WO2024060463A1 - 改进的人凝血因子viii基因表达盒及其应用 - Google Patents

改进的人凝血因子viii基因表达盒及其应用 Download PDF

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WO2024060463A1
WO2024060463A1 PCT/CN2022/143456 CN2022143456W WO2024060463A1 WO 2024060463 A1 WO2024060463 A1 WO 2024060463A1 CN 2022143456 W CN2022143456 W CN 2022143456W WO 2024060463 A1 WO2024060463 A1 WO 2024060463A1
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seq
fviii
factor viii
gene expression
aav
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PCT/CN2022/143456
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English (en)
French (fr)
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胡晗阳
李丹丹
徐悦
陈晨
王天翼
袁龙辉
王新涛
杜增民
蒋威
吴侠
郑静
肖啸
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上海勉亦生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • 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

  • the present disclosure relates to optimized polynucleotide sequences encoding Factor VIII (FVIII) polypeptides.
  • the present disclosure also relates to FVIII gene expression cassettes, recombinant AAV vectors and pharmaceutical compositions comprising the optimized polynucleotide sequence, and their use in the treatment of hemophilia A or acquired factor VIII deficiency.
  • Factor VIII plays a key role in the coagulation cascade by accelerating the conversion of factor X to factor Xa.
  • Hemophilia A is a congenital X-linked bleeding disorder characterized by lack of FVIII activity. Reduced FVIII activity inhibits the positive feedback loop in the coagulation cascade.
  • hemophilia A is treated by FVIII replacement therapy, ie, administration of FVIII protein (eg, intravenous infusion of plasma-derived or recombinant FVIII protein) to hemophilia A patients. Although this treatment is effective in controlling bleeding episodes, it is inherently costly due to the requirement for frequent infusions due to FVIII's short half-life (8-12 hours).
  • Adeno-associated virus (AAV)-based gene therapy is an attractive strategy to ultimately cure this disease.
  • AAV Adeno-associated virus
  • the progress of using AAV to deliver the FVIII gene is less than ideal, and the expression efficiency of FVIII in vivo is low.
  • the present disclosure provides optimized FVIII polynucleotide sequences with improved expression levels and FVIII gene expression cassette constructs with improved in vivo expression levels.
  • the inventor unexpectedly discovered that compared with the FVIII gene expression cassette with more complete elements, the FVIII gene expression cassette with partial regulatory elements (for example, enhancers, introns, especially 5'UTR) deleted was delivered via AAV vector to The present invention can be completed by maintaining a comparable or even significantly improved FVIII expression level after intracellular administration.
  • the present disclosure provides a polynucleotide encoding a Factor VIII (FVIII) polypeptide, wherein the nucleotide sequence of the polynucleotide is consistent with SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO :6 or the nucleotide sequence shown in SEQ ID NO:7 has at least 80% identity, preferably at least 85%, 90%, 95%, 99% identity.
  • FVIII Factor VIII
  • the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
  • the nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.
  • the above-described polynucleotide expresses a B domain deleted Factor VIII polypeptide (FVIII-BDD).
  • the optimized FVIII polynucleotide sequence of the present disclosure can significantly increase the expression level of FVIII protein compared to an unoptimized FVIII polynucleotide sequence.
  • the present disclosure provides a factor VIII gene expression cassette, which includes the following elements, preferably consists of the following elements: a promoter, an optional kozak sequence, a polynucleotide encoding a factor VIII polypeptide with a B domain deletion (e.g., a polynucleotide according to the first aspect), polyadenylic acid (poly A), and two ITRs located at both ends.
  • a promoter preferably consists of the following elements: a promoter, an optional kozak sequence, a polynucleotide encoding a factor VIII polypeptide with a B domain deletion (e.g., a polynucleotide according to the first aspect), polyadenylic acid (poly A), and two ITRs located at both ends.
  • the elements in the Factor VIII gene expression cassette are directly connected or indirectly connected through a linker.
  • the promoter is a ubiquitous expression promoter, a constitutive liver-specific promoter, or a synthetic liver-specific promoter.
  • the promoter is a synthetic liver-specific promoter.
  • the promoter is the liver-specific promoter shown in SEQ ID NO: 9 or SEQ ID NO: 10.
  • each of the two ITRs is independently a normal ITR or a truncated ITR.
  • the two ITRs are each independently the normal ITR shown in SEQ ID NO: 14 or the truncated ITR shown in SEQ ID NO: 15.
  • both ITRs are truncated ITRs shown in SEQ ID NO: 15.
  • the Factor VIII gene expression cassette further includes more than one of the following elements: enhancer, 5' untranslated region (5'UTR), and intron.
  • the enhancer is a constitutive enhancer or a synthetic enhancer; the enhancer shown in SEQ ID NO: 8 is preferred.
  • the intron is a constitutive intron or a synthetic intron; preferably the intron shown in SEQ ID NO: 12.
  • nucleotide sequence of the Factor VIII gene expression cassette is as shown in SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.
  • the present disclosure provides a recombinant AAV vector, which includes: the Factor VIII gene expression cassette according to the second aspect and an AAV capsid protein.
  • the AAV capsid protein is a natural AAV capsid protein or an artificially modified AAV capsid protein; preferably, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11.
  • the present disclosure provides use of the recombinant AAV vector according to the third aspect in the preparation of a medicament for preventing or treating hemophilia A or acquired factor VIII deficiency.
  • the present disclosure provides a medicament comprising the recombinant AAV vector according to the third aspect and optional excipients.
  • excipients include salts, organics, and surfactants.
  • the drug is administered by systemic or local routes, such as oral, rectal, transmucosal, intranasal, inhalation, intrabuccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intradermal, intrapleural, intracerebral, and intraarticular), intralymphatic, topical, intralesional administration.
  • systemic or local routes such as oral, rectal, transmucosal, intranasal, inhalation, intrabuccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intradermal, intrapleural, intracerebral, and intraarticular), intralymphatic, topical, intralesional
  • the present disclosure provides a method for preventing or treating hemophilia A or acquired factor VIII deficiency, comprising administering to a subject in need thereof a therapeutically effective amount of a polynucleotide according to the first aspect , the Factor VIII gene expression cassette according to the second aspect, the recombinant AAV vector according to the third aspect, the medicine according to the fifth aspect, and/or the host cell according to the eighth aspect.
  • the present disclosure provides a method for delivering Factor VIII gene into target cells, including: 1) packaging the Factor VIII gene expression cassette according to the second aspect in an AAV capsid protein to form a method according to the second aspect.
  • the target cells are ex vivo cells. In one embodiment, the target cells are cells in vivo.
  • the present disclosure provides a host cell infected with the recombinant AAV vector according to the third aspect.
  • the present disclosure provides a method for producing a recombinant AAV vector, comprising introducing a polynucleotide according to the first aspect into a mammalian host cell, the mammalian host cell comprising an AAV rep gene, an AAV cap gene and viral replication accessory genes.
  • Figure 1 shows the components of the gene expression cassette before and after codon optimization of the FVIII gene: inverted terminal repeat (ITR) sequence (SEQ ID NO: 14), enhancer (SEQ ID NO: 8), and liver-specific promoter LXP2.
  • ITR inverted terminal repeat
  • SEQ ID NO: 14 enhancer
  • SEQ ID NO: 8 liver-specific promoter LXP2.
  • SEQ ID NO: 9 liver-specific promoter LXP3.3
  • SEQ ID NO: 10 or liver-specific promoter LXP3.3
  • SEQ ID NO: 10 or liver-specific promoter LXP3.3 (SEQ ID NO: 10), 5'UTR (SEQ ID NO: 11), intron (SEQ ID NO: 12), Kozak sequence ( GCCACC), human FVIII-encoding cDNA (FVIII-BDD, SEQ ID NO: 3) or codon-optimized human FVIII-encoding cDNA (FVIII-BDD-Opti, SEQ ID NO: 4-7), poly(A) A).
  • Figure 2 shows the expression level of FVIII protein after transfection of Huh7 cells with the FVIII gene expression cassette plasmid having the components shown in Figure 1. Shown are the coagulation activity test results of the Huh7 cell culture supernatant transfected with the gene expression cassette.
  • Figure 3 shows the FVIII gene expression cassette composed of different combinations of codon-optimized FVIII gene (SEQ ID NO: 7) and expression regulatory elements.
  • Figure 4 shows the FVIII protein expression level after transfection of Huh7 cells with the FVIII gene expression cassette plasmid shown in Figure 3. Shown are the coagulation activity test results of the Huh7 cell culture supernatant transfected with the gene expression cassette.
  • Figure 5A shows the virus yield of adeno-associated virus (AAV) packaging of each FVIII gene expression cassette shown in Figure 3 under the same production conditions.
  • AAV adeno-associated virus
  • Figure 5B shows the results of alkaline gel electrophoresis of samples produced by DNA extraction of adeno-associated virus (AAV) packaging each FVIII gene expression cassette shown in Figure 3.
  • AAV adeno-associated virus
  • FIG. 6 shows the FVIII protein expression level after infection of Huh7 cells with adeno-associated virus (AAV) packaging each FVIII gene expression cassette shown in Figure 3. Shown are the coagulation activity test results of Huh7 cell culture supernatants infected with AAV carrying gene expression cassettes.
  • AAV adeno-associated virus
  • Figure 7 shows long-term FVIII activity (as a percentage of normal human FVIII activity) in 5 FVIII knockout mice after intravenous injection of AAV32.1/LXP2.1-F8X1Co4.3.
  • nucleic acid or polynucleotide sequences listed herein are in single-stranded form and are oriented from 5' to 3', left to right.
  • the nucleotides and amino acids provided in this article adopt the format recommended by the IUPACIUB Biochemical Nomenclature Committee, and use single-letter codes or three-letter codes for amino acids.
  • the terms "patient” and “subject” are used interchangeably and in their conventional sense and include any animal suffering from or susceptible to a hemorrhagic disorder or bleeding condition that requires and/or desires control of bleeding, It can be treated, ameliorated or prevented by administering FVIII to a subject (eg, hemophilia A and acquired FVIII deficiency (eg, due to autoantibodies to FVIII or hematological malignancies)).
  • a subject eg, hemophilia A and acquired FVIII deficiency (eg, due to autoantibodies to FVIII or hematological malignancies)).
  • Such subjects are typically mammals (e.g., laboratory animals such as rats, mice, guinea pigs, rabbits, primates, etc.), farm or commercial animals (e.g., cows, horses, goats, donkeys, sheep etc.) or domestic animals (such as cats, dogs, ferrets, etc.).
  • the subject is a primate subject, a non-human primate subject (eg, a chimpanzee, baboon, monkey, gorilla, etc.), or a human.
  • a subject of the present disclosure may be a subject known or believed to have a bleeding disorder or risk of a bleeding condition in need and/or desired control.
  • subjects according to the present disclosure may also include subjects previously unknown or suspected to have a bleeding disorder or risk of a bleeding condition that requires or is desirable to control.
  • the subject may be a laboratory animal and/or an animal model of the disease.
  • Subjects include males and/or females of any age, including newborns, infants, adults and elderly subjects.
  • subjects can be infants (e.g., less than about 12 months, 10 months, 9 months, 8 months, 7 months, 6 months or less age), toddlers (e.g., at least about 12, 18 or 24 months and/or less than about 36, 30 or 24 months) or children (e.g., at least about 1, 2, 3, 4 or 5 years old and/or less than about 14 years old, 12, 10, 8, 7, 6, 5 or 4 years old).
  • the subject is a human subject of about 0 to 3, 4, 5, 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 3 to 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 6 to 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 9 to 12, 15, 18, 24, 30, 36, 48 or 60 months of age, a human subject of about 12 to 18, 24, 36, 48 or 60 months of age, a human subject of about 18 to 24, 30, 36, 48 or 60 months of age, or a human subject of about 24 to 30, 36, 48 or 60 months of age.
  • treatment includes: (1) inhibiting a condition, disease or disorder, that is, preventing, reducing or delaying the progression of the disease or its recurrence or the development of at least one clinical or subclinical symptom thereof; or (2) Alleviating a disease, that is, causing resolution of a condition, disease or condition or at least one of its clinical or subclinical symptoms.
  • a therapeutically effective amount is an amount that provides some improvement or benefit to a subject.
  • a therapeutically effective amount of a drug suitable for treating hemophilia A or acquired factor VIII deficiency may be an amount capable of preventing or ameliorating one or more symptoms associated with hemophilia A or acquired factor VIII deficiency. .
  • the term "improvement” refers to an improvement in a symptom associated with a disease, and may refer to an improvement in at least one parameter that measures or quantifies the symptom.
  • preventing a condition, disease or disorder includes preventing, delaying or reducing the incidence and/or likelihood of the occurrence of at least one clinical or subclinical symptom of the condition, disease or disorder developing in a subject,
  • the subject may have or be susceptible to the condition, disease, or disorder but has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disease, or disorder.
  • preventing hemophilia A means reducing the number and/or severity of bleeding episodes compared to the number and/or severity of bleeding episodes that would occur in the absence of preventive treatment.
  • topical administration or “topical route” refers to administration having a local effect.
  • a measurable value such as an amount of a polypeptide, dosage, time, temperature, enzyme activity or other biological activity, etc.
  • the term "about” as used herein is intended to encompass variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5% or even ⁇ 0.1% of the specified amount.
  • a polynucleotide or polypeptide sequence of the present disclosure means that it consists of the sequence (e.g., SEQ ID NO) and the 5' and/or 3' or N terminus and/or of the sequence. or a polynucleotide or polypeptide consisting of a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) additional nucleotides or amino acids on the C-terminus, wherein The additional nucleotides or amino acids do not result in a substantial change in the function of the polynucleotide or polypeptide.
  • substantially altered means an increase or decrease in the ability to express the encoded polypeptide by at least about 50% or more as compared to the expression level of the polynucleotide consisting of the sequence.
  • substantially altered as applied to a polypeptide of the present disclosure refers to an increase or decrease in coagulation-stimulating activity of at least about 50% or more as compared to the activity of the polypeptide consisting of the recited sequence.
  • the terms “enhance,” “increase,” or “improve” refer to an increase in a specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold or even 15-fold.
  • the terms “inhibit,” “reduce,” or “reduce” refer to a decrease or decrease in a specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80% , 90%, 95% or more. In one embodiment, “inhibit,” “reduce,” or “reduce” results in little or essentially undetectable activity (eg, less than about 10% or even 5%).
  • the efficacy of treating hemophilia A or acquired Factor VIII deficiency by the methods of the present disclosure can be determined by detecting clinical improvement indicated by changes in symptoms and/or clinical parameters of the subject.
  • operably linked refers to a relationship between a first nucleotide sequence (e.g., a gene) and a second nucleotide sequence (e.g., a regulatory control element) that allows the second nucleotide sequence
  • the sequence affects one or more properties (eg, transcription rate) associated with the first nucleotide sequence.
  • a promoter is operably linked to a coding sequence, such as directly (no additional nucleotides are included between the promoter and the coding sequence) or indirectly via a linker (eg, a nonfunctional linker).
  • nucleic acid As used herein, “nucleic acid,” “nucleotide sequence” and “polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (eg, chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • the terms “nucleic acid,” “nucleotide sequence,” and “polynucleotide” refer to a chain of nucleotides without regard to chain length. Nucleic acids can be double-stranded or single-stranded. When single-stranded, the nucleic acid can be either the sense strand or the antisense strand.
  • Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (eg, inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids with altered base pairing abilities or increased nuclease resistance.
  • the present disclosure also provides a nucleic acid that is the complement (which may be a complete complement or a partial complement) of a nucleic acid, nucleotide sequence or polynucleotide of the present disclosure.
  • isolated polynucleotide is a nucleotide sequence (eg, DNA or RNA) that is not directly linked to a nucleotide sequence (one at the 5' terminus and the other in the naturally occurring genome of the organism from which it is derived). one at the 3' end).
  • the isolated nucleic acid includes some or all of the 5' non-coding (eg, promoter) sequences immediately adjacent to the coding sequence.
  • the term includes recombinant DNA incorporated, for example, into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryotic or eukaryotic organism, or as a separate molecule (e.g., by PCR or restriction enzymes).
  • cDNA or genomic DNA fragments produced by nuclease treatment exist independently of other recombinant DNA sequences. It also includes recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence.
  • An isolated polynucleotide that includes a gene is not a segment of the chromosome that includes such a gene, but rather includes the coding and regulatory regions associated with the gene, but without the additional genes naturally occurring on the chromosome.
  • fragment applied to polynucleotides will be understood to refer to a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence, and comprising (substantially consisting of and/or consisting of) a nucleotide sequence of consecutive nucleotides that are consistent or almost consistent (e.g., 90%, 92%, 95%, 98%, 99% consistent) with the reference nucleic acid or nucleotide sequence.
  • nucleic acid fragments according to the present disclosure may be included in larger polynucleotides of which they are a component, where appropriate.
  • such a fragment may comprise (substantially consisting of and/or consisting of) an oligonucleotide having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more consecutive nucleotides of a nucleic acid or nucleotide sequence according to the present disclosure.
  • isolated may refer to nucleic acids, nucleic acids, nucleic acids, nucleic acids, nucleic acids, nucleic acids, nucleic acids, nucleic acids, and/or nucleic acids that are substantially free of cellular material, viral material, and/or culture media (when produced by recombinant DNA technology), or chemical precursors or other chemicals (when chemically synthesized). nucleotide sequence or polypeptide.
  • an "isolated fragment” is a fragment of a nucleic acid, nucleotide sequence, or polypeptide that is not a naturally occurring fragment and would not be found in its natural state. "Isolated” does not mean that the preparation is technically pure (homogeneous), but that it is sufficiently pure to provide the polypeptide or nucleic acid in a form useful for the intended purpose.
