WO2023078220A1 - 分离的核酸分子及其应用 - Google Patents

分离的核酸分子及其应用 Download PDF

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WO2023078220A1
WO2023078220A1 PCT/CN2022/128833 CN2022128833W WO2023078220A1 WO 2023078220 A1 WO2023078220 A1 WO 2023078220A1 CN 2022128833 W CN2022128833 W CN 2022128833W WO 2023078220 A1 WO2023078220 A1 WO 2023078220A1
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nucleic acid
acid molecule
isolated nucleic
vector
gene
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PCT/CN2022/128833
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English (en)
French (fr)
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余腾辉
杨舟
张江红
申立军
饶毅
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苏州华毅乐健生物科技有限公司
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Priority to CN202280071109.7A priority Critical patent/CN118139980A/zh
Publication of WO2023078220A1 publication Critical patent/WO2023078220A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This application relates to the field of biomedicine, in particular to an isolated nucleic acid molecule and its application.
  • liver gene therapy has been used experimentally and clinically to treat a series of conditions and diseases, including related diseases caused by dysfunction of protein synthesis in the liver. Due to the post-translational modification function of the liver, it is an important target organ for gene therapy of inherited blood diseases. Liver gene therapy can introduce functional genes into individuals with gene defects and make them express efficiently in vivo for a long time. However, the main factor limiting liver gene therapy at present is the low level of gene expression in the liver. One of the reasons for the low level of expression is that the transcriptional activity is not strong. required.
  • the gene of interest is typically included in a construct that also contains various regulatory elements necessary for expression of the gene of interest.
  • These regulatory elements can include, for example, promoters, enhancers, initiation signals, termination signals, and other regulatory elements.
  • the ideal combination of elements can not only promote the stable expression of the target gene in the host cell, but also its expression level is sufficient to realize its function or activity. Promoters and other regulatory elements determine cell type specificity, transduction efficacy, and expression levels and duration. Stable and efficient expression of target genes in host cells may be difficult to achieve. Therefore, there is an urgent need to develop nucleic acid molecules or vectors that are conducive to the stable and high-efficiency expression of target genes in host cells.
  • the application provides an isolated nucleic acid molecule, which may comprise a liver-specific expression regulatory element, a gene of interest (for example, a polynucleotide encoding a B domain-deleted FVIII variant) and a polyadenylation signal sequence (for example, Ks13poly A), the target gene in the isolated nucleic acid molecule has a higher expression level and/or activity.
  • a gene of interest for example, a polynucleotide encoding a B domain-deleted FVIII variant
  • a polyadenylation signal sequence for example, Ks13poly A
  • the target gene in the isolated nucleic acid molecule has a higher expression level and/or activity.
  • the liver-specific expression control element of the present application can increase the expression level and/or activity of the target gene.
  • the polyadenylation signal sequence using a nucleotide sequence such as SEQ ID NO: 8 the polyadenylation signal sequence of the present application can increase the expression level and/
  • the present application provides an isolated nucleic acid molecule, the isolated nucleic acid molecule comprises a liver-specific expression regulatory element and a target gene operably linked to the liver-specific expression regulatory element in a 5' to 3' direction, wherein Compared with the expression control element with a nucleotide sequence as shown in SEQ ID NO: 10, the liver-specific expression control element increases the expression level and/or activity of the target gene by at least 50%.
  • the liver-specific expression regulatory element comprises: a) a promoter derived from dog serpinA1 gene or a functional fragment thereof; and b) a promoter derived from Xenopus albumin gene or a functional fragment thereof Fragment;
  • the liver-specific expression regulatory element sequentially comprises in the direction of 5' to 3': the promoter derived from the dog serpinA1 gene or a functional fragment thereof, and the promoter derived from the Xenopus albumin The promoter of a gene or a functional fragment thereof.
  • the liver-specific expression regulatory element comprises the nucleotide sequence shown in SEQ ID NO:1.
  • the isolated nucleic acid molecule comprises a polyA signal sequence located 3' to the gene of interest.
  • the polyadenylation signal can increase the expression level and/or activity of the target gene by at least 20% (for example, at least 25%, at least 30% %, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 99%, or More).
  • the polyadenylation signal can increase the expression level and/or activity of the target gene by at least 20%.
  • the polyA signal is derived from the polyA signal of Common vole polyomavirus.
  • the polyA signal is derived from the polyA signal of Common vole polyomavirus isolate KS13.
  • the polyadenylation signal comprises the nucleotide sequence shown in SEQ ID NO:3.
  • the target gene encodes a target protein
  • the target protein includes a reporter protein, a therapeutic protein and/or a preventive protein.
  • the gene of interest encodes a B-domain deleted Factor VIII (FVIII) variant. In some embodiments, the gene of interest encodes GFP. In certain embodiments, the gene of interest encodes luciferase.
  • the FVIII variant comprises the amino acid sequence shown in SEQ ID NO:7.
  • the target gene comprises the nucleotide sequence shown in SEQ ID NO: 2 or 6.
  • the isolated nucleic acid molecule sequentially comprises in the 5' to 3' direction: the liver-specific expression regulatory element, the target gene, and the polyadenylation signal sequence.
  • the isolated nucleic acid molecule further comprises an AAV inverted terminal repeat ITR located 5' to the liver-specific expression regulatory element and 3' to the polyadenylation signal sequence .
  • the AAV ITR is derived from an AAV serotype selected from the group consisting of AAV5 and AAV2.
  • the AAV ITR comprises the nucleotide sequence shown in any one of SEQ ID NO: 4-5.
  • the isolated nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:9.
  • the application provides a vector comprising said isolated nucleic acid molecule.
  • the vector is a viral vector or a polynucleotide vector.
  • the vector is a plasmid, cosmid or transposon.
  • the vector is a viral vector
  • the viral vector includes an AAV vector
  • the AAV vector is an AAV8 vector.
  • the present application provides a host cell comprising said isolated nucleic acid molecule and/or said vector.
  • the present application provides a diagnostic or pharmaceutical composition comprising said isolated nucleic acid molecule, said vector, and/or said host cell.
  • the present application provides a method for expressing a target gene, the method comprising introducing the isolated nucleic acid molecule or the vector into a host cell, and allowing the target gene to be expressed in the host cell.
  • the present application provides a kit, said kit comprising said isolated nucleic acid molecule, said vector, and/or said host cell.
  • the present application provides a method for delivering said isolated nucleic acid molecule to a mammal or a mammalian cell, said method comprising administering said isolated nucleic acid molecule or said carrier to said mammal or mammalian cell , or contacting said mammal or mammalian cell with said isolated nucleic acid molecule or said vector.
  • the present application provides the use of the isolated nucleic acid molecule, the vector, and/or the host cell in the preparation of a medicament for treating, alleviating and/or preventing factors related to FVIII disease or condition.
  • the disease or condition comprises hemophilia A, thrombocytopenia, and/or coagulopathy.
  • the present application provides a method for treating, alleviating and/or preventing a disease or condition related to FVIII factor, said method comprising administering said isolated nucleic acid molecule, said carrier, and /or said host cell.
  • the disease or condition comprises hemophilia A, thrombocytopenia, and/or coagulopathy.
  • the present application provides the isolated nucleic acid molecule, the vector, and/or the host cell for treating, alleviating and/or preventing diseases or conditions related to FVIII factor.
  • the disease or condition comprises hemophilia A, thrombocytopenia, and/or coagulopathy.
  • Figure 1 shows that the isolated nucleic acid molecules described herein are capable of restoring FVIII expression in hemophilia A mice.
  • Figure 2 shows the expression activity of the target gene FVIII under different regulatory elements.
  • GT001 represents the specific expression vector group of this application
  • D2 represents the expression vector group after polyA replacement
  • D1 represents the expression vector group after promoter replacement
  • the blank control represents the FVIII knockout group
  • the wild type control represents the wild mouse group.
  • Figure 3 shows the expression activity of the expression nucleic acid molecule of the present application for optionally expressing a target gene.
  • the wild type control means the wild mouse group
  • the blank control means the FVIII knockout group
  • D1-F9 means the expression vector after the promoter replacement is used to express the FIX group
  • GT001-F9 means the specific expression vector of this application is used to express the FIX group .
  • isolated generally refers to a biological component (such as a nucleic acid molecule, protein, virus, or cell) that has been purified to be substantially free of other cellular components (such as other chromosomal and extrachromosomal components) with which it is naturally associated. DNA and RNA, proteins and cells). Nucleic acid molecules and proteins that have been isolated include those purified by standard purification methods. The term also encompasses nucleic acid molecules and proteins produced by recombinant expression in host cells as well as chemically synthesized nucleic acid molecules and proteins.
  • dog serpinA1 gene generally refers to the gene encoding ⁇ 1-antitrypsin.
  • the dog may be a dog with the Latin name Canis lupus familiaris.
  • the "dog serpinA1 gene” may comprise the nucleotide sequence shown at position 63,388,498 to 63,400,377 of chromosome 8 (for the sequence, refer to NCBI accession number NC_006590.3) or a variant thereof.
  • the promoter of the "dog serpinA1 gene” may comprise the nucleotide sequence shown at position 63,398,647 to 63,398,492 of the reverse strand of chromosome 8 or a variant thereof.
  • Xenopus albumin gene generally refers to the nucleic acid sequence comprising GenBank accession number Z26825.1. Wherein, its promoter may comprise the nucleotide sequence from the 1540th to the 1605th position of the above sequence.
  • the term "codon” generally refers to an oligonucleotide consisting of three nucleotides encoding a defined amino acid. Due to the degeneracy of the genetic code, some amino acids are encoded by more than one codon. These different codons encoding the same amino acid have different relative usage frequencies in individual host cells. Thus, a particular amino acid can be encoded by a different set of codons. Likewise, the amino acid sequence of a polypeptide can be encoded by different nucleic acids. Thus, a particular amino acid may be encoded by a different set of codons, where each of these codons has a frequency of usage in a given host cell.
  • the term “codon-optimized” means replacing one, at least one, or more than one codon in a polypeptide-encoding nucleic acid with a different codon having a different usage frequency in the corresponding cell.
  • the term “codon-optimized polynucleotide” means that one, at least one, or more than one codon, a nucleic acid encoding a polypeptide that has been modified to have improved expression in a cell.
  • FVIII blood coagulation factor VIII
  • FVIII is a protein required for effective blood coagulation, and can function as a cofactor in coagulation.
  • a FVIII concentration of approximately 100 ng/ml in the blood is considered to be in the normal range.
  • Deficiency in FVIII is associated with hemophilia A, which results in severe disease when a subject has less than about 1 ng of FVIII per milliliter of blood.
  • FVIII variant generally refers to a protein that has undergone certain modifications on the basis of wild-type FVIII, such modification may be the deletion of its B domain.
  • FVIII variant refers to the FVIII protein with B domain deletion.
  • promoter generally refers to an essential nucleic acid sequence for driving transcription of a downstream (3' end) or upstream (5' end) nucleic acid sequence.
  • a promoter is usually located near the gene it transcribes. Promoters also include terminal enhancer or repressor elements that can be located several thousand base pairs from the start site of transcription.
  • the term "functional fragment” generally refers to a fragment of a polypeptide or polynucleotide that retains the same activity or capability as its larger counterpart (eg, a promoter).
  • the level of activity of a functional fragment may be the same, or less or greater than that of the larger counterpart.
  • a functional fragment of a promoter may consist of fewer polynucleotides than the promoter, but still retain the ability to drive transcription.
