WO2022268110A1 - 用于治疗b型血友病的组合物和方法 - Google Patents

用于治疗b型血友病的组合物和方法 Download PDF

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WO2022268110A1
WO2022268110A1 PCT/CN2022/100382 CN2022100382W WO2022268110A1 WO 2022268110 A1 WO2022268110 A1 WO 2022268110A1 CN 2022100382 W CN2022100382 W CN 2022100382W WO 2022268110 A1 WO2022268110 A1 WO 2022268110A1
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sequence
promoter
polynucleotide
factor
seq
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PCT/CN2022/100382
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English (en)
French (fr)
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施中东
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英斯培瑞有限公司
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Priority to JP2023579436A priority Critical patent/JP2024521537A/ja
Priority to EP22827602.8A priority patent/EP4361278A1/en
Priority to AU2022298827A priority patent/AU2022298827A1/en
Priority to CA3225312A priority patent/CA3225312A1/en
Priority to CN202280047035.3A priority patent/CN117693593A/zh
Publication of WO2022268110A1 publication Critical patent/WO2022268110A1/zh

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    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/644—Coagulation factor IXa (3.4.21.22)
    • A—HUMAN NECESSITIES
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Definitions

  • Hemophilia B is a rare inherited bleeding disorder that is usually caused by changes (mutations) in the Factor IX (also known as FIX protein) gene in an individual. Depending on the level of Factor IX activity in the patient, hemophilia B can be further classified as mild, moderate, or severe. In mild cases, bleeding occurs only after surgery, injury, or dental procedures. In some moderate and severe cases, bleeding symptoms may occur after a minor injury or even without any external trigger.
  • Clotting factors are a class of proteins required for blood to clot.
  • Factor IX Factor IX, or Christmas factor, FIX protein
  • FIX protein is a serine protease in the blood coagulation system, which belongs to the peptidase family S1. Because the gene for factor IX is located on the X chromosome (Xq27.1-q27.2), hemophilia B occurs more frequently in males.
  • compositions and methods provided by the present invention address the above needs.
  • the present invention provides a composition
  • a composition comprising: (1) a first polynucleotide sequence comprising a first parvovirus capsid operably linked to a first promoter A first open reading frame (ORF) of a gene and a second open reading frame of a second parvovirus capsid gene operably linked to a second promoter, wherein said first ORF comprises an intron comprising said second promoter sequence; and (2) a second polynucleotide sequence comprising a transgene sequence encoding a human Factor IX (FIX) protein.
  • ORF open reading frame
  • FIX human Factor IX
  • the first promoter and the second promoter are suitable for expression in insect cells or mammalian cells.
  • the first ORF, the first promoter, the second ORF and the second promoter are in the following order from 3' to 5': first promoter, first ORF comprising the second promoter, A second ORF, wherein the second ORF comprises at least one translation initiation codon and overlaps the 3' portion of the first ORF.
  • the insect cells are Sf9 cells.
  • the mammalian cells are HEK293 cells or derivatives thereof.
  • the derivatives are HEK293T cells.
  • said first promoter or said second promoter is a Polh promoter.
  • the first promoter or the second promoter is the p10 promoter.
  • the first polynucleotide sequence further comprises a poly A sequence at the 3' end.
  • the parvovirus is an adeno-associated virus (AAV).
  • the first ORF encodes an adeno-associated virus (AAV) cap protein.
  • the second ORF encodes an adeno-associated virus (AAV) rep protein.
  • the AAV cap protein is an AAV serotype 5 protein.
  • the AAV cap protein is a VP1, VP2 and/or VP3 protein.
  • the AAV rep protein is an AAV serotype 2 protein.
  • the AAV rep protein is a Rep78 and/or Rep52 protein.
  • the second polynucleotide sequence comprises a promoter.
  • the promoter of the second polynucleotide is a liver-specific promoter.
  • the promoter is the APO-HCR-hATT promoter.
  • the promoter comprises the sequence of SEQ ID NO:4.
  • the second polynucleotide sequence further comprises a second promoter.
  • the second promoter of the second polynucleotide is a modified APO-HCR-hAAT promoter.
  • the second promoter of the second polynucleotide comprises the sequence of SEQ ID NO:5.
  • the second polynucleotide sequence comprises an intron.
  • the intron is the SV40 intron or the modified first intron of human Factor IX.
  • the SV40 intron is a modified SV40 intron.
  • the modified SV40 intron comprises the sequence of SEQ ID NO:6.
  • the modified human Factor IX first intron comprises the sequence of SEQ ID NO:7.
  • said transgene sequence encoding said human Factor IX is codon optimized. In some embodiments, the transgenic sequence comprises less than 5 CG dinucleotides. In some embodiments, the human Factor IX comprises one or more amino acid substitutions compared to wild-type human Factor IX. In some embodiments, the human Factor IX comprises an amino acid substitution R338X compared to the sequence of SEQ ID NO: 1, wherein X can be any amino acid except arginine. In some embodiments, the human Factor IX comprises an amino acid substitution R338L, R338D or R338Q compared to the sequence of SEQ ID NO: 1.
  • the human Factor IX comprises the amino acid substitution R338L compared to the sequence of SEQ ID NO: 1.
  • the transgenic sequence is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:3.
  • the transgenic sequence comprises the sequence of SEQ ID NO:3.
  • the second polynucleotide sequence further comprises a stuffer sequence.
  • the present invention provides a cell comprising the composition of the present invention.
  • the cells are insect cells.
  • the insect cells are Sf9 cells.
  • the present invention provides a mammalian cell comprising the composition of the present invention.
  • the mammalian cells are HEK293 cells or derivatives thereof.
  • the derivatives are HEK293T cells.
  • the present invention provides a polynucleotide sequence comprising a transgenic sequence encoding human Factor IX, wherein said transgenic sequence has at least 50%, 60%, 70%, 80%, 85%, 90% or 95% identity. In some embodiments, the transgenic sequence is at least 96%, 97%, 98%, or 99% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises less than 5 CG dinucleotides. In some embodiments, the transgenic sequence does not comprise a CG dinucleotide. In some embodiments, the transgenic sequence comprises the sequence of SEQ ID NO:3. In some embodiments, the polynucleotide sequence comprises a promoter.
  • the promoter is a liver-specific promoter. In some embodiments, the promoter is the APO-HCR-hATT promoter. In some embodiments, the promoter comprises the sequence of SEQ ID NO:4. In some embodiments, the polynucleotide sequence further comprises a modified APO-HCR-hATT promoter. In some embodiments, the modified APO-HCR-hATT promoter comprises the sequence of SEQ ID NO:5.
  • the present invention provides an adeno-associated virus (AAV) virus particle comprising the polynucleotide of the present invention.
  • AAV viral particle is derived from AAV serotype 5.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the polynucleotide or AAV virus particle of the present invention.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a method for treating hemophilia B, which comprises administering the polynucleotide, AAV virus particle, or pharmaceutical composition of the present invention to a subject in need, wherein the The transgene sequence is expressed in the subject, thereby treating hemophilia B.
  • the subject is a human.
  • the transgene sequence is expressed in the subject's liver.
  • the administration is via intravenous injection.
  • the administered dose is 10 10 vg/kg to 10 15 vg/kg.
  • the present invention provides a kit for treating hemophilia B, comprising the composition of the present invention, the polynucleotide, the AAV virus particle, or the pharmaceutical combination items, and instructions.
  • the instructions are for a method of administering the composition, the polynucleotide, the AAV viral particle, or the pharmaceutical composition to treat hemophilia B.
  • the composition, the polynucleotide, the AAV virus particle, the pharmaceutical composition, the method, or the kit of the invention wherein the The transgene sequence stably expresses the FIX protein in vivo for at least 4 weeks, 6 weeks, or 8 weeks, and the expressed FIX protein has biological activity.
  • the composition, the polynucleotide, the AAV virus particle, the pharmaceutical composition, the method, or the kit of the invention wherein the The transgenic sequence is SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
  • composition the composition, the polynucleotide, the AAV virus particle, the pharmaceutical composition, the method, or the kit of the invention, wherein the The transgene sequence is SEQ ID NO:15.
  • Figure 1 shows the FIX expression of rAAV5-FIX in a mouse model of hemophilia B.
  • Each polynucleotide sequence encoding FIX i.e. the second polynucleotide sequence 1-6 of the test, see SEQ ID NOs:10-15
  • Different polynucleotide sequences have different levels of expression and stable expression at different times.
  • the test second polynucleotide sequence 6 (SEQ ID NO: 15) continuously and stably expresses FIX at a high level on the 29th day, the 43rd day, and the 57th day.
  • the second polynucleotide 6 (SEQ ID NO: 15) is the best expression vector design.
  • Figure 2 shows that rAAV5-FIX has stable activity of FIX expressed in the mouse model of hemophilia B. The activity was detected by coagulation method. Wherein, compared with other sequences, the FIX expressed by the test second polynucleotide sequence 6 (SEQ ID NO: 15) on the 43rd day and the 57th day continued to have high activity. Experiments show that the second polynucleotide 6 (SEQ ID NO: 15) is the best expression vector design.
  • FIG. 3 shows that rAAV5-FIX is active in FIX expressed in hemophilia B mice.
  • FIX activity was detected by a chromogenic activity assay.
  • the FIX activity obtained by this method was consistent with the activity detected by coagulation method.
  • rAAV particle includes one or more rAAV particles.