  • fragment as applied to a polypeptide will be understood to mean an amino acid sequence that is reduced in length relative to a reference polypeptide or amino acid sequence and contains (consists essentially of and/or consists of) the same sequence of amino acids as said reference polypeptide or amino acid sequence.
  • Amino acid sequences of contiguous amino acids whose sequences are identical or nearly identical (eg, 90%, 92%, 95%, 98%, 99% identical).
  • Such polypeptide fragments according to the present disclosure may, where appropriate, be included within the larger polypeptide of which they are a constituent. In some embodiments, such fragments may comprise (consist essentially of and/or consist of) at least about 4, 6, 8, 10, 12, 15, Peptides of 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200 or more consecutive amino acid lengths.
  • a “vector” is any nucleic acid molecule used for cloning and/or transferring nucleic acids into cells.
  • a vector can be a replicon to which another nucleotide sequence can be attached to allow replication of the attached nucleotide sequence.
  • a “replicon” may be any genetic element (eg, plasmid, phage, cosmid, chromosome, viral genome) that is an autonomous unit of nucleic acid replication in vivo (ie, capable of replicating under its own control).
  • the term “vector” includes viral and non-viral (eg, plasmid) nucleic acid molecules that introduce nucleic acids into cells in vitro, ex vivo, and/or in vivo.
  • a wide variety of vectors known in the art can be used to manipulate nucleic acids, incorporate response elements and promoters into genes, and the like. For example, insertion of nucleic acid fragments corresponding to the response element and promoter into a suitable vector can be achieved by ligating the suitable nucleic acid fragment into a selected vector with complementary binding termini. Alternatively, the ends of the nucleic acid molecules can be enzymatically modified, or any site can be created by joining nucleotide sequences (linkers) to the nucleic acid ends.
  • Such vectors can be engineered to contain sequences encoding a selectable marker, which facilitates selection of cells containing the vector and/or cells that have incorporated the nucleic acid of the vector into the cellular genome.
  • a "recombinant" vector refers to a viral or non-viral vector that contains one or more heterologous nucleotide sequences (i.e., a transgene), e.g., two, three, four, five or more heterologous nucleotides sequence.
  • Viral vectors have been used in a variety of gene delivery applications in cells as well as in living animal subjects.
  • Viral vectors that may be used include, but are not limited to, retroviral, lentiviral, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpesvirus, Epstein-Barr virus, and adenovirus vectors.
  • Non-viral vectors include plasmids, liposomes, charged lipids (cytofectins), nucleic acid-protein complexes and biopolymers.
  • the vector may also contain one or more regulatory regions and/or selectable markers that can be used to select, measure and monitor the outcome of nucleic acid transfer (delivery to specific tissues, duration of expression, etc.).
  • the vector can be introduced into the desired cells by methods known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome) fusion), using gene guns or nucleic acid vector transporters (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al. Canadian Patent Application No. 2,012,311 filed March 15, 1990).
  • methods known in the art such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome) fusion
  • gene guns or nucleic acid vector transporters see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J
  • nucleic acids in vivo
  • cationic oligopeptides e.g., WO95/21931
  • peptides derived from nucleic acid binding proteins e.g., WO96/25508
  • cationic polymers e.g., WO96/25508
  • Vectors that are naked nucleic acids can also be introduced in vivo (see U.S. Patent Nos. 5,693,622, 5,589,466, and 5,580,859).
  • Receptor-mediated nucleic acid delivery methods can also be used (Curiel et al., Hum. Gene Ther. 3:147 (1992); Wu et al., J. Biol. Chem. 262:4429 (1987)).
  • protein and “polypeptide” as used herein are used interchangeably and include both peptides and proteins.
  • a "fusion protein” is when two heterologous nucleotide sequences encoding two (or more) different polypeptides, or fragments thereof, not found fused together in nature, are fused together in the correct translation reading frame , the resulting peptides.
  • Illustrative fusion polypeptides include polypeptides of the present disclosure (or fragments thereof) and glutathione-S-transferase, maltose-binding protein, or reporter proteins (e.g., green fluorescent protein, beta-glucuronidase, beta-galactopyranoside enzyme, luciferase, etc.), hemagglutinin, c-myc, FLAG epitope, etc., or all or part of the fusion.
  • a “functional” polypeptide or “functional fragment” is a substance that substantially retains at least one biological activity normally associated with the polypeptide (e.g., angiogenic activity, protein binding, ligand or receptor binding). In one embodiment, a “functional" polypeptide or “functional fragment” substantially retains all activities possessed by an unmodified peptide. By “substantially retaining" biological activity, it is meant that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 99% or more of the biological activity of the native polypeptide (and may even have a higher level of activity than the native polypeptide).
  • non-functional polypeptide is a polypeptide that exhibits little or substantially no detectable biological activity normally associated with a polypeptide (e.g., at most an insignificant amount, such as less than about 10% or even 5%). Biological activities such as protein binding and angiogenic activity can be measured using assays well known in the art and as described herein.
  • expressing means that the sequence is transcribed and optionally translated. Generally, in accordance with the present disclosure, expression of a coding sequence of the disclosure will result in the production of a polypeptide of the disclosure. Whole-body expressed polypeptides or fragments can also function in intact cells without the need for purification.
  • AAV adeno-associated virus
  • AAV type 1 includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV 8 type, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV and any other AAV now known or later discovered.
  • BERNARD N. FIELDS et al. VIROLOGY, Volume 2, Chapter 69 (4th ed., Lippincott-Raven Publishers).
  • a number of additional AAV serotypes and clades have been identified (see, eg, Gao et al., (2004) J. Virol. 78:6381-6388), which are also encompassed by the term "AAV”.
  • genomic sequences of various AAVs and autonomous parvoviruses as well as the sequences of ITRs, Rep proteins and capsid subunits are known in the art. These sequences can be found in the literature or in public databases, such as GenBank, see, for example, GenBank Accession Nos.
  • a “recombinant AAV vector genome” or “rAAV genome” is an AAV genome (i.e., vDNA) comprising at least one inverted terminal repeat (e.g., one, two, or three inverted terminal repeat sequences) and one or more heterologous nucleotide sequences.
  • rAAV vectors typically retain the 145 base terminal repeats (TRs) in cis to produce the virus; however, modified AAV TRs and non-AAV TRs (including partially or completely synthetic sequences) can also be used for this purpose. All other viral sequences are unnecessary and can be provided in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158: 97).
  • rAAV vectors optionally contain two TRs (e.g., AAV TRs), which will typically be at the 5’ and 3’ ends of the heterologous nucleotide sequence, but not necessarily adjacent to it. TRs can be identical to or different from each other.
  • the vector genome may also contain a single ITR at its 3’ or 5’ end.
  • terminal repeat includes any viral terminal repeat or synthesis that forms a hairpin structure and serves as an inverted terminal repeat (i.e., mediates desired functions such as replication, viral packaging, integration and/or proviral rescue, etc.) sequence.
  • TR can be AAV TR or non-AAV TR.
  • non-AAV TR sequences such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or sequences of the SV40 hairpin that serve as the origin of SV40 replication, Can be used as TRs, they can be further modified by truncation, substitution, deletion, insertion and/or addition.
  • TR may be partially or fully synthetic, such as the "double D sequence" described in Samulski et al., US Pat. No. 5,478,745.
  • AAV terminal repeat or “AAV TR” may be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or those now known or hereafter discovered Any other AAV (see e.g. Table 1).
  • AAV terminal repeats need not have native terminal repeat sequences (e.g., native AAV TR sequences can be altered by insertions, deletions, truncations, and/or missense mutations) as long as the terminal repeats mediate the desired function such as replication, viral packaging , integration and/or original virus rescue, etc.
  • rAAV particle and "rAAV viral particle” are used interchangeably herein.
  • rAAV particles or "rAAV viral particles” comprise the rAAV vector genome packaged within an AAV capsid.
  • the AAV capsid structure is described in more detail in BERNARD N. FIELDS et al., VIROLOGY, Volume 2, Chapters 69 & 70 (4th edition, Lippincott-Raven Publishers).
  • biological activity or “activity” is determined by reference to a standard, e.g., from human plasma.
  • a standard e.g., from human plasma.
  • the standard may be (CSL Behring). The biological activity of this standard was taken as 100%.
  • Factor VIII protein or “FVIII protein” includes wild-type FVIII proteins as well as naturally occurring or man-made proteins (eg, B domain deleted proteins).
  • the FVIII proteins of the present disclosure may further include mutant forms of FVIII known in the literature.
  • the FVIII proteins of the present disclosure also include any other naturally occurring human FVIII proteins or artificial human FVIII proteins now known or later identified, as well as their derivatives and active fragments/active domains known in the art.
  • FVIII amino acid sequences of FVIII from various mammalian species are available from sequence databases such as GenBank. Examples of FVIII sequences are shown in the table below.
  • the FVIII proteins of the present disclosure also include pharmacologically active forms of FVIII, which are molecules from which the signal peptide has been removed and the B domain has been deleted by the action of a protease (or engineered from the protein by removing it at the nucleic acid level ionized out), producing two discontinuous polypeptide chains (light chain and heavy chain) of FVIII that fold into functional FVIII coagulation factor.
  • pharmacologically active forms of FVIII are molecules from which the signal peptide has been removed and the B domain has been deleted by the action of a protease (or engineered from the protein by removing it at the nucleic acid level ionized out), producing two discontinuous polypeptide chains (light chain and heavy chain) of FVIII that fold into functional FVIII coagulation factor.
  • B domain deleted forms of human FVIII are known, including the frequently used SQ version in which the residues between S743 and Q1638 are deleted.
  • the amino acid sequence of the human FVIII protein is well known in the art and can be found in GenBank accession number AAA52484.
  • the human FVIII protein is 2351 amino acids in length and consists of a signal peptide (residues 1-19), a heavy chain (residues 20-759), a B domain (residues 760-1332), and a light chain (residues 1668-1668). 2351) composition.
  • the amino acid sequence of the human FVIII protein without a signal peptide is disclosed below (SEQ ID NO: 1).
  • half-life is a broad term that includes the usual and customary meaning as well as the usual and customary meaning found in the FVIII scientific literature. Specifically included in this definition are measurements of parameters related to FVIII, which define the post-infusion time taken to reduce from the initial value measured at the time of infusion to half of the initial value.
  • the half-life of FVIII can be measured in blood and/or blood components in various immunoassays using antibodies to FVIII, as is well known in the art and described herein.
  • half-life can be measured as a decrease in FVIII activity using functional assays including standard coagulation assays, as is well known in the art and as described herein.
  • a "transformed" cell is a cell that has been transformed, transduced and/or transfected with a nucleic acid molecule encoding the FVIII protein of the present disclosure, including but not limited to FVIII protein vectors constructed using recombinant DNA technology.
  • bleeding disorder reflects any defect of cellular, physiological or molecular origin, congenital, acquired or induced, in which bleeding occurs.
  • coagulation factor deficiencies e.g., hemophilia A and B or deficiencies of coagulation factors XI, VII, VIII or IX
  • coagulation factor inhibitors e.g., platelet insufficiency, thrombocytopenia, von Willebrand's disease (von Willebrand's disease) or bleeding caused by surgery or trauma.
  • Excessive bleeding also occurs in subjects with a normal functioning blood coagulation cascade (without coagulation factor deficiencies or inhibitors to any coagulation factor) and may be caused by platelet insufficiency, thrombocytopenia, or von Willebrand syndrome disease caused.
  • the bleeding may be similar to that caused by hemophilia because the hemostatic system (as in hemophilia) lacks or has an abnormality in the necessary clotting "compounds" (such as platelets or von Willebrand factor protein) , causing major bleeding.
  • normal hemostatic mechanisms may be overwhelmed by the need for immediate hemostasis, and bleeding may occur despite normal hemostatic mechanisms.
  • Radical retropubic prostatectomy is a routine surgery for subjects with localized prostate cancer. Surgery is often complicated by significant and sometimes massive blood loss. Considerable blood loss during prostatectomy is primarily related to the complex anatomy, which has various densely vascularized sites where surgical hemostasis is not readily available, and may result in large areas of diffuse bleeding. Furthermore, intracerebral hemorrhage is the least treatable form of stroke and is associated with high mortality and hematoma growth in the first hours after intracerebral hemorrhage. Another situation that may cause problems in the setting of poor hemostasis is when subjects with normal hemostatic mechanisms are treated with anticoagulation to prevent thromboembolic disease. Such treatments may include heparin, other forms of proteoglycans, warfarin, or other forms of vitamin K antagonists, as well as aspirin and other platelet aggregation inhibitors.
  • the bleeding is associated with hemophilia. In another embodiment, the bleeding is associated with acquired inhibitor hemophilia. In another embodiment, the bleeding is associated with thrombocytopenia. In another embodiment, the bleeding is associated with von Willebrand’s disease. In another embodiment, the bleeding is associated with severe tissue damage. In another embodiment, the bleeding is associated with severe trauma. In another embodiment, the bleeding is related to surgery. In another embodiment, the bleeding is associated with laparoscopic surgery. In another embodiment, the bleeding is associated with hemorrhagic gastritis. In another embodiment, the bleeding is massive uterine bleeding. In another embodiment, the bleeding occurs in an organ with limited possibilities for mechanical hemostasis. In another embodiment, the bleeding occurs in the brain, inner ear area, or eye. In another embodiment, the bleeding is associated with the procedure of taking a biopsy. In another embodiment, the bleeding is associated with anticoagulant therapy.
  • the present disclosure relates to a codon-optimized polynucleotide sequence encoding FVIII-BDD (nucleic acid optimized FVIII-BDD-Opti) for expression in humans.
  • the B domain-deleted FVIII (FVIII-BDD) protein is a modified FVIII protein with increased glycosylation (International Patent WO2016127057; amino acid sequence SEQ ID NO: 2 and nucleotide sequence SEQ ID NO: 3).
  • a codon-optimized sequence includes, consists essentially of, or consists of a sequence that is at least 90% identical to one of SEQ ID NO: 4 and/or SEQ ID NO: 6, e.g., to SEQ ID NO: 4
  • the nucleotide sequences of NO:4 and/or SEQ ID NO:6 are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical sex.
  • the polynucleotide sequence optimizer has 95%-100% sequence identity with any one of SEQ ID NO:4 and/or SEQ ID NO:6.
  • the nucleic acid optimized to encode FVIII-BDD has a reduced CpG content compared to the wild-type nucleic acid encoding FVIII-BDD (SEQ ID NO: 3). In certain embodiments, the nucleic acid optimized has at least 20 fewer CpGs than the wild-type nucleic acid encoding FVIII-BDD.
  • the nucleic acid optimized has no more than 10 CpGs, no more than 9 CpGs, no more than 8 CpGs, no more than 7 CpGs, no more than 6 CpGs, no more than 5 CpGs, no more than 4 CpGs, no more than 3 CpGs, no more than 2 CpGs, or no more than 1 CpG. In certain embodiments, the nucleic acid optimized has at most 4 CpGs, 3 CpGs, 2 CpGs, or 1 CpG. In certain embodiments, the nucleic acid optimized has no CpGs.
  • an optimized nucleic acid encoding FVIII-BDD has reduced CpG content compared to a wild-type nucleic acid encoding FVIII-BDD, and such reduced CpG nucleic acid variant is consistent with SEQ ID NO: 5 and/or or any one of SEQ ID NO:7 having 90% or greater sequence identity.
  • the CpG-reduced nucleic acid variant is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher sequence identity.
  • the CpG-reduced nucleic acid optimizer has 90%-95% sequence identity with any one of SEQ ID NO:5 and/or SEQ ID NO:7.
  • the CpG-reduced nucleic acid optimizer has 95%-100% sequence identity with any one of SEQ ID NO:5 and/or SEQ ID NO:7.
  • an optimized nucleic acid encoding a reduced CpG of FVIII-BDD is set forth in any one of SEQ ID NO: 5 and/or SEQ ID NO: 7.
  • a nucleic acid variant encoding a FVIII-BDD protein is at least 75% identical to a B domain deleted wild-type human FVIII nucleic acid. In certain embodiments, a nucleic acid variant encoding a FVIII-BDD protein is about 75-95% identical to a B domain deleted wild-type human FVIII nucleic acid, e.g., about 75%, 76%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% same .
  • the nucleic acids and variants encoding FVIII-BDD proteins are mammalian, such as human.
  • Such mammalian nucleic acids and nucleic acid variants encoding FVIII-BDD proteins include human forms, which may be based on wild-type human FVIII deleted in the B domain.
  • One aspect of the present disclosure relates to a gene expression cassette comprising, preferably consisting of, an optional enhancer, a promoter, an optional 5' untranslated region (5'UTR), an optional intron, Optional kozak sequence, B domain deleted human FVIII (FVIII-BDD) protein encoding cDNA, poly(A), and two ITRs at both ends.
  • a gene expression cassette comprising, preferably consisting of, an optional enhancer, a promoter, an optional 5' untranslated region (5'UTR), an optional intron, Optional kozak sequence, B domain deleted human FVIII (FVIII-BDD) protein encoding cDNA, poly(A), and two ITRs at both ends.
  • the gene expression cassette of the present disclosure is a gene expression cassette composed of the following elements: a promoter, a cDNA encoding a B domain-deleted human FVIII (FVIII-BDD) protein, a poly(A) A) and two ITRs located at both ends.
  • the gene expression cassette of the present invention is a gene expression cassette composed of the following elements: a promoter, a kozak sequence, a cDNA encoding a B-domain deleted human FVIII (FVIII-BDD) protein, polyadenylic acid (poly A), and two ITRs located at both ends.
  • the enhancer comprises or consists of the nucleotide sequence shown in SEQ ID NO: 8.
  • the promoter comprises or consists of the nucleotide sequence of the liver-specific promoter LXP2.1 (SEQ ID NO: 9) or LXP3.3 (SEQ ID NO: 10).