  • gene of interest generally refers to the exogenous DNA or cDNA contained in a nucleic acid molecule, vector, host cell or kit that can encode a gene product, which can be a polypeptide, protein or itself and has a function or active polynucleotides (eg, antisense DNA, miRNA, siRNA and shRNA).
  • a gene product which can be a polypeptide, protein or itself and has a function or active polynucleotides (eg, antisense DNA, miRNA, siRNA and shRNA).
  • reporter protein generally refers to a protein that, by its biochemical nature, provides an analytically identifiable signal, thereby allowing detection.
  • reporter proteins can be enzymes, fluorophores, chemiluminescent or electrochemiluminescent proteins.
  • therapeutic protein generally refers to a protein that can be used to treat, prevent or ameliorate a disease, condition or disorder.
  • prophylactic protein generally refers to a protein that can be used to prevent a disorder or disease before any symptoms of the disorder or disease are detected.
  • operably linked generally refers to a first nucleic acid sequence being operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • polyA signal sequence generally refer to nucleic acid sequences used to induce cleavage and polyadenylation of primary transcripts of specific nucleic acid sequence segments.
  • the polyadenylation signal sequence may be selected from the group derived from SV40, bovine growth hormone (BGH) gene, immunoglobulin gene, polyomavirus gene and/or thymidine kinase gene (tk, e.g. herpes simplex virus thymidine kinase polyadenylation polyadenylation signal sequence).
  • BGH bovine growth hormone
  • tk thymidine kinase gene
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host cell, which transfers an inserted nucleic acid molecule into and/or between host cells.
  • the vectors may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and vectors mainly used for expression of transcription and/or translation of DNA or RNA.
  • the carrier may also include a carrier having various functions as described above.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce the desired expression product by culturing an appropriate host cell containing the vector.
  • AAV vector or "viral vector” generally refers to vectors derived from naturally occurring and available adeno-associated viruses as well as artificial AAV.
  • the AAV may comprise different serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, as well as any AAV variant or mixture.
  • ITRs inverted terminal repeats
  • the term "host cell” generally refers to a cell, such as a mammalian cell, which has been transformed or is capable of being transformed with a nucleic acid sequence and thus expresses a gene of interest.
  • the "host cell” includes parent cells and their progeny cells, regardless of whether the progeny is completely identical to the original parent in terms of morphology or genetic structure, as long as the target gene can be transferred or the target gene already exists.
  • the present application includes variants of genes and proteins (eg, variants of the polynucleotides encoding proteins described herein) that retain one or more biological activities.
  • variants of proteins or polypeptides include proteins or polypeptides that have been or can be modified using recombinant DNA techniques such that the protein or polypeptide has altered or additional properties, for example, variants that confer enhanced protein stability on the protein in plasma Or impart enhanced activity to the protein.
  • a variant may differ from a reference sequence, such as from a naturally occurring polynucleotide, protein or peptide.
  • naturally occurring variant genes and non-naturally occurring variant genes will typically be at least about 50% identical, more typically at least about 70% identical, and even more typically at least about 80% identical to a reference gene Identical (90% or greater identity).
  • naturally occurring variant proteins and non-naturally occurring variant proteins will typically be at least about 70% identical, more typically at least about 80% identical, and even more typically at least about 90% or more identical to the reference protein.
  • the sequence is at least 60%, 70%, 75% or more identical (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% identical to a reference sequence) %, 99% or higher identity).
  • Procedures for introducing nucleotide and amino acid changes in polynucleotides, proteins or polypeptides are known to those skilled in the art (see, e.g., Sambrook et al. (1989)).
  • sequence similarity generally refers to the fact that two or more entities are identical in sequence when they are “aligned”.
  • identity generally refers to the fact that two or more entities are identical in sequence when they are “aligned”.
  • two polypeptide sequences are identical, they have the same amino acid sequence within at least the region or portion to which they are referenced.
  • two polynucleotide sequences are identical, they have the same polynucleotide sequence at least within the region or portion referred to.
  • Identity may be to a defined region (region or domain) of sequence.
  • a “region” or “region” of identity refers to the same portion of two or more referenced entities.
  • two protein or nucleic acid sequences have identity over one or more sequence regions or regions if they are identical over that region.
  • An "aligned" sequence refers to a plurality of polynucleotide or protein (amino acid) sequences, often containing corrective deletions or additional bases or amino acids (gaps), compared to a reference sequence.
  • the degree of identity (homology) between two sequences can be determined using computer programs and mathematical algorithms.
  • Such algorithms that calculate percent sequence identity (homology) typically calculate sequence gaps and mismatches for compared regions or regions.
  • the BLAST e.g., BLAST 2.0
  • search algorithm see, e.g., Altschul et al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI
  • kits generally refers to a combination of reagents and other materials. Kits are contemplated to include reagents such as buffers, protein stabilization reagents, signal generating systems (eg, fluorescent signal generating systems), antibodies, control proteins, and test containers (eg, microtiter plates, etc.). The term “kit” is not intended to be limited to a specific combination of reagents and/or other materials.
  • the kit further comprises instructions for using the reagents. Test kits may be packaged in any suitable manner, and generally have the components in a single container or, if necessary, in multiple containers, and an instruction sheet for performing the test. In some embodiments, the kit preferably also includes a positive control sample. Kits can be prepared in a variety of ways known in the art.
  • the term "administration” generally refers to delivering a substance (such as the isolated nucleic acid molecule or the vector described herein) to a human or animal in need thereof by any means known in the art.
  • Pharmaceutically acceptable carriers and formulations or compositions are also well known in the art.
  • Routes of administration may include: intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral or mucosal.
  • these terms may refer to the delivery of an isolated nucleic acid molecule or vector as described herein to a cell or cultured cell and/or to a cell or organ of a subject. Such administration or introduction can occur in vivo, in vitro or ex vivo.
  • the isolated nucleic acid molecules or vectors described herein can be introduced into cells by transfection, which generally refers to, by physical means (e.g., calcium phosphate transfection, electroporation, microinjection, or lipotransfer) infection) the insertion of heterologous DNA into cells; infection, which generally refers to the introduction by means of an infectious agent (i.e., a virus); or transduction, which generally refers to the stable infection of a cell by a virus, or the passage of genetic material by a virus The transfer of an agent (eg, phage) from one microorganism to another.
  • transfection which generally refers to, by physical means (e.g., calcium phosphate transfection, electroporation, microinjection, or lipotransfer) infection) the insertion of heterologous DNA into cells
  • infection which generally refers to the introduction by means of an infectious agent (i.e., a virus)
  • transduction which generally refers to the stable infection of a cell by a virus, or the passage of
  • the term "contacting" generally refers to introducing a substance (such as the isolated nucleic acid molecule described herein or the vector) into a subject (such as a mammal or mammalian cells), and bringing the substance into contact with the cells in vivo.
  • a substance such as the isolated nucleic acid molecule described herein or the vector
  • the contact can be direct or indirect.
  • direct injection of cells can be performed by microinjection.
  • the substance can be brought into contact with the cells in vivo by being provided in a medium surrounding the cells, or administered to a subject.
  • hemophilia generally refers to a coagulation disorder.
  • Hemophilia A generally refers to the recessive X-linked disorder in individuals lacking functional Factor VIII.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides an isolated nucleic acid molecule, which may comprise a liver-specific expression regulatory element, and a target gene operably linked to the liver-specific expression regulatory element, wherein the nucleoside
  • the liver-specific expression control element can increase the expression level and/or activity of the target gene by at least 50%.
  • the liver-specific expression regulatory element can increase the expression level or activity of the target gene by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 95%, or at least 99%.
  • the expression level can be measured in liver or hepatocytes, and can also be measured in blood.
  • expression control element generally refers to a nucleic acid sequence, such as an enhancer and a promoter, which affects the expression of an operably linked polynucleotide. These elements can operate in cis as well as in trans. Expression regulation can function at the levels of transcription, translation, splicing, message stability, etc. Typically, an expression regulatory element that regulates transcription is juxtaposed near the 5' end of (ie, "upstream") the transcribed polynucleotide. Expression control elements can also be located 3' to the transcribed sequence (ie, "downstream") or within the transcript (eg, within an intron).
  • tissue-specific expression regulatory element generally refers to an expression regulatory element active in a specific tissue or cell, such as active in liver, brain, central nervous system, spinal cord, eye or lung.
  • the liver-specific expression regulatory element may comprise a promoter, and the promoter may be located at the 5' end of the target gene and be operably linked with the target gene.
  • the promoter can affect the expression of the isolated nucleic acid molecules described herein.
  • the promoter can function at the level of transcription, translation, splicing, message stability, etc., thereby being able to drive the expression of the isolated nucleic acid molecules described herein in many different cell types.
  • the promoter may be a liver-specific promoter.
  • the liver-specific promoter refers to a promoter active in liver tissues or cells.
  • the liver-specific expression regulatory elements described herein may comprise a promoter derived from the dog serpinA1 gene or a functional fragment thereof.
  • the promoter derived from the dog serpinA1 gene may be a modified variant of the promoter of the wild-type dog serpinA1 gene.
  • the expression regulatory elements described herein may comprise a promoter derived from Xenopus vitellogenin A2 gene or a functional fragment thereof.
  • the expression regulatory element may comprise a promoter derived from the Xenopus albumin gene or a functional fragment thereof.
  • the promoter derived from the Xenopus albumin gene may be a modified variant of the promoter of the wild-type Xenopus albumin gene.
  • the liver-specific expression regulatory element described in the present application may comprise a) a promoter derived from dog serpinA1 gene or a functional fragment thereof; and b) a promoter derived from Xenopus albumin gene or a functional fragment thereof; and
  • the liver-specific expression regulatory element sequentially comprises in the direction of 5' to 3': the promoter derived from the dog serpinA1 gene or a functional fragment thereof, and the promoter derived from the Xenopus albumin gene promoter or functional fragments thereof.
  • the isolated nucleic acid molecule may further comprise an enhancer, and the enhancer may affect the expression of the isolated nucleic acid molecule described in the present application.
  • the enhancers can function at the level of transcription, translation, splicing, message stability, etc., thereby being able to drive expression of the isolated nucleic acid molecules described herein in many different cell types.
  • the enhancer may be located at the 5' end of the isolated nucleic acid molecule described herein.
  • the enhancer can be located 5' to the promoter and operably linked to the promoter.
  • the liver-specific expression regulatory element may comprise the nucleotide sequence shown in SEQ ID NO:1.
  • the liver-specific expression regulatory element may comprise at least 90% (such as at least 91%, at least 92%, at least 93%) of the nucleotide sequence shown in SEQ ID NO: 1 , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) homologous nucleic acid sequences.
  • the isolated nucleic acid molecule may comprise a polyadenylation signal sequence, for example derived from SV40, bovine growth hormone (BGH) gene, rabbit beta globulin (RBG) gene, polyoma virus and thymidine
  • BGH bovine growth hormone
  • RBG rabbit beta globulin
  • TK kinase gene
  • the polyadenylation signal sequence may be a polyadenylation signal derived from Common vole polyomavirus.
  • the polyA signal is derived from the polyA signal of Common vole polyomavirus isolate KS13.
  • the polyadenylation signal sequence may comprise the nucleotide sequence shown in SEQ ID NO: 3, or comprise at least 90% (such as at least 91%) of the nucleotide sequence shown in SEQ ID NO: 3 , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) homologous nucleic acid sequences.
  • the polyadenylation signal sequence (for example, KS13-polyA signal sequence) can increase the expression level and/or activity of the target gene in the vector by at least 20%.
  • the polyadenylation can increase the expression level or activity of the target gene by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.
  • the expression level can be measured in liver or hepatocytes, and can also be measured in blood.