  • the term “about” or “approximately” means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value was measured or determined, ie, limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, according to the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly for biological systems or processes, the term can mean an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of a value. Where specific values are described in the application and claims, unless otherwise indicated, the term “about” should be assumed to mean within an acceptable error range for the specific value.
  • polypeptide As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acids of any length.
  • a polymer can be linear, cyclic, or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids.
  • amino acid polymers that have been modified, for example, by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic treatment, phosphorylation, iso Pennylation, racemization, selenylation, transfer RNA-mediated addition of amino acids to proteins (eg, arginylation), ubiquitination, or any other manipulation, such as conjugation to a labeling component.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics.
  • a polypeptide or amino acid sequence "derived from” a given protein refers to the origin of the polypeptide.
  • the polypeptide has an amino acid sequence substantially identical to that of the polypeptide encoded in the sequence, or a portion thereof, wherein the portion consists of at least 10-20 amino acids or at least 20-30 amino acids or at least 30-50 amino acids, or It can be immunologically identified by the polypeptide encoded in the sequence.
  • the term also includes polypeptides expressed from a specified nucleic acid sequence.
  • domain refers to a portion of a protein that is physically or functionally distinct from other portions of the protein or peptide.
  • Physically defined domains include extremely hydrophobic or hydrophilic amino acid sequences, such as those membrane bound or cytoplasmic bound. Domains can also be defined by internal homology e.g. caused by gene duplication. Functionally defined domains have distinct biological functions.
  • an antigen-binding domain refers to an antigen-binding unit or the portion of an antibody that binds to an antigen.
  • a functionally defined domain need not be encoded by a contiguous amino acid sequence, and a functionally defined domain may contain one or more physically defined domains.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including but not limited to D or L optical isomers, as well as amino acid analogs and peptidomimetics. Standard one-letter or three-letter codes are used to designate amino acids.
  • amino acids are generally referred to by one-letter and three-letter abbreviations well known in the art.
  • alanine can be represented by A or Ala.
  • sequence in the context of a polypeptide is the order of the amino acids in the polypeptide in the direction from the amino-terminus to the carboxy-terminus, wherein residues adjacent to each other in the sequence are important in the primary structure of the polypeptide. in is continuous.
  • sequence may also be a linear sequence of a portion of a polypeptide known to contain additional residues in one or both directions.
  • identity refers to the relationship between two or more polynucleotide sequences or between two or more polypeptide sequences Sequence similarity or interchangeability.
  • sequence identity refers to the relationship between two or more polynucleotide sequences or between two or more polypeptide sequences Sequence similarity or interchangeability.
  • programs such as Emboss Needle or BestFit to determine sequence identity, similarity, or homology between two different amino acid sequences
  • the default settings can be used, or an appropriate scoring matrix, such as blosum45 or blosum80, can be selected to optimize Identity, similarity or homology score.
  • homologous polynucleotides are those that hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably Polynucleotides with 97%, more preferably 98% and even more preferably 99% sequence identity.
  • Homologous polypeptides preferably have at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98% sequence identity when optimally aligned for sequences of comparable length, or have at least 99% sequence identity.
  • percent sequence identity is defined after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, and not considering any conservative substitutions as As part of sequence identity, the percentage of amino acid residues in a query sequence that are identical to those of a second, reference polypeptide sequence, or portion thereof. Alignments to determine percent amino acid sequence identities can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software . Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Percent identity may be measured over the length of the entire defined polypeptide sequence, or may be measured over shorter lengths, e.g., over the length of fragments taken from a larger, defined polypeptide sequence, such as A fragment of at least 5, at least 10, at least 15, at least 20, at least 50, at least 100 or at least 200 contiguous residues. These lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown in the Tables, Figures, or Sequence Listing herein can be used to describe the length over which percent identity can be measured.
  • a protein described herein may have one or more modifications relative to a reference sequence.
  • the modifications may be deletions, insertions or additions, or substitutions or replacements of amino acid residues.
  • “Deletion” refers to a change in amino acid sequence due to the absence of one or more amino acid residues.
  • Insertion or “addition” refers to an amino acid sequence change that results in the addition of one or more amino acid residues compared to a reference sequence.
  • substitution or “replacement” refers to the replacement of one or more amino acids with a different amino acid.
  • mutations of an antigen binding unit relative to a reference sequence can be determined by comparing the antigen binding unit to the reference sequence. Optimal alignment of sequences for comparison can be performed according to any method known in the art.
  • isolated means separated from cellular and other components with which polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are normally associated in nature. Associated. Those of skill in the art appreciate that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof do not need to be “isolated” to be distinguished from their naturally occurring counterparts. Additionally, a “concentrated”, “isolated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof is distinguishable from its naturally occurring counterpart by the concentration or number of molecules per unit volume Greater than (“enriched") or less than its naturally occurring counterpart (“isolated”). Enrichment can be measured on an absolute basis, such as weight per unit volume of solution, or it can be measured relative to a second, potentially interfering substance present in the source mixture.
  • polynucleotide refers to polymeric forms of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide can have any three-dimensional structure and can perform any known or unknown function.
  • polynucleotides coding or non-coding regions of genes or gene fragments, loci determined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomes RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, oligonucleotides, or synthetic DNA .
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotide structure if present, can be imparted before or after polymer assembly.
  • sequence of nucleotides may be interrupted by non-nucleotide components.
  • Polynucleotides may be further modified after polymerization, for example by conjugation with labeling components.
  • Recombinant when applied to a polynucleotide means that the polynucleotide is the result of cloning, restriction digestion and/or ligation steps, and other procedures that produce constructs different from polynucleotides found in nature. combined product.
  • gene or “gene fragment” are used interchangeably herein. They refer to polynucleotides containing at least one open reading frame capable of encoding a specific protein after transcription and translation.
  • a gene or gene fragment can be genomic, cDNA or synthetic, so long as the polynucleotide contains at least one open reading frame, which can cover the entire coding region or a segment thereof.
  • operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter sequence is operably linked to a coding sequence if the promoter sequence facilitates the transcription of the coding sequence.
  • expression refers to the process by which a polynucleotide is transcribed into mRNA, and/or the transcribed mRNA (also referred to as “transcript”) is subsequently translated into a peptide, polypeptide or protein. Transcripts and encoded polypeptides are collectively referred to as gene products. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in eukaryotic cells.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as ⁇ Phage or M13 phage and animal viruses, etc.
  • YAC yeast artificial chromosomes
  • BAC bacterial artificial chromosomes
  • PAC artificial chromosomes
  • bacteriophages such as ⁇ Phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg
  • codon optimization refers to exploiting redundancy in the genetic code to alter a nucleotide sequence while keeping the sequence of the protein it encodes identical.
  • codon optimization can be performed to promote increased or decreased expression of the encoded protein by targeting the codon usage bias of a particular cell type, such as the relative abundance of tRNAs in the cell type, making the nucleotide sequence The codons are adapted to this preference.
  • expression can also be reduced by selecting codons for tRNAs known to be rare in particular cell types.
  • codon optimization can also increase the fidelity of sequence replication, ie, fewer mutations occur during cycles of polynucleotide replication, such as during cloning.
  • the term "host cell” refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. , insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • an "effective amount” as used herein refers to at least the minimum amount required to achieve a measurable amelioration or prevention of a particular condition.
  • the effective amount herein may vary with factors such as the patient's disease state, age, sex, and body weight.
  • An effective amount is also one in which any toxic or adverse effects of treatment are outweighed by the therapeutic beneficial effects.
  • an effective amount of the drug may have the following effects: reduce the number of cancer cells, reduce tumor size, inhibit cancer cell infiltration into peripheral organs, inhibit tumor metastasis, inhibit tumor growth to a certain extent and/or in Alleviation to some extent of one or more symptoms associated with a condition.
  • An effective amount can be administered in one or more administrations.
  • the terms "recipient,” “individual,” “subject,” “host” and “patient” are used interchangeably herein and refer to a subject for whom diagnosis, treatment, or therapy is desired. Any mammalian subject, especially a human.
  • treatment refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and/or therapeutic in terms of partially or completely stabilizing or curing the disease and/or adverse effects attributable to the disease.
  • Treatment encompasses any treatment of disease in mammals such as mice, rats, rabbits, pigs, primates, including humans and other apes, especially humans, and The term includes: (a) preventing the occurrence of a disease or condition in a subject who may be susceptible to the disease or condition but has not yet been diagnosed; (b) inhibiting the symptoms of the disease; (c) arresting the development of the disease; (d) ameliorating the disease symptoms; (e) causing disease or symptom regression; or any combination thereof.
  • kit refers to a combination packaged for common use or commercial sale.
  • a kit of the disclosure can comprise a composition of the disclosure, along with instructions for using the composition or kit.
  • the term "instructions" refers to the descriptive insert commonly included in commercial packages of therapeutic products that contain information regarding the indications, use, dosage, administration, combination therapy, contraindications and/or warnings regarding the use of such therapeutic products.
  • biological activity refers to any activity that would normally be ascribed to the protein by one of skill in the art.
  • activity of Factor XI refers to the activity of the native or naturally occurring Factor XI protein that is capable of leading to a biological function as understood by those skilled in the art. Activity can be detected by coagulation or substrate chromogenic methods.
  • Hemophilia B also known as hemophilia B or Christmas disease, is a genetic disease caused by a genetic mutation in blood clotting factor IX. Hemophilia B was first identified in the patient Stephen Christmas in 1952, so it is also known as Christmas disease.
