo exon is as SEQ ID No: As shown in 1, and the sequence at the junction of the pseudo exon and the second intron is AGAGTA, where AGA belongs to the pseudo exon and GTA belongs to the second intron;
- the first The sequence of the exon is SEQ ID No: 2 or contains SEQ ID No: 2 or has at least 95% (preferably 96%, 97%, 98%, 99%, or 99.5%) identity with SEQ ID No: 2
- the sequence of the first exon is TAG, and the sequence of the first intron is TAG; the sequence of the first intron is
- sequence of the variable splicing regulatory element is SEQ ID No: 6 or comprises SEQ ID No: 6.
- nucleic acid sequence between the 3' end of the alternative splicing regulatory element and the 5' end of the target gene.
- the number of bases of the sequence encoding the protease cleavage site between the 3' end of the alternative splicing regulatory element and the 5' end of the target gene should be 3n, where n is an integer greater than or equal to 1, to keep the reading frame of the target gene from being changed or moved.
- a promoter sequence is operably linked to the 5' end of the alternative splicing regulatory element.
- the promoter sequence is a mammalian cell constitutive promoter.
- the promoter is CMV, CAG, CBG, EF1a, PGK1 or Ubc.
- the promoter sequence is a mammalian cell-specific promoter.
- the promoter is Tnnt2 (cardiomyocyte), Nppa (atrial cardiomyocyte), Myl2 (ventricular cardiomyocyte), Mck (skeletal muscle cell), Nkx2.5 (cardiac progenitor cell), Syn (neuron), Mecp2 (neurons), TBG (hepatocytes), Pdx1 (pancreatic cells), K14 (skin keratinocytes), Rpe65 (retinal cells) or SP-C (pulmonary epithelial cells).
- the promoter sequence is a mammalian non-coding RNA promoter.
- the promoter is U6 or H1.
- the promoter sequence is a prokaryotic promoter.
- the promoter is T7, T3, or SP6.
- the nucleic acid molecule of the invention further comprises a post-transcriptional regulatory element.
- the post-transcriptional regulatory elements comprise genes derived from hepatitis B (HPRE), bat (BPRE), ground squirrel (GSPRE), arctic squirrel (ASPRE), duck (DPRE), chimpanzee (CPRE) and long PRE of woolly monkey (WMPRE) or woodchuck (WPRE), optionally wherein the post-transcriptional regulatory element is provided at the 3' end of the gene of interest.
- the nucleic acid molecule of the invention further comprises a polyadenylation signal (polyA), optionally wherein the polyA is disposed at the 3' end of the gene of interest.
- polyA signal is SV40 polyA, human growth hormone (HGH) polyA, or bovine growth hormone (BGH) polyA, ⁇ -globin polyA, ⁇ -globin polyA, ovalbumin polyA, kappa-light chain polyA, and synthesize polyA.
- the alternative splicing regulatory element can be combined with an alternative splicing regulatory small molecule drug.
- the alternative splicing regulating small molecule drug is LMI070, or a derivative having the same binding site with LMI070 on the alternative splicing regulating element, or a pharmaceutically acceptable salt of LMI070, or the variable
- the splicing regulating small molecule drug is Risdiplam, or a derivative having the same binding site with Risdiplam on the alternative splicing regulating element, or a pharmaceutically acceptable salt of Risdiplam.
- the gene encoding the protein is a gene encoding a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a Cas9, Cas12, Cas13 protein or a functional derivative thereof, and/ or genes encoding therapeutic proteins.
- TALEN transcription activator-like effector nuclease
- ZFN zinc finger nuclease
- Cas9, Cas12, Cas13 protein or a functional derivative thereof and/ or genes encoding therapeutic proteins.
- the gene encoding RNA is a gene encoding miRNA, shRNA, or lncRNA.
- the transcript of the nucleic acid molecule according to the present invention in the presence of an alternative splicing regulating small molecule drug is provided; in another embodiment, the nucleic acid molecule according to the present invention is provided in the presence of an alternative splicing modulating small molecule drug.
- the alternative splicing-regulating small molecule drug is LMI070, or a derivative with the same binding site as LMI070 on the alternative splicing regulatory element, or LMI070 is pharmaceutically available Acceptable salts; or the alternative splicing regulating small molecule drug is Risdiplam, or a derivative having the same binding site with Risdiplam on the alternative splicing regulating element, or a pharmaceutically acceptable salt of Risdiplam.
- the nucleic acid molecule is a DNA molecule and the transcript is an RNA molecule.
- the invention provides a vector comprising a nucleic acid molecule according to the invention or a transcript according to the invention.
- the vector is a DNA or RNA vector.
- the vector is a circular vector. More preferably, the vector is a plasmid.
- the vector is double-stranded or single-stranded; preferably, the vector is double-stranded.
- the vector is a viral vector.
- the viral vector is an adeno-associated virus (AAV) vector, a chimeric AAV vector, an adenoviral vector, a retroviral vector, a lentiviral vector, a DNA viral vector, a herpes simplex virus vector, a baculovirus vector, or the like. any mutants or derivatives.
- the viral vector is a recombinant AAV vector, a self-complementing AAV (scAAV) vector, or a single-stranded AAV (ssAAV) vector.
- the recombinant AAV vector includes one or more inverted terminal repeats (ITRs), optionally wherein the ITR is an AAV2 ITR, optionally wherein the AAV vector includes two ITRs.
- ITRs inverted terminal repeats
- the invention provides a recombinant virus comprising the nucleic acid molecule or transcript according to the first aspect of the invention, or the vector according to the second aspect of the invention.
- the recombinant virus is an adeno-associated virus (AAV), chimeric AAV, adenovirus, retrovirus, lentivirus, DNA virus, herpes simplex virus, baculovirus, or any mutant or derivative thereof things.
- AAV adeno-associated virus
- chimeric AAV adenovirus
- retrovirus retrovirus
- lentivirus DNA virus
- herpes simplex virus baculovirus
- baculovirus baculovirus
- the AAV includes one or more of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh36, AAVrh37, AAV- DJ, AAV-DJ/8, AAV.Anc80, AAV.Anc80L65, AAV-PHP.B, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.A, AAV-PHP.eB, AAV-PHP. S, AAV2i8, MyoAAV, AAVMYO capsid serotypes, or variants thereof, such as a combination of capsids from more than one AAV serotype.
- the AAV is AAV9.
- the invention provides a cell comprising one or more nucleic acid molecules or transcripts according to the first aspect of the invention, or one or more nucleic acid molecules or transcripts according to the first aspect of the invention.
- the vector of the second aspect may comprise one or more recombinant viruses according to the third aspect of the present invention.
- the cells are human cells.
- the human cells are heart cells, muscle cells, neurons, liver cells, lung cells, or kidney cells.
- the expression level of the target gene is higher than the expression level of the target gene when the alternative splicing regulating small molecule drug is not present in the cell.
- the level is 1-100 times higher, such as 1-80 times, 1-70 times, 1-60 times, 1-50 times, 1-40 times, 1-30 times, 1-20 times, 2-10 times, 2 -5 times; optionally, when the alternative splicing modulating small molecule drug is not present in the cell, the expression level of the target gene cannot be detected.
- a pharmaceutical composition comprising one or more nucleic acid molecules or transcripts according to the first aspect of the present invention, or one or more nucleic acid molecules or transcripts according to the first aspect of the present invention.
- a method for regulating the expression of a target gene based on alternative splicing-regulated small molecule drugs includes: making one or more nucleic acids according to the first aspect of the present invention Molecule or transcript, or one or more vectors according to the second aspect of the invention, or one or more recombinant viruses according to the third aspect of the invention, or one or more vectors according to the third aspect of the invention.
- the cells described in the fourth aspect are in contact with one or more alternative splicing-regulating small molecule drugs, wherein when the alternative splicing-regulating small molecule drug is present, the expression level of the target gene is higher than when the alternative splicing is not present.
- the target gene expression level when regulating small molecule drugs is 1-100 times higher, such as 1-80 times, 1-70 times, 1-60 times, 1-50 times, 1-40 times, 1-30 times, 1- 20 times, 2-10 times, 2-5 times; optionally, when the alternative splicing regulating small molecule drug is not present, the expression level of the target gene cannot be detected.
- a gene therapy method for treating a subject in need comprising administering to the subject one or more nucleic acid molecules or transcripts according to the first aspect of the present invention, or one or more vectors according to the second aspect of the present invention, or one or more recombinant viruses according to the third aspect of the present invention, or one or more cells according to the fourth aspect of the present invention, or one or more pharmaceutical compositions according to the fifth aspect of the present invention, and one or more alternative splicing regulating small molecule drugs; wherein, in the presence of alternative splicing regulating small molecule drugs, the expression level of the target gene is 1-100 times higher than the expression level of the target gene in the absence of the alternative splicing regulating small molecule drugs, for example, 1-80 times, 1-70 times, 1-60 times, 1-50 times, 1-40 times, 1-30 times, 1-20 times, 2-10 times, 2-5 times higher; optionally, when the alternative splicing regulating small molecule drugs are not present
- kits comprising: (1) one or more nucleic acid molecules or transcripts according to the first aspect of the present invention, or one or more A vector according to the second aspect of the present invention, or one or more recombinant viruses according to the third aspect of the present invention, or one or more cells according to the fourth aspect of the present invention, or one or A variety of pharmaceutical compositions according to the fifth aspect of the present invention; and (2) one or more alternative splicing modulating small molecule drugs.
- the ninth aspect of the present invention there is provided the use of a substance for regulating the expression level of a target gene.
- the substance is a nucleic acid molecule or transcript according to the first aspect of the present invention, or a method according to the second aspect of the present invention.
- a substance for preparing a gene therapy drug for treating a subject in need the substance being a nucleic acid molecule or transcript according to the first aspect of the present invention, or The vector according to the second aspect of the present invention, or the recombinant virus according to the third aspect of the present invention, or the cell according to the fourth aspect of the present invention, or the pharmaceutical composition according to the fifth aspect of the present invention, and alternative splicing modulating small molecule drugs.
- a method for detecting the nucleic acid molecule or transcript according to the first aspect of the present invention, or the vector according to the second aspect of the present invention, or the recombinant virus according to the third aspect of the present invention, or the cell according to the fourth aspect of the present invention, or the pharmaceutical composition according to the fifth aspect of the present invention is provided, wherein the method uses PCR, and the sequence shown in SEQ ID NO:7 is used as the upstream primer in the PCR.
- the alternative splicing regulating small molecule drug is a small molecule drug capable of binding the junction sequence AGAGTA of the pseudo exon and the second intron, and the The alternative splicing regulating small molecule drug has the following effects: when the alternative splicing regulating small molecule drug is present, the mature mRNA generated by the nucleic acid molecule according to the first aspect of the present invention contains the pseudo exon; when it does not exist When the alternative splicing regulates small molecule drugs, the pseudo exon is not included in the mature mRNA generated from the nucleic acid molecule according to the first aspect of the present invention.
- variable splicing regulating small molecule drug is LMI070, or a derivative having the same binding site as LMI070 on the variable splicing regulatory element, or a pharmaceutically acceptable salt of LMI070, or the variable splicing regulating small molecule drug is Risdiplam, or a derivative having the same binding site as Risdiplam on the variable splicing regulatory element, or a pharmaceutically acceptable salt of Risdiplam.