  • the 5'UTR comprises or consists of the nucleotide sequence shown in SEQ ID NO: 11.
  • the intron comprises or consists of the nucleotide sequence shown in SEQ ID NO: 12.
  • the kozak sequence contains or consists of GCCACC.
  • amino acid sequence of FVIII deleted from the B domain is, for example, SEQ ID NO: 2.
  • the B domain-deleted FVIII can be encoded by a polynucleotide comprising the nucleotide sequence shown in any one of SEQ ID NOs: 4 to 7 or having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, or consisting essentially of, or consisting of.
  • polyadenylate comprises or consists of the nucleotide sequence shown in SEQ ID NO: 13.
  • the ITR comprises or consists of the nucleotide sequence shown in SEQ ID NO: 15.
  • vectors such as expression vectors, comprising polynucleotides of the disclosure.
  • the vector may be any type of vector known in the art, including but not limited to plasmid vectors and viral vectors.
  • the viral vector is a retroviral or lentiviral vector.
  • the viral vector is an AAV vector from any known AAV serotype, including, but not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV types 7, AAV 8, AAV 9, AAV 10, AAV 11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV and any other AAV now known or later discovered.
  • the AAV vector is AAVXL32.1 (see International Patent Publication WO 2019241324A1).
  • cells eg, isolated cells, transformed cells, recombinant cells, etc.
  • cells comprising polynucleotides and/or vectors of the disclosure.
  • some embodiments of the present disclosure relate to recombinant host cells containing vectors (eg, expression cassettes). Such cells can be isolated and/or present in transgenic animals.
  • Another aspect of the disclosure relates to transgenic animals comprising polynucleotides, vectors, and/or transformed cells of the disclosure.
  • kits comprising polynucleotides, vectors and/or cells of the present disclosure, and/or reagents and/or instructions for using the kit.
  • One aspect of the present disclosure relates to methods of treating hemophilia A or acquired factor VIII deficiency in a subject using the FVIII gene expression cassette of the present disclosure, comprising delivering to the subject a therapeutically effective amount of a polynucleotide of the present disclosure, Vectors and/or transformed cells, thereby treating hemophilia A or acquired factor VIII deficiency in a subject.
  • Another aspect of the present disclosure relates to methods for improving long-term high-efficiency expression of FVIII polypeptides in a subject using codon- and CpG-optimized nucleotide sequences of the present disclosure, comprising delivering an effective amount of a polynucleotide of the present disclosure to the subject , vectors and/or transformed cells, thereby improving long-term high-efficiency expression of FVIII polypeptides in subjects.
  • One aspect of the present disclosure relates to a method of producing a FVIII polypeptide in the liver of a subject, comprising delivering to the subject an optimized polynucleotide, a vector, and/or a transformed cell encoding a B domain deleted human FVIII polypeptide of the present disclosure, FVIII polypeptide is thereby produced in the subject's liver.
  • Another aspect of the present disclosure relates to a method of treating hemophilia A or acquired factor VIII deficiency in a subject, comprising delivering to the subject a therapeutically effective amount of an optimized polypeptide encoding a B domain deleted human FVIII polypeptide of the present disclosure.
  • the nucleotides, vectors and/or transformed cells are used to treat hemophilia A or acquired FVIII deficiency in a subject.
  • Bleeding disorders treatable according to the methods of the present disclosure include any condition treatable with FVIII, such as hemophilia A and acquired FVIII deficiency.
  • Such treatment strategies and dosing regimens for administering or delivering the FVIII proteins of the disclosure and/or polynucleotides encoding the FVIII proteins of the disclosure to a subject are within the art. Well known.
  • the dosage of the vector of the present disclosure can be an amount that achieves a therapeutic plasma concentration of the FVIII protein.
  • the therapeutic concentration of the FVIII protein is considered to be 1% higher than the normal level of a healthy individual, which is measured on an average of 100%, i.e., one international unit (IU) of FVIII in 1 mL of normal human plasma.
  • IU international unit
  • compositions of the present disclosure may be used for prophylactic and/or therapeutic administration.
  • the composition is administered to a subject already suffering from the disease as described above in an amount sufficient to cure, alleviate or partially prevent the disease and its complications.
  • An amount sufficient to achieve this goal is defined as a "therapeutically effective amount.”
  • the amount effective for this purpose will depend on the severity of the disease or injury and the weight and general condition of the subject.
  • an AAV vector is used to deliver the FVIII gene expression cassette of the present disclosure to a subject.
  • the present disclosure also provides an AAV virus particle (ie, a virion) comprising a FVIII gene expression cassette, wherein the virus particle packages a vector genome.
  • the virosome is a recombinant vector (e.g., for delivery to a cell) comprising a FVIII gene expression cassette. Therefore, the recombinant vector of the present disclosure can be used to deliver polynucleotides to cells in vitro, ex vivo, and in vivo. In representative embodiments, the recombinant vector of the present disclosure can be advantageously used to deliver or transfer the FVIII gene expression cassette of the present disclosure into animal (e.g., mammalian) cells.
  • animal e.g., mammalian
  • the present disclosure also provides methods of producing viral vectors.
  • the present disclosure provides a method of producing a recombinant viral vector, the method comprising providing to a cell in vitro (a) a template comprising (i) a polynucleotide of interest and (ii) sufficient to A packaging signal sequence (e.g., one or more (e.g., two) terminal repeats, e.g., an AAV terminal repeat) that encapsulates the AAV template into the virion; and (b) is sufficient to enable replication of the template and loading of the template into the virion.
  • AAV sequences such as AAV rep and AAV cap sequences).
  • the template and AAV replication and capsid sequences are provided under conditions such that recombinant viral particles comprising the template packaged within the capsid are produced in the cell.
  • the method may also include the step of collecting viral particles from said cells. Viral particles can be collected from the culture medium and/or by lysing cells.
  • the present disclosure provides a method of producing rAAV particles comprising an AAV capsid, the method comprising: providing to a cell in vitro a nucleic acid encoding an AAV capsid, an AAV rep coding sequence, a nucleic acid containing a target polynucleus nucleotides of the AAV vector genome, as well as helper functions for generating productive AAV infections; and allowing the assembly of AAV particles containing the AAV capsid and packaging of the AAV vector genome.
  • the cells are typically cells that are permissive for AAV viral replication. Any suitable cell known in the art may be used, such as a mammalian cell. Also suitable are trans-complementing packaging cell lines that provide functions missing from the replication-defective helper virus, such as 293 cells or other E1a trans-complementing cells.
  • AAV replication and capsid sequences can be provided by any method known in the art. Current protocols typically express the AAV rep/cap genes on a single plasmid. There is no need to provide AAV replication and packaging sequences together.
  • AAV rep and/or cap sequences can be provided by any viral or non-viral vector.
  • the rep/cap sequence may be provided by a hybrid adenoviral or herpesviral vector (eg, inserted into the E1a or E3 region of a deleted adenoviral vector).
  • EBV vectors can also be used to express AAV cap and rep genes.
  • One advantage of this approach is that the EBV vector is episomal but will maintain a high copy number throughout successive cell divisions (i.e. stably integrated into the cell as an extrachromosomal element, designated EBV-based nuclear episomal body(episome)).
  • rep/cap sequences can be stably carried within the cell.
  • AAV rep/cap sequences are not flanked by AAV packaging sequences (e.g., AAV ITR) to prevent rescue and/or packaging of these sequences.
  • AAV packaging sequences e.g., AAV ITR
  • the template (eg, rAAV vector genome) can be provided to the cell using any method known in the art.
  • the template can be provided by a non-viral (eg plasmid) or viral vector.
  • the template is provided by a herpesvirus or adenovirus vector (eg, inserted into a deleted E1a or E3 region of the adenovirus).
  • Palombo et al., (1998) J. Virol. 72:5025 describes baculovirus vectors carrying a reporter gene flanked by AAV ITRs. Templates can also be delivered using EBV vectors, as described above for the rep/cap gene.
  • the template is provided by a replicating rAAV virus.
  • the AAV provirus is stably integrated into the chromosome of the cell.
  • helper viral functions such as adenovirus or herpesvirus
  • helper viral sequences required for AAV replication are known in the art.
  • these sequences are provided by helper adenovirus or herpesvirus vectors.
  • adenoviral or herpesvirus sequences can be provided by another non-viral or viral vector, e.g. as a non-infectious adenoviral miniplasmid carrying all the helper genes required for efficient AAV production, such as Ferrari et al., ( 1997) Nature Med. 3:1295 and U.S. Patent Nos. 6,040,183 and 6,093,570.
  • helper virus function can be provided by packaging cells with helper genes integrated in the chromosome or maintained as stable extrachromosomal elements.
  • the helper viral sequences are not packaged within the AAV virion, e.g., are not flanked by the AAV ITR.
  • helper construct may be a non-viral or viral construct, but alternatively is a hybrid adenovirus or hybrid herpesvirus containing the AAV rep/cap gene.
  • the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector.
  • This vector also contains rAAV template.
  • AAV rep/cap sequences and/or rAAV templates can be inserted into deleted regions of adenovirus (e.g., E1a or E3 regions).
  • the AAV rep/cap sequences and adenoviral helper sequences are provided by a single adenoviral helper vector.
  • the rAAV template is provided as a plasmid template.
  • AAV rep/cap sequences and adenoviral helper sequences are provided from a single adenoviral helper vector, and the rAAV template is integrated into the cell as a provirus.
  • the rAAV template is provided by the EBV vector maintained intracellularly as an extrachromosomal element (eg as "EBV-based nuclear episome", see Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67).
  • the AAV rep/cap sequence and adenoviral helper sequence are provided by a single adenoviral helper.
  • the rAAV template is provided as a separate replicating viral vector.
  • the rAAV template can be provided by a rAAV particle or a second recombinant adenoviral particle.
  • hybrid adenovirus vectors typically contain adenovirus 5' and 3' cis-sequences (i.e., adenovirus terminal repeats and PAC sequences) sufficient for adenovirus replication and packaging.
  • AAV rep/cap sequences and (if present) rAAV template are embedded in the adenoviral backbone and flanked by the 5' and 3' cis sequences, allowing these sequences to be packaged into adenoviral capsids.
  • adenoviral helper sequences and AAV rep/cap sequences are not flanked by AAV packaging sequences (e.g., AAV ITR), such that these sequences are not packaged into AAV virions.
  • Herpes viruses can also be used as helper viruses in AAV packaging methods. Hybrid herpesviruses encoding AAV rep proteins may advantageously facilitate AAV vector production protocols. Hybrid herpes simplex virus type 1 (HSV-1) vectors expressing AAV-2 rep and cap genes have been described (Conway et al. (1999) Gene Therapy 6:986 and WO 00/17377, the disclosures of which are incorporated herein in their entirety) .
  • HSV-1 hybrid herpes simplex virus type 1
  • the viral vectors of the present disclosure can be produced in insect cells using baculovirus vectors to deliver the rep/cap gene and rAAV template, as described in Urabe et al. (2002) Human Gene Therapy 13:1935-43 narrate.
  • AAV vector stocks free of contaminating helper virus can be obtained by any method known in the art.
  • AAV and helper viruses can be easily distinguished based on their size.
  • AAV can also be isolated from helper viruses based on affinity for heparin substrate (Zolotukhin et al. (1999) Gene Therapy 6:973).
  • a deleted replication-deficient helper virus is used such that any contaminating helper virus is incapable of replication.
  • adenoviral helpers lacking late gene expression can be used, since only adenoviral early gene expression is required to mediate packaging of AAV viruses.
  • Adenovirus mutants defective in late gene expression are known in the art (eg, ts100K and ts149 adenovirus mutants).
  • the packaging methods of the present disclosure can be used to generate high titer viral particle stocks.
  • the viral stock has a titer of at least about 10 5 transduction units (tu)/ml, at least about 10 6 tu/ml, at least about 10 7 tu/ml, at least about 10 8 tu/ml, at least about 10 9 tu/ml or at least about 10 10 tu/ml.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the viral vector of the present disclosure and optional other medical agents, agents, stabilizers, buffers, carriers, adjuvants in a pharmaceutically acceptable carrier. agents, thinners, etc.
  • the carrier is usually a liquid.
  • the carrier may be solid or liquid.
  • the carrier will be respirable, and preferably will be in solid or liquid particulate form.
  • pharmaceutically acceptable refers to a substance that is not toxic or otherwise undesirable, ie, the substance can be administered to a subject without causing any undesirable biological effects.
  • Viral vectors can be introduced into cells at the appropriate multiplicity of infection according to standard transduction methods applicable to the specific target cell.
  • the titer of the viral vector or capsid used for administration can vary depending on the target cell type and number and the specific viral vector or capsid, and can be determined by one skilled in the art without undue experimentation.
  • at least about 10 3 infectious units, more preferably at least about 10 5 infectious units are introduced into the cell.
  • the cell that can introduce viral vector can be any type, including but not limited to neural cell (including peripheral and central nervous system cells, in particular, brain cells such as neurons, oligodendrocytes, glial cells, astrocytes), lung cells, eye cells (including retinal cells, retinal pigment epithelium and corneal cells), epithelial cells (such as intestinal and respiratory epithelial cells), skeletal muscle cells (including myoblasts, myotubes and myofibers), diaphragm muscle cells, dendritic cells, pancreatic cells (including islet cells), hepatocytes, gastrointestinal cells (including smooth muscle cells, epithelial cells), heart cells (including cardiomyocytes), osteocytes (such as bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, joint cells (including such as cartilage, meniscus, synovium and bone marrow), germ cells, etc.
  • neural cell
  • the cell can be any progenitor cell.
  • the cell can be a stem cell (such as a neural stem cell, a liver stem cell).
  • the cell can be a cancer or tumor cell.
  • the cell can be from a species in any source.
  • Viral vectors can be introduced into cells in vitro for the purpose of administering the modified cells to a subject.
  • cells have been removed from the subject, a viral vector has been introduced therein, and the cells have been replaced back into the subject.
  • Methods of removing cells from a subject for ex vivo treatment and then introducing them back into the subject are known in the art (see, eg, U.S. Patent No. 5,399,346).
  • the recombinant viral vector is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof. tester.
  • Suitable cells for ex vivo gene therapy are as described above.
  • the dosage of cells administered to a subject will vary depending on the age, condition and species of the subject, the type of cell, the nucleic acid expressed by the cell, the mode of administration, and the like. Typically, at least about 10 2 to about 10 8 or about 10 3 to about 10 6 cells per dose will be administered in a pharmaceutically acceptable carrier.
  • cells transduced with a viral vector are administered to a subject in an effective amount in combination with a pharmaceutical carrier.
  • Another aspect of the present disclosure relates to a method of administering a viral vector of the present disclosure to a subject.
  • the method comprises a method of delivering a target polynucleotide to an animal subject, the method comprising: administering an effective amount of a viral vector according to the present disclosure to an animal subject.
  • the viral vector of the present disclosure can be administered to a human subject or animal in need thereof by any method known in the art.
  • a viral vector in a pharmaceutically acceptable carrier is delivered at an effective dose.
  • the dose of the viral vector to be administered to a subject will depend on the mode of administration, the disease or disorder to be treated, the condition of the individual subject, the specific viral vector and the nucleic acid to be delivered, and can be determined in a routine manner.
  • Exemplary doses for achieving a therapeutic effect are at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 transduction units or higher viral titers, preferably about 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 transduction units, and more preferably about 10 12 transduction units.
  • more than one administration may be used at variously spaced time periods (e.g., daily, weekly, monthly, yearly, etc.) Achieve desired levels of gene expression.
  • Exemplary modes of administration include oral, rectal, transmucosal, topical, intranasal, inhalation (e.g., via aerosol), intrabuccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to bone, diaphragm, and/or myocardium], intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., cutaneous and mucosal surfaces, including airway surfaces and transdermal administration), intralymphatic, etc., as well as direct tissue or organ injection (e.g., liver, skeletal muscle, myocardium, diaphragm muscle, or brain). Administration may also be given to tumors (eg, in or near tumors or lymph nodes). The most appropriate route in any given case will depend on the nature and severity of the condition being treated and
  • Delivery to any of these tissues may also be achieved by delivering a depot containing the viral vector, which may be implanted in the tissue or the tissue may be in contact with a membrane or other matrix containing the viral vector.
  • a depot containing the viral vector which may be implanted in the tissue or the tissue may be in contact with a membrane or other matrix containing the viral vector. Examples of such implantable matrices or substrates are described in US Patent No. 7,201,898.
  • the methods of the present disclosure may be used to treat disorders of tissues or organs.
  • the methods of the present disclosure can be performed to deliver nucleic acids to tissues or organs for the production of Factor VIII polypeptides that typically circulate in the blood or are delivered systemically to other tissues to treat hemophilia.
  • compositions may be presented in unit/dose or multi-dose containers, for example in sealed ampoules and vials, and may be stored under freeze-dried (lyophilized) conditions requiring only the addition of a sterile liquid carrier immediately before use, e.g. Saline or water for injection.
  • a sterile liquid carrier e.g. Saline or water for injection.
  • a vector is a replicable DNA construct.
  • Vectors are used herein to amplify a nucleic acid encoding a FVIII protein and/or to express a nucleic acid encoding a FVIII protein.
  • An expression vector is a replicable nucleic acid construct in which a nucleotide sequence encoding a FVIII protein is operably linked to a suitable control sequence that enables expression of the nucleotide sequence in a suitable host cell to produce the FVIII protein. .
  • suitable control sequence that enables expression of the nucleotide sequence in a suitable host cell to produce the FVIII protein.
  • control sequences include a transcriptional promoter, optional operator sequences for controlling transcription, sequences encoding appropriate mRNA ribosome binding sites, and sequences that control termination of transcription and translation.
  • Vectors include plasmids, viruses (eg, AAV, adenovirus, cytomegalovirus), phage, and integrable DNA fragments (i.e., fragments that are integrated into the host cell genome by recombination).