  • hemophilia B can vary from person to person. Ranges include mild, moderate, and severe. Factor IX levels are 5% to 40% of normal in mild hemophilia; 1% to 5% of normal in moderate hemophilia; below normal in severe hemophilia 1% of the level. In mild cases of hemophilia B, individuals may experience bruising and bleeding after surgery, dental procedures, injuries, or trauma. Individuals with hemophilia B typically experience longer bleeding episodes, although some bleeding also occurs in normal individuals after injury or trauma. Many people with mild hemophilia B may not be diagnosed until after surgery or injury.
  • Spontaneous bleeding may include bleeding into muscles and joints, causing pain and swelling and limiting joint movement. If left untreated, it can cause long-term damage, including inflammation of the lining of the joints (synovitis) and joint disease (arthritis), muscle weakness and/or swelling, tightness, and limited movement of the affected joints, and permanent joint damage damage. Spontaneous joint bleeding is the most common symptom of severe hemophilia B. Other symptoms affecting individuals with severe hemophilia B include frequent and severe bruising and bleeding from muscles, gastrointestinal tract, and central nervous system.
  • Bleeding in individuals with moderate or severe hemophilia may also infiltrate other organs, including the kidneys, stomach, intestines, and brain. Bleeding in the kidneys or in the stomach and intestines may result in blood in the urine (called hematuria) and in the stool (called melena or hematochezia, respectively). Intracerebral hemorrhage may cause headache, stiff neck, vomiting, seizures, and altered mental status, including excessive sleepiness and arousal, which may lead to death if left untreated.
  • hemophilia B A common treatment for hemophilia B is the administration of replacement factor IX to achieve adequate blood clotting and prevent disease-related complications.
  • replacement of Factor IX to adequate levels is usually accomplished using recombinant products or products derived from human blood or plasma.
  • Factor IX also known as Christmas factor or FIX protein
  • FIX protein is one of the serine proteases of the coagulation system and belongs to the peptidase family S1. A deficiency of this protein causes hemophilia B. Because the gene for factor IX is located on the X chromosome (Xq27.1-q27.2), hemophilia B occurs more frequently in males.
  • factor IX factor IX recombinant product preparations for the treatment of hemophilia B include: BeneFix, Idelvion, Alprolix and Refixia.
  • Adeno-associated virus is a single-stranded DNA (ssDNA) virus belonging to the Parvoviridae family.
  • the genome of AAV is about 4.7 kilobases in full length, including inverted terminal repeats (ITRs) located at both ends of the DNA strand and two open reading frames (ORFs) called rep and cap.
  • ITRs inverted terminal repeats
  • ORFs open reading frames
  • the "AAV inverted terminal repeat (ITR)" sequence is a sequence of approximately 145 nucleotides that occurs at both ends of the native single-stranded AAV genome.
  • the ITR is a symmetrical nucleic acid sequence in the adeno-associated virus genome for efficient replication that serves as the origin of replication for viral DNA synthesis and is an essential structural component of recombinant AAV vectors.
  • rep comprises the polynucleotide sequence encoding the four rep proteins rep78, rep68, rep52 and rep40 required for the AAV life cycle.
  • cap comprises polynucleotide sequences encoding AAV cap proteins VP1, VP2 and VP3, wherein AAV cap proteins VP1, VP2 and VP3 can interact to form a twenty-four-sided symmetrical AAV capsid.
  • AAV is capable of efficiently infecting dividing as well as non-dividing human cells, and its genome can integrate into a single chromosomal locus in the host cell genome. Most importantly, although AAV is present in many people, current research does not suggest that AAV is associated with any disease. Based on its high safety, low immunogenicity, wide host range, and ability to mediate long-term stable expression of foreign genes in animals, AAV has become the most promising vector system in gene therapy.
  • AAV1-AAV13 13 different AAVs have been identified, named AAV1-AAV13, according to the AAV serotype or the tissue or cells infected. Also, as shown in Table 1 below, different AAVs have been developed as advantageous vector systems for transfection of specific cell types.
  • serotype 2 AAV2
  • AAV2 is the most widely studied and used one, which can infect retinal epithelium, photoreceptor cells, skeletal muscle, central nervous system and liver cells, etc. in a clinical study.
  • AAV serotype delivery organization AAV1, AAV2, AAV4, AAV5, AAV8, AAV9 central nervous system AAV1, AAV8, AAV9 heart AAV2 kidney AAV2, AAV5, AAV7, AAV8, AAV9 liver AAV4, AAV5, AAV6, AAV9 lung AAV8 pancreas AAV2, AAV5, AAV8 photoreceptor cells AAV1, AAV2, AAV4, AAV5, AAV8 retinal epithelium AAV1, AAV6, AAV7, AAV8, AAV9 skeletal muscle
  • rAAV vector refers to a polynucleotide vector containing one or more heterologous sequences (i.e., non-AAV derived nucleic acid sequences) flanked by two AAV inverted terminal repeats ( ITR).
  • ITR AAV inverted terminal repeats
  • a “recombinant AAV (rAAV) virus” or “rAAV virion” refers to an AAV virion consisting of at least one AAVcap protein encapsulating an rAAV vector.
  • rAAV virus particles can be produced by host cells (HEK293, SF9, Hela, and A549) or HSV systems. Host cells currently commonly used for rAAV virus particle production are cell types derived from mammals, such as HEK293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines. rAAV virions can be produced in said mammalian cell culture system by providing an rAAV plasmid.
  • Baculovirus belongs to the baculoviridae family and is a double-stranded circular DNA virus with a genome size between 90kb and 230kb. Baculoviruses exclusively parasitize arthropods and are known to infect more than 600 species of insects. In 1983, Smith et al. used Autographa Californica Multicapsid Nuclear Polyhedrosis Virus (AcMNPV) to successfully express human ⁇ -interferon in the Spodoptera frugiperda cell line Sf9, creating the first baculovirus Expression system (Mol Cell Biol, 1983, 3:2156-2165).
  • AcMNPV Autographa Californica Multicapsid Nuclear Polyhedrosis Virus
  • Urabe et al. confirmed that Sf9 insect cells infected with baculovirus can support the replication of AAV. They used three recombinant baculoviruses carrying AAV rep gene, Cap gene and ITR core expression elements to co-infect Sf9 cells. And successfully prepared rAAV virus particles. On this basis, researchers have successively developed a system that is more suitable for large-scale preparation of rAAV virus particles.
  • the main process of using the two baculovirus system to prepare rAAV virus particles is to integrate the rep gene and Cap gene of AAV into one baculovirus genome, and integrate the ITR core expression element and the target gene of interest into another baculovirus genome, and then use the above two recombinant baculoviruses to co-infect host cells to produce rAAV virus particles carrying the target gene.
  • the main process of preparing rAAV virus particles using a baculovirus system that relies on packaging cell lines is to first establish a packaging cell line that induces the expression of the rep gene and the Cap gene, and this packaging cell line integrates the rep gene and the Cap gene expression elements, wherein The rep gene and the Cap gene were respectively placed under the control of the strong promoter PH of the baculovirus late gene expression, and the hr2 enhancer sequence and the AAV rep protein binding sequence were further added upstream of the PH promoter. After infection with a recombinant baculovirus containing the AAV ITR and the gene of interest, the rep gene and the Cap gene in the packaging cell line were induced to express, thereby producing rAAV virus particles integrated with the gene of interest.
  • the rAAV vector used to carry the gene of interest in the rAAV virus particle may also include one or more "expression regulatory elements".
  • expression control element refers to a nucleic acid sequence that affects the expression of an operably linked polynucleotide, including polynucleotide sequences that facilitate transcription and translation of a heterologous polynucleotide.
  • Expression regulatory elements that can be used in the present invention include, but are not limited to, promoters, enhancers, intron splicing signals, polyA (polyA), inverted terminal repeats (ITR), and the like.
  • a “promoter” is a DNA sequence located adjacent to a heterologous polynucleotide sequence encoding a product of interest, usually operably linked to the adjacent sequence, eg, heterologous polynucleotide.
  • a promoter generally increases the amount of heterologous polynucleotide expressed compared to the amount expressed in the absence of the promoter.
  • an “enhancer” is a sequence that increases the activity of a promoter. Unlike a promoter, an enhancer does not have promoter activity and generally can function independently of its position relative to the promoter (ie, upstream or downstream of the promoter).
  • enhancer elements or portions thereof that may be used in the present invention include baculovirus enhancers and enhancer elements found in insect cells.
  • “Stuffer sequence” refers to a nucleotide sequence contained within a larger nucleic acid molecule, such as a vector, typically used to create a desired spacing between two nucleic acid features, such as between a promoter and a coding sequence, or A nucleic acid molecule is extended to have a desired length.
  • a stuffer sequence does not contain protein coding information and may be of unknown/synthetic origin and/or unrelated to other nucleic acid sequences within a larger nucleic acid molecule.
  • the present invention provides a composition
  • a composition comprising: (1) a first polynucleotide sequence comprising a first parvovirus capsid operably linked to a first promoter A first open reading frame (ORF) of a gene and a second open reading frame of a second parvovirus capsid gene operably linked to a second promoter, wherein said first ORF comprises an intron comprising said second promoter sequence; and (2) a second polynucleotide sequence comprising a transgene sequence encoding a human Factor IX (FIX) protein.
  • ORF open reading frame
  • FIX human Factor IX
  • the first ORF, the first promoter, the second ORF and the second promoter may be any suitable for expressing the genes encoded by the first ORF and the second ORF respectively sorted in order.