- the technical solution of the present invention has good technical effects, which are at least reflected in: (1) the number of base pairs of the alternative splicing regulatory elements contained in the nucleic acid molecules provided by the present invention is more Small, only 417 bp. Compared with existing elements such as the X on system element (560 bp) reported by Monteys, AM, etc., its size is reduced by more than 25%.
- the alternative splicing regulatory elements contained in the nucleic acid molecules provided by the invention are derived from organs in the body, and are more suitable for and adaptable to the physiological and pathological environment in the body; (3) the alternative splicing regulatory elements contained in the nucleic acid molecules provided by the invention are combined with specific initiation can achieve targeted gene therapy for specific organs and reduce the possibility of toxic side effects in undesired organs; (4) the alternative splicing regulatory elements contained in the nucleic acid molecules provided by the invention can be used in a wider range Modulation of alternative splicing modulation by small molecule drugs, including regulation by Risdiplam, which has been approved for marketing, has a wider range of applications and advantages in treatment selection.
- Figure 1 shows the discovery of alternatively spliced fragments regulated by LMI070 in mouse hearts.
- A Experimental flow chart. After 48 hours of oral administration of 10 mg/kg LMI070 to wild-type adult C57BL/6 mice, ventricular tissue was taken for full transcriptome sequencing and quantitative analysis using CASH software to discover differentially spliced DNA elements.
- B Sashimi diagram of Stradb site. Arcs and numbers mark the sequencing sequence across exons, and black arrows mark false exon sites.
- Figure 2 shows engineering of the Stradb locus and in vitro validation of the DM st element.
- A The engineering strategy of the Stradb site is to retain the sequences required for splicing in the Stradb gene sequence, delete other sequences, set a unique start codon ATG on the pseudo exon, and remove other exons and introns. ATG in the intron sequence.
- B Construct a GFP-luciferase reporter plasmid containing the DM st element, transfect it into HEK293T cells, and observe the GFP signal after 24 hours of LMI070 treatment.
- C Use a microplate reader to quantitatively analyze the GFP signal.
- PC positive control
- PC refers to the positive control, which is the component in the X on system reported by Monteys, AM, etc.
- D DM st -GFP-luciferase reporter plasmid was transfected into HEK293T cells and treated with 1 ⁇ M LMI070, RG7800 or Risdiplam for 24 hours to observe the GFP signal.
- E Quantitative analysis of the GFP signal in Figure 2D using a microplate reader.
- Figure 3 shows the in vivo validation and evaluation of DM st element-controlled AAV vectors.
- A Design of AAV reporter gene vector controlled by DM st element and flow chart of mouse experiment.
- B Western Blot analysis of GFP-Luci. expression level 2 days after LMI070 administration.
- PC positive control
- C RT-PCR analysis of pseudoexon splicing changes 2 and 4 days after LMI070 administration, dpi, days post injection, refers to the number of days after injection.
- D In vivo imaging analysis of luciferase activity in small animals. The same mouse was treated with the same LMI070 twice: the first time by intraperitoneal injection (IP injection) and the second time by gavage feeding.
- IP injection intraperitoneal injection
- Figure 4 shows in vivo validation of cardiomyocyte-specific AAV-DM st vectors.
- A Schematic diagram of cardiomyocyte-specific AAV-DM st vector and in vivo verification experiments.
- B Quantitative analysis of RNA splicing changes in DM st by qPCR in myocardial tissue.
- C GFP fluorescence signal in myocardial tissue.
- D GFP signal of other tissues after AAV-DM st -GFP+LMI070 treatment. cTnnt2, chicken Tnnt2 promoter; hTnnt2, human Tnnt2 promoter.
- polynucleotide and “nucleic acid” are used interchangeably herein and refer to a polymeric form of nucleotides of any length. They may include one or more ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or DNA containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non- Polymers of natural or derivatized nucleotide bases, such as locked nucleic acids (LNA) and peptide nucleic acids (PNA).
- LNA locked nucleic acids
- PNA peptide nucleic acids
- polypeptide refers to compounds consisting of amino acid residues covalently linked by peptide bonds.
- a protein or peptide typically contains at least two amino acids or amino acid variants, and there is no limit to the maximum number of amino acids that may be included in a protein or peptide sequence.
- Polypeptides include any peptide or protein containing two or more amino acids or variants linked to each other by peptide bonds. These terms include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of a polypeptide, modified polypeptides, derivatives, analogs, fusion proteins, and the like.
- Polypeptides include natural peptides, recombinant peptides, or combinations thereof.
- sequence identity and “homology” are used interchangeably herein when used to describe a polynucleotide or polypeptide sequence to mean that when two sequences of a polypeptide or polynucleotide are compared or aligned, they are identical. The percentage of bases or amino acids that are in the same relative position. Sequence identity can be determined in a number of different ways. For example, sequences can be aligned using various methods and computer programs (eg, BLAST, T-COFFEE, MUSCLE, MAFFT, etc.).
- the nucleic acid molecule described in the present invention may, for example, be an isolated nucleic acid molecule.
- isolated when used with respect to a nucleic acid molecule or protein molecule refers to a nucleic acid or protein that has been separated from one or more components with which it is normally found in the natural environment. Separation may include detachment from a larger nucleic acid (eg, from a gene or chromosome) or from other proteins or molecules with which the nucleic acid or protein normally comes into contact. The terms encompass but do not require a complete separation.
- operably linked refers to a functional relationship between two or more polynucleotide (eg, DNA) segments.
- the term refers to the functional relationship of a transcriptional regulatory sequence to the sequence to be transcribed.
- a promoter or enhancer sequence is operably linked to a coding sequence if the promoter or enhancer sequence stimulates or modulates the transcription of the coding sequence, such as in a suitable host cell or other expression system.
- promoter transcriptional regulatory sequences operably linked to a sequence are contiguous with the sequence or separated by short spacer sequences, ie, they are cis-acting.
- some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequence for which they enhance transcription.
- the nucleic acid molecule provided by the present invention includes an alternative splicing regulatory element and a target gene encoding a target molecule (eg, a target protein), wherein the target gene is operably connected to the 3' end of the alternative splicing regulatory element.
- a target molecule eg, a target protein
- connection between the gene of interest and the alternative splicing regulatory element may be a tight connection, that is, there is no nucleic acid sequence between the gene of interest and the alternative splicing regulatory element;
- the connection between the gene of interest and the alternative splicing regulatory element may also be non-tightly connected, that is, there are other nucleic acid sequences between the gene of interest and the alternative splicing regulatory element, as long as the other nucleic acid sequences do not
- the purpose of the present invention can be met by affecting the expression of the target gene without affecting the function of the alternative splicing regulatory element.
- the other nucleic acid sequence does not contain a stop codon or a start codon (such as ATG), At the same time, the number of nucleotides in the other nucleic acid sequences is a multiple of 3, thereby not affecting the reading frame of the target gene.
- the nucleic acid molecules provided by the present invention can be any type of nucleic acid molecules, as long as the nucleic acid molecules can achieve the purpose of the present invention.
- the nucleic acid molecule of the present invention can be a single-stranded or double-stranded deoxyribonucleic acid molecule (ie, DNA molecule), or it can be a single-stranded or double-stranded ribonucleic acid molecule (ie, RNA molecule); the nucleic acid molecule of the present invention can be Linear or circular.
- the alternative splicing regulatory element provided by the present invention includes multiple introns and exons and at least one alternative splicing regulating small molecule drug binding site nucleic acid sequence.
- the alternative splicing regulatory element is operably connected to the gene of interest, and the gene of interest is located downstream of the alternative splicing regulatory element.
- the alternative splicing regulatory element provided by the present invention is used in combination with one or more alternative splicing regulatory small molecule drugs to regulate (for example, turn on) the expression of the target gene.
- the regulation provided by the present invention based on the combined use of alternative splicing regulatory elements and one or more alternative splicing regulating small molecule drugs exhibits dose dependence, that is, can be controlled by administering different doses of alternative splicing regulating small molecule drugs. target gene expression level.
- the regulation provided by the present invention based on the combined use of alternative splicing regulatory elements and one or more alternative splicing regulatory small molecule drugs exhibits time correlation, that is, the alternative splicing regulatory small molecule drugs can be administered at different times. Control the expression level of the target gene.
- the alternative splicing regulatory element includes at least in sequence from the 5' to the 3' end: a first exon, a first intron, a pseudoexon, a second intron, and a second exon.
- the alternative splicing regulatory element is, from 5' to 3', a first exon, a first intron, a pseudoexon, a second intron, and a second exon.
- a sequence in which one or more alternative splicing modulating small molecule drugs bind to the junction of the pseudo exon and the second intron.
- the pseudo exon in the presence of the alternative splicing regulating small molecule drug, the pseudo exon is included in the mRNA product of the nucleic acid; and in the absence of the alternative splicing regulating small molecule drug, The pseudoexons are not included in the mRNA product of the nucleic acid.
- the pseudoexon of the alternative splicing regulatory element has a unique start codon (eg, an ATG sequence).
- the alternative splicing-modulating small molecule drugs used in the present invention refer to compounds that can mediate alternative splicing.
- the alternative splicing modulating small molecule drug modulates (particularly increases) the mRNA product comprising the pseudoexon.
- the alternative splicing modulating small molecule drug binds to a sequence at a specific position in the alternative splicing modulating element.
- the specific position in the alternative splicing regulatory element that regulates small molecule drug binding is located at the junction of the 3' end of the pseudo exon and the 5' end of the second exon. at.
- the specific position in the alternative splicing regulatory element that regulates the binding of small molecule drugs is located between the 3' end of the pseudo exon and the 5' end of the second exon.
- the alternative splicing regulating small molecule drug used in the present invention is LMI070, or a derivative having the same binding site as LMI070 on the alternative splicing regulating element, or Pharmaceutically acceptable salts of LMI070, where LMI070 is also known as Branaplam or NVS-SM1, and is a compound with the structure shown in the following formula (1).
- Derivatives that have the same binding site as LMI070 on the alternative splicing regulatory element refer to derivative compounds obtained by replacing the compounds of formula (1) with groups commonly used in the art, and the derivative compounds are the same as LMI070 Bind the same sequence fragment to the alternative splicing regulatory element; as a means of testing whether it belongs to the derivatives defined in the present invention, those skilled in the art can determine through simple experiments, for example, for the alternative splicing described in the present invention Whether the regulatory element can also produce the same effect as LMI070, that is, if adding a certain derivative of LMI070 can also cause the generated mRNA to contain the pseudo exon, then this derivative belongs to the same group as LMI070 according to the present invention. Derivatives having the same binding site on the alternative splicing regulatory element.
- Examples of pharmaceutically acceptable salts of LMI070 include, but are not limited to, inorganic and organic acid salts such as hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate, fumarate acid salts, mandelates and oxalates; and inorganic and organic base salts with bases such as sodium hydroxyl, tris(hydroxymethyl)aminomethane (TRIS, tromethamine) and N-methylglucamine .
- inorganic and organic acid salts such as hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate, fumarate acid salts, mandelates and oxalates
- inorganic and organic base salts with bases such as sodium hydroxyl, tris(hydroxymethyl)aminomethane (TRIS, tromethamine) and N-methylglucamine .
- the alternative splicing regulating small molecule drug used in the present invention is Risdiplam, or a derivative having the same binding site as Risdiplam on the alternative splicing regulating element, or Pharmaceutically acceptable salts of Risdiplam, wherein Risdiplam is a compound having the structure shown in the following formula (2).