  • the vector can replicate and function independently of the host cell genome (e.g., by transient expression), or can integrate into the host cell genome itself (e.g., stable integration).
  • An expression vector may contain a promoter and RNA binding site operably linked to the nucleic acid molecule to be expressed and operable in a host cell and/or organism.
  • DNA regions or nucleotide sequences are operably linked or operably associated when they are functionally related to each other.
  • a promoter is operably linked to a coding sequence if the promoter controls the transcription of the coding sequence; or a ribosome binding site is operably linked to a coding sequence if the promoter is positioned to allow translation of the coding sequence. this coding sequence.
  • Transcriptional and translational control sequences in expression vectors used in transforming vertebrate cells are often provided by viral sources.
  • Non-limiting examples include promoters derived from polyomavirus, adenovirus 2, and simian virus 40 (SV40). See, for example, US Patent No. 4,599,308.
  • Ubiquitous expression promoters refer to a type of strong promoters with broad activity in animal cells, tissues and cell cycles, including CMV, EF1A, EFS , CAG, CBh, CBA, SFFV, MSCV, SV40, mPGK, hPGK, UBC, etc.
  • the coding sequences of the present disclosure may encode FVIII from any species, including mouse, rat, dog, opossum, rabbit, cat, pig, horse, sheep, cow, guinea pig, opossum, platypus, and human, but preferably encode Human FVIII protein. Also included are FVIII-encoding nucleic acids that hybridize to the protein-encoding nucleic acids disclosed herein. This can be done in standard in situ hybridization assays, under reduced stringency conditions or even under stringent conditions (e.g.
  • a cis-acting regulatory region that is "active" in breast tissue may be used because the promoter is more active in breast tissue than in other tissues under the physiological conditions of synthetic milk.
  • These promoters include, but are not limited to, short and long whey acidic protein (WAP), short and long alpha, beta and kappa casein, alpha-lactalbumin and beta-lactoglobulin ("BLG”) promoters.
  • WAP short and long whey acidic protein
  • beta and kappa casein alpha-lactalbumin and beta-lactoglobulin
  • BLG beta-lactoglobulin
  • Signal sequences that secrete the expressed protein directly into other body fluids, particularly blood and urine may also be used in accordance with the present disclosure. Examples of these sequences include the signal peptides of secreted coagulation factors, including those of FVIII, protein C, and tissue plasminogen activator.
  • useful sequences for regulating transcription include enhancers, splicing signals, transcription termination signals, polyadenylation sites, buffer sequences, RNA processing sequences, and other sequences that regulate transgene expression.
  • the expression system or construct contains the 3' untranslated region downstream of the nucleotide sequence encoding the recombinant protein. This region increases transgene expression.
  • useful 3' untranslated regions are sequences that provide the poly A signal.
  • Suitable heterologous 3'-untranslated sequences may be derived from, for example, the SV40 small t antigen, the casein 3' untranslated region, or other 3' untranslated sequences well known in the art.
  • the ribosome binding site is also important for increasing the expression efficiency of FVIII.
  • sequences that modulate post-translational modifications of FVIII are useful in the present disclosure.
  • the coding sequence of the human FVIII-BDD gene FVIII-BDD-Opti was completely synthesized according to the human codon usage efficiency. Its purpose It maximizes the use of more efficient codons to produce fully synthetic human FVIII-BDD genes F8X1Co1 (SEQ ID NO: 4) and F8X1Co3 (SEQ ID NO: 6).
  • FVIII-BDD human FVIII-BDD protein in hepatocytes
  • two synthetic liver-specific promoters were selected: 188 nt LXP2.1 and 200 nt LXP3.3.
  • a synthetic transcription enhancing element with a length of only 71 nt was selected.
  • a small synthetic human intron was selected to further enhance FVIII gene expression.
  • a synthetic polyadenylate of only 48 nt was selected.
  • liver-specific promoter 5' non-coding region, intron, human FVIII-BDD encoding cDNA and polyadenylation were sequentially assembled into a gene expression cassette ( Figure 1) mediated by PEI.
  • Liver Huh7 cells were transfected in vitro, and the cell culture supernatant 3 days after transfection was used to detect human FVIII coagulation activity.
  • the gene expression cassette needs to contain a variety of regulatory elements (such as promoters, enhancers, introns, 5'UTR) to enhance gene transcription and promote gene expression.
  • regulatory elements such as promoters, enhancers, introns, 5'UTR
  • UTRs are known to play a crucial role in the post-transcriptional regulation of gene expression.
  • the inventors simplified the expression cassettes (LXP2.1-F8X1Co4 and LXP3.3-F8X1Co4) constructed in Example 1, thereby constructing a new FVIII gene expression cassette, and studied each Expression efficiency of FVIII gene expression cassette.
  • the enhancer, intron and 5'UTR in the LXP2.1-F8X1Co4 expression cassette were deleted in sequence, and the ITR was further truncated (the D sequences of the ITR at both ends were truncated by 10nt), thus Four human FVIII gene expression cassettes were obtained: LXP2.1-F8X1Co4.1, LXP2.1-F8X1Co4.2, LXP2.1-F8X1Co4.3 and LXP2.1-F8X1Co4.4 ( Figure 3).
  • the gene expression regulatory elements of LXP2.1-F8X1Co4.3 and LXP2.1-F8X1Co4.4 only retain the promoter and polyadenylic acid (poly A).
  • LXP2.1-F8X1Co4.4 in addition to removing the above elements, the D sequence of the ITR at both ends was truncated by 10 nt, and the CAGATCT sequence immediately adjacent to the D sequence was also deleted.
  • the enhancer, intron and 5’UTR in the LXP3.3-F8X1Co4 expression cassette were simultaneously deleted, thereby obtaining LXP3.3-F8X1Co4.1 ( Figure 3).
  • Each of the above expression cassettes was transfected into liver Huh7 cells in vitro through PEI mediation, and the cell culture supernatant 3 days after transfection was used to detect human FVIII coagulation activity.
  • the gene expression cassette LXP2.1-F8X1Co4.1 with the enhancer deleted did not show obvious reduction in gene expression activity, but after deleting the intron or 5'UTR, LXP2.1-F8X1Co4.2, LXP2 .1-F8X1Co4.3, LXP2.1-F8X1Co4.4 and LXP3.3-F8X1Co4.1 gene expression activities were significantly reduced, especially LXP2.1-F8X1Co4.3 and LXP2.1- whose 5'UTR was deleted.
  • F8X1Co4.4 and LXP3.3-F8X1Co4.1 their gene expression activity was reduced by even more than 80%. This result is consistent with the general expectations of those skilled in the art. It is generally believed that the removal of regulatory elements, especially the removal of the 5’ UTR that plays an important role in gene expression, will lead to a reduction in gene expression efficiency.
  • Example 3 Virus yield and genome integrity of adeno-associated virus (AAV) packaging FVIII gene expression cassette
  • human liver-tropic AAV32.1 was used to package the FVIII gene expression cassette involved in Example 2, and the virus integrity and virus yield were detected.
  • the recombinant adeno-associated viruses AAV32.1/LXP2.1-F8X1Co4, AAV32.1/LXP2.1-F8X1Co4.1, and AAV32.1/LXP2 produced by the three-plasmid suspension HEK293 system in Example 3 were used.
  • .1-F8X1Co4.2, AAV32.1/LXP2.1-F8X1Co4.3, AAV32.1/LXP2.1-F8X1Co4.4, AAV32.1/LXP3.3-F8X1Co4 and AAV32.1/LXP3.3-F8X1Co4 .1 Separate and purify through iodixanol two-step ultracentrifugation method.
  • the purified viruses were used to infect liver Huh7 cells at an MOI of 5E+5vg/cell, and the cell culture supernatant 5 days after transfection was used to detect the coagulation activity of human factor VIII.
  • AAV32.1/LXP2.1-F8X1Co4.3, AAV32.1/LXP2.1-F8X1Co4.4 and AAV32.1/LXP3.3-F8X1Co4 were found to have fewer regulatory elements in the expression cassette. 1
  • the gene expression activity produced by AAV32.1/LXP2.1-F8X1Co4, AAV32.1/LXP2.1-F8X1Co4.1, AAV32.1/LXP2.1-F8X1Co4.2 and AAV32.1 is more complete than that of gene regulatory elements.
  • /LXP3.3-F8X1Co4 is significantly higher.
  • the long-term effectiveness of the FVIII gene expression cassette LXP2.1-F8X1Co4.3 in vivo was tested.
  • Male mice with 2 to 3 months of FVIII gene knockout (hemophilia A animal model commonly used in the art) were used.
  • a standard vector dose of 2.5E+11vg/mouse was selected, and recombinant adeno-associated virus AAV32.1/LXP2.1-F8X1Co4.3 was injected into 5 model mice via the tail vein.
  • Untreated age- and sex-matched FVIII knockout mice were used as negative controls.
  • Plasma was collected via the retroorbital vein using standard methods, and plasma was collected weekly within 6 weeks after injection; plasma was collected every 4 weeks thereafter.
  • FVIII gene expression In order to quantitatively evaluate FVIII gene expression, purified recombinant human FVIII was diluted stepwise and added to the plasma of severe hemophilia A patients, as a standard curve for FVIII activity, and conventional APTT tests were performed on plasma samples.
  • the above experimental results show that the FVIII gene expression cassette of the present invention not only has a higher expression level of FVIII in vivo but also can be expressed stably and long-term in vivo.

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Abstract

本发明涉及编码人凝血因子VIII(FVIII)多肽的优化多核苷酸序列。本公开还涉及包含该优化多核苷酸序列的FVIII基因表达盒、重组AAV载体和药物组合物，以及它们在治疗血友病A或获得性因子VIII缺乏症中的应用。本发明的优化的多核苷酸序列和FVIII基因表达盒具有改善的FVIII表达水平。

Description

改进的人凝血因子VIII基因表达盒及其应用 技术领域
本公开涉及编码因子VIII(FVIII)多肽的优化多核苷酸序列。本公开还涉及包含该优化多核苷酸序列的FVIII基因表达盒、重组AAV载体和药物组合物，以及它们在治疗血友病A或获得性因子VIII缺乏症中的应用。
背景技术
因子VIII(FVIII)通过加速因子X转化为因子Xa而在凝血级联(coagulation cascade)中起关键作用。血友病A是一种以缺乏FVIII活性为特征的先天性X连锁性出血性病症。降低的FVIII活性抑制了凝血级联中的正反馈回路。常规地，通过FVIII替代疗法来治疗血友病A，即，向血友病A患者施用FVIII蛋白(例如静脉输注血浆源的或重组的FVIII蛋白)。尽管这种治疗能有效控制出血发作，但由于FVIII的半衰期短(8-12小时)，频繁输注的要求使其固有成本高昂。
基于腺相关病毒(AAV，adeno-associated virus)的基因疗法是最终治愈这种疾病的有吸引力的策略。然而，使用AAV递送FVIII基因的进展不太理想，FVIII在体内的表达效率较低。已有一些试图改变FVIII密码子以提高蛋白表达水平的尝试，但取得的成功有限。
发明内容
鉴于现有技术的上述缺陷与不足，本公开提供了表达水平提高的优化的FVIII多核苷酸序列以及体内表达水平提高的FVIII基因表达盒构建体。本发明人意外地发现，与具有较完整元件的FVIII基因表达盒相比，剔除部分调控元件(例如，增强子、内含子、特别是5’UTR)的FVIII基因表达盒经AAV载体递送至细胞内后仍可以保持相当的甚至是显著改善的FVIII表达水平，由此完成了本发明。
在第一方面，本公开提供一种编码因子VIII(FVIII)多肽的多核苷酸，其中，所述多核苷酸的核苷酸序列与SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：7所示的核苷酸序列具有至少80％的同一性，优选具有至少85％、90％、95％、99％的同一性。
在一个实施方案中，上述多核苷酸包含SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：7所示的核苷酸序列。在一个优选实施方案中，上述多核苷酸的核苷酸 序列如SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：7所示。
在一个实施方案中，上述多核苷酸表达B结构域缺失的因子VIII多肽(FVIII-BDD)。