  • the first ORF, the first promoter, the second ORF and the second promoter are in the following order from 3' to 5': first promoter, first ORF comprising the second promoter, Second ORF.
  • said second ORF comprises at least one translation initiation codon and overlaps with the 3' portion of said first ORF.
  • the second ORF comprises at least one translation initiation codon and does not overlap the 3' portion of the first ORF.
  • the first ORF, the first promoter, the second ORF and the second promoter are in the following order from 3' to 5': first promoter, first ORF comprising the second promoter, A second ORF, wherein the second ORF comprises at least one translation initiation codon and overlaps the 3' portion of the first ORF.
  • the parvovirus is an adeno-associated virus (AAV).
  • the first ORF encodes an adeno-associated virus (AAV) cap protein.
  • the cap protein may be any structural protein known in the art capable of forming a functional AAV capsid (ie capable of packaging DNA and infecting target cells).
  • the cap proteins include VP1, VP2, and VP3.
  • the cap protein does not need to include all of VP1, VP2, VP3, as long as it can generate a functional AAV capsid.
  • the cap proteins include VP1 and VP2.
  • the cap proteins include VP1 and VP3. In some embodiments, the cap proteins include VP2 and VP3. In some embodiments, the cap protein includes VP1. In some embodiments, the cap protein includes VP2. In some embodiments, the cap protein includes VP3.
  • Said VP1, VP2, VP3 may be derived from any AAV serotype.
  • the VP1 may be derived from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3, including serotypes 3A and 3B), AAV serotype 4 (AAV4) , AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV Serotype 11 (AAV11), AAV serotype 12 (AAV12), AAV serotype 13 (AAV13), AAV-Rh10, AAV-Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 and any other known AAV.
  • the VP1 is derived from AAV serotype 2 (AAV1), AAV
  • the VP1 is derived from AAV1, AAV2, AAV3, (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV - wild-type VP1 in Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59 or LK03 has at least 75%, 80%, 85%, 90%, 95% or more identity. In some embodiments, the VP1 is at least 75%, 80%, 85%, 90%, 95% or more identical to wild-type VP1 derived from AAV2 or AAV5.
  • the VP1 is derived from AAV1, AAV2, AAV3, (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, Wild-type VP1 in AAV-Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59 or LK03 has one or more amino acid substitutions, deletions and/or additions. In some embodiments, the VP1 has one or more amino acid substitutions, deletions and/or additions compared to wild-type VP1 derived from AAV2 or AAV5.
  • the VP2 may be derived from AAV1, AAV2, AAV3, (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV -Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 and any other AAV known.
  • the VP2 is derived from AAV serotype 2 (AAV2).
  • the VP2 is derived from AAV serotype 5 (AAV5).
  • the VP2 is derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV- Wild-type VP2 in Rh74 or AAV-2i8 is at least 75%, 80%, 85%, 90%, 95% or more identical. In some embodiments, the VP2 is at least 75%, 80%, 85%, 90%, 95% or more identical to wild-type VP1 derived from AAV2 or AAV5.
  • the VP2 is derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV - wild-type VP2 in Rh74 or AAV-2i8 has one or more amino acid substitutions, deletions and/or additions. In some embodiments, the VP2 has one or more amino acid substitutions, deletions and/or additions compared to wild-type VP1 derived from AAV2 or AAV5.
  • the VP3 may be derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 and any other AAV known.
  • the VP3 is derived from AAV serotype 2 (AAV2).
  • the VP3 is derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV- Wild-type VP3 in Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 has at least 75%, 80%, 85%, 90%, 95% or more identity. In some embodiments, the VP3 is at least 75%, 80%, 85%, 90%, 95% or more identical to wild-type VP1 derived from AAV2 or AAV5.
  • the VP3 is derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV - Wild-type VP3 in Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 has one or more amino acid substitutions, deletions and/or additions. In some embodiments, the VP3 has one or more amino acid substitutions, deletions and/or additions compared to wild-type VP1 derived from AAV2 or AAV5.
  • the cap protein comprises VP1, VP2 and/or VP3 derived from AAV of the same serotype. In some embodiments, the cap protein may comprise VP1, VP2 and/or VP3 derived from AAV2. In some embodiments, the cap protein may comprise VP1, VP2 and/or VP3 derived from AAV5.
  • the cap comprises VP1, VP2, and/or VP3 derived from AAV of different serotypes
  • the cap may comprise VP1, VP2, and/or VP3 derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, VP1, VP2 and/or any one or more of AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 or VP3.
  • the first ORF is operably linked to a first promoter.
  • the first promoter can be any suitable promoter known in the art capable of driving the expression of the cap.
  • the first promoter may be a tissue specific promoter, a constitutive promoter, a regulatable promoter.
  • the first promoter can be selected from different sources, for example, the first promoter can be a viral promoter, a plant promoter and a mammalian promoter.
  • the first promoter examples include, but are not limited to, human cytomegalovirus (CMV) immediate early enhancer/promoter, SV40 early enhancer/promoter, JC polyomavirus promoter, myelin basic protein (MBP ) or glial fibrillary acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latency-associated promoter (LAP), Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, neuronal Specific promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, melanin concentrating hormone (MCH) promoter, CBA, matrix metal protein promoter (MPP), chicken ⁇ -actin promoter, CAG, MNDU3, PGK and EF1a promoters.
  • CMV human cytomegalovirus
  • MBP myelin basic protein
  • GFAP glial fibrillary acidic protein
  • HSV-1 herpes simplex virus
  • the first promoter is a promoter suitable for expression in insect cells.
  • the promoters suitable for expression in insect cells include, but are not limited to, PolH promoter, p10 promoter, basic promoter, inducible promoter, E1 promoter or ⁇ E1 promoter.
  • the first promoter is a PolH promoter.
  • the first promoter is the p10 promoter.
  • the 3' end of the first ORF further comprises a polyadenylation sequence or "poly A sequence.”
  • the polyadenylation sequence or "poly A sequence” may range in length from about 1-500 bp.
  • the polyadenylation sequence or "poly A sequence” can be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 in length. , 50, 10, 200 or 500 nucleotides.
  • the second ORF encodes an AAV rep protein, wherein the rep protein can be a replication protein necessary for replication and packaging of any rAAV vector into rAAV virions.
  • the rep proteins include rep78, rep68, rep52, and rep40.
  • the rep protein need not include all of rep78, rep68, rep52, and rep40, so long as it is capable of allowing rAAV vectors to replicate and package into rAAV virions.
  • the rep protein includes any three of rep78, rep68, rep52, and rep40.
  • the rep protein includes any two of rep78, rep68, rep52, and rep40.
  • the rep protein includes any one of rep78, rep68, rep52, and rep40. In some embodiments, the rep proteins include rep78 and rep52. In some embodiments, the rep proteins include rep78 and rep40. In some embodiments, the rep proteins include rep68 and rep52. In some embodiments, the rep proteins include rep68 and rep40.
  • the rep78, rep68, rep52 and rep40 may be derived from any AAV serotype.
  • the rep78 can be derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV- Rh74, AAV-2i8 and any other AAV known.
  • the rep78 can be derived from AAV2 or AAV5.
  • the rep78 is derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV- Wild-type rep78 in Rh74 or AAV-2i8 is at least 75%, 80%, 85%, 90%, 95% or more identical. In some embodiments, the rep78 is at least 75%, 80%, 85%, 90%, 95% or more identical to wild-type rep78 derived from AAV2 or AAV5.
  • the rep78 is derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV - wild-type rep78 in Rh74 or AAV-2i8 has one or more amino acid substitutions, deletions and/or additions. In some embodiments, the rep78 has one or more amino acid substitutions, deletions and/or additions compared to wild-type rep78 derived from AAV2 or AAV5.
  • the rep68 may be derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV- Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 and any other AAV known.
  • the rep68 can be derived from AAV2 or AAV5.
  • the rep68 is derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV- Wild-type rep68 in Rh74, AAV-2i8, DJ, KP1, NP59, LK03 has at least 75%, 80%, 85%, 90%, 95% or more identity. In some embodiments, the rep68 is at least 75%, 80%, 85%, 90%, 95% or more identical to wild-type rep68 derived from AAV2 or AAV5.
  • the rep68 is derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV - Wild-type rep68 in Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 has one or more amino acid substitutions, deletions and/or additions. In some embodiments, the rep68 has one or more amino acid substitutions, deletions and/or additions compared to wild-type rep68 derived from AAV2 or AAV5.
  • the rep52 can be derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV- Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 and any other AAV known.
  • the rep52 can be derived from AAV2 or AAV5.
  • the rep52 is derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV- Wild-type rep52 in Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 has at least 75%, 80%, 85%, 90%, 95% or more identity. In some embodiments, the rep52 is at least 75%, 80%, 85%, 90%, 95% or more identical to wild-type rep52 derived from AAV2 or AAV5.
  • the rep52 is derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV - wild-type rep52 in Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 has one or more amino acid substitutions, deletions and/or additions. In some embodiments, the rep52 has one or more amino acid substitutions, deletions and/or additions compared to wild-type rep52 derived from AAV2 or AAV5.
  • the rep40 may be derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV- Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 and any other AAV known.
  • the rep40 can be derived from AAV2 or AAV5.
  • the rep40 is derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV- Wild-type rep40 in Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 has at least 75%, 80%, 85%, 90%, 95% or more identity. In some embodiments, the rep40 is at least 75%, 80%, 85%, 90%, 95% or more identical to wild-type rep40 derived from AAV2 or AAV5.