- the derivative having the same binding site as Risdiplam on the alternative splicing regulatory element refers to a derivative compound obtained by replacing the group conventionally used in the art on the basis of the compound of formula (2), and the derivative compound binds to the same sequence fragment as Risdiplam on the alternative splicing regulatory element; as a means of testing whether it belongs to the derivative defined in the present invention, those skilled in the art can determine it through simple experiments, for example, whether the alternative splicing regulatory element described in the present invention can also produce the same effect as Risdiplam, that is, if a certain derivative of Risdiplam is added, the generated mRNA can also contain the pseudo exon, then this kind of derivative belongs to the derivative having the same binding site as Risdiplam on the alternative splicing regulatory element described in the present invention.
- Examples of pharmaceutically acceptable salts of Risdiplam include, but are not limited to, inorganic and organic acid salts such as hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate, fumarate acid salts, mandelates and oxalates; and inorganic and organic base salts with bases such as sodium hydroxyl, tris(hydroxymethyl)aminomethane (TRIS, tromethamine) and N-methylglucamine .
- inorganic and organic acid salts such as hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate, fumarate acid salts, mandelates and oxalates
- inorganic and organic base salts with bases such as sodium hydroxyl, tris(hydroxymethyl)aminomethane (TRIS, tromethamine) and N-methylglucamine .
- the nucleic acid molecules of the invention optionally include one or more sequences encoding a cleavage site located between the alternative splicing regulatory element and the gene of interest, For the purpose of cutting and separating the alternative splicing regulatory element and the target gene.
- the cleavage site may be an autocleavage site, a protease cleavage site, or a combination of both.
- the present invention controls the expression of the target gene through the provided alternative splicing regulatory element.
- the target gene is a gene encoding a protein or nucleic acid whose expression is desired to be increased or decreased or controlled in the body or cells for gene therapy or other purposes.
- skilled artisans can use the alternative splicing regulatory elements of the present invention to construct nucleic acid molecules containing any gene of interest.
- the gene of interest may be a gene encoding a protein, such as a gene encoding an antibody or functional binding fragment, a receptor, an enzyme, etc.
- the gene of interest may be a gene encoding a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a Cas9 protein.
- the gene of interest can be a gene encoding RNA, such as a gene encoding inhibitory RNA, such as a gene encoding miRNA, shRNA, or lncRNA.
- the number of target genes contained in the nucleic acid molecules provided by the present invention can be more than one or more.
- the nucleic acid molecules provided by the present invention include alternative splicing regulatory elements and target genes, wherein the composition of the alternative splicing regulatory elements has the restrictive conditions described in the present invention, but those skilled in the art can understand that the selection range of the target gene It is broad and not fixed, and different target genes can be selected according to the different purposes of using the nucleic acid molecule of the present invention.
- RT-PCR for the purpose of qualitative or quantitative detection to determine the presence of the nucleic acid molecules of the present invention, methods such as RT-PCR can be used for detection, in which the upstream primer of the primer pair used (or called forward primer, forward primer ) is designed based on the alternative splicing regulatory element, such as the sequence shown in SEQ ID NO:7, but the downstream primer (or reverse primer, forward primer) needs to be designed according to the target gene.
- the principles, tools, and methods of primer design known in the art can be used to design and screen downstream primers.
- the relevant experiments of the present invention use wild-type C57BL/6 mice as animal models. Animal production, breeding, and experiments were completed at the Department of Laboratory Animal Science, Peking University School of Medicine. The relevant experimental operations involved were approved by the Biomedical Ethics Committee of Peking University (Approval No.: 2022412).
- LMI070 (Cayman, 26757) was selected as a small molecule splicing regulator, dissolved in DMSO at a concentration of 10 mg/ml, and stored in a -20°C refrigerator for long-term use.
- the small molecule splicing regulator was diluted in corn oil. For animal administration.
- mice Weigh the body weight of the mice and administer the drug at a dose of 10 mg/kg.
- Adult mice aged 6-10 weeks are administered a small molecule splicing regulator orally while they are awake. After 48 hours of administration, the mice are anesthetized with isoflurane. After sacrifice, the cardiac apex tissue was quickly frozen at -80°C for RNA sequencing experiments.
- the primers used to identify endogenous alternative splicing of the mouse Stradb gene are 5′-GAACAGAGAAGACTGAGGAG-3′ (SEQ ID NO:7) and 5′-CTGTAAAGCTTTCAGACGTTCTTCA-3′ (SEQ ID NO:8).
- the natural Stradb splicing regulatory sequence is 3339bp long.
- the inventors determined the sequences necessary for mRNA splicing, including the connection sequence between the first exon and the first intron, and the sequence in the first intron.
- CMV-DM st -GFP-2A-luciferase reporter plasmid In order to verify the function of the engineered DM st sequence, the inventor constructed a CMV-DM st -GFP-2A-luciferase reporter plasmid. Among them, CMV is a constitutive expression promoter, and the GFP-2A-luciferase gene deletes its own ATG and uses the same reading frame as the ATG in DM st . At the same time, as a positive control group (PC), according to the sequence reported by Monteys, AM, etc. (see Monteys, AM et al., Regulated control of gene therapies by drug-induced splicing.
- PC positive control group
- the inventor constructed a CMV-X on -GFP-2A-luciferase reporter plasmid containing the X on system, in which the length of the alternative splicing regulatory element in the X on system is 560 bp.
- the absolute value of the induction intensity of the DM st sequence of the present invention is higher, and can reach a change factor similar to that of the X on system.
- the DM st sequence still has advantages in loading vectors or recombinant viruses. .
- the inventors packaged AAV9 viral vectors containing CMV-DM st -GFP-2A-luciferase element or CMV-X on -GFP-2A-luciferase element.
- the reported X on system is as Positive control (PC).
- the AAV plasmid construction method is as follows: the DNA fragments of DM st sequence and X on sequence are entrusted to Suzhou GENEWIZ Biotechnology Co., Ltd. (GENEWIZ, Suzhou, Jiangsu, China) for gene synthesis. CMV, GFP and luciferase sequences were obtained from Addgene public data sources. DNA splicing and plasmid construction are constructed through seamless cloning reactions.
- the AAV packaging method is as follows:
- the plasmid used for AAV packaging is 70 ⁇ g AAV plasmid (containing CMV-DM st -GFP-2A-luciferase element or CMV-X on -GFP-2A-luciferase element), 70 ⁇ g AAV9Rep/Cap plasmid and 160 ⁇ g pHelper auxiliary plasmid. These plasmids were transfected into HEK293T cells cultured in 15cm dishes through PEI transfection reagent (Yeasen, 40816ES03).
- AAV9 in the cell culture medium was precipitated with 40% PEG8000 (Sigma, P2139) in 2.5 M NaCl solution, resuspended in lysis buffer, and mixed with the cell lysate.
- AAV in the lysate was purified in an Optiprep (Sigma, D1556-250ML) density gradient by 70 Ti rotor ultracentrifugation (Beckman, XPN-100), followed by using a 100 kDa centrifugal filter tube (Millipore, UFC910096) to exchange AAV into 0.001% pluronic F68/PBS (Caisson, PFL01-100ML) solution and concentrated.
- AAV titers were measured by qPCR technology using primers 5′-AAGCTGACCCTGAAGTTCATCTGC-3′ (SEQ ID NO:9) and 5′-CTTGTAGTTGCCGTCGTCCTTGAA-3′ (SEQ ID NO:10) that recognize the GFP sequence.
- the operation method of Western Blot is: after washing the fresh tissue with cold PBS, in RIPA buffer (25mM Tris PH7.0 ⁇ 8.0, 150mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100 , protease inhibitor (Solarbio, A8260)) using a homogenizer to fully disrupt the tissue. After lysis on ice for 10 minutes, the lysate was centrifuged at 12,000 rpm for 15 minutes at 4°C, the supernatant was collected, and the protein concentration was determined using the BCA method (TransGen, DQ111-01).
- the inventors treated AAV-infected mice with LMI070 multiple times, and detected the dynamic changes in DM st activity through mouse in vivo imaging experiments (Figure 3D).
- the steps for in vivo imaging of small animals are as follows: first prepare fluorescein, dissolve the fluorescein powder (Yeasen, 40903ES03) with 500mM NaOH solution, add HCl to adjust the pH to 7.4, aliquot in the dark, freeze at -80°C, and take out when needed Preheat to 37°C and set aside.
- mice to be tested were injected intraperitoneally with luciferase substrate 15 minutes before imaging, and were anesthetized with isoflurane and placed in a supine position for exposure testing.
- the results showed that the luciferase reporter gene signal in mice reached a peak 2 days after LMI070 administration and returned to the background level after 1 week.
- the second LMI070 administration again enhanced the reporter gene signal.
- results of this example show that the DM st element of the present invention can control the expression of target genes through multiple administrations, and both intraperitoneal injection and oral administration of small molecule drugs can control the expression of target genes through DM st elements, such as enhanced reporting genetic signals.
- DM st element it is advantageous to specifically express the gene of interest in a specific tissue or organ.
- the inventors connected the Tnnt2 promoter, DM st element and GFP reporter gene specifically expressed in cardiomyocytes in tandem, packaged a new AAV9 vector, and conducted in vivo studies in mice ( Figure 4A).
- the method of AAV plasmid construction is as follows: The DM st sequence DNA fragment was entrusted to Suzhou GENEWIZ Biotechnology Co., Ltd. (GENEWIZ, Suzhou, Jiangsu, China) for gene synthesis. cTnnt2/hTnnt2 and GFP sequences were obtained from Addgene’s public data source. DNA splicing and plasmid construction are constructed through seamless cloning reactions.
- RT-qPCR uses Perfect Start Green qPCR Super Mix (+DveII) (TransGen, AQ602-24) reagent.
- the qPCR machine was AriaMx Real-Time PCR System (Agilent Technologies).
- the primers for GAPDH are 5′-CAACTCCCTCAAGATTGTCAGCAA-3′ (SEQ ID NO: 11) and 5′-GGCATGGACTGTGGTCATGA-3′ (SEQ ID NO: 12); the primers for detecting DM st -GFP reporter system are 5′- GAGGAGCAATGCAGCAGTCC-3' (SEQ ID NO: 13) and 5'-CAAGTTGTCAAACCCTCTTC-3' (SEQ ID NO: 14).
- LMI070 increased the degree of DM st element pseudo-exon splicing in the heart by about six times (Figure 4B).