  • polyadenylation signal sequences derived from SV40, bovine growth hormone (BGH) gene, rabbit beta globulin (RBG) gene, polyoma virus, and thymidine kinase gene (TK) or variants thereof
  • BGH bovine growth hormone
  • RBG rabbit beta globulin
  • TK thymidine kinase gene
  • the polyadenylation signal sequence can increase the expression level and/or activity of the target gene in the vector by at least 20%.
  • the isolated polynucleotide may comprise the liver-specific expression regulatory element and the polyadenylation signal sequence, and the liver-specific expression regulatory element may comprise SEQ ID NO:1
  • the nucleotide sequence shown, the polyadenylation signal sequence may comprise the nucleotide sequence shown in SEQ ID NO:3.
  • the isolated nucleic acid molecule may further comprise a target gene, which is operably linked to the liver-specific expression regulatory element.
  • the liver-specific expression regulatory element and the target gene may be directly adjacent, or separated by an inserted nucleotide.
  • the liver-specific expression regulatory element and the gene of interest may be separated by an intron, such as an intron described herein. In some cases, no intron is included between the liver-specific expression regulatory element and the gene of interest.
  • the target gene described in this application can encode polypeptide, protein, or polynucleic acid, or itself can be transcribed into a polynucleic acid with functionality or activity, for example, antisense nucleic acid or inhibitory oligonucleotide, including antisense DNA and RNA (eg, miRNA, siRNA and shRNA).
  • the gene of interest may encode a protein of interest, which may include a reporter protein.
  • the reporter protein can be an enzyme, a fluorescent label, a molecule containing a radioactive isotope, and a chemiluminescent or electrochemiluminescent molecule.
  • Exemplary reporter proteins include, but are not limited to, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), cyan fluorescent protein (CFP), enhanced Cyan fluorescent protein (eCFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), MmGFP (Zernicka-Goetz et al., Development, 124:1133-1137, 1997), dsRed, luciferase and ⁇ -galactosidase (lacZ).
  • GFP green fluorescent protein
  • eGFP enhanced green fluorescent protein
  • YFP yellow fluorescent protein
  • eYFP enhanced yellow fluorescent protein
  • CFP cyan fluorescent protein
  • eCFP enhanced Cyan fluorescent protein
  • BFP blue fluorescent protein
  • eBFP enhanced blue fluorescent protein
  • MmGFP Zernicka-Goetz et al., Development, 124:1133-1137, 1997)
  • dsRed luciferase and ⁇
  • the gene of interest may be a therapeutic gene.
  • the therapeutic gene may encode a therapeutic peptide, therapeutic polypeptide, therapeutic protein, or therapeutic polynucleic acid.
  • the therapeutic peptide, therapeutic polypeptide, or therapeutic protein can be a peptide, polypeptide or protein that can be used to restore or replace the function of a defective endogenous peptide, polypeptide or protein.
  • the therapeutic protein or therapeutic polynucleic acid can be used to alter the expression level and/or activity of one or more proteins or polynucleic acid in a host cell.
  • the gene of interest may be a preventive gene.
  • the prophylactic gene may encode a prophylactic peptide, a prophylactic polypeptide, a prophylactic protein, or a prophylactic polynucleic acid.
  • the prophylactic peptide, prophylactic polypeptide, or prophylactic protein may be a peptide, polypeptide or protein that can be used to restore or replace the function of a defective endogenous peptide, polypeptide or protein.
  • the prophylactic protein or polynucleic acid can be used to alter the expression level and/or activity of one or more proteins or polynucleic acid in the host cell.
  • the target protein may participate in or affect cell metabolism, immune response, hematopoietic function, inflammatory response, cell growth and/or proliferation, cell differentiation, and/or stress response.
  • Exemplary proteins of interest may include, but are not limited to: Factor FVIII, Factor FIX, Factor FVII, Factor FX, Interferon- ⁇ , Interferon- ⁇ , Interferon- ⁇ , Interferon- ⁇ , Interleukin 1 (IL-1), Interleukin Interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin IL-7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin IL-12, chemokine (CXC motif) ligand 5 (CXCL5), granulocyte colony-stimulating factor (G-C
  • the target gene may encode factor VIII (FVIII).
  • the gene of interest may encode a B-domain deleted Factor VIII (FVIII) variant.
  • the FVIII variant may comprise the amino acid sequence shown in SEQ ID NO: 7, or comprise an amino acid sequence having at least 90% (such as at least 91%, at least 92%, at least 93%) of the amino acid sequence shown in SEQ ID NO: 7 , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) homologous amino acid sequences.
  • the gene of interest may comprise the nucleotide sequence shown in SEQ ID NO: 6, or comprise at least 90% (such as at least 91%, at least 90%) of the nucleotide sequence shown in SEQ ID NO: 6 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) homologous nucleotide sequences.
  • the target gene may be a codon-optimized polynucleotide.
  • the gene of interest may comprise the nucleotide sequence shown in SEQ ID NO: 2, or comprise at least 90% (such as at least 91%, at least 90%) of the nucleotide sequence shown in SEQ ID NO: 2 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) homologous nucleotide sequences.
  • the isolated polynucleotide may sequentially comprise the liver-specific expression regulatory element, the target gene, and the polyadenylation signal sequence, and the liver
  • the specific expression control element may comprise the nucleotide sequence shown in SEQ ID NO:1
  • the polyadenylation signal sequence may comprise the nucleotide sequence shown in SEQ ID NO:3.
  • the isolated polynucleotide may sequentially comprise the liver-specific expression regulatory element, the target gene, and the polyadenylation signal sequence, and the liver
  • the specific expression control element can comprise the nucleotide sequence shown in SEQ ID NO:1
  • the polyadenylation signal sequence can comprise the nucleotide sequence shown in SEQ ID NO:3
  • the target gene can encode FVIII protein.
  • the target gene may comprise the nucleotide sequence shown in SEQ ID NO: 2 or 6.
  • certain components of the isolated nucleic acid molecule eg, expression regulatory elements and/or target genes
  • vectors eg, AAV vectors
  • certain components (e.g., expression regulatory elements and/or genes of interest) of the isolated nucleic acid molecule can be packaged into AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12 or AAV13 vectors.
  • certain components of the isolated nucleic acid molecule eg, expression regulatory elements and/or a gene of interest
  • the isolated nucleic acid molecule described herein may further comprise an AAV inverted terminal repeat ITR, which may be located 5' to the enhancer and 3' to the polyA signal sequence.
  • the AAV ITR can be derived from any serotype of AAV, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13, and their native or artificial variants.
  • the nucleic acid sequence contained in the AAV ITR described in the present application may be a nucleic acid sequence through nucleotide insertion, deletion, and/or substitution.
  • the AAV ITRs of the isolated nucleic acid molecule may be different or derived from different serotypes, as long as they have the desired function (eg, in gene therapy, capable of replicating and packaging the gene of interest).
  • the AAV ITRs described herein may be derived from an AAV serotype selected from the group consisting of AAV5 and AAV2.
  • the AAV ITR can comprise the nucleotide sequence shown in any one of SEQ ID NO: 4-5.
  • the nucleotide sequence can be prepared by cloning techniques, or produced synthetically. It can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (TAS), self-sustaining sequence replication system (3SR). Many methods of cloning, synthesis, amplification are known in the art.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self-sustaining sequence replication system
  • the isolated nucleic acid molecule described in the present application may sequentially comprise: the liver-specific expression regulatory element, the target gene and the polyadenylation signal sequence in the 5' to 3' direction.
  • the polyA signal sequence can be a KS13-polyA signal sequence.
  • the isolated nucleic acid molecule described in the present application may sequentially comprise: the liver-specific expression regulatory element, the target gene, and the KS13-polyA signal sequence in the 5' to 3' direction.
  • an isolated nucleic acid molecule described herein can comprise the nucleotide sequence shown in SEQ ID NO:9.
  • the present application also provides a vector, which may comprise the isolated nucleic acid molecule described in the present application.
  • the vector may be a viral vector, eg, an AAV vector (eg, an AAV8 vector), lentivirus, retrovirus, adenovirus, herpes virus, or hepatitis virus vector.
  • the vector may also be a polynucleotide vector, eg, a plasmid, cosmid or transposon.
  • vectors suitable for use have been extensively described and are well known in the art.
  • a vector described herein may include a prokaryotic replicon, ie, a nucleotide sequence having the ability to direct the host's own replication and maintenance in a prokaryotic host cell (eg, a bacterial host cell).
  • a prokaryotic host cell eg, a bacterial host cell
  • the vector may contain a shuttle element that renders the vector suitable for replication and integration in prokaryotes and eukaryotes.
  • the vector may also include a gene capable of expressing a detectable marker (eg, a drug resistance gene).
  • the vector may also have a reporter gene, eg, a gene that encodes a fluorescent or other detectable protein.
  • the vector may be a viral vector, eg, AAV, lentivirus, retrovirus, adenovirus, herpes virus, and hepatitis virus.
  • viral vectors comprising nucleic acid molecules (eg, the isolated nucleic acid molecules described herein) as part of the vector genome are well known in the art and will not require undue experimentation by those skilled in the art.
  • the vector may be a recombinant AAV virion packaging the isolated nucleic acid molecule described herein.
  • Methods of producing recombinant AAV can include introducing the isolated nucleic acid molecule described herein into a packaging cell line, the AAV cap and rep genes providing helper functions, and recovering the recombinant AAV from the packaging cell line.
  • Various types of cells can be used as packaging cell lines.
  • the present application also provides a host cell, which may comprise the isolated nucleic acid molecule or the vector described in the present application.
  • the host cell can be used to amplify, replicate, package and/or purify the nucleic acid molecule or vector.
  • the host cell can be used to express a gene of interest contained in the isolated nucleic acid molecule or vector.
  • an isolated nucleic acid molecule or vector described herein can be introduced into a host cell, eg, a liver cell.
  • a host cell eg, a liver cell.
  • the method can be an in vivo or in vitro method.
  • the host cells can include prokaryotic cells and eukaryotic cells.
  • the host cell can be a mammalian host cell.
  • the host cells may also be transfected with one or more plasmids or infected with one or more viruses that provide the necessary vectors for packaging. auxiliary molecules.
  • the host cell can stably express one or more accessory molecules from the genome.
  • suitable host cells for amplification, replication, packaging and/or purification of the vectors described in this application.
  • the host cell can be a liver-derived cell, such as HUH7 and HepG2 cells, or a liver cell isolated from a subject.
  • the present application also provides a method for expressing a target gene, the method comprising introducing the isolated nucleic acid molecule described in the present application or the vector described in the present application into a host cell, and making the target gene expressed in the expressed in the above host cells.
  • the present application also provides a diagnostic or pharmaceutical composition, which comprises the isolated nucleic acid molecule described in the present application, the vector, or the host cell.
  • AAV vectors and other compositions, agents, drugs, biologics (proteins) can be incorporated into the diagnostic and pharmaceutical compositions, eg, pharmaceutically acceptable carriers, excipients, diluents and adjuvants.
  • Carriers, excipients, diluents, and adjuvants may include buffers, antioxidants, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides, and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions, and / or nonionic surfactants).
  • the composition comprises a nucleic acid molecule or a nucleic acid molecule carrier (such as a plasmid)
  • the nucleic acid molecule or nucleic acid molecule carrier may be present as "naked DNA” or prepared in a delivery vehicle, such as microparticles or nanoparticles, including liposomes, gels, bundles, lipid particles, ceramic/inorganic particles, and virus-like particles.
  • compositions, methods and uses described in this application can be administered to subjects in need in sufficient or effective amounts.