  • the rep40 is derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV -
  • the wild-type rep40 in Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 has one or more amino acid substitutions, deletions and/or additions.
  • the rep40 has one or more amino acid substitutions, deletions and/or additions compared to wild-type rep40 derived from AAV2 or AAV5.
  • the rep comprises rep78, rep68, rep52 and/or rep40 derived from AAV of the same serotype.
  • the rep may comprise rep78, rep68, rep52 and/or rep40 derived from AAV2.
  • the rep may comprise rep78 and/or rep52 derived from AAV2.
  • the rep may comprise rep78, rep68, rep52 and/or rep40 derived from AAV5.
  • the rep may comprise rep78 and/or rep52 derived from AAV5.
  • the rep comprises rep78, rep68, rep52 and/or rep40 derived from AAV of different serotypes
  • the rep may comprise reps derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, Wild-type and any other AAV known in AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 Any one or more of rep78, rep68, rep52 and/or rep40.
  • the second ORF encoding the rep protein is operably linked to a second promoter.
  • the second promoter can be any suitable promoter known in the art capable of driving the expression of the cap.
  • the second promoter can be a tissue-specific promoter, a constitutive promoter, a regulatable promoter.
  • the second promoter can be selected from different sources, for example, the second promoter can be a viral promoter, a plant promoter, and a mammalian promoter.
  • the second promoter examples include, but are not limited to, human cytomegalovirus (CMV) immediate early enhancer/promoter, SV40 early enhancer/promoter, JC polyomavirus promoter, myelin basic protein (MBP ) or glial fibrillary acidic protein (GFAP) promoter, herpes simplex virus (HSV-1) latency-associated promoter (LAP), Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter, neuronal Specific promoter (NSE), platelet-derived growth factor (PDGF) promoter, hSYN, melanin concentrating hormone (MCH) promoter, CBA, matrix metal protein promoter (MPP), chicken ⁇ -actin promoter, CAG, MNDU3, PGK and EF1a promoters.
  • CMV human cytomegalovirus
  • MBP myelin basic protein
  • GFAP glial fibrillary acidic protein
  • HSV-1 herpes simplex virus
  • the second promoter is a promoter suitable for expression in insect cells.
  • the promoters suitable for expression in insect cells include, but are not limited to, PolH promoter, p10 promoter, basic promoter, inducible promoter, E1 promoter or ⁇ E1 promoter.
  • the second promoter is the PolH promoter.
  • the second promoter is the p10 promoter.
  • the 3' end of the second ORF further comprises a polyadenylation sequence or "poly A sequence.”
  • the polyadenylation sequence or "poly A sequence” may range in length from about 1-500 bp.
  • the polyadenylation sequence or "poly A sequence” can be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30 in length. , 50, 10, 200 or 500 nucleotides.
  • said cap and said rep may be derived from the same AAV serotype.
  • the cap and rep can be derived from the same AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV- Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 or any other AAV known.
  • the cap and the rep can be derived from different AAV serotypes, for example, the cap and the rep can be derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, respectively , AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 or any other AAV known.
  • the cap can be derived from AAV2 and the rep is derived from AAV5.
  • the first and second promoters may be the same promoter.
  • both the first promoter and the second promoter are any one selected from PolH promoter, p10 promoter, basic promoter, inducible promoter, E1 promoter or ⁇ E1 promoter.
  • the first promoter and the second promoter are both PolH promoters.
  • the first promoter and the second promoter are both p10 promoters.
  • the first and second promoters may be different promoters.
  • the first promoter and the second promoter can be any two promoters selected from PolH promoter, p10 promoter, basic promoter, inducible promoter, E1 promoter or ⁇ E1 promoter respectively .
  • the first promoter is a PolH promoter and the second promoter is a p10 promoter.
  • said first promoter is a p10 promoter and said second promoter is a PolH promoter.
  • the second ORF comprises at least one translation initiation codon and overlaps the 3' portion of said first ORF.
  • the first ORF comprises an intron sequence comprising the second promoter.
  • said first ORF is linked to said second ORF by a sequence encoding a linker.
  • the linker is a cleavable linker.
  • the cleavable linker is a sequence comprising a 2A peptide.
  • the 2A peptide may be selected from 2A peptides derived from aphthviruses or cardioviruses, for example from foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), Thoseaasigna virus (TaV) or 2A peptide of porcine Czech Republic virus (PTV-1).
  • FMDV foot-and-mouth disease virus
  • EAV equine rhinitis A virus
  • TaV Thoseaasigna virus
  • PTV-1 porcine Czech Republic virus
  • the second polynucleotide in the compositions described herein comprises a transgene operably linked to a CMV, CAG, MNDU3, PGK, EF1a promoter, or a liver-specific promoter.
  • the liver-specific promoter includes one or more of the following: minimal TTR promoter (minimal TTR promoter, TTRm), hAAT promoter, albumin (ALB) promoter, apolipoprotein (APO) promoter, apolipoprotein A (apolipoprotein A, APOA1) promoter, apolipoprotein C3 (apolipoprotein C3, APOC3) promoter, complement factor B (complement factor B, CFB) promoter, ketohexokinase ( ketohexokinase (KHK) promoter, hemopexin (HPX) promoter, nicotinamide N-methyltransferase (icotinamide N-methyltransferase, NNMT)
  • the liver-specific promoter further includes other regulatory sequences.
  • the regulatory sequences include enhancer sequences.
  • the enhancer sequence comprises a liver-specific hepatic control region (HCR) enhancer.
  • the liver-specific promoter is selected from the group consisting of Apo-hAAT, HCR-hAAT, APO-HCR-hAAT, APOA1-HCR-hAAT, APOC3-HCR-hAAT, and modified variants thereof.
  • the promoter of the second polynucleotide comprises a polynucleotide sequence at least 75% identical to SEQ ID NO:4. In some embodiments, the promoter of the second polynucleotide comprises a polynucleotide sequence at least 80% identical to SEQ ID NO:4. In some embodiments, the promoter of the second polynucleotide comprises a polynucleotide sequence at least 85% identical to SEQ ID NO:4. In some embodiments, the promoter of the second polynucleotide comprises a polynucleotide sequence at least 90% identical to SEQ ID NO:4.
  • the promoter of the second polynucleotide comprises a polynucleotide sequence at least 95% identical to SEQ ID NO:4. In some embodiments, the promoter of the second polynucleotide comprises a polynucleotide sequence at least 96% identical to SEQ ID NO:4. In some embodiments, the promoter of the second polynucleotide comprises a polynucleotide sequence at least 97% identical to SEQ ID NO:4. In some embodiments, the promoter of the second polynucleotide comprises a polynucleotide sequence at least 98% identical to SEQ ID NO:4.
  • the promoter of the second polynucleotide comprises a polynucleotide sequence at least 99% identical to SEQ ID NO:4. In some embodiments, the promoter of the second polynucleotide comprises the polynucleotide sequence of SEQ ID NO:4.
  • the promoter of the second polynucleotide comprises a polynucleotide sequence at least 75% identical to SEQ ID NO:5. In some embodiments, the promoter of the second polynucleotide comprises a polynucleotide sequence at least 80% identical to SEQ ID NO:5. In some embodiments, the promoter of the second polynucleotide comprises a polynucleotide sequence at least 85% identical to SEQ ID NO:5. In some embodiments, the promoter of the second polynucleotide comprises a polynucleotide sequence at least 90% identical to SEQ ID NO:5.
  • the promoter of the second polynucleotide comprises a polynucleotide sequence at least 95% identical to SEQ ID NO:5. In some embodiments, the promoter of the second polynucleotide comprises a polynucleotide sequence at least 96% identical to SEQ ID NO:5. In some embodiments, the promoter of the second polynucleotide comprises a polynucleotide sequence at least 97% identical to SEQ ID NO:5. In some embodiments, the promoter of the second polynucleotide comprises a polynucleotide sequence at least 98% identical to SEQ ID NO:5.
  • the promoter of the second polynucleotide comprises a polynucleotide sequence at least 99% identical to SEQ ID NO:5. In some embodiments, the promoter of the second polynucleotide comprises the polynucleotide sequence of SEQ ID NO:5.
  • the second polynucleotide further comprises a second promoter.
  • the second promoter of the second polynucleotide may be any promoter known in the art suitable for expression of the transgene or a modified variant thereof.
  • the second promoter of the second polynucleotide includes, but is not limited to, a CMV, CAG, MNDU3, PGK, EF1a promoter, or a liver-specific promoter.
  • the second promoter of the second polynucleotide is a liver-specific promoter
  • the liver-specific promoter includes one or more of the following: minimal TTR promoter ( minimal TTR promoter, TTRm), human ⁇ -1 antitrypsin promoter (human alpha 1-antitrypsin, hAAT), albumin (ALB) promoter, apolipoprotein (APO) promoter, apolipoprotein A (apolipoprotein A , APOA1) promoter, apolipoprotein C3 (apolipoprotein C3, APOC3) promoter, complement factor B (complement factor B, CFB) promoter, ketohexokinase (ketohexokinase, KHK) promoter, hemopexin (hemopexin, HPX) promoter, nicotinamide N-methyltransferase (icotinamide N-methyltransferase, NNMT) promoter or minimal promoter, (liver) carb
  • the second promoter of the second polynucleotide is a liver-specific promoter, and the liver-specific promoter further includes other regulatory sequences.
  • the regulatory sequences include enhancer sequences.
  • the enhancer sequence includes a liver-specific hepatic control region (HCR) enhancer of apolipoprotein E (apolipoprotein E, ApoE).