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Abstract
本发明公开了包含可变剪接调节元件和位于该可变剪接调节元件3'端的目的基因的核酸分子，该核酸分子的转录物，以及包含该核酸分子或转录物的载体、重组病毒、细胞、药物组合物，及其用于调节目的基因表达量的方法和在基因治疗中的应用。
Description
本发明总体上涉及分子生物学和医学领域，具体涉及一种包含基于小分子药物的可变剪接调节元件的核酸分子。
已知人类的多种疾病和基因缺陷相关，解决这些与基因缺陷相关疾病的一种有前景的治疗方式是基因治疗。基因治疗是将外源性遗传物质通过多种方法转移到靶细胞中，例如可通过导入正常基因以替代缺失或异常突变的基因、或抑制非正常的内源性基因的功能，从而实现在核酸水平上对细胞生理机能进行干预，并最终用于治疗特定的疾病(刘健，基因治疗中的核酸药物及非病毒递送载体的研究进展，基础医学与临床，第42卷第1期，第41-50页，2022年1月)。然而，基因治疗在过去20年的研究重点主要集中于基因的递送系统，例如腺相关病毒(AAV)衣壳经工程化改造后提高了其靶向性(Ravindra Kumar等,Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types.Nat Methods 17,541–550(2020))，对于被运载的治疗基因本身却关注和研究甚少，主要集中于对启动子工程、核糖开关、以及限制特定细胞类型表达的3'调节元件等。
当治疗基因通过递送系统被运载至靶组织或细胞后，治疗基因的表达水平将成为影响基因治疗效果的重要因素。如果治疗基因表达水平不足或不表达，则无法获得预期的治疗效果；另一方面，如果治疗基因的表达水平过高，即过表达，则很可能对患者造成不期望的毒副作用。如何调节被递送至体内的治疗基因的表达水平是基因治疗方案中必须考虑的方面。2021年，Monteys,A.M等研究者报导了通过药物诱导剪接来调控基因治疗的方法，其通过构建可药物诱导的开关X
on系统从而精细控制蛋白质的翻译。该系统控制RNA的可变剪接，能够实现成熟mRNA中包含或不包含特定外显子。特别是，X
on系统无需使用外源的调节元件，而是利用口服后人体可生物利用的药物LMI070来诱导含起始密码子的外显子被剪接在成熟mRNA中，并由此进行基因表达的精细调控(Monteys,A.M等,Regulated control of gene therapies by drug-induced splicing.Nature 596,291–295(2021))。
WO2020033473A1公开了基因表达的可变剪接调控及治疗方法，其使用嵌合反式激活子小基因，其中小基因的可变剪接决定反式激活子是否表达，反式激活子的表达可导致启动子序列控制下的靶基因转录，或者提供嵌合靶基因小基因，其中小基因的可变剪接直接决定靶基因是否表达。
WO2021163556A1提供了嵌合小基因，其中小基因的可变剪接决定了编码基因是否表达，小基因响应于剪接调节药物而被可变剪接，使得编码基因仅在存在剪接调节药物时表达。
WO2021014428A1公开了一种可调节的表达系统，具体提供了一种包含具有剪接调节剂结合序列的小基因、可用于可调节的基因表达的组合物、及其系统和使用方法。
尽管如此，现有的可变剪接调节元件仍存在需要改进之处，至少包括：(1)本领域技术人员已知AAV等载体载荷有限，然而，现有文献报导的可变剪接调节元件的碱基对数量较多(例如Monteys,A.M等报导的X
on系统的元件大小达560bp)，限制了目的基因的大小，不利于通过AAV等载体将目的基因递送至靶细胞、组织或器官；(2)现有文献报导的可变剪接调节元件来源于体外培养的细胞，而非体内器官，缺乏针对体内生理病理环境的适应和优化；(3)现有元件的应用缺乏器官特异性，不利于靶细胞或组织存在于特定器官时基因治疗的靶向性，以及有可能在不期望的器官中出现毒副作用。此外，扩大可变剪接调节小分子药物的选择范围在治疗手段的选择方面也是有利的。
发明内容
为解决现有技术中存在的上述问题，提供了本发明的技术方案。
在本发明的第一方面，提供了一种核酸分子，所述核酸分子包含可变剪接调节元件和位于所述可变剪接调节元件3'端的目的基因；其中所述可变剪接调节元件从5'端至3'端依次包含第一外显子、第一内含子、假外显子、第二内含子、第二外显子；所述假外显子的序列如SEQ ID No:1所示，并且所述假外显子和所述第二内含子的交界处序列为AGAGTA，其中AGA属于所述假外显子，GTA属于所述第二内含子；所述第一外显子的序列为SEQ ID No:2或包含SEQ ID No:2或为与SEQ ID No:2具有至少95％(优选96％、97％、98％、99％、或99.5％)同一性的序列，并且所述第一外显子的序列紧接所述第一内含子的序列为TAG；所述第一内含子的序列为SEQ ID No:3或包含SEQ ID No:3或为与SEQ ID No:3具有至少95％(优选96％、97％、98％、99％、或99.5％)同一性的序列，并且所述第一内含子的序列紧接所述第一外显子的序列为GTA，所述第一内含子的序列紧接所述假外显子的序列为CAG；所述第二内含子的序列为SEQ ID No:4或包含SEQ ID No:4或为与SEQ ID No:4具有至少95％(优选96％、97％、98％、99％、或99.5％)同一性的序列，并且所述第二内含子的序列紧接所述第二外显子的序列为CAG；所述第二外显子的序列为SEQ ID No:5或包含SEQ ID No:5或为与SEQ ID No:5具有至少95％(优选96％、97％、98％、99％、或99.5％)同一性的序列，并且所述第二外显子的序列紧接所述第二内含子的序列为GAA；所述第一外显子、所述第一内含子、所述第二内含子、所述第二外显子中均不含有ATG序列；其中所述目的基因为一个或多个编码蛋白的基因和/或编码RNA的基因，并且所述编码蛋白的基因和/或所述编码RNA的基因中的第一个起始密码子ATG被删除。
在一个实施例中，所述可变剪接调节元件的序列为SEQ ID No:6或包含SEQ ID No:6。
在一个实施例中，所述可变剪接调节元件的3'端与所述目的基因的5'端之间不存在任何核酸序列。
在一个实施例中，所述可变剪接调节元件的3'端与所述目的基因的5'端之间存在编码蛋白酶切割位点的序列，其中所述蛋白酶切割位点被哺乳动物蛋白酶切割或被自切割。本领域技术人员将理解，在该实施例中，可变剪接调节元件的3'端与目的基因的5'端之间存在的编码蛋白酶切割位点的序列的碱基数应该是3n个，n为大于等于1的整数，以保持目的基因的阅读框没有被改变或移动。
在一个实施例中，所述可变剪接调节元件的5'端可操作地连接有启动子序列。
在一个实施例中，所述启动子序列为哺乳动物细胞组成型启动子。优选地，所述启动子为CMV、CAG、CBG、EF1a、PGK1或Ubc。
在一个实施例中，所述启动子序列为哺乳动物细胞的特异性启动子。优选地，所述启动子为Tnnt2(心肌细胞)、Nppa(心房心肌细胞)、Myl2(心室心肌细胞)、Mck(骨骼肌细胞)、Nkx2.5(心肌祖细胞)、Syn(神经元)、Mecp2(神经元)、TBG(肝细胞)、Pdx1(胰细胞)、K14(皮肤角质细胞)、Rpe65(视网膜细胞)或SP-C(肺上皮细胞)。
在一个实施例中，所述启动子序列为哺乳动物非编码RNA启动子。优选地，所述 启动子为U6、H1。
在一个实施例中，所述启动子序列为原核细胞启动子。优选地，所述启动子为T7、T3、SP6。
在一个实施例中，本发明的核酸分子进一步包含转录后调节元件。优选地，所述转录后调节元件(PRE)包含衍生自乙型肝炎(HPRE)、蝙蝠(BPRE)、地松鼠(GSPRE)、北极松鼠(ASPRE)、鸭(DPRE)、黑猩猩(CPRE)和长毛猴(WMPRE)或土拨鼠(WPRE)的PRE，任选地其中所述转录后调节元件设置在所述目的基因的3'端。
在一个实施例中，本发明的核酸分子进一步包含多腺苷酸化信号(polyA)，任选地其中所述polyA设置在所述目的基因的3'端。优选地，所述polyA信号是SV40polyA、人生长激素(HGH)polyA、或牛生长激素(BGH)polyA、β-珠蛋白polyA、α-珠蛋白polyA、卵清蛋白polyA、κ-轻链polyA、和合成polyA。
在一个实施例中，所述可变剪接调节元件能够与可变剪接调节小分子药物结合。优选地，所述可变剪接调节小分子药物为LMI070、或与LMI070在所述可变剪接调节元件上具有相同结合位点的衍生物、或LMI070药学上可接受的盐，或者所述可变剪接调节小分子药物为Risdiplam、或与Risdiplam在所述可变剪接调节元件上具有相同结合位点的衍生物、或Risdiplam药学上可接受的盐。
在一个实施例中，所述编码蛋白的基因为编码转录激活样效应因子核酸酶(TALEN)、锌指核酸酶(ZFN)或者Cas9、Cas12、Cas13蛋白或其相同功能衍生物的基因，和/或编码治疗蛋白的基因。
在一个实施例中，所述编码RNA的基因为编码miRNA、shRNA、lncRNA的基因。
在一个实施例中，提供了根据本发明所述的核酸分子在可变剪接调节小分子药物存在时的转录物；在另一个实施例中，提供了根据本发明所述的核酸分子在可变剪接调节小分子药物不存在时的转录物；其中，可变剪接调节小分子药物为LMI070、或与LMI070在所述可变剪接调节元件上具有相同结合位点的衍生物、或LMI070药学上可接受的盐；或者可变剪接调节小分子药物为Risdiplam、或与Risdiplam在所述可变剪接调节元件上具有相同结合位点的衍生物、或Risdiplam药学上可接受的盐。
在一个实施例中，所述核酸分子为DNA分子，所述转录物为RNA分子。
在本发明的第二方面，本发明提供了一种载体，包含根据本发明所述的核酸分子或者根据本发明所述的转录物。
在一个实施例中，所述载体是DNA或RNA载体。优选地，所述载体是环状载体。更优选地，所述载体是质粒。
在一个实施例中，所述载体是双链的或单链的；优选地，所述载体是双链的。
在一个实施例中，所述载体是病毒载体。优选地，所述病毒载体是腺相关病毒(AAV)载体、嵌合AAV载体、腺病毒载体、逆转录病毒载体、慢病毒载体、DNA病毒载体、单纯疱疹病毒载体、杆状病毒载体、或其任何突变体或衍生物。优选地，所述病毒载体是重组AAV载体、自互补AAV(scAAV)载体、或单链AAV(ssAAV)载体。优选地，所述重组AAV载体包括一个或多个反向末端重复(ITR)，可选地其中所述ITR是AAV2ITR，可选地其中所述AAV载体包括两个ITR。
在本发明的第三方面，本发明提供了一种重组病毒，所述重组病毒包含根据本发明第一方面所述的核酸分子或转录物、或根据本发明第二方面所述的载体。