本公开的优化的FVIII多核苷酸序列相比于未经优化的FVIII多核苷酸序列可显著提高FVIII蛋白的表达水平。
在第二方面，本公开提供一种因子VIII基因表达盒，其包括以下元件，优选由以下元件组成：启动子、可选的kozak序列、编码B结构域缺失的因子VIII多肽的多核苷酸(例如根据第一方面所述的多核苷酸)、多聚腺苷酸(poly A)，以及位于两端的两个ITR。
在一个实施方案中，因子VIII基因表达盒中各元件之间直接连接或通过接头间接连接。
在一个实施方案中，启动子为泛表达启动子、组成型肝特异性启动子或人工合成型肝特异性启动子。在一个优选实施方案中，启动子为人工合成型肝特异性启动子。在一个更优选实施方案中，启动子为SEQ ID NO：9或SEQ ID NO：10所示的肝特异性启动子。
在一个实施方案中，两个ITR各自独立地为正常ITR或截短ITR。在一个优选实施方案中，两个ITR各自独立地为SEQ ID NO：14所示的正常ITR或SEQ ID NO：15所示的截短ITR。在一个更优选实施方案中，两个ITR均为SEQ ID NO：15所示的截短ITR。
在一个实施方案中，因子VIII基因表达盒还包括以下元件中的一种以上：增强子、5’非翻译区(5’UTR)和内含子。
在一个实施方案中，增强子为组成型增强子或人工合成型增强子；优选SEQ ID NO：8所示的增强子。
在一个实施方案中，内含子为组成型内含子或人工合成型内含子；优选SEQ ID NO：12所示的内含子。
在一个实施方案中，因子VIII基因表达盒的核苷酸序列如SEQ ID NO：16、SEQ ID NO：17或SEQ ID NO：18所示。
在第三方面，本公开提供一种重组AAV载体，其包括：根据第二方面所述的因子VIII基因表达盒和AAV衣壳蛋白。
在一个实施方案中，AAV衣壳蛋白为天然AAV衣壳蛋白或人工改造的AAV衣壳蛋白；优选地，AAV选自：AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10和AAV11。
在第四方面，本公开提供根据第三方面所述的重组AAV载体在制备用于预防或治疗血友病A或获得性因子VIII缺乏症的药物中的应用。
在第五方面，本公开提供一种药物，其包含根据第三方面所述的重组AAV载体和可 选的赋形剂。
在一个实施方案中，赋形剂包括：盐、有机物和表面活性剂。
在一个实施方案中，药物通过全身途径或局部途径施用，例如口服、经直肠、经粘膜、鼻内、吸入、颊内(例如舌下)、阴道、鞘内、眼内、透皮、子宫内(或卵内)、肠胃外(例如静脉内、皮下、皮内、肌内、皮内、胸膜内、脑内和关节内)、淋巴内、局部接触、病灶内施用。
在第六方面，本公开提供一种预防或治疗血友病A或获得性因子VIII缺乏症的方法，包括向有需要的受试者施用治疗有效量的根据第一方面所述的多核苷酸、根据第二方面所述的因子VIII基因表达盒、根据第三方面所述的重组AAV载体、根据第五方面所述的药物和/或根据第八方面所述的宿主细胞。
在第七方面，本公开提供一种将因子VIII基因递送至靶细胞中的方法，包括：1)将根据第二方面所述的因子VIII基因表达盒包装于AAV衣壳蛋白中，形成根据第三方面所述的重组AAV载体；以及2)使所述靶细胞与所述重组AAV载体接触。
在一个实施方案中，靶细胞是离体细胞。在一个实施方案中，靶细胞是体内细胞。
在第八方面，本公开提供一种宿主细胞，其用根据第三方面所述的重组AAV载体感染。
在第九方面，本公开提供一种生产重组AAV载体的方法，包括将根据第一方面所述的多核苷酸引入哺乳动物宿主细胞中，所述哺乳动物宿主细胞包含AAV rep基因、AAV cap基因和病毒复制辅助基因。
在下面的描述中更详细地阐述了本公开的上述和其它方面。
附图说明
图1显示了FVIII基因密码子优化前后的基因表达盒组成元件：反向末端重复(ITR)序列(SEQ ID NO：14)、增强子(SEQ ID NO：8)、肝特异性启动子LXP2.1(SEQ ID NO：9)或肝特异性启动子LXP3.3(SEQ ID NO：10)、5’UTR(SEQ ID NO：11)、内含子(SEQ ID NO：12)、Kozak序列(GCCACC)、人FVIII编码cDNA(FVIII-BDD，SEQ ID NO：3)或密码子优化的人FVIII编码cDNA(FVIII-BDD-Opti，SEQ ID NO：4～7)、多聚腺苷酸(poly A)。
图2显示了具有图1所示组成元件的FVIII基因表达盒质粒转染Huh7细胞后FVIII蛋白表达水平。显示的是经基因表达盒转染的Huh7细胞培养上清的凝血活性检测结果。
图3显示了密码子优化的FVIII基因(SEQ ID NO：7)与表达调控元件的不同组合构成的FVIII基因表达盒。
图4显示了图3所示的FVIII基因表达盒质粒转染Huh7细胞后FVIII蛋白表达水平。显示的是经基因表达盒转染的Huh7细胞培养上清的凝血活性检测结果。
图5A显示了腺相关病毒(AAV)包装图3所示的各个FVIII基因表达盒在相同生产条件下的病毒产量。
图5B显示了包装图3所示的各个FVIII基因表达盒的腺相关病毒(AAV)经DNA抽提产生的样品进行碱性胶电泳的结果。
图6显示了包装图3所示的各个FVIII基因表达盒的腺相关病毒(AAV)感染Huh7细胞后FVIII蛋白表达水平。显示的是经携带基因表达盒的AAV感染的Huh7细胞培养上清的凝血活性检测结果。
图7显示了在静脉注射AAV32.1/LXP2.1-F8X1Co4.3后，5只FVIII敲除小鼠中的长期FVIII活性(作为正常人类FVIII活性的百分比)。
具体实施方式
除非另有定义，否则本文使用的所有技术和科学术语具有与本公开所属领域的普通技术人员的通常理解相同的含义。在本文中本公开的描述中使用的术语仅用于描述特定实施方案的目的，而不旨在限制本发明。
除非另有说明，否则本文列出的核酸或多核苷酸序列是单链形式，方向是从5'至3'，从左至右。本文提供的核苷酸和氨基酸采用IUPACIUB生化命名委员会建议的格式，对于氨基酸采用单字母代码或三字母代码。
除非另有说明，本领域技术人员可使用已知的标准方法用于克隆基因、扩增和检测核酸等。这些技术是本领域技术人员已知的。参见例如Sambrook等，Molecular Cloning：A Laboratory Manual，第2版(Cold Spring Harbor，纽约，1989)；Ausubel等，Current Protocols in Molecular Biology(Green Publishing Associates，Inc.和John Wiley&Sons，Inc.，纽约)。
定义
如本文所用，术语“和/或”涉及并涵盖一个以上的所列项目的任何和所有可能组合。
在本文中，术语“包含”、“具有”、“包括”和“含有”应被解释为开放式术语(即意味着“包括但不限于”)。
在本文中，术语“患者”和“受试者”可互换使用并且以其常规意义使用，包括患有或易感出血性病症或出血病况的任何动物，其需要和/或期望控制出血，其可以通过向受试者给 药FVIII来治疗、改善或预防(例如血友病A和获得性FVIII缺乏症(例如，由于针对FVIII或血液恶性肿瘤的自身抗体引起的))。这样的受试者通常是哺乳动物(例如，实验室动物，如大鼠、小鼠、豚鼠、兔、灵长类动物等)、农场或商业动物(例如母牛、马、山羊、驴、绵羊等)或家畜(例如猫、狗、白鼬等)。在一个实施方案中，受试者是灵长类受试者、非人灵长类受试者(例如黑猩猩、狒狒、猴、大猩猩等)或人类。本公开的受试者可以是已知或相信具有需要和/或期望控制的出血性病症或出血病况风险的受试者。可替代地，根据本公开所述的受试者，还可以包括先前未知或怀疑具有需要或期望控制的出血性病症或出血病况风险的受试者。作为另一个选择，受试者可以是实验室动物和/或疾病的动物模型。
受试者包括任何年龄的男性和/或女性，包括新生儿、幼年、成年和老年受试者。关于人类受试者，在代表性的实施方案中，受试者可以是婴儿(例如小于约12个月、10个月、9个月、8个月、7个月、6个月或更小的年龄)、幼儿(例如至少约12、18或24个月和/或小于约36、30或24个月)或儿童(例如至少约1、2、3、4或5岁和/或小于约14岁、12、10、8、7、6、5或4岁)。在本公开的实施方案中，受试者是约0至3、4、5、6、9、12、15、18、24、30、36、48或60个月龄的人类受试者，约3至6、9、12、15、18、24、30、36、48或60个月龄的人类受试者，约6至9、12、15、18、24、30、36、48或60个月龄的人类受试者，约9至12、15、18、24、30、36、48或60个月龄的人类受试者，约12至18、24、36、48或60个月龄的人类受试者，约18至24、30、36、48或60个月龄的人类受试者，或约24至30、36、48或60个月龄的人类受试者。
在本文中，术语“治疗”包括：(1)抑制病状、疾病或者病症，即，阻止、减少或者延迟疾病的发展或其复发或者其至少一种临床或者亚临床症状的发展；或者(2)缓解疾病，即，引起病状、疾病或者病症或者其临床或者亚临床症状中的至少一种消退。
在本文中，术语“治疗有效量”是为受试者提供一些改善或益处的量。例如，适用于治疗血友病A或获得性因子VIII缺乏症的药物的治疗有效量可为能够预防或改善与血友病A或获得性因子VIII缺乏症相关的一种或多种症状的量。
在本文中，术语“改善”指与疾病有关的症状的改善，并且可以指至少一种衡量或定量该症状的参数的改善。
在本文中，术语“预防”病状、疾病或者病症包括：预防、延迟或者减少受试者中发展的病状、疾病或者病症的至少一种临床或者亚临床症状出现的发生率和/或可能性，该受试者可能患有或易患该病状、疾病或者病症但尚未经历或者表现出该病状、疾病或者病症的 临床或亚临床症状。例如，“预防”血友病A是指与在不存在预防性治疗时发生的出血发作的次数和/或严重程度相比，出现出血发作的次数和/或严重程度的降低。
在本文中，术语“局部施用”或“局部途径”是指具有局部作用的给药。
当涉及可测量的值，例如多肽的量、剂量、时间、温度、酶活性或其他生物活性等时，如本文所使用的术语“约”意在涵盖指定数量的±20％、±10％、±5％、±1％、±0.5％或甚至±0.1％的变化。
过渡性短语“基本上由…组成”或“由…组成”是指权利要求的范围应被解释为包括权利要求中所述的指定材料或步骤以及不会实质上影响所要求保护的发明的特征的那些。
应用于本公开的多核苷酸或多肽序列的术语“基本上由…组成”是指由所述序列(例如SEQ ID NO)和在所述序列的5′和/或3′或N末端和/或C末端上的总计为十个或更少(例如1、2、3、4、5、6、7、8、9或10个)附加核苷酸或氨基酸组成的多核苷酸或多肽，其中所述附加核苷酸或氨基酸不会导致所述多核苷酸或多肽的功能实质上改变。应用于本公开的多核苷酸的术语“实质上改变”，是指与由所述序列组成的多核苷酸的表达水平相比，表达编码的多肽的能力增加或降低至少约50％或更多。应用于本公开的多肽的术语“实质上改变”，是指与由所述序列组成的多肽的活性相比，凝结刺激活性的增加或降低至少约50％或更多。
本文所用的术语“增强”、“增加”或“改善”是指指定参数的增加至少为约1.25倍、1.5倍、2倍、3倍、4倍、5倍、6倍、8倍、10倍、12倍或甚至15倍。
本文所用的术语“抑制”、“减少”或“降低”是指指定水平或活性的降低或减少至少约15％、25％、35％、40％、50％、60％、75％、80％、90％、95％或更多。在一个实施方案中，“抑制”、“减少”或“降低”导致很少或基本上不可检测的活性(例如小于约10％或甚至5％)。
如本领域技术人员众所周知的，可以通过检测受试者的症状和/或临床参数的变化所指示的临床改善来确定通过本公开的方法治疗血友病A或获得性因子VIII缺乏症的功效。
如本文所用，术语“可操作地连接”是指第一核苷酸序列(例如基因)与第二核苷酸序列(例如调控控制元件)之间的关系，所述关系允许第二核苷酸序列影响一种或多种与第一核苷酸序列相关的性质(例如转录速率)。例如，启动子可操作地连接于编码序列，如直接连接(启动子与编码序列之间不包含额外的核苷酸)或通过接头(如无功能接头)间接连接。
如本文所使用的，“核酸”、“核苷酸序列”和“多核苷酸”可互换使用并且涵盖RNA和 DNA两者，包括cDNA、基因组DNA、mRNA、合成(例如化学合成的)DNA或RNA以及RNA和DNA的嵌合体。术语“核酸”、“核苷酸序列”和“多核苷酸”是指不考虑链长度的核苷酸的链。核酸可以是双链或单链的。当单链时，核酸可以是有义链或反义链。可以使用寡核苷酸类似物或衍生物(例如肌苷或硫代磷酸酯核苷酸)合成核酸。这样的寡核苷酸可以用于例如制备具有改变的碱基配对能力或增加的核酸酶抗性的核酸。本公开还提供了一种核酸，其为本公开的核酸、核苷酸序列或多核苷酸的互补体(可以是完全互补体或部分互补体)。
“分离的多核苷酸”是一核苷酸序列(例如DNA或RNA)，其没有直接与其在所来源的生物体的天然存在的基因组中直接连接的核苷酸序列(一个在5'末端和一个在3'末端)相连接。因此，在一个实施方案中，分离的核酸包括紧邻编码序列的5′非编码(例如启动子)序列的一些或全部。因此，该术语包括例如并入载体中、并入自主复制的质粒或病毒中、或并入原核生物或真核生物的基因组DNA中的重组DNA，或作为单独分子(例如通过PCR或限制性内切核酸酶处理产生的cDNA或基因组DNA片段)存在的独立于其他序列的重组DNA。它还包括作为编码附加多肽或肽序列的杂交核酸的一部分的重组DNA。包括基因的分离的多核苷酸，不是包括此种基因的染色体的片段，而是包括与该基因相关的编码区和调节区，但是没有该染色体上天然存在的额外基因。
应用于多核苷酸的术语“片段”将被理解为是指相对于参考核酸或核苷酸序列，长度减小的核苷酸序列，并且包含(基本上其组成和/或由其组成)与所述参考核酸或核苷酸序列一致或几乎一致(例如90％、92％、95％、98％、99％一致)的连续核苷酸的核苷酸序列。根据本公开所述的这种核酸片段在适当的情况下可以被包括在其作为组成的更大的多核苷酸中。在一些实施方案中，此种片段可以包含(基本由其组成和/或由其组成)具有根据本公开所述的核酸或核苷酸序列的至少约8、10、12、15、20、25、30、35、40、45、50、75、100、150、200或更多连续核苷酸长度的寡核苷酸。
术语“分离的”可以指基本上不含细胞材料、病毒材料、和/或培养基(当通过重组DNA技术产生时)、或化学前体或其它化学物质(当化学合成时)的核酸、核苷酸序列或多肽。此外，“分离的片段”是核酸、核苷酸序列或多肽的片段，其不是天然存在的片段，并且在天然状态下不会被发现。“分离的”并不意味着制备在技术上是纯的(均匀的)，但是其足够纯以提供可用于预期目的形式的多肽或核酸。
应用于多肽的术语“片段”将被理解为是指相对于参考多肽或氨基酸序列，长度减少的氨基酸序列，并且包含(基本上由其组成和/或由其组成)与所述参考多肽或氨基酸序列一 致或几乎一致(例如90％、92％、95％、98％、99％一致)的连续氨基酸的氨基酸序列。根据本公开所述的这种多肽片段在适当的情况下可以被包含在其作为组成的较大多肽中。在一些实施方案中，此种片段可以包含(基本上由其组成和/或由其组成)具有根据本公开所述的多肽或氨基酸序列的至少约4、6、8、10、12、15、20、25、30、35、40、45、50、75、100、150、200或更多连续氨基酸长度的肽。
“载体”是用于将核酸克隆和/或转移到细胞中的任何核酸分子。载体可以是可以附接另一个核苷酸序列以允许所附接的核苷酸序列复制的复制子。“复制子”可以是在体内作为核酸复制的自主单元(即能够在其自身控制下复制)的任何遗传元件(例如质粒、噬菌体、粘粒、染色体、病毒基因组)。术语“载体”包括在体外、离体和/或体内将核酸引入细胞的病毒和非病毒(例如质粒)核酸分子。可以使用本领域已知的大量载体来操纵核酸、将响应元件和启动子合并到基因中等。例如，将对应于响应元件和启动子的核酸片段插入合适的载体中，可以通过将合适的核酸片段连接到具有互补结合末端的选定载体中来实现。可替代地，核酸分子的末端可以被酶促修饰，或者可以通过将核苷酸序列(接头)连接到核酸末端来产生任何位点。可以将这样的载体工程化成含有编码选择性标记的序列，这便于含有所述载体的细胞和/或已将所述载体的核酸合并到细胞基因组中的细胞的选择。这样的标记允许鉴定和/或选择合并且表达由所述标记编码的蛋白质的宿主细胞。“重组”载体是指包含一个或多个异源核苷酸序列(即转基因)的病毒或非病毒载体，例如包含两个、三个、四个、五个或更多个异源核苷酸序列。
病毒载体已经用于在细胞中以及活的动物受试者中的各种基因递送应用中。可以使用的病毒载体包括但不限于逆转录病毒、慢病毒、腺相关病毒、痘病毒、甲病毒、杆状病毒、痘苗病毒、疱疹病毒、爱泼斯坦-巴尔病毒和腺病毒载体。非病毒载体包括质粒、脂质体、带电荷的脂质(细胞转染剂(cytofectin))、核酸-蛋白复合物和生物聚合物。除目标核酸之外，载体还可以包含一个或多个调节区和/或可用于选择、测量和监测核酸转移结果(递送到特定组织、表达持续时间等)的可选择标记。
可以通过本领域已知的方法将载体引入到所需的细胞中，例如转染、电穿孔、微注射、转导、细胞融合、DEAE葡聚糖、磷酸钙沉淀、脂质转染(溶酶体融合)、使用基因枪或核酸载体转运蛋白(参见例如Wu等，J.Biol.Chem.267：963(1992)；Wu等，J.Biol.Chem.263：14621(1988)；和Hartmut等于1990年3月15日提交的加拿大专利申请号2,012,311)。在各种实施方案中，可用其他分子来促进核酸在体内的递送，诸如阳离子寡肽(例如WO95/21931)、衍生自核酸结合蛋白的肽(例如WO96/25508)和/或阳离子聚合物(例如 WO95/21931)。也可以在体内引入作为裸核酸的载体(参见美国专利号5,693,622、5,589,466和5,580,859)。还可以使用受体介导的核酸递送方法(Curiel等，Hum.GeneTher.3：147(1992)；Wu等，J.Biol.Chem.262：4429(1987))。
除非另有说明，如本文使用的术语“蛋白质”和“多肽”可互换使用并且包括肽和蛋白质。
“融合蛋白”是当自然界中未发现融合在一起的编码两种(或更多种)不同多肽的两个异源核苷酸序列或其片段，在正确的翻译阅读框中被融合在一起时，产生的多肽。说明性的融合多肽包括本公开的多肽(或其片段)与谷胱甘肽-S-转移酶、麦芽糖结合蛋白或报告蛋白(例如绿色荧光蛋白、β-葡萄糖醛酸酶、β-半乳糖苷酶、荧光素酶等)、血凝素、c-myc、FLAG表位等的全部或部分的融合物。
如本文所使用的，“功能性”多肽或“功能性片段”是基本上保留至少一种通常与该多肽相关的生物活性(例如血管生成活性、蛋白结合、配体或受体结合)的物质。在一个实施方案中，“功能性”多肽或“功能性片段”基本上保留由未修饰的肽所具有的所有活性。“基本保留”生物活性，是指多肽保留天然多肽的至少约20％、30％、40％、50％、60％、75％、85％、90％、95％、97％、99％或更多的生物活性(并且甚至可以具有比天然多肽更高的活性水平)。“非功能性”多肽是表现出很少或基本上没有通常与多肽相关的可检测的生物活性的多肽(例如，至多只有不明显的量，例如小于约10％或甚至5％)。生物活性如蛋白质结合和血管生成活性可以使用本领域熟知的和如本文所述的测定法来测量。
术语“表达”多核苷酸编码序列，其意思是将该序列转录并可选地翻译。通常，根据本公开，本公开的编码序列的表达将导致本公开的多肽的产生。整体表达的多肽或片段也可以在完整的细胞中起作用而不需要纯化。
在本公开的上下文中，术语“腺相关病毒”(AAV)包括但不限于此AAV1型、AAV2型、AAV3型(包括3A和3B型)、AAV4型、AAV5型、AAV6型、AAV7型、AAV8型、AAV9型、AAV10型、AAV11型、鸟AAV、牛AAV、犬AAV、马AAV和羊AAV以及现在已知或以后发现的任何其他AAV。参见例如BERNARD N.FIELDS等，VIROLOGY，第2卷，第69章(第4版，Lippincott-Raven Publishers)。已经鉴定了一些额外的AAV血清型和进化枝(clade)(参见例如Gao等，(2004)J.Virol.78：6381-6388)，其也被术语“AAV”所涵盖。
各种AAV和自主细小病毒的基因组序列，以及ITR、Rep蛋白和衣壳亚基的序列是本领域已知的。这些序列可以在文献或公共数据库中找到，例如GenBank，参见例如，GenBank登录号NC 002077、NC 001401、NC 001729、NC 001863、NC 001829、NC 001862、NC 000883、 NC 001701、NC 001510、AF063497、U89790、AF043303、AF028705、AF028704、J02275、J01901、J02275、X01457、AF288061、AH009962、AY028226、AY028223、NC 001358、NC001540、AF513851、AF513852、AY530579、AY631965、AY631966；其公开内容全部并入本文。还参见例如Srivistava等，(1983)J.Virol.45：555；Chiorini等，(1998)J.Virol.71：6823；Chiorini等，(1999)J.Virol.73：1309；Bantel-Schaal等，(1999)J.Virol.73：939；Xiao等，(1999)J.Virol.73：3994；Muramatsu等，(1996)Virology 221：208；Shade等，(1986)J.Virol.58：921；Gao等，(2002)Proc.Nat.Acad.Sci.USA 99：11854；国际专利公布WO 00/28061、WO 99/61601、WO 98/11244；美国专利号6,156,303；它们的公开内容全部并入本文。Xiao，X.，(1996)，“Characterization of Adeno-associated virus(AAV)DNA replication andintegration”，Ph.D.Dissertation，University of Pittsburgh，Pittsburgh，PA(其整体并入本文)提供了AAV1、AAV2和AAV3末端重复序列的早期描述。
“重组AAV载体基因组”或“rAAV基因组”是包含至少一个反向末端重复(例如一个、两个或三个反向末端重复序列)和一个或多个异源核苷酸序列的AAV基因组(即vDNA)。rAAV载体通常以顺式(cis)保留145个碱基末端重复(TR)以产生病毒；然而，修饰的AAV TR和非AAV TR(包括部分或完全合成序列)也可以用于该目的。所有其他病毒序列是不必要的，可以以反式(trans)提供(Muzyczka，(1992)Curr.Topics Microbiol.Immunol.158：97)。rAAV载体可选地包含两个TR(例如AAV TR)，其通常将处于异源核苷酸序列的5’和3’端，但不必与其相邻近。TR可以彼此相同或彼此不同。载体基因组还可以在其3’或5’端含有单个ITR。
术语“末端重复”或“TR”包括形成发夹结构并用作反向末端重复(即介导所需功能例如复制、病毒包装、整合和/或原病毒拯救等等)的任何病毒末端重复或合成序列。TR可以是AAV TR或非AAV TR。例如，非AAV TR序列，例如其他细小病毒(例如犬细小病毒(CPV)、小鼠细小病毒(MVM)、人细小病毒B-19)的那些序列或作为SV40复制起点的SV40发夹的序列，可以被用作TR，它们可以进一步通过截短、取代、缺失、插入和/或添加来修饰。此外，TR可以是部分或完全合成的，诸如Samulski等的美国专利号5,478,745中所描述的“双D序列”。
“AAV末端重复”或“AAV TR”可以来自任何AAV，包括但不限于血清型1、2、3、4、5、6、7、8、9、10或11或现在已知或以后发现的任何其他AAV(参见例如表1)。AAV末端重复不需要具有天然末端重复序列(例如天然AAV TR序列可以通过插入、缺失、截短和/或错义突变而改变)，只要所述末端重复介导所需的功能例如复制、病毒包装、整合 和/或原病毒拯救等即可。
术语“rAAV颗粒”和“rAAV病毒颗粒”在这里可互换使用。“rAAV颗粒”或“rAAV病毒颗粒”包含包装在AAV衣壳内的rAAV载体基因组。BERNARD N.FIELDS等，VIROLOGY，第2卷，第69&70章(第4版，Lippincott-Raven Publishers)中更详细地描述了AAV衣壳结构。
如本文所用的，“生物活性”或“活性”是参照例如来自人类血浆的标准物来确定的。对于FVIII，该标准物可以是
Figure PCTCN2022143456-appb-000001
(CSL Behring)。该标准物的生物活性取为100％。
本文所用的术语“因子VIII蛋白”或“FVIII蛋白”包括野生型FVIII蛋白以及天然存在的或人造的蛋白(例如B结构域缺失的蛋白)。本公开的FVIII蛋白可以进一步包括文献中已知的突变形式的FVIII。本公开的FVIII蛋白，还包括现在已知的或稍后鉴定的任何其它天然存在的人类FVIII蛋白或人造的人类FVIII蛋白，以及本领域已知的它们的衍生物和活性片段/活性结构域。
来自多种哺乳动物物种的FVIII的氨基酸序列，可从诸如GenBank的序列数据库获得。FVIII序列的例子如下表所示。
物种 GenBank登录号.