  • the second promoter of the second polynucleotide is a liver-specific promoter, and the liver-specific promoter is selected from the group consisting of Apo-hAAT, HCR-hAAT, APO-HCR- hAAT, APOA1-HCR-hAAT, APOC3-HCR-hAAT, and modified variants thereof.
  • the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 75% identical to SEQ ID NO:4. In some embodiments, the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 80% identical to SEQ ID NO:4. In some embodiments, the second promoter of the second polynucleotide comprises a polynucleotide sequence that is at least 85% identical to SEQ ID NO:4. In some embodiments, the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 90% identical to SEQ ID NO:4.
  • the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 95% identical to SEQ ID NO:4. In some embodiments, the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 96% identical to SEQ ID NO:4. In some embodiments, the second promoter of the second polynucleotide comprises a polynucleotide sequence that is at least 97% identical to SEQ ID NO:4. In some embodiments, the second promoter of the second polynucleotide comprises a polynucleotide sequence that is at least 98% identical to SEQ ID NO:4.
  • the second promoter of the second polynucleotide comprises a polynucleotide sequence that is at least 99% identical to SEQ ID NO:4. In some embodiments, the second promoter of the second polynucleotide comprises the polynucleotide sequence of SEQ ID NO:4.
  • the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 75% identical to SEQ ID NO:5. In some embodiments, the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 80% identical to SEQ ID NO:5. In some embodiments, the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 85% identical to SEQ ID NO:5. In some embodiments, the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 90% identical to SEQ ID NO:5.
  • the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 95% identical to SEQ ID NO:5. In some embodiments, the second promoter of the second polynucleotide comprises a polynucleotide sequence that is at least 96% identical to SEQ ID NO:5. In some embodiments, the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 97% identical to SEQ ID NO:5. In some embodiments, the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 98% identical to SEQ ID NO:5.
  • the second promoter of the second polynucleotide comprises a polynucleotide sequence at least 99% identical to SEQ ID NO:5. In some embodiments, the second promoter of the second polynucleotide comprises the polynucleotide sequence of SEQ ID NO:5.
  • the transgene encodes a human Factor IX (FIX) protein sequence.
  • the Factor IX described herein may be Factor IX and variants thereof derived from any mammal.
  • the mammal includes, but is not limited to, primates (eg, humans), cows, dogs, cats, rodents (eg, guinea pigs, rats, or mice).
  • the Factor IX described herein is human-derived Factor IX or a variant thereof.
  • Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 75% identical to human Factor IX. In some embodiments, said transgene-encoded Factor IX in said second polynucleotide comprises a sequence at least 80% identical to human Factor IX. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 85% identical to human Factor IX. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 90% identical to human Factor IX.
  • said transgene-encoded Factor IX in said second polynucleotide comprises a sequence at least 95% identical to human Factor IX. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 96% identical to human Factor IX. In some embodiments, said transgene encoded Factor IX in said second polynucleotide comprises a sequence at least 97% identical to human Factor IX. In some embodiments, said transgene-encoded Factor IX in said second polynucleotide comprises a sequence at least 98% identical to human Factor IX.
  • said transgene-encoded Factor IX in said second polynucleotide comprises a sequence at least 99% identical to human Factor IX.
  • Factor IX encoded by said transgene in said second polynucleotide comprises the sequence of human Factor IX.
  • said transgene-encoded Factor IX in said second polynucleotide comprises a sequence having one or more amino acid mutations, substitutions, deletions or additions compared to human Factor IX.
  • Factor IX encoded by said transgene in said second polynucleotide comprises the sequence of SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 75% identical to SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 80% identical to SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 85% identical to SEQ ID NO: 1.
  • Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 90% identical to SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 96% identical to SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 97% identical to SEQ ID NO: 1.
  • Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 98% identical to SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 99% identical to SEQ ID NO: 1. In some embodiments, said transgene-encoded Factor IX in said second polynucleotide comprises a sequence having one or more amino acid mutations, substitutions, deletions or additions compared to SEQ ID NO: 1.
  • said transgene-encoded Factor IX in said second polynucleotide comprises an amino acid sequence having at least one amino acid substitution compared to the sequence of SEQ ID NO: 1.
  • the amino acid substitution comprises R338X, where X can be any amino acid except arginine.
  • the X is selected from H, K, D, E, S, T, N, Q, C, G, P, A, V, I, L, M, F, Y or W.
  • the X is selected from L, D or Q.
  • the amino acid substitution comprises R338L, R338D or R338Q. In some embodiments, the amino acid substitution is R338L.
  • the second polynucleotide comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 300 CG dinucleotides. In some embodiments, the second polynucleotide comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 250 CG dinucleotides. In some embodiments, the second polynucleotide comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 200 CG dinucleotides.
  • the second polynucleotide comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 150 CG dinucleotides. In some embodiments, the second polynucleotide comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 100 CG dinucleotides. In some embodiments, the second polynucleotide comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 50 CG dinucleotides.
  • the second polynucleotide comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 25 CG dinucleotides. In some embodiments, the second polynucleotide comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 20 CG dinucleotides. In some embodiments, the second polynucleotide comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 15 CG dinucleotides.
  • the second polynucleotide comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 10 CG dinucleotides. In some embodiments, the second polynucleotide comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 5 CG dinucleotides. In some embodiments, the second polynucleotide comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX does not comprise a CG dinucleotide.
  • the second polynucleotide sequence comprises a sequence encoding human Factor IX. In some embodiments, the second polynucleotide comprises a sequence at least 50% identical to SEQ ID NO:2. In some embodiments, the second polynucleotide comprises a sequence at least 55% identical to SEQ ID NO:2. In some embodiments, the second polynucleotide comprises a sequence at least 60% identical to SEQ ID NO:2. In some embodiments, the second polynucleotide comprises a sequence at least 65% identical to SEQ ID NO:2. In some embodiments, the second polynucleotide comprises a sequence at least 70% identical to SEQ ID NO:2.
  • the second polynucleotide comprises a sequence at least 75% identical to SEQ ID NO:2. In some embodiments, the second polynucleotide comprises a sequence at least 80% identical to SEQ ID NO:2. In some embodiments, the second polynucleotide comprises a sequence at least 85% identical to SEQ ID NO:2. In some embodiments, the second polynucleotide comprises a sequence at least 90% identical to SEQ ID NO:2. In some embodiments, the second polynucleotide comprises a sequence at least 95% identical to SEQ ID NO:2. In some embodiments, the second polynucleotide comprises a sequence at least 96% identical to SEQ ID NO:2.
  • the second polynucleotide comprises a sequence at least 97% identical to SEQ ID NO:2. In some embodiments, the second polynucleotide comprises a sequence at least 98% identical to SEQ ID NO:2. In some embodiments, the second polynucleotide comprises a sequence at least 99% identical to SEQ ID NO:2. In some embodiments, the second polynucleotide comprises the sequence of SEQ ID NO:2. In some embodiments, the second polynucleotide comprises a sequence having one or more nucleotide mutations, substitutions, deletions or additions compared to SEQ ID NO:2.
  • the second polynucleotide comprises a sequence encoding human Factor IX and is codon optimized. In some embodiments, the second polynucleotide comprises a sequence at least 50% identical to SEQ ID NO:3. In some embodiments, the second polynucleotide comprises a sequence at least 55% identical to SEQ ID NO:3. In some embodiments, the second polynucleotide comprises a sequence at least 60% identical to SEQ ID NO:3. In some embodiments, the second polynucleotide comprises a sequence at least 65% identical to SEQ ID NO:3. In some embodiments, the second polynucleotide comprises a sequence at least 70% identical to SEQ ID NO:3.
  • the second polynucleotide comprises a sequence at least 75% identical to SEQ ID NO:3. In some embodiments, the second polynucleotide comprises a sequence at least 80% identical to SEQ ID NO:3. In some embodiments, the second polynucleotide comprises a sequence at least 85% identical to SEQ ID NO:3. In some embodiments, the second polynucleotide comprises a sequence at least 90% identical to SEQ ID NO:3. In some embodiments, the second polynucleotide comprises a sequence at least 95% identical to SEQ ID NO:3. In some embodiments, the second polynucleotide comprises a sequence at least 96% identical to SEQ ID NO:3.
  • the second polynucleotide comprises a sequence at least 97% identical to SEQ ID NO:3. In some embodiments, the second polynucleotide comprises a sequence at least 98% identical to SEQ ID NO:3. In some embodiments, the second polynucleotide comprises a sequence at least 99% identical to SEQ ID NO:3. In some embodiments, the second polynucleotide comprises the sequence of SEQ ID NO:3. In some embodiments, the second polynucleotide comprises a sequence having one or more nucleotide mutations, substitutions, deletions or additions compared to SEQ ID NO:3.
  • the second polynucleotide further comprises an intron.
  • the intron is the SV40 intron or the modified first intron of human Factor IX.
  • the SV40 intron is a modified SV40 intron.
  • the modified SV40 intron contains the coding sequence of SEQ ID NO:6.
  • the second polynucleotide further comprises other regulatory sequences including, but not limited to, inverted terminal repeats (ITRs), enhancers, splicing signals, polyadenylation signals, stuffer Sequences, terminators, protein degradation signals, internal ribosome entry elements (IRES), 2A sequences, etc.
  • the second polynucleotide further comprises an enhancer region.
  • the enhancer region comprises an SV40 enhancer, an immediate early cytomegalovirus enhancer, an IRBP enhancer, an enhancer derived from an immunoglobulin gene, or a liver-specific hepatic control region. region, HCR) enhancer.