在一个实施例中，所述重组病毒是腺相关病毒(AAV)、嵌合AAV、腺病毒、逆转录病毒、慢病毒、DNA病毒、单纯疱疹病毒、杆状病毒、或其任何突变体或衍生物。优选地，所述重组病毒是AAV。更优选地，所述AAV包含以下的一种或多种：AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAVrh8、AAVrh10、AAVrh36、AAVrh37、AAV-DJ、AAV-DJ/8、AAV.Anc80、AAV.Anc80L65、AAV-PHP.B、AAV-PHP.B2、AAV-PHP.B3、AAV-PHP.A、AAV-PHP.eB、AAV-PHP.S、AAV2i8、MyoAAV、AAVMYO衣壳血清型、或其变体，例如来自一个以上AAV血清型的衣壳的组合。在一个实施例中，所述AAV是AAV9。
在本发明的第四方面，本发明提供了一种细胞，所述细胞包含一种或多种根据本发明第一方面所述的核酸分子或转录物、或者包含一种或多种根据本发明第二方面所述的载体、或者包含一种或多种根据本发明第三方面所述的重组病毒。
在一个实施例中，所述细胞为人细胞。优选地，所述人细胞为心脏细胞、肌肉细胞、神经元、肝细胞、肺细胞、或肾细胞。
在一个实施例中，当所述细胞中存在可变剪接调节小分子药物时，所述目的基因的表达水平比当所述细胞中不存在所述可变剪接调节小分子药物时的目的基因表达水平高1-100倍，例如高1-80倍、1-70倍、1-60倍、1-50倍、1-40倍、1-30倍、1-20倍、2-10倍、2-5倍；任选地，当所述细胞中不存在所述可变剪接调节小分子药物时，无法检测到目的基因表达水平。
在本发明的第五方面，提供了一种药物组合物，所述药物组合物包含一种或多种 根据本发明第一方面所述的核酸分子或转录物、或者一种或多种根据本发明第二方面所述的载体、或者一种或多种根据本发明第三方面所述的重组病毒、或一种或多种根据本发明第四方面所述的细胞。
在本发明的第六方面，提供了一种基于可变剪接调节小分子药物调节目的基因的表达量的方法，所述方法包括：使一种或多种根据本发明第一方面所述的核酸分子或转录物、或者一种或多种根据本发明第二方面所述的载体、或者一种或多种根据本发明第三方面所述的重组病毒、或一种或多种根据本发明第四方面所述的细胞与一种或多种可变剪接调节小分子药物接触，其中，当存在可变剪接调节小分子药物时，所述目的基因的表达水平比当不存在所述可变剪接调节小分子药物时的目的基因表达水平高1-100倍，例如高1-80倍、1-70倍、1-60倍、1-50倍、1-40倍、1-30倍、1-20倍、2-10倍、2-5倍；任选地，当不存在所述可变剪接调节小分子药物时，无法检测到目的基因表达水平。
在本发明的第七方面，提供了一种治疗有需要的受试者的基因治疗方法，所述基因治疗方法包括向所述受试者施用一种或多种根据本发明第一方面所述的核酸分子或转录物、或者一种或多种根据本发明第二方面所述的载体、或者一种或多种根据本发明第三方面所述的重组病毒、或一种或多种根据本发明第四方面所述的细胞、或一种或多种根据本发明第五方面所述的药物组合物，以及一种或多种可变剪接调节小分子药物；其中，当存在可变剪接调节小分子药物时，所述目的基因的表达水平比当不存在所述可变剪接调节小分子药物时的目的基因表达水平高1-100倍，例如高1-80倍、1-70倍、1-60倍、1-50倍、1-40倍、1-30倍、1-20倍、2-10倍、2-5倍；任选地，当不存在所述可变剪接调节小分子药物时，无法检测到目的基因表达水平。
在本发明的第八方面，提供了一种试剂盒，所述试剂盒包含：(1)一种或多种根据本发明第一方面所述的核酸分子或转录物、或者一种或多种根据本发明第二方面所述的载体、或者一种或多种根据本发明第三方面所述的重组病毒、或一种或多种根据本发明第四方面所述的细胞、或一种或多种根据本发明第五方面所述的药物组合物；和(2)一种或多种可变剪接调节小分子药物。
在本发明的第九方面，提供了物质用于调节目的基因的表达量的方法的用途，所述物质为根据本发明第一方面所述的核酸分子或转录物、或者根据本发明第二方面所述的载体、或者根据本发明第三方面所述的重组病毒、或根据本发明第四方面所述的细胞、或根据本发明第五方面所述的药物组合物，以及可变剪接调节小分子药物。
在本发明的第十方面，提供了物质用于制备用于治疗有需要的受试者的基因治疗药物的用途，所述物质为根据本发明第一方面所述的核酸分子或转录物、或者根据本发明第二方面所述的载体、或者根据本发明第三方面所述的重组病毒、或根据本发明第四方面所述的细胞、或根据本发明第五方面所述的药物组合物，以及可变剪接调节小分子药物。
在本发明的第十一方面，提供了一种用于检测根据本发明第一方面所述的核酸分子或转录物、或者根据本发明第二方面所述的载体、或者根据本发明第三方面所述的重组病毒、或根据本发明第四方面所述的细胞、或根据本发明第五方面所述的药物组合物的方法，其中所述方法使用了PCR，所述PCR中使用了如SEQ ID NO:7所示的序列作为上游引物。
在本发明所述的任何一方面中，所述可变剪接调节小分子药物为能够结合所述假外显子和所述第二内含子的交界处序列AGAGTA的小分子药物，并且所述可变剪接调节小分子药物具有以下作用：当存在所述可变剪接调节小分子药物时，由根据本发明第一方面的核酸分子生成的成熟mRNA中包含所述假外显子；当不存在所述可变剪接调节小分子药物时，由根据本发明第一方面的核酸分子生成的成熟mRNA中不包含所述假外显子。
优选地，所述可变剪接调节小分子药物为LMI070、或与LMI070在所述可变剪接调节元件上具有相同结合位点的衍生物、或LMI070药学上可接受的盐，或者所述可变剪接调节小分子药物为Risdiplam、或与Risdiplam在所述可变剪接调节元件上具有相同结合位点的衍生物、或Risdiplam药学上可接受的盐。
相较于已报导的可变剪接调节元件，本发明的技术方案具有良好的技术效果，至少体现在：(1)本发明提供的核酸分子中包含的可变剪接调节元件的碱基对数量更少，仅有417bp，相对于现有元件例如Monteys,A.M等报导的X
on系统元件(560bp)其大小减少超过25％，这种改进有利于更大的目的基因的调控和递送；(2)本发明提供的核酸分子中包含的可变剪接调节元件来源于体内器官，更匹配和适应体内生理病理环境；(3)本发明提供的核酸分子中包含的可变剪接调节元件，结合特异性启动子，能够实现对特定器官基因治疗的靶向性，降低在不期望的器官中出现毒副作用的可能性；(4)本发明提供的核酸分子中包含的可变剪接调节元件可以被更大范围的可变剪接调节小分子药物调节，包括被已批准上市的Risdiplam调控，具有更广泛的应用范围和在治疗手段选择方面的优势。
附图用来提供对本申请技术方案的进一步理解，并且构成说明书的一部分，与本申请的实施例一起用于解释本申请的技术方案，但并不构成对本申请技术方案的限制。
图1显示小鼠心脏中受LMI070调控的可变剪接片段的发现。A：实验流程图，野生型成年C57BL/6小鼠灌胃给药10mg/kg LMI070药物48小时后，取心室组织进行全转录组测序，并通过CASH软件定量分析，发现差异剪接的DNA元件。B：Stradb位点的sashimi图，弧线和数字标注跨外显子的测序序列，黑色箭头标出假外显子位点。C：反转录PCR(RT-PCR)的结果图，在假外显子片段上设计引物，使只有受药物诱导产生剪接变化的片段才能被有效扩增，PCR产物通过琼脂糖凝胶电泳的结果(n＝3)。
图2显示Stradb位点的工程改造和DM
st元件的体外验证。A：Stradb位点的工程改造策略，在Stradb基因序列上保留剪接所需的序列，删减其他序列，在假外显子上设置唯一的起始密码子ATG，移除其他外显子和内含子序列中的ATG。B：构建含DM
st元件的GFP-luciferase报告基因质粒，转染HEK293T细胞，LMI070处理24小时后观察GFP信号。C：用酶标仪对GFP信号定量分析，PC(positive control)指阳性对照，即Monteys,A.M等已报导的X
on系统中的元件。D：将DM
st-GFP-luciferase报告基因质粒转染HEK293T细胞，用1μM LMI070、RG7800或者Risdiplam处理24小时后观察GFP信号。E：用酶标仪对图2D中的GFP信号进行定量分析。
图3显示DM
st元件控制的AAV载体的体内验证与评估。A：DM
st元件控制的AAV报告基因载体设计和小鼠实验流程图。B：LMI070给药2天后Western Blot分析GFP-Luci.表达量，PC(positive control)指阳性对照，即Monteys,A.M等已报导的X
on系统中的元件。C：LMI070给药2天和4天后，RT-PCR分析假外显子剪接变化，d.p.i.,days post injection，指注射后天数。D：小动物活体成像分析荧光素酶活性，同一只小鼠两次给相同的LMI070处理：第一次腹腔注射(IP injection)，第二次灌胃给药(Gavage feeding)。
图4显示心肌细胞特异性AAV-DM
st载体的体内验证。A：心肌细胞特异性AAV-DM
st载体和体内验证实验示意图。B：心肌组织qPCR定量分析DM
st的RNA剪接变化。C：心肌组织GFP荧光信号。D：AAV-DM
st-GFP+LMI070处理后的其他组织的GFP信号。cTnnt2，鸡Tnnt2启动子；hTnnt2，人Tnnt2启动子。
在本文中，单数形式“一个/种(a/an)”以及“所述(the)”包括复数形式，除非上下文另外明确地指示。如技术人员从本文包含的教导中显而易见的是，当在数值和范围的语境中使用时，术语“约”或“大约”是指近似或接近所指定的值或范围的值或范围，使得实施例可以按照预期执行。在一些实施例中，“约”意指数值量±10％。
术语“多核苷酸”和“核酸”在本文中可互换使用，并且是指任何长度的核苷酸的聚合形式。它们可以包括一种或多种核糖核苷酸或脱氧核糖核苷酸。因此，该术语包括但不限于单链、双链或多链DNA或RNA，基因组DNA，cDNA，DNA-RNA杂合体，或包含嘌呤和嘧啶碱基或其他天然的、化学或生化修饰的、非天然的、或衍生化的核苷酸碱基的聚合物，例如锁核酸(LNA)、肽核酸(PNA)。
术语“肽”、“多肽”和“蛋白”可互换使用，并且是指由通过肽键共价连接的氨基酸残基构成的化合物。蛋白或肽典型地含有至少两个氨基酸或氨基酸变体，并且对可包含蛋白或肽序列的氨基酸的最大数目没有限制。多肽包括包含由肽键彼此相连的两个或更多个氨基酸或变体的任何肽或蛋白。这些术语包括例如生物活性片段、基本上同源的多肽、寡肽、同源二聚体、异源二聚体、多肽的变体、经修饰的多肽、衍生物、类似物、融合蛋白等。多肽包括天然肽、重组肽、或其组合。
术语“同一性”和“同源性”在本文中可互换使用，其在用于描述多核苷酸或多肽序列时是指当比较或比对多肽或多核苷酸的两个序列时，相同且处于相同相对位置的碱基或氨基酸的百分比。序列同一性可以以多种不同的方式确定。例如，可以使用各种方法和计算机程序(例如，BLAST、T-COFFEE、MUSCLE、MAFFT等)比对序列。
本发明描述的核酸分子例如可以是一种分离的核酸分子。术语“分离的”在用于核酸分子或蛋白分子时是指已与在自然环境中通常存在的与其相关的一种或多种组分分开的核酸或蛋白。分开可以包括从较大的核酸(例如，从基因或染色体)或从通常与所述核酸或蛋白接触的其他蛋白或分子脱离。所述术语涵盖但不要求完全分离。