人类(Homo sapiens) AAA52484.1
小家鼠(Mus musculus) NP_032003.2
野猪(Sus scrofa) AAB06705.1
黄牛(Bos Taurus) NP_001138980.1
家犬(Canis lupus familiaris) NP-001003212.1
褐家鼠(Rattus norvegicus) ADU79112.1
本公开的FVIII蛋白还包括FVIII的药理活性形式，其是已除去了信号肽的分子，并且已通过蛋白酶的作用缺失了B结构域(或通过在核酸水平上除去其而将其从蛋白质中工程化出来)，产生折叠为功能性FVIII凝血因子的FVIII的两个不连续的多肽链(轻链和重链)。已知多个B结构域缺失形式的人类FVIII，包括经常使用的SQ版本，其中S743和Q1638之间的残基被缺失。具体地说，具有增加糖基化程度的修饰的FVIII蛋白在广义上被具体地包括在内。
人类FVIII蛋白的氨基酸序列是本领域熟知的，并且可以在GenBank登录号AAA52484 中找到。人类FVIII蛋白的长度为2351个氨基酸，并且由信号肽(残基1-19)、重链(残基20-759)、B结构域(残基760-1332)和轻链(残基1668-2351)组成。以下公开了不具有信号肽的人FVIII蛋白的氨基酸序列(SEQ ID NO：1)。
术语“半衰期”是一个广义术语，其包括通常和惯用的含义以及在FVIII科学文献中发现的通常和惯用意义。该定义中具体包括与FVIII相关的参数的测量，其定义了从输注时测量的初始值减少到初始值的一半所用的输注后的时间。在一些实施方案中，可以在各种免疫测定中使用FVIII的抗体在血液和/或血液成分中测量FVIII的半衰期，如本领域熟知的和本文所述的。可替代地，可以使用包括标准凝血测定在内的功能测定法，以FVIII活性的降低来测量半衰期，如本领域公知的和如本文所述的。
如本文所用的，“转化的”细胞是已经用编码本公开的FVIII蛋白的核酸分子转化、转导和/或转染的细胞，包括但不限于使用重组DNA技术构建的FVIII蛋白载体。
如本文所用的，术语“出血性病症”反映了出血表现出的细胞、生理或分子起源的任何缺陷、先天性、获得性或诱导性。例子是凝血因子缺陷(例如，血友病A和B或凝血因子XI、VII、VIII或IX的缺陷)、凝血因子抑制剂、血小板功能不全、血小板减少症、血管性血友病(冯·威利布兰德疾病)或由外科手术或外伤引起的出血。
过度出血也发生在具有正常功能的血液凝血级联(无凝血因子缺乏症或针对任何凝血因子的抑制剂)的受试者中，并且可能由血小板功能不全、血小板减少症、或血管性血友病引起。在这种情况下，出血可能与血友病引起的出血相似，因为止血系统(如在血友病中)缺少或具有异常的必要凝血“化合物”(如血小板或血管性血友病因子蛋白)，导致重大出血。在经历与手术或创伤相关的广泛组织损伤的受试者中，正常的止血机制可能会由于立即止血的需求而不堪重负，因此尽管有正常的止血机制也仍可能出现出血。在诸如脑、内耳区域和眼睛等器官中出血时，外科止血的可能性有限，实现令人满意的止血也是一个问题。在各种器官(肝、肺、肿瘤组织、胃肠道)以及腹腔镜手术中进行活组织检查的过程中也可能出现同样的问题。所有这些情况的共同之处在于，通过手术技术(缝线、夹子等)难以提供止血，在出血为扩散性时(出血性胃炎和大量子宫出血)也是如此。急性和大量出血也可能发生在抗凝治疗的受试者中，在受试者中通过给予的治疗诱发了有缺陷的止血。在必须迅速抵消抗凝作用的情况下，这些受试者可能需要手术干预。根治性耻骨前列腺缺失术(radical retropubic prostatectomy)是针对具有局限性前列腺癌的受试者的常规手术。手术经常因显著且有时大量失血而复杂化。前列腺缺失术过程中相当大的失血主要与复杂的解剖情况有关，其具有不容易获得手术止血的各种密集血管化的部位，并且可能 导致大面积的弥漫性出血。此外，大脑内出血是中风中最不可治疗的形式，并且与脑内出血后头几个小时的高死亡率和血肿生长有关。在止血不良的情况下可能引起问题的另一种情形是，当具有正常止血机制的受试者进行抗凝治疗以预防血栓栓塞性疾病时的情形。这样的治疗可以包括肝素、其他形式的蛋白聚糖、杀鼠灵或其他形式的维生素K拮抗剂以及阿司匹林和其他血小板聚集抑制剂。
在本公开的一个实施方案中，出血与血友病有关。在另一个实施方案中，出血与获得性抑制剂的血友病有关。在另一个实施方案中，出血与血小板减少症有关。在另一个实施方案中，出血与血管性血友病(von Willebrand’s disease)有关。在另一个实施方案中，出血与严重的组织损伤有关。在另一个实施方案中，出血与严重创伤有关。在另一个实施方案中，出血与手术有关。在另一个实施方案中，出血与腹腔镜手术有关。在另一个实施方案中，出血与出血性胃炎有关。在另一个实施方案中，出血是大量的子宫出血。在另一个实施方案中，出血发生在机械止血可能性有限的器官中。在另一个实施方案中，出血发生在脑、内耳区域或眼睛中。在另一个实施方案中，出血与采取活组织检查的过程有关。在另一个实施方案中，出血与抗凝治疗有关。
优化的FVIII基因
本公开的一个方面涉及密码子优化的编码FVIII-BDD的多核苷酸序列(核酸优化体FVIII-BDD-Opti)，用于在人中表达。在一些实施方案中，B结构域缺失的FVIII(FVIII-BDD)蛋白是糖基化程度增加的修饰的FVIII蛋白(国际专利WO2016127057；氨基酸序列SEQ ID NO：2和核苷酸序列SEQ ID NO：3)。
在某些实施方案中，密码子优化的序列包括、基本上由或由与SEQ ID NO：4和/或SEQ ID NO：6其中之一具有至少90％同一性的序列组成，例如与SEQ ID NO：4和/或SEQ ID NO：6的核苷酸序列具有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。在某些实施方案中，多核苷酸序列优化体与SEQ ID NO：4和/或SEQ ID NO：6中的任一个具有95％-100％的序列同一性。
在某些实施方案中，与编码FVIII-BDD的野生型核酸(SEQ ID NO：3)相比，编码FVIII-BDD的核酸优化体具有降低的CpG含量。在某些实施方案中，与编码FVIII-BDD的野生型核酸相比，核酸优化体的CpG至少少20个。在某些实施方案中，核酸优化体具有不超过10个CpG，不超过9个CpG，不超过8个CpG，不超过7个CpG，不超过6个CPG，不超过5个CpG，不超过4个CpG，不超过3个CpG，不超过2个CpG或不超过1个CpG。在某些实施方案中，核酸优化体具有至多4个CpG、3个CpG、2个CpG或1 个CpG。在某些实施方案中，核酸优化体不具有CpG。
在某些实施方案中，与编码FVIII-BDD的野生型核酸相比，编码FVIII-BDD的核酸优化体具有降低的CpG含量，并且此类CpG减少的核酸变体与SEQ ID NO：5和/或SEQ ID NO：7中的任一个具有90％或更高的序列同一性。在某些实施方案中，CpG减少的核酸变体与SEQ ID NO：5和/或SEQ ID NO：7中的任一个具有91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％或更高的序列同一性。在某些实施方案中，CpG减少的核酸优化体与SEQ ID NO：5和/或SEQ ID NO：7中的任一个具有90％-95％的序列同一性。在某些实施方案中，CpG减少的核酸优化体与SEQ ID NO：5和/或SEQ ID NO：7中的任一个具有95％-100％的序列同一性。在某些实施方案中，编码FVIII-BDD的CpG减少的核酸优化体如SEQ ID NO：5和/或SEQ ID NO：7中的任一个所示。
在某些实施方案中，编码FVIII-BDD蛋白的核酸变体与B结构域缺失的野生型人FVIII核酸至少75％相同。在某些实施方案中，编码FVIII-BDD蛋白的核酸变体与B结构域缺失的野生型人FVIII核酸约75-95％相同，例如，约75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％相同。
在某些实施方案中，编码FVIII-BDD蛋白的核酸和变体是哺乳动物的，例如人的。这样的编码FVIII-BDD蛋白的哺乳动物核酸和核酸变体包括人形式，其可以基于B结构域缺失的野生型人FVIII。
表达盒
本公开的一个方面涉及包含以下元件，优选由以下元件组成的基因表达盒：可选的增强子、启动子、可选的5’非编码区(5’UTR)、可选的内含子、可选的kozak序列、B结构域缺失的人FVIII(FVIII-BDD)蛋白编码cDNA、多聚腺苷酸(poly A)以及位于两端的两个ITR。
在一个优选的实施方案中，本公开的基因表达盒为由以下元件组成的基因表达盒：启动子、B结构域缺失的人FVIII(FVIII-BDD)蛋白编码cDNA、多聚腺苷酸(poly A)以及位于两端的两个ITR。
在一个更优选的实施方案中，本公开的基因表达盒为由以下元件组成的基因表达盒：启动子、kozak序列、B结构域缺失的人FVIII(FVIII-BDD)蛋白编码cDNA、多聚腺苷酸(poly A)以及位于两端的两个ITR。
在一个优选的实施方案中，增强子包含或由SEQ ID NO：8所示的核苷酸序列组成。
在一个优选的实施方案中，启动子包含或由肝特异性启动子LXP2.1(SEQ ID NO：9)或LXP3.3(SEQ ID NO：10)的核苷酸序列组成。
在一个优选的实施方案中，5’UTR包含或由SEQ ID NO：11所示的核苷酸序列组成。
在一个优选的实施方案中，内含子包含或由SEQ ID NO：12所示的核苷酸序列组成。
在一个优选的实施方案中，kozak序列包含或由GCCACC组成。
在一个实施方案中，B结构域缺失的FVIII的氨基酸序列例如SEQ ID NO：2。
在一个优选的实施方案中，B结构域缺失的FVIII可以由多核苷酸编码，所述多核苷酸包含SEQ ID NO：4～7中任一项所示的核苷酸序列或与其具有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，或基本上由其组成，或由其组成。
在一个优选的实施方案中，多聚腺苷酸(poly A)包含或由SEQ ID NO：13所示的核苷酸序列组成。
在一个优选的实施方案中，ITR包含或由SEQ ID NO：15所示的核苷酸序列组成。
本公开的另一方面涉及包含本公开的多核苷酸的载体，例如表达载体。载体可以是本领域已知的任何类型的载体，包括但不限于质粒载体和病毒载体。在一些实施方案中，病毒载体是逆转录病毒或慢病毒载体。在一些实施方案中，病毒载体是来自任何已知AAV血清型的AAV载体，包括但不限于AAV1型、AAV2型、AAV3型(包括3A型和3B型)、AAV4型、AAV5型、AAV6型、AAV7型、AAV8型、AAV9型、AAV10型、AAV11型、鸟AAV、牛AAV、犬AAV、马AAV和羊AAV以及现在已知或稍后发现的任何其他AAV。在一些实施方案中，AAV载体是AAVXL32.1(参见国际专利公布WO 2019241324A1)。
本公开的另一方面涉及包含本公开的多核苷酸和/或载体的细胞(例如，分离的细胞、转化细胞、重组细胞等)。因此，本公开的一些实施方案涉及含有载体(例如表达盒)的重组宿主细胞。这样的细胞可以是分离的和/或存在于转基因动物中。
本公开的另一方面涉及包含本公开的多核苷酸、载体和/或转化细胞的转基因动物。
本公开的多核苷酸、载体和/或细胞可以被包括在药物组合物中。一些实施方案涉及试剂盒，其包含本公开的多核苷酸、载体和/或细胞，和/或使用该试剂盒的试剂和/或说明书。
方法
本公开的一个方面涉及使用本公开的FVIII基因表达盒治疗受试者的血友病A或获得性因子VIII缺乏症的方法，包括向受试者递送治疗有效量的本公开的多核苷酸、载体和/或转化细胞，由此治疗受试者的血友病A或获得性因子VIII缺乏症。
本公开的另一方面涉及使用本公开的密码子和CpG优化的核苷酸序列提高FVIII多肽 在受试者中长期高效表达的方法，包括向受试者递送有效量的本公开的多核苷酸、载体和/或转化细胞，由此提高受试者中FVIII多肽的长期高效表达。
本公开的一个方面涉及在受试者的肝脏中产生FVIII多肽的方法，包括向受试者递送本公开的编码B结构域缺失的人FVIII多肽的优化多核苷酸、载体和/或转化细胞，从而在受试者的肝脏中产生FVIII多肽。
本公开的另一方面涉及治疗受试者的血友病A或获得性因子VIII缺乏症的方法，包括向受试者递送治疗有效量本公开的编码B结构域缺失的人FVIII多肽的优化多核苷酸、载体和/或转化细胞，由此治疗受试者的血友病A或获得性FVIII缺乏症。
根据本公开的方法可以治疗的出血性病症包括可用FVIII治疗的任何病症，诸如血友病A和获得性FVIII缺乏症。用于向受试者(例如有此需要的受试者)给药或递送本公开的FVIII蛋白和/或本公开的编码FVIII蛋白的多核苷酸的此种治疗策略和给药方案是本领域熟知的。
在本公开的实施方案中，本公开的载体(例如，病毒载体或其他核酸载体)的剂量可以是使达到FVIII蛋白的治疗血浆浓度的量。FVIII蛋白的治疗浓度被认为是高于健康个体的正常水平的1％，其是在平均100％上测量的，即1mL正常人类血浆中的一个国际单位(IU)的FVIII。本领域技术人员将能够确定给定受试者和给定病况的最佳剂量。
本公开的药物组合物可以用于预防性和/或治疗性给药。在治疗性应用中，以足以治愈、减轻或部分阻止如上所述的疾病及其并发症的量，将组合物给药于已经患有所述疾病的受试者。足以实现这一目标的量被定义为“治疗有效量”。如本领域技术人员所理解的，有效于此目的量将取决于疾病或损伤的严重程度以及受试者的体重和一般状态。
在一些实施方案中，使用AAV载体向受试者递送本公开的FVIII基因表达盒。因此，本公开还提供了包含FVIII基因表达盒的AAV病毒颗粒(即病毒体)，其中病毒颗粒包装载体基因组。
在一个实施方案中，病毒体是包含FVIII基因表达盒的重组载体(例如用于递送至细胞)。因此，本公开的重组载体可用于在体外、离体和体内将多核苷酸递送至细胞。在代表性的实施方案中，本公开的重组载体可有利地用于将本公开的FVIII基因表达盒递送或转移至动物(例如哺乳动物)细胞中。
本公开还提供了生产病毒载体的方法。在代表性的实施方案中，本公开提供了一种生产重组病毒载体的方法，该方法包含在体外向细胞提供(a)模板，该模板包含(i)目标多核苷酸和(ii)足以将AAV模板封装入病毒颗粒的包装信号序列(例如一个或多个(例 如两个)末端重复序列，例如AAV末端重复序列)；和(b)足以使模板复制和将模板装入到病毒颗粒中的AAV序列(例如AAV rep和AAV cap序列)。在使得在细胞中生产包含包装在衣壳内的模板的重组病毒颗粒的条件下，提供所述模板和AAV复制和衣壳序列。该方法还可以包括从所述细胞收集病毒颗粒的步骤。可以从培养基和/或通过裂解细胞来收集病毒颗粒。
在一个示例性的实施方案中，本公开提供了一种生产包含AAV衣壳的rAAV颗粒的方法，该方法包含：在体外向细胞提供编码AAV衣壳的核酸、AAV rep编码序列、包含目标多核苷酸的AAV载体基因组，以及用于产生生产性AAV感染的辅助子功能；和允许组装包含AAV衣壳并且包装AAV载体基因组的AAV颗粒。
细胞通常是允许AAV病毒复制的细胞。可以使用本领域已知的任何合适的细胞，例如哺乳动物细胞。同样合适的是，提供从复制缺陷型辅助病毒缺失的功能的反式互补包装细胞系(trans-complementing packaging cell line)，例如293细胞或其他E1a反式互补细胞(trans-complementing cell)。
可以通过本领域已知的任何方法提供AAV复制和衣壳序列。目前的方案通常是在单个质粒上表达AAV rep/cap基因。不需要一起提供AAV复制和包装序列。AAV rep和/或cap序列可以由任何病毒载体或非病毒载体提供。例如，rep/cap序列可以由杂交腺病毒或疱疹病毒载体提供(例如插入到缺失的腺病毒载体的E1a或E3区中)。也可以使用EBV载体来表达AAV cap和rep基因。该方法的一个优点是，EBV载体是附加型的(episomal)，但是在整个连续的细胞分裂中将保持高拷贝数(即作为染色体外元件稳定地整合到细胞中，指定为基于EBV的核附加体(episome))。
作为另一替代方案，细胞内可稳定地携带rep/cap序列(附加型或整合的)。
通常，AAV rep/cap序列不会被AAV包装序列侧接(例如，AAV ITR)，以防止这些序列的拯救和/或包装。
可以使用本领域已知的任何方法将模板(例如rAAV载体基因组)提供给细胞。例如，模板可以由非病毒(例如质粒)或病毒载体提供。在一个实施方案中，模板由疱疹病毒或腺病毒载体提供(例如插入到缺失的腺病毒的E1a或E3区域中)。作为另一说明，Palombo等，(1998)J.Virol.72：5025描述了携带侧翼为AAV ITR的报告基因的杆状病毒载体。也可以使用EBV载体递送模板，如上文关于rep/cap基因所述。