  • Effective amount or “sufficient amount” means to provide, in single or multiple doses, alone or in combination with one or more other compositions (therapeutic agents such as drugs), treatments, regimens or regimens, pharmaceutical agents, Detectable responses of any duration (long-term or short-term), any measurable or detectable degree, or any duration (e.g., lasting minutes, hours, days, months, years, or cure) desired or desirable outcome or amount of benefit.
  • the route of administration is not particularly limited.
  • a therapeutically effective amount of a nucleic acid molecule or carrier can be administered, e.g., intramuscularly, intravaginally, intravenously, intraperitoneally, subcutaneously, epidermally, intradermally, rectally, intraocularly, pulmonary, intracranially, intraosseously, orally, orally, or nasally. , given to the subject.
  • the nucleic acid molecules or vectors may be administered in single or multiple doses and at different intervals.
  • kits comprising the isolated nucleic acid molecule, the vector, or the host cell.
  • the kits also typically include a label or insert including a description of the components or instructions for use of the components therein in vitro, in vivo or ex vivo.
  • the present application also provides a method for delivering the isolated nucleic acid molecule described in the present application to a mammal or a mammalian cell, the method comprising administering the nucleic acid molecule described in the present application to the mammal or a mammalian cell.
  • the isolated nucleic acid molecule or said vector, or said mammal or mammalian cell is contacted with said isolated nucleic acid molecule or said vector.
  • the method comprises administering the isolated nucleic acid molecule described herein or the vector to said mammal or mammalian secretory cells, or contacting said mammal or mammalian secretory cells with said isolated nucleic acid molecule.
  • the nucleic acid molecule or the vector comprises administering the isolated nucleic acid molecule or the vector described herein to the mammal or the endothelial cells of the mammal, or contacting the mammal or the endothelial cells of the mammal with the The isolated nucleic acid molecule or said vector.
  • the administration may include: intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral or mucosal administration.
  • the mode of administration may also include transfection, infection or transduction.
  • the contact can be direct or indirect.
  • direct injection of cells can be performed by microinjection.
  • the substance can be brought into contact with the cells in vivo by being provided in a medium surrounding the cells, or administered to a subject.
  • the present application also provides the application of the isolated nucleic acid molecule described in the present application, the vector, or the host cell in the preparation of medicines, and the medicines are used for treating, alleviating and/or preventing A disease or condition associated with FVIII factor.
  • the disease or condition may include hemophilia A, thrombocytopenia and/or coagulopathy.
  • the present application also provides a method for treating, alleviating and/or preventing diseases or conditions related to FVIII factors, the method comprising administering the isolated nucleic acid molecule described in the present application to a subject, said The vector, or the host cell.
  • the disease or condition may include hemophilia A, thrombocytopenia and/or coagulopathy.
  • the present application also provides the isolated nucleic acid molecule, the vector, or the host cell described in the present application, which can treat, alleviate and/or prevent diseases or conditions related to FVIII factor.
  • the disease or condition may include hemophilia A, thrombocytopenia and/or coagulopathy.
  • the isolated nucleic acid molecules described in the application are as follows from 5' to 3' directions:
  • the liver-specific expression control element (SEQ ID NO: 1)
  • the target gene (SEQ ID NO:2),
  • the KS13-polyA signal sequence (SEQ ID NO:3),
  • the 5' end of the liver-specific expression regulatory element and the 3' end of the KS13-polyA respectively have AAV ITR (the nucleic acid sequences are respectively 5'ITR: SEQ ID NO: 4, 3'ITR: SEQ ID NO: 5 ).
  • the nucleic acid molecule was named GT001.
  • comparison nucleic acid molecule D1 the liver-specific expression regulatory element in GT001 was replaced with a promoter whose nucleotide sequence was SEQ ID NO:10.
  • Construct comparison nucleic acid molecule D2 replace the KS13-polyA signal sequence in GT001 with poly A whose nucleotide sequence is SPA, SV 40 or BGH.
  • HEK293 cells were inoculated onto plates of DMEM medium containing 10% FBS at a concentration of 4x10 6 /100ml diameter, and cultured overnight at 37°C in a humidified environment containing 5% CO 2 .
  • HEK-293 cells containing rAAV8 were subjected to three freeze-thaw cycles, 50 U/mL Benzonase was added and treated at 37°C for 30 minutes to remove unencapsulated DNA, centrifuged at 3000g for 10 minutes to pellet the cells, and the supernatant was transferred for ultracentrifugation. Equipped with an iodixanol centrifugal system, add four gradient iodixanol solutions to a 33ml Optiseal tube (Beckman) sequentially in the order of 17%, 25%, 40%, and 60% with a 10ml syringe.
  • Example 2 The repair ability of the isolated nucleic acid molecule described in the application to the hemophilia A mouse model
  • the recombinant AAV8 vector obtained in Example 1 was injected into the hemophilia A disease model mouse through the tail vein at a dose of 4 ⁇ 10 11 vg/kg, and the FVIII activity in the serum was measured 2W after the injection to detect the symptoms of the mice.