术语“可操作地连接”是指两个或更多个多核苷酸(例如，DNA)区段之间的功能性关系。典型地，该术语是指转录调节序列与待转录序列的功能性关系。例如，如果启动子或增强子序列例如在适当的宿主细胞或其他表达系统中刺激或调节编码序列的转录，则启动子或增强子序列与编码序列可操作地连接。通常，与序列可操作地连接的启动子转录调节序列与该序列邻接或由短间隔子序列分开，即他们是顺式作用的。然而，一些转录调节序列如增强子不需要在物理上邻接或位于极为接近这些转录调节 序列增强其转录的编码序列的位置。
下面将参照附图来详细描述本发明的各种示例性实施例。对示例性实施例的描述仅仅是说明性的，并不作为对本发明及其应用或使用的任何限制。本发明可以以许多不同的形式实现，不限于这里所述的实施例。提供这些实施例是为了使本发明透彻且完整，并且向本领域技术人员充分表达本发明的范围。应注意到：除非另外具体说明，否则在这些实施例中描述的技术手段应被解释为仅是示例性的，而非限制性的。
核酸分子
本发明提供的核酸分子包含可变剪接调节元件和编码目的分子(例如目的蛋白)的目的基因，其中所述目的基因可操作地连接于所述可变剪接调节元件的3'端。本领域技术人员将理解，所述目的基因与所述可变剪接调节元件之间的连接可以是紧密连接的，即所述目的基因与所述可变剪接调节元件之间不存在任何核酸序列；所述目的基因与所述可变剪接调节元件之间的连接也可以是非紧密连接的，即所述目的基因与所述可变剪接调节元件之间存在其他核酸序列，只要所述其他核酸序列不影响目的基因的表达并且不影响所述可变剪接调节元件的功能即可满足本发明的目的，例如所述其他核酸序列内不含有终止密码子，也不含有起始密码子(例如ATG)，同时所述其他核酸序列的核苷酸数目为3的倍数，从而不会影响目的基因的读码框。
本发明提供的核酸分子可以是任何类型的核酸分子，只要所述核酸分子能够实现本发明的目的。例如，本发明的核酸分子可以是单链或双链的脱氧核糖核酸分子(即DNA分子)，也可以是单链或双链的核糖核酸分子(即RNA分子)；本发明的核酸分子可以是线性或环状的。
可变剪接调节元件
本发明提供的可变剪接调节元件包含多个内含子和外显子以及至少一个可变剪接调节小分子药物结合位点的核酸序列。在实施例中，所述可变剪接调节元件与所述目的基因可操作地连接，所述目的基因位于所述可变剪接调节元件的下游位置。本发明提供的可变剪接调节元件与一种或多种可变剪接调节小分子药物结合使用，能够调节(例如打开)所述目的基因的表达。本发明提供的基于可变剪接调节元件与一种或多种可变剪接调节小分子药物的结合使用的调节呈现出剂量相关性，即能够通过施用不同剂量的可变剪接调节小分子药物来控制目的基因表达水平。另外，本发明提供的基于可变剪接调节元件与一种或多种可变剪接调节小分子药物的结合使用的调节呈现出时间相关性，即能够通过不同时间施用可变剪接调节小分子药物来控制目的基因表达 水平。
在多个方面，可变剪接调节元件从5'至3'端至少依次包含：第一外显子、第一内含子、假外显子、第二内含子、第二外显子。在多个方面，可变剪接调节元件从5'至3'端为第一外显子、第一内含子、假外显子、第二内含子、第二外显子。其中一种或多种可变剪接调节小分子药物结合于所述假外显子和所述第二内含子的交界处的序列。其中，在所述可变剪接调节小分子药物存在的情况下，所述假外显子包括在所述核酸的mRNA产物中；并且在所述可变剪接调节小分子药物不存在的情况下，所述假外显子不包括在所述核酸的mRNA产物中。
在本发明中，可变剪接调节元件的假外显子具有唯一的起始密码子(例如ATG序列)。
可变剪接调节小分子药物
本发明所使用的可变剪接调节小分子药物是指能够介导可变剪接的化合物。在一些实施方式中，所述可变剪接调节小分子药物调节(特别是增加)mRNA产物包含所述假外显子。在一些实施方式中，所述可变剪接调节小分子药物结合所述可变剪接调节元件中的特定位置的序列。优选地，在一些实施方式中，所述可变剪接调节小分子药物结合的可变剪接调节元件中的特定位置位于所述假外显子的3'端与第二外显子5'端的接合处。更优选地，在一些实施方式中，所述可变剪接调节小分子药物结合的可变剪接调节元件中的特定位置位于所述假外显子的3'端与第二外显子5'端的接合处的AGAGTA序列，其中AGA为所述假外显子3'端的最后3个核苷酸残基，GTA为所述第二内含子5'端的第1-3个核苷酸残基。
在可变剪接调节小分子药物的第一个方面，本发明使用的可变剪接调节小分子药物为LMI070、或与LMI070在所述可变剪接调节元件上具有相同结合位点的衍生物、或LMI070药学上可接受的盐，其中LMI070又名Branaplam或NVS-SM1，是具有如下式(1)所示结构的化合物。
与LMI070在所述可变剪接调节元件上具有相同结合位点的衍生物是指在式(1)化合物的基础上通过本领域常规使用的基团替换所得到的衍生化合物，并且衍生化合物与LMI070在所述可变剪接调节元件上结合相同的序列片段；作为是否属于本发明限定的衍生物的检验手段，本领域技术人员可通过简单的试验加以确定，例如针对本发明所述的可变剪接调节元件是否也能够产生和LMI070相同的效果，即如果加入LMI070的某种衍生物后也可以使生成的mRNA包含所述假外显子，则该种衍生物即属于本发明所述的与LMI070在所述可变剪接调节元件上具有相同结合位点的衍生物。
LMI070药学上可接受的盐的例子包括但不限于无机和有机酸盐，例如盐酸盐、氢溴酸盐、硫酸盐、柠檬酸盐、乳酸盐、酒石酸盐、马来酸盐、富马酸盐、扁桃酸盐和草酸盐；以及与碱例如钠羟基、三(羟基甲基)胺基甲烷(TRIS，胺丁三醇)和N-甲基葡糖胺形成的无机和有机碱盐。
在可变剪接调节小分子药物的第二个方面，本发明使用的可变剪接调节小分子药物为Risdiplam、或与Risdiplam在所述可变剪接调节元件上具有相同结合位点的衍生物、或Risdiplam药学上可接受的盐，其中Risdiplam是具有如下式(2)所示结构的化合物。
与Risdiplam在所述可变剪接调节元件上具有相同结合位点的衍生物是指在式(2)化合物的基础上通过本领域常规使用的基团替换所得到的衍生化合物，并且衍生化合物与Risdiplam在所述可变剪接调节元件上结合相同的序列片段；作为是否属于 本发明限定的衍生物的检验手段，本领域技术人员可通过简单的试验加以确定，例如针对本发明所述的可变剪接调节元件是否也能够产生和Risdiplam相同的效果，即如果加入Risdiplam的某种衍生物后也可以使生成的mRNA包含所述假外显子，则该种衍生物即属于本发明所述的与Risdiplam在所述可变剪接调节元件上具有相同结合位点的衍生物。
Risdiplam药学上可接受的盐的例子包括但不限于无机和有机酸盐，例如盐酸盐、氢溴酸盐、硫酸盐、柠檬酸盐、乳酸盐、酒石酸盐、马来酸盐、富马酸盐、扁桃酸盐和草酸盐；以及与碱例如钠羟基、三(羟基甲基)胺基甲烷(TRIS，胺丁三醇)和N-甲基葡糖胺形成的无机和有机碱盐。
切割位点
在一些实施方案中，本发明的核酸分子可选地包括一个或多个编码切割位点的序列，所述编码切割位点的序列位于所述可变剪接调节元件和所述目的基因之间，用于实现将所述可变剪接调节元件和所述目的基因切割分离的目的。所述切割位点可以是自切割位点、蛋白酶切割位点或二者的组合。
目的基因
本发明通过提供的可变剪接调节元件控制目的基因的表达。目的基因是出于基因治疗或者其他目的需要在体内或细胞内希望增加或减少或受控表达的蛋白质或核酸的编码基因。不受理论的限制，技术人员可使用本发明的可变剪接调节元件构建包含任何目的基因的核酸分子。在一些实施方案中，目的基因可以是编码蛋白的基因，如编码抗体或功能性结合片段、受体、酶等的基因。在一些实施方案中，目的基因可以是编码转录激活样效应因子核酸酶(TALEN)、锌指核酸酶(ZFN)或者Cas9蛋白的基因。在一些实施方案中，目的基因可以是编码RNA的基因，例如是编码抑制RNA的基因，例如是编码miRNA、shRNA、lncRNA的基因。本发明提供的核酸分子中所包含的目的基因的数量可多于一种或多种。
包含可变剪接调节元件的核酸分子的检测
本发明提供的核酸分子包括可变剪接调节元件和目的基因，其中可变剪接调节元件的组成具有如本发明所述的限制性条件，但本领域技术人员能够理解的是，目的基因的选择范围是广泛且不固定的，可根据使用本发明核酸分子的目的不同而选择不同的目的基因。基于此，出于确定本发明所述的核酸分子存在的定性或定量检测的目的，能够使用例如RT-PCR的方法进行检测，其中所用引物对的上游引物(或称为正向引 物，forward primer)根据可变剪接调节元件设计，例如为SEQ ID NO:7所示的序列，但下游引物(或称为反向引物，forward primer)需要根据目的基因的不同而进行设计。可采用本领域已知的引物设计的原则、工具、方法进行下游引物的设计和筛选。
以下实施例说明性地例示了本发明的技术方案，但不构成对本发明保护范围的任何限制。
本发明相关实验采用野生型C57BL/6小鼠作为动物模型。动物生产、繁育和实验在北京大学医学部实验动物科学部完成。涉及到的相关实验操作获得北京大学生物医学伦理委员会批准(批准号：2022412)。
实施例1响应LMI070的剪接调节元件的发现
发现小鼠心脏中受LMI070调控的可变剪接片段的过程参见图1A。选用LMI070(Cayman，26757)作为小分子剪接调节剂，以10mg/ml的浓度溶解于DMSO中，于-20℃冰箱长期保存备用，在实验中，将小分子剪接调节剂稀释于玉米油中以用于动物给药。
称量小鼠体重，并按照10mg/kg的剂量给药，6-10周龄成年小鼠在清醒状态下灌胃施用小分子剪接调节剂，给药48小时后，将小鼠异氟烷麻醉处死后迅速取心尖组织冻于-80℃，用于RNA测序实验。
通过以下方法提取RNA：小鼠组织首先使用匀浆机(DREMEL F6/10)破碎组织，然后使用Trizol(TransGen，ER501-01)提取RNA，分装并保存于-80℃。提取的RNA质检后取1mg反转录cDNA，使用KAPA Hyper Prep Kits文库构建试剂盒(KAPA，KK8504)完成Bulk转录组文库构建，经过Fragment Analyzer 1.0.2.9质检、qPCR定量之后，通过Illumina Nova-seq测序平台进行PE150双末端测序(二代测序)，返回的测序数据使用生物信息学技术进行分析。
将上述二代测序结果使用Trim Galore软件做预处理，并用FastQC软件对处理后数据进行质量控制，随后通过STAR软件将质控后的测序结果与小鼠mm10参考基因组比对，然后使用CASH软件进行可变剪接分析，筛选得到潜在的可变剪接候选位点(Stradb基因位点，图1B)。最后结合统计学分析结果进行筛选，确定包含假外显子的可变剪接片段并利用如下RT-PCR方法进行验证。
通过RT-PCR验证LMI070对Stradb mRNA在心脏中的剪接调节作用：提取的RNA测定浓度后，取1μg使用逆转录试剂盒(TransGen，AH311-03)逆转录为cDNA，以逆转录后的cDNA为模板，使用2XTaq酶(TIANGEN，KT211)进行PCR扩增后， 将产物进行凝胶电泳并记录条带。RT-PCR中，用于鉴定小鼠Stradb基因内源可变剪接的引物为5′-GAACAGAGAAGACTGAGGAG-3′(SEQ ID NO:7)和5′-CTGTAAAGCTTTCAGACGTTCTTCA-3′(SEQ ID NO:8)，RT-PCR产物通过琼脂糖凝胶电泳的结果如图1C所示(n＝3)。