  • the enhancer region is located upstream of the promoter of the second polynucleotide.
  • the enhancer region is located downstream of the promoter of the second polynucleotide.
  • the enhancer region is located upstream of the second promoter of the second polynucleotide.
  • the enhancer region is located downstream of the second promoter of the second polynucleotide.
  • said second polynucleotide further comprises an inverted terminal repeat (ITR). In some embodiments, said second polynucleotide comprises at least one inverted terminal repeat (ITR). In some embodiments, said second polynucleotide comprises two inverted terminal repeats (ITRs). In some embodiments, the two ITRs are identical to each other. In some embodiments, the two ITRs are different from each other. In some embodiments, the inverted terminal repeat (ITR) is an AAV-derived ITR.
  • the ITRs may be derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV- ITRs for Rh74, AAV-2i8, and any other AAV known.
  • the ITRs are derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV - Wild-type ITRs of Rh74, AAV-2i8, and any other AAV known to have mutations, insertions, or deletions of one or more bases, as long as they retain the desired terminal repeat function, such as replication of the gene of interest, viral particles packaging and/or integration etc.
  • the second polynucleotide further comprises one or more stuffer sequences.
  • the stuffer sequence is located upstream of the promoter of the second polynucleotide. In some embodiments, the stuffer sequence is located downstream of the promoter of the second polynucleotide. In some embodiments, the stuffer sequence is located upstream of the second promoter of the second polynucleotide. In some embodiments, the stuffer sequence is located downstream of the second promoter of the second polynucleotide. In some embodiments, the stuffer sequence is located 5' to the 5'ITR sequence. In some embodiments, the stuffer sequence is located 3' to the 5'ITR sequence. In some embodiments, the stuffer sequence is located 5' to the 5'ITR sequence. In some embodiments, the stuffer sequence is located 5' to the 3'ITR sequence. In some embodiments, the stuffer sequence is located 3' to the 3'ITR sequence. In some embodiments, the stuffer sequence is located 3' to the 3'ITR sequence.
  • the length of the filler sequence may be about 0.1kb-5kb, such as but not limited to 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb , 1kb, 1.1kb, 1.2kb, 1.3kb, 1.4kb, 1.5kb, 1.6kb, 1.7kb, 1.8kb, 1.9kb, 2kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2.5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3kb, 3.1kb, 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, 3.8kb, 3.9kb, 4.0kb, 4.1kb, 4.2kb, 4.3kb, 4.4kb, 4.5kb, 4.6kb,
  • the present invention provides a polynucleotide sequence comprising a transgene sequence encoding human Factor IX, wherein the transgene sequence has at least 50% identity to SEQ ID NO:2.
  • the transgenic sequence comprises a sequence at least 55% identical to SEQ ID NO:2.
  • the transgenic sequence comprises a sequence at least 60% identical to SEQ ID NO:2.
  • the transgenic sequence comprises a sequence at least 65% identical to SEQ ID NO:2.
  • the transgenic sequence comprises a sequence at least 70% identical to SEQ ID NO:2.
  • the transgenic sequence comprises a sequence at least 75% identical to SEQ ID NO:2.
  • the transgenic sequence comprises a sequence at least 80% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 85% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 90% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 95% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 96% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 97% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 98% identical to SEQ ID NO:2.
  • the transgenic sequence comprises a sequence at least 99% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises the sequence of SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence having one or more nucleotide mutations, substitutions, deletions or additions compared to SEQ ID NO:2.
  • the encoded amino acid sequence of human Factor IX comprises a sequence at least 75% identical to human Factor IX.
  • said transgene-encoded Factor IX in said second polynucleotide comprises a sequence at least 80% identical to human Factor IX.
  • Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 85% identical to human Factor IX.
  • Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 90% identical to human Factor IX.
  • said transgene-encoded Factor IX in said second polynucleotide comprises a sequence at least 95% identical to human Factor IX. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 96% identical to human Factor IX. In some embodiments, said transgene encoded Factor IX in said second polynucleotide comprises a sequence at least 97% identical to human Factor IX. In some embodiments, said transgene-encoded Factor IX in said second polynucleotide comprises a sequence at least 98% identical to human Factor IX.
  • said transgene-encoded Factor IX in said second polynucleotide comprises a sequence at least 99% identical to human Factor IX.
  • Factor IX encoded by said transgene in said second polynucleotide comprises the sequence of human Factor IX.
  • said transgene-encoded Factor IX in said second polynucleotide comprises a sequence having one or more amino acid mutations, substitutions, deletions or additions compared to human Factor IX.
  • Factor IX encoded by said transgene in said second polynucleotide comprises the sequence of SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide consists of the sequence of SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 75% identical to SEQ ID NO: 1. In some embodiments, said transgene-encoded Factor IX in said second polynucleotide comprises a sequence at least 80% identical to SEQ ID NO: 1.
  • Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 85% identical to SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 90% identical to SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 96% identical to SEQ ID NO: 1.
  • Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 97% identical to SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 98% identical to SEQ ID NO: 1. In some embodiments, Factor IX encoded by said transgene in said second polynucleotide comprises a sequence at least 99% identical to SEQ ID NO: 1. In some embodiments, said transgene-encoded Factor IX in said second polynucleotide comprises a sequence having one or more amino acid mutations, substitutions, deletions or additions compared to SEQ ID NO: 1.
  • said transgene-encoded Factor IX in said second polynucleotide comprises an amino acid sequence having at least one amino acid substitution compared to the sequence of SEQ ID NO: 1.
  • the amino acid substitution comprises R338X, where X can be any amino acid except arginine.
  • the X is selected from H, K, D, E, S, T, N, Q, C, G, P, A, V, I, L, M, F, Y or W.
  • the X is selected from L, D or Q.
  • the amino acid substitution comprises R338L, R338D or R338Q. In some embodiments, the amino acid substitution is R338L.
  • the transgenic sequence comprises a sequence encoding human Factor IX. In some embodiments, the transgenic sequence comprises a sequence at least 50% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 55% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 60% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 65% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 70% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 75% identical to SEQ ID NO:2.
  • the transgenic sequence comprises a sequence at least 80% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 85% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 90% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 95% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 96% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 97% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence at least 98% identical to SEQ ID NO:2.
  • the transgenic sequence comprises a sequence at least 99% identical to SEQ ID NO:2. In some embodiments, the transgenic sequence comprises the sequence of SEQ ID NO:2. In some embodiments, the transgenic sequence consists of the sequence of SEQ ID NO:2. In some embodiments, the transgenic sequence comprises a sequence having one or more nucleotide mutations, substitutions, deletions or additions compared to SEQ ID NO:2.
  • the transgenic sequence comprises a sequence encoding human Factor IX and is codon optimized. In some embodiments, the transgenic sequence comprises a sequence at least 50% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises a sequence at least 55% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises a sequence at least 60% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises a sequence at least 65% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises a sequence at least 70% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises a sequence at least 75% identical to SEQ ID NO:3.
  • the transgenic sequence comprises a sequence at least 80% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises a sequence at least 85% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises a sequence at least 90% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises a sequence at least 95% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises a sequence at least 96% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises a sequence at least 97% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises a sequence at least 98% identical to SEQ ID NO:3.
  • the transgenic sequence comprises a sequence at least 99% identical to SEQ ID NO:3. In some embodiments, the transgenic sequence comprises the sequence of SEQ ID NO:3. In some embodiments, the transgenic sequence consists of the sequence of SEQ ID NO:3. In some embodiments, the transgenic sequence comprises a sequence having one or more nucleotide mutations, substitutions, deletions or additions compared to SEQ ID NO:3.
  • the transgenic sequence comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 300 CG dinucleotides. In some embodiments, the transgenic sequence comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 250 CG dinucleotides. In some embodiments, the transgenic sequence comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 200 CG dinucleotides.
  • the transgenic sequence comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 150 CG dinucleotides. In some embodiments, the transgenic sequence comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 100 CG dinucleotides. In some embodiments, the transgenic sequence comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 50 CG dinucleotides. In some embodiments, the transgenic sequence comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 25 CG dinucleotides.
  • the transgenic sequence comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 20 CG dinucleotides. In some embodiments, the transgenic sequence comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 15 CG dinucleotides. In some embodiments, the transgenic sequence comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 10 CG dinucleotides. In some embodiments, the transgenic sequence comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX comprises less than 5 CG dinucleotides. In some embodiments, the transgenic sequence comprises a sequence encoding human Factor IX, and wherein the sequence encoding human Factor IX does not comprise a CG dinucleotide.
  • the polynucleotide further comprises a promoter.
  • the promoter of the polynucleotide may be any promoter known in the art suitable for the expression of the transgene or a modified variant thereof.
  • the promoter of the polynucleotide includes, but is not limited to, a CMV, CAG, MNDU3, PGK, EF1a promoter, or a liver-specific promoter.
  • the promoter of the polynucleotide is a liver-specific promoter
  • the liver-specific promoter includes one or more of the following: minimal TTR promoter (minimal TTR promoter, TTRm ), human alpha 1-antitrypsin promoter (human alpha 1-antitrypsin, hAAT), albumin (ALB) promoter, apolipoprotein (APO) promoter, apolipoprotein A (apolipoprotein A, APOA1) promoter , Apolipoprotein C3 (apolipoprotein C3, APOC3) promoter, complement factor B (complement factor B, CFB) promoter, ketohexokinase (ketohexokinase, KHK) promoter, hemagglutinin (hemopexin, HPX) promoter, Nicotinamide N-methyltransferase (icotinamide N-methyltransferase, NNMT) promoter or minimal promoter, (hepatic)
  • the promoter of the polynucleotide is a liver-specific promoter, and the liver-specific promoter further includes other regulatory sequences.