  • the results shown in Figure 1 show that the isolated nucleic acid molecule GT001 described in this application can restore the expression of FVIII in hemophilia A mice.
  • the comparison nucleic acid molecules D1 and D2 obtained in Example 1, and the isolated nucleic acid molecule GT001 described in the present application were packaged with AAV8, and the recombinant AAV8 vector was injected into the hemophilia A disease model mouse through the tail vein for recovery. FVIII was expressed, and the results showed that the expression activity of the FVIII variant of the isolated nucleic acid molecule GT001 described in the present application was increased compared to the comparison nucleic acid molecules D1 and D2.
  • Figure 2 shows the expression activity of the target gene FVIII under different regulatory elements.
  • the expression effect of the D1 obtained by replacing the promoter of the present application with a control promoter is significantly lower than that of the specific expression vector of the present application; for another example, replacing the polyA signal sequence of the present application with a control polyA, such as SPA polyA, The expression effect of the obtained D2 is significantly lower than that of the specific expression vector of this application.
  • the results show that the isolated nucleic acid molecule GT001 specified in the present application is capable of restoring FVIII expression.
  • the expressed nucleic acid molecule of the present application can exhibit a similar effect of increasing expression activity.
  • Figure 3 shows the expression activity of the expression nucleic acid molecule of the present application for optionally expressing a target gene.
  • Various molecules were packaged with AAV8, and the recombinant AAV8 vector was injected into hemophilia B disease model mice through the tail vein at a dose of 6 ⁇ 10 11 vg/kg, and the FIX activity in serum was measured 2 weeks after the injection.

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Abstract

一种分离的核酸分子及其应用，该核酸分子包含肝脏特异性表达调控元件以及目的基因，且所述肝脏特异性表达调控元件能使所述目的基因的表达量和/或活性增加。还提供了一种包含所述分离的核酸分子的载体，诊断或药物组合物，及其应用。

Description

分离的核酸分子及其应用 技术领域
本申请涉及生物医药领域，具体地涉及一种分离的核酸分子及其应用。
背景技术
随着分子生物学的发展，基因疗法已经在实验和临床中用于治疗一系列病症和疾病，包括由于肝脏的蛋白合成功能障碍造成的相关疾病。由于肝脏所具有的蛋白翻译后的修饰功能使其成为基因治疗遗传性血液病的重要靶器官，肝脏基因疗法能将功能基因导入基因缺陷的个体并使其在体内长期高效表达。但目前限制肝脏基因治疗的主要因素是基因在肝脏内表达水平较低，造成低水平表达的原因之一是转录活性不强，为加强基因在体内的高水平表达，构建肝脏高效表达调控序列成为必需。
为了促进目的基因在宿主细胞中的表达，通常将目的基因包含在也含有表达目的基因所必需的各种调控元件的构建体中。这些调控元件可包括，例如，启动子、增强子、起始信号、终止信号和其他调节元件。理想的元件之间的共同作用不仅能促进目的基因在宿主细胞中的稳定表达，而且其表达水平足以实现其功能或活性。启动子以及其他调控元件决定了细胞类型的特异性、转导功效以及表达水平和持续时间。目的基因在宿主细胞中的稳定高效表达可能难以实现。因此，亟需开发有助于目的基因在宿主细胞中稳定且高效表达的核酸分子或载体。
发明内容
本申请提供了分离的核酸分子，所述核酸分子可包含肝脏特异性表达调控元件、目的基因(例如，编码B结构域缺失的FVIII变体的多核苷酸)和多聚腺苷酸信号序列(例如，Ks13poly A)，所述分离的核酸分子中的目的基因具有较高的表达量和/或活性。与使用核苷酸序列如SEQ ID NO:10所示的表达调控元件相比，本申请的肝脏特异性表达调控元件能够增加所述目的基因的表达量和/或活性。与使用核苷酸序列如SEQ ID NO:8所示的多聚腺苷酸信号序列相比，本申请的多聚腺苷酸信号序列能够增加所述目的基因的表达量和/或活性。
一方面，本申请提供了分离的核酸分子，所述分离的核酸分子以5’至3’方向包含肝脏特异性表达调控元件以及与所述肝脏特异性表达调控元件可操作连接的目的基因，其中与使用核苷酸序列如SEQ ID NO:10所示的表达调控元件相比，所述肝脏特异性表达调控元件使得所述目的基因的表达量和/或活性增加至少50％。
在某些实施方式中，所述肝脏特异性表达调控元件包含：a)源自狗serpinA1基因的启动子或其功能性片段；以及b)源自爪蟾白蛋白基因的启动子或其功能性片段；
在某些实施方式中，所述肝脏特异性表达调控元件以5’至3’的方向依次包含：所述源自 狗serpinA1基因的启动子或其功能性片段，所述源自爪蟾白蛋白基因的启动子或其功能性片段。
在某些实施方式中，所述肝脏特异性表达调控元件包含SEQ ID NO:1所示的核苷酸序列。
在某些实施方式中，所述分离的核酸分子包含多聚腺苷酸信号序列，所述多聚腺苷酸信号序列位于所述目的基因的3’端。
与现有技术中的多聚腺苷酸信号序列相比，所述多聚腺苷酸信号能够使得所述目的基因的表达量和/或活性增加至少20％(例如，至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少90％、至少99％或更多)。与BGH poly A和/sv40poly A相比，所述多聚腺苷酸信号能够使得所述目的基因的表达量和/或活性增加至少20％。
在某些实施方式中，所述多聚腺苷酸信号源自普通田鼠多瘤病毒(Common vole polyomavirus)的多聚腺苷酸信号。
在某些实施方式中，所述多聚腺苷酸信号源自普通田鼠多瘤病毒(Common vole polyomavirus)分离株KS13的多聚腺苷酸信号。
在某些实施方式中，所述多聚腺苷酸信号包含SEQ ID NO:3所示的核苷酸序列。
在某些实施方式中，所述目的基因编码目的蛋白质，且所述目的蛋白质包括报告蛋白质、治疗性蛋白质和/或预防性蛋白质。
在某些实施方式中，所述目的基因编码B结构域缺失的因子VIII(FVIII)变体。在某些实施方式中，所述目的基因编码GFP。在某些实施方式中，所述目的基因编码荧光素酶。
在某些实施方式中，所述FVIII变体包含SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述目的基因包含SEQ ID NO:2或6所示的核苷酸序列。
在某些实施方式中，所述的分离的核酸分子以5’至3’方向依次包含：所述肝脏特异性表达调控元件、所述目的基因以及所述多聚腺苷酸信号序列。
在某些实施方式中，所述分离的核酸分子还包含AAV末端反向重复ITR，所述ITR位于肝脏特异性表达调控元件的5’端以及所述多聚腺苷酸信号序列的3’端。
在某些实施方式中，所述AAV ITR源自选自下组的AAV血清型：AAV5和AAV2。
在某些实施方式中，所述AAV ITR包含SEQ ID NO:4-5中任一项所示的核苷酸序列。
在某些实施方式中，所述的分离的核酸分子其包含SEQ ID NO:9所示的核苷酸序列。
另一方面，本申请提供载体，所述载体包含所述分离的核酸分子。
在某些实施方式中，所述载体为病毒载体或多核苷酸载体。
在某些实施方式中，所述载体为质粒，cosmid或转座子。
在某些实施方式中，所述载体为病毒载体，且所述病毒载体包括AAV载体。
在某些实施方式中，所述AAV载体为AAV8载体。
另一方面，本申请提供宿主细胞，所述宿主细胞包含所述分离的核酸分子和/或所述载体。
另一方面，本申请提供诊断或药物组合物，所述诊断或药物组合物包含所述分离的核酸分子，所述载体，和/或所述宿主细胞。
另一方面，本申请提供表达目的基因的方法，所述方法包括将所述的分离的核酸分子或所述的载体引入宿主细胞，并使得所述目的基因在所述宿主细胞中表达。
另一方面，本申请提供试剂盒，所述试剂盒包含所述的分离的核酸分子，所述的载体，和/或所述的宿主细胞。
另一方面，本申请提供将所述分离的核酸分子传递到哺乳动物或哺乳动物细胞中的方法，所述方法包括向所述哺乳动物或哺乳动物细胞施用所述的分离核酸分子或所述载体，或使所述哺乳动物或哺乳动物细胞接触所述的分离核酸分子或所述载体。
另一方面，本申请提供所述分离的核酸分子，所述的载体，和/或所述的宿主细胞在制备药物中的应用，所述药物用于治疗、缓解和/或预防与FVIII因子相关的疾病或病症。
在某些实施方式中，所述疾病或病症包括血友病A、血小板减少症和/或凝血病。
另一方面，本申请提供治疗、缓解和/或预防与FVIII因子相关的疾病或病症的方法，所述方法包括向有需要的受试者施用所述分离的核酸分子，所述的载体，和/或所述的宿主细胞。
在某些实施方式中，所述疾病或病症包括血友病A、血小板减少症和/或凝血病。
另一方面，本申请提供所述分离的核酸分子，所述的载体，和/或所述的宿主细胞，用于治疗、缓解和/或预防与FVIII因子相关的疾病或病症。
在某些实施方式中，所述疾病或病症包括血友病A、血小板减少症和/或凝血病。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请所述分离的核酸分子能够恢复血友病A小鼠的FVIII表达。
图2显示的是不同调控元件下，目标基因FVIII的表达活性。其中GT001表示本申请特定的表达载体组，D2表示polyA替换后的表达载体组，D1表示启动子替换后的表达载体组，空白对照表示FVIII敲除组，野生型对照表示野生小鼠组。
图3显示的是本申请的表达核酸分子用于任选地表达目的基因的表达活性。其中野生型对照表示野生小鼠组，空白对照表示FVIII敲除组，D1-F9表示启动子替换后的表达载体用于表达FIX组，GT001-F9表示本申请特定的表达载体用于表达FIX组。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“分离的”通常指生物组分(如核酸分子、蛋白质、病毒或细胞)被纯化至基本上不含有在天然状态下与其相关的其它细胞成分(如其他染色体和染色体外的DNA和RNA、蛋白质和细胞)。已经分离的核酸分子和蛋白质包括由标准纯化方法纯化的那些。该术语还包含通过在宿主细胞中重组表达制得的核酸分子和蛋白质以及化学合成的核酸分子和蛋白质。