实验结果显示，只有受药物诱导产生剪接变化的片段才能被有效扩增，没有施用LMI070药物的对照组不会出现扩增，这一结果证明了LMI070对Stradb mRNA在心脏中的剪接调节作用。
实施例2 Stradb剪接调节元件的工程改造
天然Stradb剪接调节序列长3339bp。考虑到有利于载体或重组病毒的载荷，如图2A所示，发明人确定其中mRNA剪接所必须的序列，包括第一外显子-第一内含子的连接序列、第一内含子中剪接所需的分支点序列、第一内含子-假外显子的连接序列、假外显子-第二内含子的连接序列、第二内含子中剪接所需的分支点序列、第二内含子-第二外显子的连接序列。
在假外显子上设置唯一的ATG作为起始密码子，删减其他组分中的ATG和CTG等起始密码子序列，并且删除其他非必须的序列，将体积缩小到417bp，得到最终的DM
st序列(Drug-elicitable alternative-splicing modulator-Stradb)，即本发明所述的可变剪接调节元件，其序列为：
其中，所述可变剪接调节元件的各区域划分如表1所示。
表1可变剪接调节元件的各区域序列
为了验证工程改造后DM
st序列的功能，发明人构建了CMV-DM
st-GFP-2A-luciferase报告质粒。其中CMV为组成型表达启动子，GFP-2A-luciferase基因删除自身的ATG，并且与DM
st中的ATG使用同一个读码框。同时，作为阳性对照组(positive control group,PC)，根据Monteys,A.M等已报导的序列(参见Monteys,A.M等,Regulated control of gene therapies by drug-induced splicing.Nature 596,291–295(2021))，发明人构建了包含X
on系统的CMV-X
on-GFP-2A-luciferase报告质粒，其中X
on系统中可变剪接调节元件的长度为560bp。
将上述两种质粒分别转染HEK293T细胞，然后分别用0μM、0.1μM、1μM的LMI070处理，24小时后观察GFP荧光信号(图2B)并定量分析(图2C)。结果显示，LMI070处理组具有增强的GFP荧光且荧光强度与LMI070浓度正相关，即DM
st 序列具有基于小分子药物诱导的可变剪接调控目的基因表达的功能，并且这种功能呈现出与给药剂量的正相关性。另外，与已报导的X
on系统相比，本发明的DM
st序列的诱导强度的绝对值更高，并且可以达到和X
on系统类似的变化倍数。然而，考虑到DM
st序列的调节元件的长度更小(已报导的X
on系统为560bp DM
st序列为417bp，降低幅度超过25％)，DM
st序列在载体或重组病毒的负载方面仍然具有优势。
另外，为了验证其他小分子是否也能够对剪接调节元件发挥作用，使用相同浓度的另外两种具有类似功能的小分子剪接调节剂Risdiplam和RG7800分别处理含上述报告质粒的细胞，结果发现Risdiplam与LMI070激活DM
st元件的效果相似，而RG7800的激活能力较弱(图2D-E)。
实施例3构建DM
st元件控制的AAV载体
为验证DM
st元件在体内是否有效，发明人包装了含CMV-DM
st-GFP-2A-luciferase元件或者CMV-X
on-GFP-2A-luciferase元件的AAV9病毒载体，已报导的X
on系统作为阳性对照(PC)。其中，AAV质粒构建的方法如下：DM
st序列和X
on序列的DNA片段委托苏州金唯智生物科技有限公司(GENEWIZ，中国，江苏苏州)进行基因合成。CMV、GFP和luciferase序列来自Addgene公开的数据源。DNA拼接和质粒构建通过无缝克隆反应构造。
AAV包装方法如下：AAV包装使用的质粒为70μg AAV质粒(含CMV-DM
st-GFP-2A-luciferase元件或者CMV-X
on-GFP-2A-luciferase元件)、70μg AAV9Rep/Cap质粒和160μg pHelper辅助质粒。这些质粒通过PEI转染试剂(Yeasen，40816ES03)转染到15cm皿培养的HEK293T细胞中，转染60小时后，刮下细胞并重悬于裂解缓冲液(20mM Tris pH＝8，150mM NaCl，1mM MgCl2，50μg/ml苯甲酸酶(Yeasen，20156ES60))中，三次反复冻融裂解细胞，将AAV9释放到溶液中。同时，用40％PEG8000(Sigma，P2139)在2.5M NaCl溶液中沉淀细胞培养基中的AAV9，再用裂解缓冲液重悬，并与细胞裂解液混合。裂解液中的AAV在Optiprep(Sigma，D1556-250ML)密度梯度中通过70Ti转子超速离心(Beckman，XPN-100)纯化，接下来使用100kDa离心过滤管(Millipore，UFC910096)，将AAV换入0.001％pluronic F68/PBS(Caisson，PFL01-100ML)溶液中并浓缩。利用识别GFP序列的引物5′-AAGCTGACCCTGAAGTTCATCTGC-3′(SEQ ID NO:9)和5′-CTTGTAGTTGCCGTCGTCCTTGAA-3′(SEQ ID NO:10)， 通过qPCR技术测量AAV滴度。
在小鼠出生后一天(P1)，将小鼠用异氟烷麻醉后，通过皮下注射，将包含DM
st元件或X
on元件的AAV9病毒载体注入体内。第6天对小鼠腹腔注射50mg/kg LMI070，给药2天后取心脏和肝脏组织进行Western blot分析(图3A-B)，GAPDH为内参，评估DM
st元件和X
on系统的工作能力(n＝4/组)。
其中，Western Blot的操作方法为：用冷PBS清洗新鲜组织后，在RIPA缓冲液(25mM Tris PH7.0～8.0，150mM NaCl，0.1％SDS，0.5％脱氧胆酸钠，1％Triton X-100，蛋白酶抑制剂(Solarbio，A8260))中使用匀浆机充分破碎组织。冰上裂解10分钟后，裂解液在4℃以12000rpm离心15分钟，收集上清液，使用BCA方法(TransGen，DQ111-01)测定蛋白质浓度。
测定样品蛋白浓度后，用RIPA缓冲液将裂解液中的蛋白浓度调节至相同水平，并在4x SDS样品缓冲液中稀释(Solarbio，P1016)。稀释液煮沸10分钟后，准备好4-15％梯度凝胶(TransGen，DG101-01)，从每个样品中取20μg蛋白上样跑胶，之后转移到PVDF膜上。在含5％脱脂牛奶的TBST中室温孵育一小时后，孵一抗(Ms-Anti-GAPDH，1:5000，TransGen，HC301；Ms-Anti-GFP，1:5000，TransGen，HT801)4℃过夜。次日，使用TBST漂洗三次，每次五分钟后，孵二抗(HRP-Gt-Anti-Ms,1:5000,BBI,D110087-0100)1小时。再使用TBST同样漂洗三次，每次五分钟后，在膜上均匀淋上ECL底物(Solarbio，PE0010)，使用iBright CL1500成像系统(Thermo Fisher Scientific))拍摄图像。
本实验结果显示，LMI070存在时DM
st元件能在心脏和肝脏中增强报告基因GFP的表达，在心脏中DM
st的作用强于X
on系统。
进一步在LMI070注射2天和4天后，通过RT-PCR检测假外显子剪接入mRNA的程度，发现对应条带在注射2天后可明显检测到，但是在注射4天后明显减少(图3C，对照组为未施用LMI070的小鼠在2天后的样品)。因此，单次LMI070注射增强DM
st元件的作用不超过4天。
发明人用LMI070多次处理AAV侵染的小鼠，通过小鼠活体成像实验检测DM
st活性的动态变化(图3D)。小动物活体成像的操作步骤如下：首先配置荧光素，用500mM NaOH溶液溶解荧光素粉末(Yeasen，40903ES03)后，加HCl调节PH至7.4后避光分装，冻于-80℃，需要时取出预热到37℃备用。使用小动物光学成像仪(Perkin Elmer，IVIS Spectrum)，待检测的小鼠在成像前15分钟腹腔注射荧光素 酶底物，使用异氟烷麻醉后取仰卧位进行曝光检测。结果显示，小鼠体内荧光素酶报告基因信号在LMI070给药2天后达到高峰，1周后恢复本底水平。第一次LMI070给药一个月后，第二次LMI070给药能再次增强报告基因信号。
本实施例的结果显示，本发明的DM
st元件可通过多次给药的方式控制目的基因表达，且腹腔注射和口服施用小分子药物均能通过DM
st元件控制目的基因的表达，如增强报告基因信号。
实施例4心脏特异性的DM
st元件激活
在某些情况下，目的基因在特定的组织或器官中特异性表达是有利的。为了实现体内DM
st元件在组织或器官中特异性表达，发明人串联心肌细胞特异性表达的Tnnt2启动子、DM
st元件和GFP报告基因，包装了新的AAV9载体，进行小鼠体内研究(图4A)。
AAV质粒构建的方法如下：DM
st序列DNA片段委托苏州金唯智生物科技有限公司(GENEWIZ，中国，江苏苏州)进行基因合成。cTnnt2/hTnnt2和GFP序列来自Addgene公开的数据源。DNA拼接和质粒构建通过无缝克隆反应构造。
用上述AAV对P1小鼠腹腔注射，六天后腹腔注射50mg/kg LMI070，给LMI0702天后取心脏组织，进行qPCR分析。qPCR的方法如下，RT-qPCR使用Perfect Start Green qPCR Super Mix(+DveII)(TransGen，AQ602-24)试剂。qPCR机器为AriaMx Real-Time PCR System(Agilent Technologies)。qPCR中，GAPDH的引物为5′-CAACTCCCTCAAGATTGTCAGCAA-3′(SEQ ID NO:11)和5′-GGCATGGACTGTGGTCATGA-3′(SEQ ID NO:12)；检测DM
st-GFP报告系统的引物为5′-GAGGAGCAATGCAGCAGTCC-3′(SEQ ID NO:13)和5′-CAAGTTGTCAAACCCTCTTC-3′(SEQ ID NO:14)。qPCR的结果显示，在mRNA剪接水平，LMI070在心脏中提高DM
st元件假外显子剪入程度约六倍(图4B)。
同时取心脏、肝脏、脾脏、肺、肾、脑、骨骼肌等器官，用4％多聚甲醛固定，用OCT包埋，然后制备冰冻切片，用DAPI染细胞核，进行组织荧光成像分析。心脏组织切片成像结果显示LMI070增强GFP的表达(图4C)，而在肝脏、脾脏、肺、肾、脑、骨骼肌等器官中，没有检测到LMI070增强的GFP信号(图4D)。
因此，根据上述实验和图4B、图4D的结果，可以确认本实施例的AAV载体在小鼠体内实现了心脏特异性的DM
st元件激活。
Claims (30)