在另一代表性实施方案中，模板由复制rAAV病毒提供。在一个实施方案中，AAV原病毒稳定地整合到细胞的染色体中。
为了获得最大的病毒滴度，通常向细胞提供对生产性AAV感染重要的辅助病毒功能(例如腺病毒或疱疹病毒)。AAV复制所需的辅助病毒序列是本领域已知的。通常，这些序列由辅助腺病毒或疱疹病毒载体提供。可替代地，腺病毒或疱疹病毒序列可以由另一种非病毒或病毒载体提供，例如作为携带有效AAV生产所需的所有辅助基因的非感染性腺病毒迷你质粒(miniplasmid)，如Ferrari等，(1997)Nature Med.3：1295以及美国专利号6,040,183和6,093,570所述。
此外，辅助病毒功能(helper virus function)可以由包装细胞提供，该包装细胞具有整合在染色体中或维持为稳定的染色体外元件的辅助基因。在代表性的实施方案中，辅助病毒序列不包装在AAV病毒体中，例如不侧接有AAV ITR。
本领域技术人员将理解，在单个辅助构建体上提供AAV复制和衣壳序列以及辅助病毒序列(例如腺病毒序列)可能是有利的。该辅助构建体可以是非病毒或病毒构建体，但可选地是包含AAV rep/cap基因的杂交腺病毒或杂交疱疹病毒。
在一个实施方案中，AAV rep/cap序列和腺病毒辅助序列由单个腺病毒辅助载体提供。该载体还含有rAAV模板。可以将AAV rep/cap序列和/或rAAV模板插入到腺病毒的缺失区域(例如E1a或E3区域)中。
在另一个实施方案中，AAV rep/cap序列和腺病毒辅助序列由单个腺病毒辅助载体提供。rAAV模板作为质粒模板提供。
在另一个说明性实施方案中，AAV rep/cap序列和腺病毒辅助序列由单个腺病毒辅助载体提供，并且rAAV模板被整合到细胞中作为原病毒。可替代地，rAAV模板由作为染色体外元件保持在细胞内的EBV载体提供(例如作为“基于EBV的核附加体”，参见Margolski，(1992)Curr.Top.Microbiol.Immun.158：67)。
在另一个示例性实施方案中，AAV rep/cap序列和腺病毒辅助序列由单个腺病毒辅助子提供。rAAV模板作为单独的复制病毒载体提供。例如，rAAV模板可以由rAAV颗粒或第二重组腺病毒颗粒提供。
根据上述方法，杂交腺病毒载体通常包含足以用于腺病毒复制和包装的腺病毒5’和3’顺式序列(即腺病毒末端重复和PAC序列)。AAV rep/cap序列和(如果存在)rAAV模板被嵌入在腺病毒骨架中，并且被所述5’和3’顺式序列侧接，使得这些序列可以被包装到腺病毒衣壳中。如上所述，在代表性的实施方案中，腺病毒辅助序列和AAV rep/cap序列不侧接AAV包装序列(例如AAV ITR)，使得这些序列不包装到AAV病毒体中。
疱疹病毒也可以用作AAV包装方法中的辅助病毒。编码AAV rep蛋白的杂交疱疹病 毒可有利地促进AAV载体的生产方案。已经描述了表达AAV-2rep和cap基因的杂交单纯疱疹病毒I型(HSV-1)载体(Conway等，(1999)Gene Therapy 6：986和WO 00/17377，它们的公开内容全部并入本文)。
作为另一种替代方案，可以使用杆状病毒载体在昆虫细胞中生产本公开的病毒载体，以递送rep/cap基因和rAAV模板，如Urabe等，(2002)Human Gene Therapy 13：1935-43所述。
生产AAV的其他方法使用稳定转化的包装细胞(参见例如美国专利号5,658,785)。
可以通过本领域已知的任何方法获得没有污染辅助病毒的AAV载体储料。例如，可以根据大小容易地区分AAV和辅助病毒。也可以基于对肝素底物的亲和力从辅助病毒中分离出AAV(Zolotukhin等，(1999)Gene Therapy 6：973)。在代表性的实施方案中，使用缺失的复制缺陷型辅助病毒，使得任何污染辅助病毒不具有复制能力。作为另外的替代方案，可以使用缺乏晚期基因表达的腺病毒辅助子，因为只需要腺病毒早期基因表达来介导AAV病毒的包装。晚期基因表达缺陷的腺病毒突变体是本领域已知的(例如ts100K和ts149腺病毒突变体)。
本公开的包装方法可用于产生高滴度的病毒颗粒储料。在具体的实施方案中，所述病毒储料具有的滴度为至少约10 5个转导单位(tu)/ml，至少约10 6tu/ml，至少约10 7tu/ml，至少约10 8tu/ml，至少约10 9tu/ml或至少约10 10tu/ml。
在一个实施方案中，本公开提供了一种药物组合物，其在药学上可接受的载体中包含本公开的病毒载体和可选的其它医疗剂、药剂、稳定剂、缓冲液、载体、佐剂、稀释剂等。对于注射，载体通常是液体。对于其它给药方式，载体可以是固体或液体。对于吸入给药，载体将是可吸入的，并且优选为固体或液体颗粒形式。
术语“药学上可接受的”是指不具有毒性或不因其它原因而不期望的物质，即可将物质给药于受试者而不引起任何不期望的生物学效应。
本公开的一个方面涉及在体外将目标多核苷酸转移至细胞的方法。根据适用于特定靶细胞的标准转导方法，可以以适当的感染多重性将病毒载体引入到细胞中。用于给药的病毒载体或衣壳的滴度可以根据靶细胞类型和数量以及特定的病毒载体或衣壳而变化，并且本领域技术人员无需过多的实验即可确定。在一个实施方案中，向细胞中引入至少约10 3个传染性单位，更优选地至少约10 5个传染性单位。
可以引入病毒载体的细胞可以是任何类型的，包括但不限于神经细胞(包括外周和中枢神经系统的细胞，特别是，脑细胞如神经元、少突胶质细胞、神经胶质细胞、星形胶质 细胞)，肺细胞，眼细胞(包括视网膜细胞、视网膜色素上皮和角膜细胞)，上皮细胞(例如肠和呼吸上皮细胞)，骨骼肌细胞(包括成肌细胞、肌管和肌纤维)，隔膜肌细胞，树突状细胞，胰腺细胞(包括胰岛细胞)，肝细胞，胃肠道细胞(包括平滑肌细胞、上皮细胞)，心脏细胞(包括心肌细胞)，骨细胞(例如骨髓干细胞)，造血干细胞，脾细胞，角质形成细胞，成纤维细胞，内皮细胞，前列腺细胞，关节细胞(包括例如软骨、半月板、滑膜和骨髓)，生殖细胞等。可替代地，细胞可以是任何祖细胞。作为另外的选择，细胞可以是干细胞(例如神经干细胞、肝干细胞)。作为另一种替代方案，细胞可以是癌症或肿瘤细胞。此外，如上所述，细胞可以来自任何来源的物种。
病毒载体可以在体外被引入细胞中，目的是将修饰的细胞给药于受试者。在一个实施方案中，已从受试者中移出细胞，将病毒载体导入其中，然后将细胞置换回受试者中。从受试者中取出细胞进行体外治疗并且然后引入回受试者的方法是本领域已知的(参见例如美国专利号5,399,346)。可替代地，将重组病毒载体引入到来自另一受试者的细胞中，引入到培养的细胞，或引入到来自任何其它适合来源的细胞中，并将所述细胞给药于有需要的受试者。
用于离体基因治疗的合适的细胞如上所述。施用于受试者的细胞的剂量将根据受试者的年龄、状况和物种、细胞类型、细胞表达的核酸、给药方式等而变化。典型地，将处于药学上可接受的载体中每剂量至少约10 2至约10 8或约10 3至约10 6个细胞给药。在一个实施方案中，用病毒载体转导的细胞以有效量与药物载体组合施用于受试者。
本公开的另一方面涉及将本公开的病毒载体向受试者给药的方法。在具体的实施方案中，所述方法包含向动物受试者递送目标多核苷酸的方法，该方法包含：向动物受试者给药有效量的根据本公开的病毒载体。可以通过本领域已知的任何方法向有此需要的人类受试者或动物给药本公开的病毒载体。可选地，以有效剂量递送处于药学上可接受的载体(carrier)中的病毒载体(vector)。
要给药于受试者的病毒载体的剂量将取决于给药方式、待治疗的疾病或病症、个体受试者的状况、特定病毒载体和待递送的核酸，并且可以常规方式确定。用于实现治疗效果的示例剂量是至少约10 5、10 6、10 7、10 8、10 9、10 10、10 11、10 12、10 13、10 14、10 15个转导单位或更高的病毒滴度，优选约10 7、10 8、10 9、10 10、10 11、10 12、10 13或10 14个转导单位，还更优选约10 12个转导单位。
在一个实施方案中，可以使用多于一次给药(例如两次、三次、四次或更多次给药)以在各个间隔的时间段内(例如每天、每周、每月、每年等)实现期望水平的基因表达。
示例性给药方式包括口服、直肠、经粘膜、局部、鼻内、吸入(例如通过气雾剂)、颊内(例如舌下)、阴道、鞘内、眼内、透皮、子宫内(或卵内)、肠胃外(例如静脉内、皮下、皮内、肌肉内[包括向骨骼、隔膜和/或心肌给药]、皮内、胸膜内、脑内和关节内)、局部(例如皮肤和粘膜表面，包括气道表面和透皮给药)、淋巴内等，以及直接组织或器官注射(例如肝、骨骼肌、心肌、隔膜肌或脑)。也可以向肿瘤给药(例如在肿瘤或淋巴结中或附近)。任何给定情况下，最合适的途径将取决于所治疗的病症的性质和严重程度以及正在使用的特定载体的性质。
通过递送包含病毒载体的贮库也可以实现向这些组织中任一种的递送，所述贮库可以被植入组织中或所述组织可以与包含病毒载体的膜或其它基质接触。这种可植入基质或底物的例子描述于美国专利号7,201,898中。
本公开的方法可用于治疗组织或器官的病症。可以实施本公开的方法以将核酸递送至组织或器官，所述组织或器官用于生产通常在血液中循环或全身递送至其他组织以治疗血友病的因子VIII多肽。
组合物可以存在于单位/剂量或多剂量容器中，例如在密封的安瓿瓶和小瓶中，并且可以在冷冻干燥(冻干)条件下储存，仅需要在使用前立即加入无菌液体载体，例如盐水或注射用水。
基因工程技术
通过基因工程生产克隆基因、重组DNA、载体、转化细胞、蛋白质和蛋白质片段是众所周知的。参见例如Bell等的美国专利号4,761,371第6栏第3行至第9栏第65行；Clark等的美国专利号4,877,729第4栏第38行至第7第6行；Schilling的美国专利号4,912,038第3栏第26行至第14栏第12行；和Wallner的美国专利号4,879,224第6栏第8行至第8栏第59行。
载体(vector)是可复制的DNA构建体。载体在本文中用于扩增编码FVIII蛋白的核酸和/或表达编码FVIII蛋白的核酸。表达载体是可复制的核酸构建体，其中编码FVIII蛋白的核苷酸序列可操作地连接到合适的控制序列，该控制序列能够在合适的宿主细胞中实现核苷酸序列的表达以产生FVIII蛋白。对这种控制序列的需要将根据所选择的宿主细胞和所选择的转化方法而变化。通常，控制序列包括转录启动子、用于控制转录的可选的操纵子序列、编码合适的mRNA核糖体结合位点的序列、以及控制转录和翻译终止的序列。
载体包含质粒、病毒(例如AAV、腺病毒、巨细胞病毒)、噬菌体和可整合的DNA片段(即可通过重组整合到宿主细胞基因组中的片段)。载体可以独立于宿主细胞基因组 复制并起作用(例如通过瞬时表达)，或可以整合入宿主细胞基因组本身(例如，稳定整合)。表达载体可以含有启动子和RNA结合位点，所述启动子和RNA结合位点可操作地连接到要表达的核酸分子并且可在宿主细胞和/或生物体中操作。
当DNA区域或核苷酸序列在功能上彼此相关时，它们被可操作地连接或可操作地相关联。例如，如果启动子控制编码序列的转录，则启动子可操作地连接到该编码序列；或者如果核糖体结合位点被定位以允许编码序列翻译，则该核糖体结合位点可操作地连接到该编码序列。
在转化脊椎动物细胞中使用的表达载体中的转录和翻译控制序列通常由病毒源提供。非限制性例子包括衍生自多瘤病毒、腺病毒2和猿猴病毒40(SV40)的启动子。参见例如美国专利号4,599,308。
另外，还可以使用动物源的泛表达启动子来控制目的基因转录，其中泛表达启动子是指在动物细胞、组织和细胞周期中具有广泛活性的一类强启动子，包括CMV、EF1A、EFS、CAG、CBh、CBA、SFFV、MSCV、SV40、mPGK、hPGK、UBC等。
本公开的编码序列可以编码任何物种来源的FVIII，包括小鼠、大鼠、狗、负鼠、兔、猫、猪、马、绵羊、母牛、豚鼠、负鼠、鸭嘴兽和人类，但优选编码人源性FVIII蛋白。可与本文公开的编码蛋白质的核酸杂交的编码FVIII的核酸也包括在内。可以在标准原位杂交测定中，在降低的严格性条件或甚至在严格条件下(例如由处于60℃或甚至70℃的0.3M NaCl、0.03M柠檬酸钠、0.1％SDS的洗涤严格度代表的严格条件)，进行此种序列与本文公开的编码FVIII蛋白的核酸的杂交。参见例如Sambrook等，Molecular Cloning，A Laboratory Manual(第2版1989)，Cold Spring Harbor Laboratory。
在一些实施方案中，可以使用在乳腺组织中具有“活性”的顺式作用调节区，因为在合成乳的生理条件下，启动子在乳腺组织中比在其它组织中更有活性。这些启动子包括但不限于短和长的乳清酸性蛋白质(WAP)，短和长α、β和κ酪蛋白，α-乳白蛋白和β-乳球蛋白(“BLG”)启动子。也可以根据本公开使用信号序列，其将表达的蛋白质直接分泌到其它体液中，特别是血液和尿液。这些序列的例子包括分泌的凝血因子的信号肽，包括FVIII、蛋白C和组织型纤溶酶原激活物的信号肽。
除了上面讨论的启动子之外，调节转录的有用序列包括增强子、剪接信号、转录终止信号、聚腺苷酸化位点、缓冲序列、RNA加工序列和调节转基因表达的其他序列。
优选地，表达系统或构建体包含编码重组蛋白的核苷酸序列下游的3’非翻译区。该区域可增加转基因的表达。在这一方面，有用的3’非翻译区是提供poly A信号的序列。
合适的异源3’-非翻译序列可以衍生自例如SV40小t抗原、酪蛋白3’非翻译区或本领域熟知的其他3’非翻译序列。核糖体结合位点对于增加FVIII的表达效率也是重要的。同样地，调节FVIII的翻译后修饰的序列在本公开中是有用的。
下面结合附图和实施例对本公开作进一步详细的说明。以下实施例仅用于说明本公开而不用于限制本公开的范围。实施例中未注明具体条件的实验方法，系按照本领域已知的常规条件，或按照制造厂商所建议的条件进行操作。
实施例
实施例1.优化B结构域缺失因子VIII(FVIII-BDD)多核苷酸
在本实施例中，为了提高体内FVIII-BDD基因的表达效率，采取了两种方法来实现这一目标。
首先，在原始人FVIII-BDD基因的编码序列(F8X1，SEQ ID NO：3)的基础上，依据人密码子使用效率完全合成了人FVIII-BDD基因的编码序列FVIII-BDD-Opti，其目的是最大限度地利用更有效的密码子，产生完全合成的人FVIII-BDD基因F8X1Co1(SEQ ID NO：4)和F8X1Co3(SEQ ID NO：6)。
接下来，减少或完全去除合成的FVIII-BDD基因中的CpG序列。根据文献记载，该基因中的CpG岛或基序可诱导先天性免疫反应(例如，Toll样受体9(TLR9)介导的免疫应答(Bauer等人，Proc.Natl.Acad.Sci.USA 98(16):9237(2001))并且也可能导致基因沉默。在FVIII-BDD基因F8X1Co1基础上，将CpG基序完全去除，获得F8X1Co2(SEQ ID NO：5)。在F8X1Co3的基础上，去除95％的CpG基序，获得F8X1Co4(SEQ ID NO：7)。
为了在肝细胞中特异性的表达人FVIII-BDD蛋白，选择了两个人工合成的肝特异性启动子：188nt的LXP2.1和200nt的LXP3.3。为了提高FVIII-BDD或FVIII-BDD-opti的mRNA转录效率，选择了一个长度仅为71nt的合成的转录增强元件。同时，选择了一个小的合成人内含子来进一步提高FVIII的基因表达。另外，为了提高FVIII-BDD或FVIII-BDD-opti的mRNA的稳定性，选择了仅48nt的合成多聚腺苷酸。将所选的增强子、肝特异性启动子、5’非编码区、内含子、人FVIII-BDD编码cDNA和多聚腺苷酸依次组装的基因表达盒(图1)分别通过PEI介导体外转染肝Huh7细胞，并取转染后3天的细胞培养上清进行人FVIII凝血活性检测。
结果如图2所示，与原始的FVIII-BDD(F8X1)相比，经过核苷酸序列优化的FVIII-BDD-Opti在相同的条件下表现出高约3倍的基因表达活性。此外，发现LXP2.1较 LXP3.3表现出明显的基因表达活性提升作用，在相同的条件下，LXP2.1比LXP3.3增加近1倍的基因表达活性。
实施例2.构建FVIII基因表达盒
通常，基因表达盒中需包含多种调控元件(如启动子、增强子、内含子、5’UTR)，以增强基因的转录作用，促进基因的表达。特别是，众所周知，UTR在基因表达的转录后调控中起着至关重要的作用。
在本实施例中，发明人对实施例1中构建的表达盒(LXP2.1-F8X1Co4和LXP3.3-F8X1Co4)进行了简化改造，由此构建了新的FVIII基因表达盒，并研究了各FVIII基因表达盒的表达效率。