在本申请中，术语“狗serpinA1基因”通常指编码α1-抗胰蛋白酶的基因。在某些情形中，所述狗可以是拉丁名为Canis lupus familiaris的狗。在另一个情形中，所述“狗serpinA1基因”可以包含染色体8(序列可参考NCBI登录号NC_006590.3)的第63,388,498位至第63,400,377位所示的核苷酸序列或其变体。其中，所述“狗serpinA1基因”的启动子可包含所述染色体8反向链的第63,398,647至第63,398,492位所示的核苷酸序列或其变体。
在本申请中，术语“爪蟾白蛋白基因”通常指包含GenBank登录号为Z26825.1所示的核酸序列。其中，其启动子可包含上述序列第1540位至1605位的核苷酸序列。
在本申请中，术语“密码子”通常指由编码限定的氨基酸的三个核苷酸组成的寡核苷酸。由于遗传密码的简并性，一些氨基酸由多于一种密码子编码。这些编码相同氨基酸的不同密码子在个体宿主细胞中具有不同的相对使用频率。因此，特定氨基酸可以由一组不同的密码子编码。同样地，多肽的氨基酸序列可以由不同核酸编码。因此，特定氨基酸可以由一组不同的密码子编码，其中这些密码子中的每个具有给定的宿主细胞中的使用频率。在本申请中，术语“密码子经优化”表示用在相应细胞中具有不同使用频率的不同密码子替换多肽编码核酸中的一个、至少一个、或一个以上密码子。在本申请中，术语“密码子经优化的多核苷酸”表示通过用在细胞中具有不同的相对使用频率的编码相同氨基酸残基的密码子，替换亲代多肽编码核酸中的一个、至少一个、或一个以上密码子，已经改良而在细胞中具有改善的表达 的编码多肽的核酸。
在本申请中，术语“FVIII”、“FVIII因子”通常指凝血因子VIII，FVIII是血液有效凝结所需的蛋白质，可在凝结中发挥辅因子的功能。血液中大约100ng/ml的FVIII浓度被认为处于正常范围。FVIII的缺乏与血友病A有关，当受试者每毫升血液含有少于约1ng的FVIII时，会导致严重的疾病。
在本申请中，术语“FVIII变体”通常是指这样的一种蛋白，其在野生型FVIII的基础上进行了一定的修饰，这种修饰可以是使其B domain缺失。即所谓FVIII变体，指的是B domain缺失的FVIII蛋白。
在本申请中，术语“启动子”通常指用于推动下游(3’端)或上游(5’端)核酸序列转录的必要核酸序列。启动子通常位于其所转录的基因附近。启动子还包括可位于离转录起始位点几千个碱基对处的末端增强子或抑物元件。
在本申请中，术语“功能性片段”通常指保留了与其更大对应物(如，启动子)相同活性或能力的多肽或多核苷酸的片段。功能性片段的活性水平可以与所述更大对应物的活性相同，或比它更小或更大。例如，启动子的功能性片段可以是由比启动子更少的多核苷酸组成，但是仍然保留了推动转录的能力。
在本申请中，术语“目的基因”通常指核酸分子、载体、宿主细胞或试剂盒中包含的能编码基因产品的外源DNA或cDNA，所述基因产品可以是多肽、蛋白质或本身且具有功能或活性的聚核苷酸(例如，反义DNA，miRNA，siRNA和shRNA)。
在本申请中，术语“报告蛋白质”通常指通过生物化学本性提供分析上可鉴定的信号，从而容许检测的蛋白质。常用的报告蛋白质可以为酶、荧光团、化学发光或电化学发光蛋白质。
在本申请中，术语“治疗性蛋白质”通常指可用于治疗、防止或改善疾病、病况或病症的蛋白质。
在本申请中，术语“预防性蛋白质”通常指指在检测疾患或疾病的任何症状之前可被用于预防此疾患或疾病的蛋白质。
在本申请中，术语“可操作连接”通常指当第一核酸序列与第二核酸序列处于功能性关系时，第一核酸序列与第二核酸序列可操作连接。例如，如果启动子影响编码序列的转录或表达，那么启动子与编码序列可操作连接。
在本申请中，术语“多聚腺苷酸信号序列”、“polyA信号序列”通常指用来诱导特定核酸序列区段的初级转录物的切割和聚腺苷酸化的核酸序列。多聚腺苷酸信号序列可以选自衍生自SV40、牛生长激素(BGH)基因、免疫球蛋白基因、多瘤病毒基因和/或胸苷激酶基因(tk，例如单纯疱疹病毒胸苷激酶聚腺苷酸化信号)的多聚腺苷酸信号序列。
在本申请中，术语“载体”通常是指能够在合适的宿主细胞中自我复制的核酸分子，其将***的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA***细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还可以包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“AAV载体”或“病毒载体”通常指来源于天然存在且可用的腺相关病毒以及人工AAV的载体。所述AAV可包括不同的血清型AAVl，AAV2，AAV3，AAV4，AAV5，AAV6，AAV7，AAV8，AAV9，AAV10，AAV11，AAV12或AAV13，以及任何AAV变体或混合物。AAV基因组两端通常有末端反向重复序列(ITR)，术语“ITR”或“末端反向重复”是指存在于AAV和/或重组AAV中的核酸序列段，其可形成完成AAV溶解和潜伏生命周期所需的T形回文结构。
在本申请中，术语“宿主细胞”通常指已经用核酸序列转化或能够用核酸序列转化并且因而表达目的基因的细胞，如哺乳动物细胞。所述“宿主细胞”包括亲本细胞及其子代细胞，且不论所述子代是否在形态上或在遗传结构上与原始亲本完全相同，只要能够转入目的基因或目的基因已存在即可。
因此，本申请包括基因和蛋白质的变体(例如，本文所述的编码蛋白质的多核苷酸的变体)，其保留一种或多种生物活性。蛋白质或多肽的此类变体包括已经或可以使用重组DNA技术进行修饰的蛋白质或多肽，使得该蛋白质或多肽具有改变的或附加的属性，例如，变体赋予蛋白质在血浆中增强的蛋白质稳定性或赋予蛋白质增强的活性。变体可以不同于参照序列，如不同于天然存在的多核苷酸、蛋白质或肽。在核苷酸序列水平，天然存在的变体基因和非天然存在的变体基因与参照基因将典型地至少约50％相同，更典型地至少约70％相同，甚至更典型地至少约80％相同(90％或更高同一性)。在氨基酸序列水平上，天然存在的变异蛋白质和非天然存在的变异蛋白质与参照蛋白质将典型地至少约70％相同，更典型地至少约80％相同，甚至更典型地至少约90％或者有更高的同一性，但在非保守区域允许有大部分非同一性的区域(例如，相同部分小于70％，诸如小于60％,小于50％或者甚至小于40％)。在其他实施方式中，所述序列与参照序列具有至少60％，70％，75％或更高的同一性(例如，80％，85％，90％，95％，96％，97％，98％，99％或更高的同一性)。对于本领域技术人员而言，在多核苷酸、蛋白或多肽中导入核苷酸和氨基酸的变化的程序是已知的(参见，例如，Sambrook et al.(1989))。
在本申请中，术语“序列相似性”、“同一性”、“同源性”及其语法变体通常指两个或 更多个实体是“对齐”的序列时，它们是相同的。因此，例如，当两个多肽序列是相同的时，它们至少所参照的区域或部分之内具有相同的氨基酸序列。如果两个多核苷酸序列是相同的，则它们至少在所参照的区域或部分之内具有相同的多核苷酸序列。同一性可以是序列的所限定的区(区域或结构域)。同一性的“区”或“区域”是指两个或两个以上所参照的实体的相同的部分。因此，如果两个蛋白质或核酸序列在一个或多个序列区或区域内是相同的，则它们在该区域内有同一性。“对齐”的序列是指多个多核苷酸或蛋白质(氨基酸)序列，相比于参照序列，常含补正缺失或附加的碱基或氨基酸(间隙)。两个序列之间的同一性(同源性)的程度可以使用计算机程序和数学算法确定。计算百分比序列同一性(同源性)的这样的算法一般计算比较区域或区的序列间隙和不匹配。例如，BLAST(例如，BLAST 2.0)搜索算法(参见，例如，Altschul et al.,J.Mol.Biol.215:403(1990),公众可通过NCBI获得)具有示范性的搜索参数如下：不匹配-2，间隙打开5，间隙延伸2。
在本申请中，术语“试剂盒”通常指试剂和其他材料的组合。试剂盒预期可包含试剂，诸如缓冲剂、蛋白稳定试剂、信号产生体系(例如，荧光信号生成体系)、抗体、对照蛋白、以及测试容器(例如，微量滴定板等)。术语“试剂盒”不旨在限于试剂和/或其他材料的特定组合。在一个实施例中，试剂盒还包含使用试剂的说明。测试试剂盒可以任何合适的方式包装，通常，其具有处于单个容器或(必要时)处于多个容器的成分以及用于进行测试的一张说明书。在一些实施例中，试剂盒还优选地包括阳性对照样本。可以本领域中已知的多种方法来制备试剂盒。
在本申请中，术语“施用”通常是指通过本领域已知的任意途径，将物质(例如本申请所述的分离的核酸分子或所述的载体)递送给有此需要的人或动物。药用载体和制剂或组合物也是本领域众所周知的。给药途径可以包括：静脉内的、肌肉内的、真皮内的、皮下的、透皮的、粘膜的、瘤内的或粘膜的。或者，这些术语可以表示，将本申请所述的分离的核酸分子或载体递送给细胞或培养的细胞和/或受试者的细胞或器官。这样的施用或导入可以在体内、在体外或先体外后体内地发生。通过下述方式，可以将本申请所述的分离的核酸分子或载体导入细胞中：转染，其通常是指，通过物理方式(例如，磷酸钙转染、电穿孔、显微注射或脂转染)将异源DNA***细胞中；感染，其通常是指，借助于传染性病原体(即病毒)的导入；或转导，其通常是指，病毒对细胞的稳定感染，或遗传物质通过病毒剂(例如，噬菌体)从一种微生物向另一种微生物的转移。
在本申请中，术语“接触”通常是指通过本领域已知的任意途径，将物质(例如本申请所述的分离的核酸分子或所述的载体)引入受试者(例如，哺乳动物或哺乳动物细胞)，并且使所述物质在体内与细胞接触。所述接触可以是直接的，也可以是间接的。例如，可以通 过显微注射进行的细胞的直接注射。又例如，可以通过在包围细胞的培养基中提供，或给受试者施用，从而使物质在体内与细胞接触。
在本申请中，术语“血友病”通常指凝血紊乱。“血友病A”通常指缺乏功能性因子VIII的个体中的隐性伴X基因紊乱。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
分离的核酸分子
肝脏特异性表达调控元件
一方面，本申请提供一种分离的核酸分子，所述分离的核酸分子可以包含肝脏特异性表达调控元件，以及与所述肝脏特异性表达调控元件可操作连接的目的基因，其中与使用核苷酸序列如SEQ ID NO:10所示的表达调控元件相比，所述肝脏特异性表达调控元件可以使得所述目的基因的表达量和/或活性增加至少50％。例如，所述肝脏特异性表达调控元件可以使得所述目的基因的表达量或者活性增加至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、或至少99％。其中，所述表达量可以是在肝脏或者肝细胞中测得的，还可以是在血液中测得的。
在本申请中，术语“表达调控元件”通常指一段核酸序列，如增强子和启动子，其影响可操作连接的多核苷酸的表达。这些元件可顺式操作，也可反式操作。表达调控可在转录、翻译、剪接、消息稳定性等层级上起作用。通常，调节转录的表达调控元件并列靠近转录的多核苷酸的5’端(即“上游”)。表达调控元件也可位于转录序列的3’端(即，“下游”)或转录物内(例如，在内含子内)。表达调控元件的一个具体的实例是启动子，启动子通常位于转录序列的5’端。表达调控元件还可包括普遍存在的或混杂的启动子，从而能够驱动多核苷酸在许多不同的细胞类型中的表达。术语“组织特异性表达调控元件”通常指在特定的组织或细胞中活跃的表达调控元件，如在肝，脑，中枢神经***，脊髓，眼或肺中活跃的。
所述肝脏特异性表达调控元件可以包含启动子，所述启动子可以位于所述目的基因的5’端且与目的基因可操作连接。所述启动子可以影响本申请所述的分离的核酸分子的表达。例如，所述启动子可以在转录、翻译、剪接、消息稳定性等层级上起作用，从而能够驱动本申请所述的分离的核酸分子在许多不同的细胞类型中的表达。在本申请中，所述启动子可以为肝脏特异性启动子。其中，肝脏特异性启动子指的是在肝脏的组织或者细胞中活跃的启动子。
在某些情形中，本申请所述肝脏特异性表达调控元件可包含源自狗serpinA1基因的启动 子或其功能性片段。所述源自狗serpinA1基因的启动子可以是野生型狗serpinA1基因的启动子经过修饰的变体。在某些情形中，本申请所述表达调控元件可包含源自爪蟾卵黄蛋白原A2基因的启动子或其功能性片段。在某些情形中，所述表达调控元件可包含源自爪蟾白蛋白基因的启动子或其功能性片段。所述源自爪蟾白蛋白基因的启动子可以是野生型爪蟾白蛋白基因的启动子经过修饰的变体。
本申请所述肝脏特异性表达调控元件可包含a)源自狗serpinA1基因的启动子或其功能性片段；以及b)源自爪蟾白蛋白基因的启动子或其功能性片段；以及
在某些情形中，所述肝脏特异性表达调控元件以5’至3’的方向依次包含：所述源自狗serpinA1基因的启动子或其功能性片段，所述源自爪蟾白蛋白基因的启动子或其功能性片段。
此外，在本申请中，所述的分离的核酸分子还可以包含增强子，所述增强子可以影响本申请所述的分离的核酸分子的表达。例如，所述增强子可以在转录、翻译、剪接、消息稳定性等层级上起作用，从而能够驱动本申请所述的分离的核酸分子在许多不同的细胞类型中的表达。在某些情形中，所述增强子可以位于本申请所述的分离的核酸分子的5’端。例如，所述增强子可以位于所述启动子的5’端，并且与所述启动子可操作地连接。