- 一种核酸分子，包含可变剪接调节元件和位于所述可变剪接调节元件3'端的目的基因；所述可变剪接调节元件从5'端至3'端依次包含第一外显子、第一内含子、假外显子、第二内含子、第二外显子；所述假外显子的序列如SEQ ID No:1所示，并且所述假外显子和所述第二内含子的交界处序列为AGAGTA，其中AGA属于所述假外显子，GTA属于所述第二内含子；所述第一外显子的序列为SEQ ID No:2或包含SEQ ID No:2或为与SEQ ID No:2具有至少95％同一性的序列，并且所述第一外显子的序列紧接所述第一内含子的序列为TAG；所述第一内含子的序列为SEQ ID No:3或包含SEQ ID No:3或为与SEQ ID No:3具有至少95％同一性的序列，并且所述第一内含子的序列紧接所述第一外显子的序列为GTA，所述第一内含子的序列紧接所述假外显子的序列为CAG；所述第二内含子的序列为SEQ ID No:4或包含SEQ ID No:4或为与SEQ ID No:4具有至少95％同一性的序列，并且所述第二内含子的序列紧接所述第二外显子的序列为CAG；所述第二外显子的序列为SEQ ID No:5或包含SEQ ID No:5或为与SEQ ID No:5具有至少95％同一性的序列，并且所述第二外显子的序列紧接所述第二内含子的序列为GAA；所述第一外显子、所述第一内含子、所述第二内含子、所述第二外显子中均不含有ATG序列；其中所述目的基因为一个或多个编码蛋白的基因和/或编码RNA的基因，并且所述编码蛋白的基因中的第一个起始密码子ATG被删除。
- 根据权利要求1所述的核酸分子，其中，所述可变剪接调节元件的序列为SEQ ID No:6或包含SEQ ID No:6。
- 根据权利要求1或2中任一项所述的核酸分子，其中，所述可变剪接调节元件的3'端与所述目的基因的5'端之间不存在任何核酸序列。
- 根据权利要求1或2中任一项所述的核酸分子，其中，所述可变剪接调节元件的3'端与所述目的基因的5'端之间存在编码蛋白酶切割位点的序列，其中所述蛋白酶切割位点被哺乳动物蛋白酶切割或被自切割，并且所述编码蛋白酶切割位点的序列的 碱基数是3的整数倍。
- 根据权利要求1或2中任一项所述的核酸分子，其中，所述可变剪接调节元件的5'端可操作地连接有启动子序列。
- 根据权利要求5所述的核酸分子，其中，所述启动子序列为哺乳动物细胞组成型启动子、哺乳动物细胞的特异性启动子、哺乳动物非编码RNA启动子、或原核细胞启动子。
- 根据权利要求6所述的核酸分子，其中所述哺乳动物细胞组成型启动子为CMV、CAG、CBG、EF1a、PGK1或Ubc；所述哺乳动物细胞的特异性启动子为Tnnt2、Nppa、Myl2、Mck、Nkx2.5、Syn、Mecp2、TBG、Pdx1、K14、Rpe65或SP-C；所述哺乳动物非编码RNA启动子为U6、H1；所述原核细胞启动子为T7、T3、SP6。
- 根据权利要求1或2中任一项所述的核酸分子，其中，所述核酸分子进一步包含转录后调节元件。
- 根据权利要求8所述的核酸分子，其中，所述转录后调节元件(PRE)包含衍生自乙型肝炎(HPRE)、蝙蝠(BPRE)、地松鼠(GSPRE)、北极松鼠(ASPRE)、鸭(DPRE)、黑猩猩(CPRE)和长毛猴(WMPRE)或土拨鼠(WPRE)的PRE，任选地，其中所述转录后调节元件设置在所述目的基因的3'端。
- 根据权利要求1或2中任一项所述的核酸分子，其中，所述核酸分子进一步包含多腺苷酸化信号(polyA)，任选地其中所述polyA设置在所述目的基因的3'端。
- 根据权利要求10所述的核酸分子，所述polyA信号是SV40polyA、人生长激素(HGH)polyA、或牛生长激素(BGH)polyA、β-珠蛋白polyA、α-珠蛋白polyA、卵清蛋白polyA、κ-轻链polyA、和合成polyA。
- 根据权利要求1或2中任一项所述的核酸分子，其中，所述可变剪接调节元件能够与可变剪接调节小分子药物结合，所述可变剪接调节小分子药物为LMI070、或与LMI070在所述可变剪接调节元件上具有相同结合位点的衍生物、或LMI070药学上可接受的盐，或者所述可变剪接调节小分子药物为Risdiplam、或与Risdiplam在所述可变剪接调节元件上具有相同结合位点的衍生物、或Risdiplam药学上可接受的盐。
- 根据权利要求1或2中任一项所述的核酸分子，其中，所述编码蛋白的基因为编码TALEN、ZFN、Cas9、Cas12、Cas13蛋白或其相同功能衍生物的基因，和/或编码治疗蛋白的基因；所述编码抑制RNA的基因为编码miRNA、shRNA、lncRNA的基因。
- 一种转录物，其为根据权利要求1-13中任一项所述的核酸分子在可变剪接调节小分子药物存在时的转录物，或者为根据权利要求1-13中任一项所述的核酸分子在可变剪接调节小分子药物不存在时的转录物；所述可变剪接调节小分子药物为LMI070、或与LMI070在所述可变剪接调节元件上具有相同结合位点的衍生物、或LMI070药学上可接受的盐，或者所述可变剪接调节小分子药物为Risdiplam、或与Risdiplam在所述可变剪接调节元件上具有相同结合位点的衍生物、或Risdiplam药学上可接受的盐。
- 一种载体，包含根据权利要求1-13中任一项所述的核酸分子或根据权利要求14中所述的转录物。
- 根据权利要求15所述的载体，其中，所述载体是DNA或RNA载体，或者所述载体是双链的或单链的。
- 根据权利要求15所述的载体，其中，所述载体是病毒载体。
- 根据权利要求17所述的载体，其中，所述病毒载体是腺相关病毒(AAV)载体、嵌合AAV载体、腺病毒载体、逆转录病毒载体、慢病毒载体、DNA病毒载体、单纯疱疹病毒载体、杆状病毒载体、或其任何突变体或衍生物。
- 一种重组病毒，包含根据权利要求1-13中任一项所述的核酸分子、或根据权利要求14中所述的转录物、或根据权利要求15-18中任一项所述的载体。
- 根据权利要求19所述的重组病毒，其中，所述重组病毒是腺相关病毒(AAV)、嵌合AAV、腺病毒、逆转录病毒、慢病毒、DNA病毒、单纯疱疹病毒、杆状病毒、或其任何突变体或衍生物。
- 根据权利要求20所述的重组病毒，其中，所述腺相关病毒为以下的一种或多种：AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAVrh8、AAVrh10、AAVrh36、AAVrh37、AAV-DJ、AAV-DJ/8、AAV.Anc80、AAV.Anc80L65、AAV-PHP.B、AAV-PHP.B2、AAV-PHP.B3、AAV-PHP.A、AAV-PHP.eB、AAV-PHP.S、AAV2i8、MyoAAV、AAVMYO衣壳血清型、或其变体。
- 一种细胞，包含一种或多种根据权利要求1-13中任一项所述的核酸分子、或根据权利要求14中所述的转录物、或一种或多种根据权利要求15-18中任一项所述的载体、或一种或多种根据权利要求19-21中任一项所述的重组病毒。
- 根据权利要求22所述的细胞，其中，所述细胞为人细胞。
- 根据权利要求23所述的细胞，其中，所述人细胞为心脏细胞、肌肉细胞、神 经元、肝细胞、肺细胞、或肾细胞。
- 一种药物组合物，所述药物组合物包含一种或多种根据权利要求1-13中任一项所述的核酸分子、或根据权利要求14中所述的转录物、或一种或多种根据权利要求15-18中任一项所述的载体、或一种或多种根据权利要求19-21中任一项所述的重组病毒、或一种或多种根据权利要求22-24中任一项所述的细胞。
- 一种基于可变剪接调节小分子药物调节目的基因的表达量的方法，包括：使一种或多种根据权利要求1-13中任一项所述的核酸分子、或根据权利要求14中所述的转录物、或一种或多种根据权利要求15-18中任一项所述的载体、或一种或多种根据权利要求19-21中任一项所述的重组病毒、或一种或多种根据权利要求22-24中任一项所述的细胞、或一种或多种根据权利要求25所述的药物组合物与一种或多种可变剪接调节小分子药物接触；其中，所述可变剪接调节小分子药物为LMI070、或与LMI070在所述可变剪接调节元件上具有相同结合位点的衍生物、或LMI070药学上可接受的盐，或者所述可变剪接调节小分子药物为Risdiplam、或与Risdiplam在所述可变剪接调节元件上具有相同结合位点的衍生物、或Risdiplam药学上可接受的盐。
- 一种治疗有需要的受试者的基因治疗方法，包括：向所述受试者施用一种或多种根据权利要求1-13中任一项所述的核酸分子、或根据权利要求14中所述的转录物、或一种或多种根据权利要求15-18中任一项所述的载体、或一种或多种根据权利要求19-21中任一项所述的重组病毒、或一种或多种根据权利要求22-24中任一项所述的细胞、或一种或多种根据权利要求25所述的药物组合物，以及一种或多种可变剪接调节小分子药物；其中，所述可变剪接调节小分子药物为LMI070、或与LMI070在所述可变剪接调节元件上具有相同结合位点的衍生物、或LMI070药学上可接受的盐，或者所述可变剪接调节小分子药物为Risdiplam、或与Risdiplam在所述可变剪接调节元件上具有相同结合位点的衍生物、或Risdiplam药学上可接受的盐。
- 一种试剂盒，包含：(1)一种或多种根据一种或多种根据权利要求1-13中任一项所述的核酸分子、或根据权利要求14中所述的转录物、或一种或多种根据权利要求15-18中任一项所述的载体、或一种或多种根据权利要求19-21中任一项所述的重组病毒、或一种或多种根据权利要求22-24中任一项所述的细胞、或一种或多种根据权利要求25所述的 药物组合物；和(2)一种或多种可变剪接调节小分子药物；其中，所述可变剪接调节小分子药物为LMI070、或与LMI070在所述可变剪接调节元件上具有相同结合位点的衍生物、或LMI070药学上可接受的盐，或者所述可变剪接调节小分子药物为Risdiplam、或与Risdiplam在所述可变剪接调节元件上具有相同结合位点的衍生物、或Risdiplam药学上可接受的盐。
- 物质用于调节目的基因表达量、或者用于制备用于治疗有需要的受试者的基因治疗药物的用途，其中，所述物质包含：(1)一种或多种根据权利要求1-13中任一项所述的核酸分子、或一种或多种根据权利要求15-18中任一项所述的载体、或一种或多种根据权利要求19-21中任一项所述的重组病毒、或一种或多种根据权利要求22-24中任一项所述的细胞、或一种或多种根据权利要求25所述的药物组合物；和(2)可变剪接调节小分子药物；其中所述可变剪接调节小分子药物为LMI070、或与LMI070在所述可变剪接调节元件上具有相同结合位点的衍生物、或LMI070药学上可接受的盐，或者所述可变剪接调节小分子药物为Risdiplam、或与Risdiplam在所述可变剪接调节元件上具有相同结合位点的衍生物、或Risdiplam药学上可接受的盐。
- 一种用于检测根据权利要求1-13中任一项所述的核酸分子、或一种或多种根据权利要求15-18中任一项所述的载体、或一种或多种根据权利要求19-21中任一项所述的重组病毒、或一种或多种根据权利要求22-24中任一项所述的细胞、或一种或多种根据权利要求25所述的药物组合物的方法，其中，所述方法使用了PCR，所述PCR中使用了如SEQ ID NO:7所示的序列作为上游引物。
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