具体地，依次剔除LXP2.1-F8X1Co4表达盒中的增强子、内含子和5’UTR，并进一步对ITR进行截短(对两端ITR的D序列均进行10nt的截短)，由此获得了4个人FVIII基因表达盒：LXP2.1-F8X1Co4.1、LXP2.1-F8X1Co4.2、LXP2.1-F8X1Co4.3和LXP2.1-F8X1Co4.4(图3)。其中LXP2.1-F8X1Co4.3和LXP2.1-F8X1Co4.4的基因表达调控元件仅保留了启动子和多聚腺苷酸(poly A)。LXP2.1-F8X1Co4.4中，除剔除了上述元件之外，两端ITR的D序列被截短了10nt，并同时删除了D序列紧临的CAGATCT序列。另外，同时剔除LXP3.3-F8X1Co4表达盒中的增强子、内含子和5’UTR，由此获得了LXP3.3-F8X1Co4.1(图3)。
将上述各个表达盒分别通过PEI介导体外转染肝Huh7细胞，并取转染后3天的细胞培养上清进行人FVIII凝血活性检测。
如图4所示，剔除增强子的基因表达盒LXP2.1-F8X1Co4.1没有表现出明显的基因表达活性降低，但剔除内含子或5’UTR后，LXP2.1-F8X1Co4.2、LXP2.1-F8X1Co4.3、LXP2.1-F8X1Co4.4和LXP3.3-F8X1Co4.1的基因表达活性均明显降低，特别是剔除了5’UTR的LXP2.1-F8X1Co4.3、LXP2.1-F8X1Co4.4和LXP3.3-F8X1Co4.1，其基因表达活性降低甚至超过80％。这一结果与本领域技术人员的普遍预期是一致的，通常认为调控元件的去除，特别是对基因表达有重要作用的5’UTR的去除，会导致基因表达效率的降低。
实施例3.包装FVIII基因表达盒的腺相关病毒(AAV)的病毒产量与基因组完整性
在本实施例中，使用人肝脏嗜性AAV32.1分别对实施例2中涉及的FVIII基因表达盒进行包装，并检测病毒完整性和病毒产量。
通过三质粒悬浮HEK293细胞包装系统，分别取AAV32.1/LXP2.1-F8X1Co4、 AAV32.1/LXP2.1-F8X1Co4.1、AAV32.1/LXP2.1-F8X1Co4.2、AAV32.1/LXP2.1-F8X1Co4.3、AAV32.1/LXP2.1-F8X1Co4.4、AAV32.1/LXP3.3-F8X1Co4和AAV32.1/LXP3.3-F8X1Co4.1重组腺相关病毒的72小时细胞培养液进行裂解。意外地发现，AAV32.1/LXP2.1-F8X1Co4.3、AAV32.1/LXP2.1-F8X1Co4.4和AAV32.1/LXP3.3-F8X1Co4.1这三个病毒的基因组滴度明显高于其他元件更完整的基因表达盒(图5A)。
同时，在相同的基因组滴度下，对上述重组腺相关病毒基因组进行抽提，并对抽提的基因组进行碱性胶鉴定。意外地发现，LXP2.1-F8X1Co4.3、LXP2.1-F8X1Co4.4和LXP3.3-F8X1Co4.1呈现出良好的基因组完整性，明显优于其他元件更完整的基因表达盒(图5B)。
实施例4.FVIII基因表达盒的FVIII表达活性
在本实施例中，将实施例3中通过三质粒悬浮HEK293系统生产的重组腺相关病毒AAV32.1/LXP2.1-F8X1Co4、AAV32.1/LXP2.1-F8X1Co4.1、AAV32.1/LXP2.1-F8X1Co4.2、AAV32.1/LXP2.1-F8X1Co4.3、AAV32.1/LXP2.1-F8X1Co4.4、AAV32.1/LXP3.3-F8X1Co4和AAV32.1/LXP3.3-F8X1Co4.1分别通过碘克沙醇两步超速离心法进行分离纯化。将纯化后的病毒分别以5E+5vg/个细胞的MOI感染肝Huh7细胞，并取转染后5天的细胞培养上清进行人因子VIII凝血活性检测。
结果如图6所示，发现表达盒中调控元件更少的AAV32.1/LXP2.1-F8X1Co4.3、AAV32.1/LXP2.1-F8X1Co4.4和AAV32.1/LXP3.3-F8X1Co4.1产生的基因表达活性较基因调控元件更完整的AAV32.1/LXP2.1-F8X1Co4、AAV32.1/LXP2.1-F8X1Co4.1、AAV32.1/LXP2.1-F8X1Co4.2和AAV32.1/LXP3.3-F8X1Co4明显更高。
实施例5.FVIII基因表达盒在血友病A小鼠模型中的体内长期高效表达
在本实施例中，测试了FVIII基因表达盒LXP2.1-F8X1Co4.3在体内的长期有效性。使用2至3个月的FVIII基因敲除的雄性小鼠(本领域常用的血友病A动物模型)。基于早期实验，选择标准载体剂量2.5E+11vg/小鼠，并通过尾静脉注射重组腺相关病毒AAV32.1/LXP2.1-F8X1Co4.3到5只模型小鼠中。使用未处理的年龄和性别匹配的FVIII敲除小鼠作为阴性对照。使用标准方法经由眶后静脉收集血浆，注射后6周内，每周收集血浆；之后每4周收集血浆。为了定量评估FVIII基因表达，将纯化的重组人FVIII逐级稀释，加入到严重血友病A患者血浆中，作为FVIII活性的标准曲线，对血浆样品进行常规APTT测试。
如图7所示，在全部5只KO小鼠中，AAV32.1/LXP2.1-F8X1Co4.3载体均成功表达了FVIII，平均表达活性达到人FVIII因子活性的正常生理水平的大约600％。并且，在长达数月(25周以上)的实验持续时间内，FVIII均在体内持续稳定表达。VIII KO小鼠中持续高水平表达VIII基因产物并未引起任何明显的不利影响。
上述实验结果表明，本发明的FVIII基因表达盒不仅具有较高的FVIII体内表达水平而且可在体内稳定长期表达。
本领域技术人员将理解，在不脱离本发明的精神的情况下，可以进行许多和各种修改。因此，应当清楚地理解，本发明的形式仅是说明性的，并不意图限制本发明的范围。
本文所提及的所有出版物、专利申请、专利、专利公开、
数据库登录号标识的序列以及其它参考文献，因与呈现该参考文献的句子和/或段落相关的教导而通过引用以其全部内容并入。
前述内容是对本发明的说明，而不应被解释为对本发明的限制。本发明由所附权利要求限定，其中包括所述权利要求的等同物。

Claims (22)

  1. 一种编码因子VIII(FVIII)多肽的多核苷酸，其中，所述多核苷酸的核苷酸序列与SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：7所示的核苷酸序列具有至少80％的同一性；优选地，所述多核苷酸的核苷酸序列与SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：7所示的核苷酸序列具有至少85％、90％、95％、99％的同一性。
  2. 根据权利要求1所述的多核苷酸，其中，所述多核苷酸包含SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：7所示的核苷酸序列；优选地，所述多核苷酸的核苷酸序列如SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：7所示。
  3. 根据权利要求1或2所述的多核苷酸，其中，所述多核苷酸表达B结构域缺失的因子VIII多肽(FVIII-BDD)。
  4. 一种因子VIII基因表达盒，其包括以下元件，优选由以下元件组成：启动子、可选的kozak序列、编码B结构域缺失的因子VIII多肽的多核苷酸(优选权利要求1至3中任一项所述的多核苷酸)、多聚腺苷酸(poly A)，以及位于两端的两个ITR。
  5. 根据权利要求4所述的因子VIII基因表达盒，其中，各元件之间直接连接或通过接头间接连接。
  6. 根据权利要求4所述的因子VIII基因表达盒，其中，所述启动子为泛表达启动子、组成型肝特异性启动子或人工合成型肝特异性启动子；优选地，启动子为人工合成型肝特异性启动子；更优选地，所述启动子为SEQ ID NO：9或SEQ ID NO：10所示的肝特异性启动子。
  7. 根据权利要求4至6中任一项所述的因子VIII基因表达盒，其中，所述两个ITR各自独立地为正常ITR或截短ITR；优选地，所述两个ITR各自独立地为SEQ ID NO：14所示的正常ITR或SEQ ID NO：15所示的截短ITR。
  8. 根据权利要求4至7中任一项所述的因子VIII基因表达盒，其中，所述因子VIII基因表达盒还包括以下元件中的一种以上：增强子、5’UTR和内含子。
  9. 根据权利要求8所述的因子VIII基因表达盒，其中，所述增强子为组成型增强子或人工合成型增强子；优选SEQ ID NO：8所示的增强子。
  10. 根据权利要求8所述的因子VIII基因表达盒，其中，所述内含子为组成型内含子或人工合成型内含子；优选SEQ ID NO：12所示的内含子。
  11. 根据权利要求4至10中任一项所述的因子VIII基因表达盒，其中，所述因子VIII基因表达盒的核苷酸序列如SEQ ID NO：16、SEQ ID NO：17或SEQ ID NO：18所示。
  12. 一种重组AAV载体，其包括：权利要求4至11中任一项所述的因子VIII基因表达盒和AAV衣壳蛋白。
  13. 根据权利要求12所述的重组AAV载体，其中，所述AAV衣壳蛋白为天然AAV衣壳蛋白或人工改造的AAV衣壳蛋白；优选地，所述AAV选自：AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10和AAV11。
  14. 权利要求12或13所述的重组AAV载体在制备用于预防或治疗血友病A或获得性因子VIII缺乏症的药物中的应用。
  15. 一种药物，其包含：权利要求12或13所述的重组AAV载体和可选的赋形剂。
  16. 根据权利要求15所述的药物，其中，所述赋形剂包括：盐、有机物和表面活性剂。
  17. 一种预防或治疗血友病A或获得性因子VIII缺乏症的方法，包括向有需要的受试者施用治疗有效量的权利要求15或16所述的药物。
  18. 根据权利要求17所述的方法，其中，所述药物通过全身途径或局部途径施用，例如口服、经直肠、经粘膜、鼻内、吸入、颊内(例如舌下)、阴道、鞘内、眼内、透皮、 子宫内(或卵内)、肠胃外(例如静脉内、皮下、皮内、肌内、皮内、胸膜内、脑内和关节内)、淋巴内、局部接触、病灶内施用。
  19. 一种将因子VIII基因递送至靶细胞中的方法，包括：
    1)将权利要求4至11中任一项所述的因子VIII基因表达盒包装于AAV衣壳蛋白中，形成权利要求12或13所述的重组AAV载体；以及
    2)使所述靶细胞与所述重组AAV载体接触。
  20. 根据权利要求19所述的方法，其中，所述靶细胞是离体细胞。
  21. 一种宿主细胞，其中，所述宿主细胞用权利要求12或13所述的重组AAV载体感染。
  22. 一种生产重组AAV载体的方法，包括将权利要求1至3中任一项所述的多核苷酸引入哺乳动物宿主细胞中，所述哺乳动物宿主细胞包含AAV rep基因、AAV cap基因和病毒复制辅助基因。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107531774A (zh) * 2015-02-06 2018-01-02 北卡罗来纳大学查珀尔希尔分校 优化的人类凝血因子viii基因表达盒及其用途
CN108884145A (zh) * 2015-11-13 2018-11-23 百深公司 用于血友病a的基因治疗的具有增加的表达的编码重组fviii变体的病毒载体
CN112538501A (zh) * 2013-09-12 2021-03-23 生物马林药物股份有限公司 腺相关病毒因子viii载体

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0911870D0 (en) * 2009-07-08 2009-08-19 Ucl Business Plc Optimised coding sequence and promoter
EP3114138B1 (en) * 2014-03-05 2021-11-17 Pfizer Inc. Improved muteins of clotting factor viii
BR112017002781A2 (pt) * 2014-08-13 2017-12-19 Sabatino Denise cassete de expressão aprimorado para acondicionamento e expressão de variantes do fator viii para o tratamento de distúrbios de hemostasia
KR20230067694A (ko) * 2014-09-24 2023-05-16 시티 오브 호프 고효율 게놈 편집을 위한 아데노-관련 바이러스 벡터 변이체 및 이의 방법
WO2017074526A1 (en) * 2015-10-28 2017-05-04 Sangamo Biosciences, Inc. Liver-specific constructs, factor viii expression cassettes and methods of use thereof
PE20231949A1 (es) * 2015-10-30 2023-12-05 Spark Therapeutics Inc VARIANTES DEL FACTOR VIII REDUCIDO CON CpG, COMPOSICIONES Y METODOS Y USOS PARA EL TRATAMIENTO DE TRASTORNOS DE LA HEMOSTASIA
CN114875051A (zh) * 2017-05-31 2022-08-09 北卡罗来纳大学教堂山分校 优化的人凝血因子ix基因表达盒及其应用
CA3071519A1 (en) * 2017-08-01 2019-02-07 Spark Therapeutics, Inc. Factor viii (fviii) gene therapy methods
JP2021533805A (ja) * 2018-08-24 2021-12-09 スパーク セラピューティクス インコーポレイテッドSpark Therapeutics, Inc. 最適化されたプロモーター配列、イントロンを含まない発現構築物及び使用方法
SG11202103614RA (en) * 2018-10-12 2021-05-28 Genzyme Corp Generation of improved human pah for treatment of severe pku by liver-directed gene replacement therapy
CN111808863B (zh) * 2020-06-23 2021-05-28 康霖生物科技（杭州）有限公司 一种密码子优化的凝血因子viii基因及其构建体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112538501A (zh) * 2013-09-12 2021-03-23 生物马林药物股份有限公司 腺相关病毒因子viii载体
CN107531774A (zh) * 2015-02-06 2018-01-02 北卡罗来纳大学查珀尔希尔分校 优化的人类凝血因子viii基因表达盒及其用途
CN108884145A (zh) * 2015-11-13 2018-11-23 百深公司 用于血友病a的基因治疗的具有增加的表达的编码重组fviii变体的病毒载体

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LI-JUN ZHONG, JIANG WEI, WANG FENG-SHAN, ZANG HENG-CHANG, : "Research Progress on Human Coagulation Factor Ⅷ Chinese Full Tex", FOOD AND DRUG, vol. 16, no. 3, 20 May 2014 (2014-05-20), pages 212 - 214, XP093149605 *
LIRON ELKOUBY ET AL.: "Preclinical Assessment of an Optimized AAV-FVIII Vector in Mice and Non-human Primates for the Treatment of Hemophilia A", MOLECULAR THERAPY - METHODS & CLINICAL DEVELOPMENT, vol. 24, 10 March 2022 (2022-03-10), pages 20 - 29, XP093069094, DOI: 10.1016/j.omtm.2021.11.005 *

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