在本申请中，所述肝脏特异性表达调控元件可以包含SEQ ID NO:1所示的核苷酸序列。在本申请中，在本申请中，所述肝脏特异性表达调控元件可以包含与SEQ ID NO:1所示的核苷酸序列具有至少90％(如至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％)同源性的核酸序列。
多聚腺苷酸信号序列
在本申请中，所述的分离的核酸分子可以包含多聚腺苷酸信号序列，例如源于SV40、牛生长激素(BGH)基因、兔β球蛋白(RBG)基因、多瘤病毒和胸苷激酶基因(TK)的多聚腺苷酸信号序列或其变体。
在本申请中，所述多聚腺苷酸信号序列可以为源自普通田鼠多瘤病毒(Common vole polyomavirus)的多聚腺苷酸信号。在某些实施方式中，所述多聚腺苷酸信号源自普通田鼠多瘤病毒(Common vole polyomavirus)分离株KS13的多聚腺苷酸信号。
例如，所述多聚腺苷酸信号序列可包含SEQ ID NO:3所示的核苷酸序列，或包含与SEQ ID NO:3所示的核苷酸序列具有至少90％(如至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％)同源性的核酸序列。
所述多聚腺苷酸信号序列(例如，KS13-polyA信号序列)可以使所述载体中的目的基因的表达量和/或活性增加至少20％。例如，所述多聚腺苷酸可以使得所述目的基因的表达量或者活性增加至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、 至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、或至少99％。其中，所述表达量可以是在肝脏或者肝细胞中测得的，还可以是在血液中测得的。
在某些情形中，与源于SV40、牛生长激素(BGH)基因、兔β球蛋白(RBG)基因、多瘤病毒和胸苷激酶基因(TK)的多聚腺苷酸信号序列或其变体相比，所述多聚腺苷酸信号序列(例如，KS13-polyA信号序列)可以使所述载体中的目的基因的表达量和/或活性增加至少20％。
在本申请中，所述分离的多核苷酸可以包含所述肝脏特异性表达调控元件和所述多聚腺苷酸信号序列，且所述肝脏特异性表达调控元件可包含SEQ ID NO:1所示的核苷酸序列，所述多聚腺苷酸信号序列可包含SEQ ID NO:3所示的核苷酸序列。
目的基因
在本申请中，所述分离的核酸分子还可包含目的基因，所述目的基因与所述肝脏特异性表达调控元件可操作连接。所述肝脏特异性表达调控元件和所述目的基因可以直接相邻，或被一段***的核苷酸分离。在某些情形中，所述肝脏特异性表达调控元件和所述目的基因可以被内含子分离，如本申请所述的内含子。在某些情形中，所述肝脏特异性表达调控元件和所述目的基因之间不包含内含子。
本申请所述目的基因可编码多肽、蛋白质，或多聚核酸，或本身可转录为具有功能性或活性的多聚核酸，例如，反义核酸或抑制性寡核苷酸，包括反义DNA和RNA(如，miRNA，siRNA和shRNA)。在某些情形中，所述目的基因可编码目的蛋白质，所述目的蛋白质可包括报告蛋白质。所述报告蛋白质可以是酶，荧光标记物，含有放射性同位素的分子，以及化学发光或电化学发光分子。示例性的报告蛋白包括但不限于绿色荧光蛋白(GFP)、增强的绿色荧光蛋白(eGFP)、黄色荧光蛋白(YFP)、增强的黄色荧光蛋白(eYFP)、青色荧光蛋白(CFP)、增强的青色荧光蛋白(eCFP)、蓝色荧光蛋白(BFP)、增强的蓝色荧光蛋白(eBFP)、MmGFP(Zernicka-Goetz et al.,Development,124:1133-1137,1997)、dsRed、荧光素酶和β-半乳糖苷酶(lacZ)。例如，可以是萤火虫荧光素酶和海肾荧光素酶。
在另一些情形中，所述目的基因可以是治疗性基因。所述治疗性基因可以编码治疗性肽，治疗性多肽，治疗性蛋白质，或治疗性多聚核酸。所述治疗性肽，治疗性多肽，或治疗性蛋白质可以是可用于恢复或替代有缺陷的内源性肽，多肽或蛋白质的功能的肽，多肽或蛋白质。在某些情形中，所述治疗性蛋白质或治疗性多聚核酸可以用于改变宿主细胞中的一种或多种蛋白质或多聚核酸的表达水平和/或活性。
在另一些情形中，所述目的基因可以是预防性基因。所述预防性基因可以编码预防性肽， 预防性多肽，预防性蛋白质，或预防性多聚核酸。所述预防性肽，预防性多肽，或预防性蛋白质可以是可用于恢复或替代有缺陷的内源性肽，多肽或蛋白质的功能的肽，多肽或蛋白质。在某些情形中，所述预防性蛋白质或预防性多聚核酸可以用于改变宿主细胞中的一种或多种蛋白质或多聚核酸的表达水平和/或活性。
在某些情形中，所述目的蛋白可以参与或影响细胞代谢，免疫应答，造血功能，炎症反应，细胞生长和/或增殖，细胞分化，和/或应激反应。示例性的目的蛋白质可包括但不限于：FVIII因子，FIX因子，FVII因子，FX因子，干扰素-α，干扰素-β，干扰素-γ，白细胞介素1(IL-1)，白细胞介素2(IL-2)，白细胞介素3(IL-3)，白细胞介素4(IL-4)，白细胞介素5(IL-5)，白细胞介素6(IL-6)，白细胞介素7(IL-7)，白细胞介素8(IL-8)，白细胞介素9(IL-9)，白细胞介素10(IL-10)，白细胞介素11(IL-11)，白细胞介素12(IL-12)，趋化因子(CXC基序)配体5(CXCL5)，粒细胞集落刺激因子(G-CSF)，粒细胞-巨噬细胞集落刺激因子(GM-CSF)，巨噬细胞集落刺激因子(M-CSF)，干细胞因子(SCF)，角质形成细胞生长因子(KGF)，单核细胞趋化蛋白-1(MCP-1)，肿瘤坏死因子(TNF)，afamin(AFM)，α-半乳糖苷酶A，aL-艾杜糖苷酶，溶酶体α-葡糖苷酶，苯丙氨酸羟化酶，脂蛋白脂酶，载脂蛋白，低密度脂蛋白受体(LDL-R)，白蛋白，葡萄糖-6-磷酸酶，抗体，纳米抗体，抗病毒显性失活蛋白，以及它们的片段，亚基或突变体。
在本申请中，所述目的基因可以编码因子VIII(FVIII)。例如，所述目的基因可以编码B结构域缺失的因子VIII(FVIII)变体。例如，所述FVIII变体可以包含SEQ ID NO:7所示的氨基酸序列，或者包含与SEQ ID NO:7所示的氨基酸序列具有至少90％(如至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％)同源性的氨基酸序列。
在本申请中，所述目的基因可以包含SEQ ID NO:6所示的核苷酸序列，或者包含与SEQ ID NO:6所示的核苷酸序列具有至少90％(如至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％)同源性的核苷酸序列。
在本申请中，所述目的基因可以是密码子经优化的多核苷酸。在本申请中，所述目的基因可以包含SEQ ID NO:2所示的核苷酸序列，或者包含与SEQ ID NO:2所示的核苷酸序列具有至少90％(如至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％)同源性的核苷酸序列。
在本申请中，所述分离的多核苷酸自5’端至3’端，可以依次包含所述肝脏特异性表达调控元件、目的基因和所述多聚腺苷酸信号序列，且所述肝脏特异性表达调控元件可包含SEQ ID NO:1所示的核苷酸序列，所述多聚腺苷酸信号序列可包含SEQ ID NO:3所示的核苷酸序 列。
在本申请中，所述分离的多核苷酸自5’端至3’端，可以依次包含所述肝脏特异性表达调控元件、目的基因和所述多聚腺苷酸信号序列，且所述肝脏特异性表达调控元件可包含SEQ ID NO:1所示的核苷酸序列，所述多聚腺苷酸信号序列可包含SEQ ID NO:3所示的核苷酸序列，所述目的基因可编码FVIII蛋白。例如，所述目的基因可以包含SEQ ID NO:2或6所示的核苷酸序列。
其他元件
在本申请中，为了便于将所述分离的核酸分子的某些组成部分(例如，表达调控元件和/或目的基因)包装入载体(例如AAV载体)。例如，可以将所述分离的核酸分子的某些组成部分(例如，表达调控元件和/或目的基因)包装入AAV1，AAV2，AAV3，AAV4，AAV5，AAV6，AAV7，AAV8，AAV9，AAV10，AAV11，AAV12或AAV13载体。例如，可以将所述分离的核酸分子的某些组成部分(例如，表达调控元件和/或目的基因)包装入AAV8载体。
本申请所述的分离的核酸分子还可以进一步包含AAV末端反向重复ITR，所述ITR可以位于所述增强子的5’端以及所述多聚腺苷酸信号序列的3’端。所述AAV ITR可源自任意一种血清型的AAV，包括但不限于，AAV1，AAV2，AAV3，AAV4，AAV5，AAV6，AAV7，AAV8，AAV9，AAV10，AAV11，AAV12或AAV13，以及其天然的或人工的变体。与野生型AAV ITR相比，本申请所述AAV ITR包含的核酸序列可以是经过核苷酸***，删除，和/或取代的核酸序列。且所述分离的核酸分子的所述AAV ITR可以是不同的或源自不同的血清型的，只要它们具有所期望的功能(如，在基因治疗中，能够复制和包装目的基因)即可。在某些情形中，本申请所述AAV ITR可以源自选自下组的AAV血清型：AAV5和AAV2。例如，所述AAV ITR可包含SEQ ID NO:4-5中任一项所示的核苷酸序列。
在一个具体的情形中，所述核苷酸序列可以通过克隆的技术来制备，或者合成生成。也可以通过扩增方法来制备。扩增方法包括聚合酶链反应(PCR)，连接酶链反应(LCR)，基于转录的扩增***(TAS)，自维持序列复制***(3SR)。许多克隆方法、合成、扩增方法是本领域公知的。
在本申请中，本申请所述分离的核酸分子以5’至3’方向依次可以包含：所述肝脏特异性表达调控元件、所述目的基因以及所述多聚腺苷酸信号序列。在某些情形中，所述多聚腺苷酸信号序列可以是KS13-polyA信号序列。例如，本申请所述分离的核酸分子以5’至3’方向依次可以包含：所述肝脏特异性表达调控元件、所述目的基因以及所述KS13-polyA信号序列。
例如，本申请所述的分离的核酸分子可以包含SEQ ID NO:9所示的核苷酸序列。
载体、宿主细胞和方法
在另一方面，本申请还提供了一种载体，其可以包含本申请所述的分离的核酸分子。所述载体可以为病毒载体，例如，AAV载体(例如，AAV8载体)、慢病毒、逆转录病毒、腺病毒、疱疹病毒或肝炎病毒载体。所述载体还可以为多核苷酸载体，例如，质粒，cosmid或转座子。
适合使用的载体已被广泛描述并且是本领域公知的。本领域技术人员将理解，包含本申请所述的分离的核酸分子的载体还可包含可用于载体在原核和/或真核细胞中复制所需的其他序列和元件。例如，本申请所述载体可包括原核复制子，即，具有在原核宿主细胞(例如，细菌宿主细胞)中引导宿主自身复制和维持的能力的核苷酸序列。所述复制子在本领域是公知的。在某些情形中，所述载体可包含穿梭元件，其使载体适于在原核生物和真核生物中复制和整合。此外，所述载体还可包括能够表达可检测标记(例如，药物抗性基因)的基因。所述载体还可具有报告基因，如，可编码荧光或其他可检测蛋白的基因。
在某些情形中，所述载体可以是病毒载体，例如，AAV、慢病毒、逆转录病毒、腺病毒、疱疹病毒和肝炎病毒。用于产生包含核酸分子(例如，本申请所述分离的核酸分子)作为载体基因组一部分的病毒载体的方法是本领域公知的，并且本领域技术人员可无需进行过多的实验。在另一些情形中，所述载体可以是包装了本申请所述分离的核酸分子的重组AAV病毒粒子。产生重组AAV的方法可包括将本申请所述分离的核酸分子引入包装细胞系，AAV cap和rep基因提供辅助功能，以及，从包装细胞系回收重组的AAV。可以使用各种类型的细胞作为包装细胞系。
另一方面，本申请还提供了一种宿主细胞，其可以包含本申请所述的分离的核酸分子或所述的载体。在某些情形中，所述宿主细胞可用于扩增，复制，包装/或纯化所述核酸分子或载体。在另一些情形中，所述宿主细胞可用于表达包含在所述分离的核酸分子或载体中的目的基因。例如，可以将本申请所述分离的核酸分子或载体导入宿主细胞，例如，肝脏细胞。本领域技术人员将理解将所述分离的核酸分子或载体导入宿主细胞所需的条件，以及支持或促进目的基因在细胞内表达的条件。此外，所述方法可以是体内或体外的方法。
所述宿主细胞可以包括原核细胞和真核细胞。在某些情况中，宿主细胞可以是哺乳动物宿主细胞。例如，当宿主细胞用于包装所述的病毒载体时，所述宿主细胞也可以用一种或多种质粒转染或用一种或多种病毒感染，所述病毒可提供用于包装的必要的辅助分子。在另一些的情况中，宿主细胞可以稳定地表达来自基因组的一种或多种辅助分子。本领域技术人员能够选择合适的宿主细胞用于本申请所述的载体的扩增，复制，包装和/或纯化。在一个特定的实例中，为了使所述分离的核酸分子或载体表达目的基因，宿主细胞可以是源自肝脏的细 胞，例如HUH7和HepG2细胞，或分离自受试者的肝细胞。
另一方面，本申请还提供了一种表达目的基因的方法，所述方法包括将本申请所述的分离的核酸分子或本申请所述的载体引入宿主细胞，并使得所述目的基因在所述宿主细胞中表达。
药物组合物和试剂盒
另一方面，本申请还提供了一种诊断或药物组合物，其包含本申请所述的分离的核酸分子，所述的载体，或所述的宿主细胞。AAV载体和其他组合物、药剂、药物、生物制剂(蛋白质)可并入所述诊断和药物组合物中，例如，药学上可接受的载体、赋形剂、稀释剂和佐剂。载体、赋形剂、稀释剂和佐剂可包括缓冲剂，抗氧化剂，蛋白质，亲水性聚合物，氨基酸，单糖，二糖和其他碳水化合物，螯合剂，糖醇，成盐抗衡离子，和/或非离子表面活性剂)。
在组合物包含核酸分子或核酸分子载体(例如质粒)的情况下，核酸分子或核酸分子载体可以作为“裸露DNA”存在或制备在递送载体中，例如微粒或纳米颗粒，包括脂质体、胶束、脂质颗粒、陶瓷/无机颗粒和病毒样颗粒。
本申请所述的药物组合物、方法和用途可以足够量或有效量给药于有此需要的受试者。“有效量”或“足够量”是指以单剂量或多剂量单独或与一种或一种以上其他组合物(诸如药物之类的治疗剂)、治疗、方案或治疗方案、药剂组合提供受试者中的任何持续时间(长期或短期)的可检测的反应、任何可测量或可检测程度或任何持续时间(例如，持续数分钟、数小时、数天、数月、数年或持续到治愈)的期望或理想的结果或者益处的量。给药途径没有特别限制。例如，治疗有效量的核酸分子或载体可以通过例如肌肉内，***内，静脉内，腹腔内，皮下，表皮，皮内，直肠，眼内，肺，颅内，骨内，口服，口腔或鼻腔，给予受试者。所述核酸分子或载体可以以单剂量或多剂量施用，并且以不同的间隔时间施用。
另一方面，本申请还提供了一种试剂盒，其包含所述分离的核酸分子，所述的载体，或所述的宿主细胞。所述试剂盒还通常包括标签或说明书，所述标签或说明书包括组分的描述或关于体外、体内或间接体内使用其中的组分的说明。
应用及用途
另一方面，本申请还提供了将本申请所述的分离的核酸分子传递到哺乳动物或哺乳动物细胞中的方法，所述方法包括向所述哺乳动物或哺乳动物细胞施用本申请所述的分离的核酸分子或所述的载体，或使所述哺乳动物或哺乳动物细胞接触所述的分离的核酸分子或所述的载体。
例如，所述方法包括向所述哺乳动物或哺乳动物的分泌细胞施用本申请所述的分离的核酸分子或所述的载体，或使所述哺乳动物或哺乳动物的分泌细胞接触所述的分离的核酸分子 或所述的载体。又例如，所述方法包括向所述哺乳动物或哺乳动物的内皮细胞施用本申请所述的分离的核酸分子或所述的载体，或使所述哺乳动物或哺乳动物的内皮细胞接触所述的分离的核酸分子或所述的载体。
所述施用可以包括：静脉内的、肌肉内的、真皮内的、皮下的、透皮的、粘膜的、瘤内的或粘膜等途径的施用。所述施用的方式还可以包括转染、感染或转导。
所述接触可以是直接的，也可以是间接的。例如，可以通过显微注射进行的细胞的直接注射。又例如，可以通过在包围细胞的培养基中提供，或给受试者施用，从而使物质在体内与细胞接触。
另一方面，本申请还提供了本申请所述的分离的核酸分子，所述的载体，或所述的宿主细胞在制备药物中的应用，所述药物用于治疗、缓解和/或预防与FVIII因子相关的疾病或病症。其中，所述疾病或病症可以包括血友病A、血小板减少症和/或凝血病。
另一方面，本申请还提供了一种治疗、缓解和/或预防与FVIII因子相关的疾病或病症的方法，所述方法包括向受试者施用本申请所述的分离的核酸分子，所述的载体，或所述的宿主细胞。其中，所述疾病或病症可以包括血友病A、血小板减少症和/或凝血病。
另一方面，本申请还提供了本申请所述的分离的核酸分子，所述的载体，或所述的宿主细胞，其治疗、缓解和/或预防与FVIII因子相关的疾病或病症。其中，所述疾病或病症可以包括血友病A、血小板减少症和/或凝血病。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的增加目的基因表达的核酸分子及其应用等，而不用于限制本申请发明的范围。
实施例
实施例1.重组AAV载体产生
核酸分子各元件核苷酸序列
本申请所述的分离的核酸分子从5’至3’方向依次为：
所述肝脏特异性表达调控元件(SEQ ID NO:1)
所述目的基因(SEQ ID NO:2)，
所述KS13-polyA信号序列(SEQ ID NO:3)，
所述肝脏特异性表达调控元件的5’端以及所述KS13-polyA的3’端分别有AAV ITR(核酸序列分别为5’ITR：SEQ ID NO:4，3’ITR：SEQ ID NO:5)。
将该核酸分子命名为GT001。
构建对比核酸分子D1：将GT001中的所述肝脏特异性表达调控元件替换为核苷酸序列为SEQ ID NO:10的启动子。
构建对比核酸分子D2：将GT001中的所述KS13-polyA信号序列替换为核苷酸序列为SPA、SV 40或BGH的poly A。
AAV8载体包装
将HEK293细胞以4x10 6/100ml直径的浓度接种至含有10％FBS的DMEM培养基的平板上，在37℃下含有5％CO 2的潮湿环境中培养过夜。第二天，制备含有本申请的核酸分子或对比例的核酸分子、AAV8衣壳蛋白和辅助质粒的PEI转染混合物，然后将转染混合物加入到细胞培养基中转染6小时后，用含有10％FBS的DMEM更换培养基，转染72h后收获细胞。用含有100mM氯化钠、2mM氯化镁和10mMTris的缓冲液(pH＝8)重悬，于-80℃保存。
AAV8载体的纯化和定量分析
将含有rAAV8的HEK-293细胞进行三次冻融循环，加入50U/mL Benzonase在37℃处理30min以除去未包封的DNA，3000g离心10min使细胞沉淀，上清转移进行超速离心。配备碘克砂醇离心体系，用10ml注射器将四种梯度的iodixanol溶液按照17％、25％、40％、60％的顺序依次加到33ml Optiseal tube(Beckman)。每种溶液都从底部慢慢加入Optiseal管中：6ml的17％，6ml的25％，5ml的40％，4ml的60％。加完后在Optiseal管顶部标记样品名称，在40％和60％的交界处划线标记。然后用试管小心将上清添加到离心管中，在14℃离心53000g，时间为2h40min。
将5ml注射器的针头沿之前标记的横线(40％和60％交界线)***Optiseal tub中，吸出40％部分的溶液(约2-3ml)至新的15ml管中。将病毒溶液加至已平衡过的100K Centrifuge filter中，补加1x PBS(10 -4F188)至约50ml线处，3500rpm,离心10min。去掉废液，再加满1x PBS(10-4F188)，3500rpm,离心10min。重复洗3次。加300-500ul的1x PBS(10-4F188)收集病毒。吸至1.5ml EP管中。然后根据说明书使用试剂盒对纯化的AVV载体基因组进行qPCR定量分析，测定病毒原液的滴度。
实施例2本申请所述分离的核酸分子对血友病A小鼠模型的修复能力
将实施例1得到的重组AAV8载体通过尾静脉注射至血友病A疾病模型小鼠，剂量为4×10 11vg/kg，分别于注射后2W测定血清中FVIII活性，检测小鼠症状。结果如图1显示本申请所述分离的核酸分子GT001能够恢复血友病A小鼠的FVIII表达。
实施例3本申请所述分离的核酸分子表达活性检测
类似的，将实施例1得到的对比核酸分子D1和D2，以及本申请所述分离的核酸分子GT001用AAV8包装，重组AAV8载体通过尾静脉注射至血友病A疾病模型小鼠，用于恢复FVIII表达，结果显示相比于对比核酸分子D1和D2，本申请所述分离的核酸分子GT001的FVIII变体的表达活性增加。
图2显示的是不同调控元件下，目标基因FVIII的表达活性。例如，将本申请的启动子替换为对照启动子构建得到的D1，其表达效果显著低于本申请特定的表达载体；又例如，将本申请的polyA信号序列替换为对照polyA，如SPA polyA，得到的D2，其表达效果显著低于本申请特定的表达载体。结果显示，本申请特定的所述分离的核酸分子GT001能够恢复FVIII表达。
实施例4本申请所述分离的核酸分子表达活性检测
将本申请的表达核酸分子中的FVIII基因，任选地替换为其它目的基因，例如FIX基因，本申请的表达核酸分子可以体现出类似的表达活性增加的效果。
图3显示的是本申请的表达核酸分子用于任选地表达目的基因的表达活性。将各种分子用AAV8包装，重组AAV8载体通过尾静脉注射至血友病B疾病模型小鼠，剂量为6×10 11vg/kg，分别于注射后2周测定血清中FIX活性。结果显示，将本申请特定的所述分离的核酸分子GT001中目的基因替换为FIX基因的GT001-F9分子，能够恢复血友病B小鼠的FIX表达；将对照分子D1(包含对照启动子)中目的基因替换为FIX基因的D1-F9分子，其表达效果显著低于本申请特定的表达载体。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (28)

  1. 分离的核酸分子，其以5’至3’方向包含肝脏特异性表达调控元件以及与所述肝脏特异性表达调控元件可操作连接的目的基因，其中与使用核苷酸序列如SEQ ID NO:10所示的表达调控元件相比，所述肝脏特异性表达调控元件使得所述目的基因的表达量和/或活性增加至少50％。
  2. 根据权利要求1所述的分离的核酸分子，其中所述肝脏特异性表达调控元件包含：
    a)源自狗serpinA1基因的启动子或其功能性片段；以及
    b)源自爪蟾白蛋白基因的启动子或其功能性片段。
  3. 根据权利要求1-2中任一项所述的分离的核酸分子，其中所述肝脏特异性表达调控元件包含SEQ ID NO:1所示的核苷酸序列。
  4. 根据权利要求1-3中任一项所述的分离的核酸分子，其包含多聚腺苷酸信号序列，所述多聚腺苷酸信号序列位于所述目的基因的3’端。
  5. 根据权利要求4所述的分离的核酸分子，其中所述多聚腺苷酸信号源自普通田鼠多瘤病毒(Common vole polyomavirus)的多聚腺苷酸信号。
  6. 根据权利要求4-5中任一项所述的分离的核酸分子，其中所述多聚腺苷酸信号源自普通田鼠多瘤病毒(Common vole polyomavirus)分离株KS13的多聚腺苷酸信号。
  7. 根据权利要求4-6中任一项所述的分离的核酸分子，其中所述多聚腺苷酸信号包含SEQ ID NO:3所示的核苷酸序列。
  8. 根据权利要求1-7中任一项所述的分离的核酸分子，其中所述目的基因编码目的蛋白质，且所述目的蛋白质包括报告蛋白质、治疗性蛋白质和/或预防性蛋白质。
  9. 根据权利要求1-8中任一项所述的分离的核酸分子，其中所述目的基因编码B结构域缺失的因子VIII(FVIII)变体。
  10. 根据权利要求9所述的分离的核酸分子，其中所述FVIII变体包含SEQ ID NO:7所示的氨基酸序列。
  11. 根据权利要求1-10中任一项所述的分离的核酸分子，其中所述目的基因包含SEQ ID NO:2或6所示的核苷酸序列。
  12. 根据权利要求1-11中任一项所述的分离的核酸分子，其以5’至3’方向依次包含：所述肝脏特异性表达调控元件、所述目的基因以及所述多聚腺苷酸信号序列。
  13. 根据权利要求1-12中任一项所述的分离的核酸分子，其还包含AAV末端反向重复ITR，所述ITR位于所述肝脏特异性表达调控元件的5’端以及所述多聚腺苷酸信号序列的3’端。
  14. 根据权利要求13所述的分离的核酸分子，其中所述AAV ITR源自选自下组的AAV血清型：AAV5和AAV2。
  15. 根据权利要求13-14中任一项所述的分离的核酸分子，其中所述AAV ITR包含SEQ ID NO:4-5中任一项所示的核苷酸序列。
  16. 根据权利要求1-15中任一项所述的分离的核酸分子，其包含SEQ ID NO:9所示的核苷酸序列。
  17. 载体，其包含权利要求1-16中任一项所述的分离的核酸分子。
  18. 根据权利要求17所述的载体，其为病毒载体或多核苷酸载体。
  19. 根据权利要求17-18中任一项所述的载体，其为质粒，cosmid或转座子。
  20. 根据权利要求17-19中任一项所述的载体，其为病毒载体，且所述病毒载体包括AAV载体。
  21. 根据权利要求20所述的载体，其中所述AAV载体为AAV8载体。
  22. 宿主细胞，其包含根据权利要求1-16中任一项所述的分离的核酸分子和/或根据权利要求17-21中任一项所述的载体。
  23. 诊断或药物组合物，其包含权利要求1-16中任一项所述的分离的核酸分子，权利要求17-21中任一项所述的载体，和/或权利要求22所述的宿主细胞。
  24. 表达目的基因的方法，所述方法包括将根据权利要求1-16中任一项所述的分离的核酸分子或根据权利要求17-21中任一项所述的载体引入宿主细胞，并使得所述目的基因在所述宿主细胞中表达。
  25. 试剂盒，其包含权利要求1-16中任一项所述的分离的核酸分子，权利要求17-21中任一项所述的载体，和/或权利要求22所述的宿主细胞。
  26. 将权利要求1-16中任一项所述的分离的核酸分子传递到哺乳动物或哺乳动物细胞中的方法，所述方法包括向所述哺乳动物或哺乳动物细胞施用权利要求1-16中任一项所述的分离的核酸分子或根据权利要求17-21中任一项所述的载体，或使所述哺乳动物或哺乳动物细胞接触权利要求1-16中任一项所述的分离的核酸分子或根据权利要求17-21中任一项所述的载体。
  27. 权利要求1-16中任一项所述的分离的核酸分子，权利要求17-21中任一项所述的载体，和/或权利要求22所述的宿主细胞在制备药物中的用途，所述药物用于治疗、缓解和/或预防与FVIII因子相关的疾病或病症。
  28. 根据权利要求27所述的用途，其中所述疾病或病症包括血友病A、血小板减少症和/或凝血病。
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