  • the regulatory sequences include enhancer sequences.
  • the enhancer sequence comprises a liver-specific hepatic control region (HCR) enhancer.
  • the promoter of the polynucleotide is a liver-specific promoter, and the liver-specific promoter is selected from the group consisting of Apo-hAAT, HCR-hAAT, APO- HCR-hAAT, APOA1-HCR-hAAT, APOC3-HCR-hAAT, and modified variants thereof.
  • the promoter of the polynucleotide comprises a polynucleotide sequence having at least 75% identity to SEQ ID NO:4. In some embodiments, the promoter of the polynucleotide is a polynucleotide sequence that is at least 80% identical to SEQ ID NO:4. In some embodiments, the promoter of the polynucleotide comprises a polynucleotide sequence at least 85% identical to SEQ ID NO:4. In some embodiments, the promoter of the polynucleotide comprises a polynucleotide sequence at least 90% identical to SEQ ID NO:4.
  • the promoter of the polynucleotide comprises a polynucleotide sequence at least 95% identical to SEQ ID NO:4. In some embodiments, the promoter of the polynucleotide comprises a polynucleotide sequence at least 96% identical to SEQ ID NO:4. In some embodiments, the promoter of the polynucleotide comprises a polynucleotide sequence at least 97% identical to SEQ ID NO:4. In some embodiments, the promoter of the polynucleotide comprises a polynucleotide sequence at least 98% identical to SEQ ID NO:4.
  • the promoter of the polynucleotide comprises a polynucleotide sequence at least 99% identical to SEQ ID NO:4. In some embodiments, the promoter of the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:4.
  • the promoter of the polynucleotide comprises a polynucleotide sequence having at least 75% identity to SEQ ID NO:5. In some embodiments, the promoter of the polynucleotide is a polynucleotide sequence that is at least 80% identical to SEQ ID NO:5. In some embodiments, the promoter of the polynucleotide comprises a polynucleotide sequence at least 85% identical to SEQ ID NO:5. In some embodiments, the promoter of the polynucleotide comprises a polynucleotide sequence at least 90% identical to SEQ ID NO:5.
  • the promoter of the polynucleotide comprises a polynucleotide sequence at least 95% identical to SEQ ID NO:5. In some embodiments, the promoter of the polynucleotide comprises a polynucleotide sequence at least 96% identical to SEQ ID NO:5. In some embodiments, the promoter of the polynucleotide comprises a polynucleotide sequence at least 97% identical to SEQ ID NO:5. In some embodiments, the promoter of the polynucleotide comprises a polynucleotide sequence at least 98% identical to SEQ ID NO:5.
  • the promoter of the polynucleotide comprises a polynucleotide sequence at least 99% identical to SEQ ID NO:5. In some embodiments, the promoter of the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:5.
  • the polynucleotide further comprises a second promoter.
  • the second promoter of the polynucleotide may be any promoter known in the art suitable for expression of the transgene or a modified variant thereof.
  • the second promoter of the polynucleotide includes, but is not limited to, a CMV, CAG, MNDU3, PGK, EF1a promoter, or a liver-specific promoter.
  • the second promoter of the polynucleotide is a liver-specific promoter
  • the liver-specific promoter includes one or more of the following: minimal TTR promoter (minimal TTR promoter , TTRm), human alpha-1 antitrypsin promoter (human alpha 1-antitrypsin, hAAT), albumin (ALB) promoter, apolipoprotein (APO) promoter, apolipoprotein A (apolipoprotein A, APOA1) Promoter, apolipoprotein C3 (apolipoprotein C3, APOC3) promoter, complement factor B (complement factor B, CFB) promoter, ketohexokinase (ketohexokinase, KHK) promoter, hemopexin (HPX) promoter promoter, nicotinamide N-methyltransferase (icotinamide N-methyltransferase, NNMT) promoter or minimal promoter, (hepatic) carboxyleste
  • the second promoter of the polynucleotide is a liver-specific promoter, and the liver-specific promoter further includes other regulatory sequences.
  • the regulatory sequences include enhancer sequences.
  • the enhancer sequence comprises a liver-specific hepatic control region (HCR) enhancer.
  • the second promoter of the polynucleotide is a liver-specific promoter, and the liver-specific promoter is selected from the group consisting of Apo-hAAT, HCR-hAAT, APO-HCR-hAAT, APOA1-HCR-hAAT, APOC3-HCR-hAAT, and modified variants thereof.
  • the second promoter of the polynucleotide comprises a polynucleotide sequence that is at least 75% identical to SEQ ID NO:5. In some embodiments, the second promoter of the polynucleotide is a polynucleotide sequence that is at least 80% identical to SEQ ID NO:5. In some embodiments, the second promoter of the polynucleotide comprises a polynucleotide sequence at least 85% identical to SEQ ID NO:5. In some embodiments, the second promoter of the polynucleotide comprises a polynucleotide sequence at least 90% identical to SEQ ID NO:5.
  • the second promoter of the polynucleotide comprises a polynucleotide sequence at least 95% identical to SEQ ID NO:5. In some embodiments, the second promoter of the polynucleotide comprises a polynucleotide sequence at least 96% identical to SEQ ID NO:5. In some embodiments, the second promoter of the polynucleotide comprises a polynucleotide sequence at least 97% identical to SEQ ID NO:5. In some embodiments, the second promoter of the polynucleotide comprises a polynucleotide sequence at least 98% identical to SEQ ID NO:5.
  • the second promoter of the polynucleotide comprises a polynucleotide sequence at least 99% identical to SEQ ID NO:5. In some embodiments, the second promoter of the polynucleotide comprises the polynucleotide sequence of SEQ ID NO:5.
  • the polynucleotide comprises an APO-HCR-hAAT promoter, an SV40 intron, an SV40 poly(A), and a gene encoding FIX. In some embodiments, the polynucleotide comprises a modified APO-HCR-hAAT promoter, an SV40 intron, an SV40 poly(A), and a gene encoding FIX. In some embodiments, the polynucleotide comprises an APO-HCR-hAAT promoter, a modified SV40 intron, an SV40 poly(A), and a gene encoding FIX.
  • the polynucleotide comprises an APO-HCR-hAAT promoter, an SV40 intron, bGH poly(A), and a gene encoding FIX. In some embodiments, the polynucleotide comprises an APO-HCR-hAAT promoter, an SV40 intron, an SV40 poly(A), and an optimized FIX-encoding gene. In some embodiments, the polynucleotide comprises a modified APO-HCR-hAAT promoter, an SV40 intron, an SV40 poly(A), and an optimized FIX-encoding gene.
  • the polynucleotide comprises an APO-HCR-hAAT promoter, a modified SV40 intron, an SV40 poly(A), and an optimized FIX-encoding gene. In some embodiments, the polynucleotide comprises an APO-HCR-hAAT promoter, an SV40 intron, bGH poly(A), and an optimized FIX-encoding gene. In some embodiments, the polynucleotide comprises a modified APO-HCR-hAAT promoter, a modified SV40 intron, an SV40 poly(A), and a gene encoding FIX.
  • the polynucleotide comprises a modified APO-HCR-hAAT promoter, a modified SV40 intron, an SV40 poly(A), and a gene encoding FIX. In some embodiments, the polynucleotide comprises a modified APO-HCR-hAAT promoter, a modified SV40 intron, bGH poly(A), and a gene encoding FIX. In some embodiments, the polynucleotide comprises a modified APO-HCR-hAAT promoter, a modified SV40 intron, an SV40 poly(A), and an optimized FIX-encoding gene.
  • the polynucleotide comprises a modified APO-HCR-hAAT promoter, a modified SV40 intron, an SV40 poly(A), and an optimized FIX-encoding gene. In some embodiments, the polynucleotide comprises a modified APO-HCR-hAAT promoter, a modified SV40 intron, bGH poly(A), and an optimized FIX-encoding gene. In some embodiments, the polynucleotide comprises a modified APO-HCR-hAAT promoter, a modified SV40 intron, bGH poly(A), and a gene encoding FIX.
  • the polynucleotide comprises a modified APO-HCR-hAAT promoter, a modified SV40 intron, bGH poly(A), and an optimized FIX-encoding gene.
  • the polynucleotide comprises a modified APO-HCR-hAAT promoter, an SV40 intron, bGH poly(A), and a gene encoding FIX.
  • the polynucleotide comprises a modified APO-HCR-hAAT promoter, a modified SV40 intron, bGH poly(A), and a gene encoding FIX.
  • the polynucleotide comprises a modified APO-HCR-hAAT promoter, a modified SV40 intron, bGH poly(A), and a gene encoding FIX. In some embodiments, the polynucleotide comprises a modified APO-HCR-hAAT promoter, an SV40 intron, bGH poly(A), and an optimized FIX-encoding gene. In some embodiments, the polynucleotide comprises a modified APO-HCR-hAAT promoter, a modified SV40 intron, bGH poly(A), and an optimized FIX-encoding gene.
  • the polynucleotide comprises a modified APO-HCR-hAAT promoter, a modified SV40 intron, bGH poly(A), and an optimized FIX-encoding gene.
  • the polynucleotide comprises a modified modified APO-HCR-hAAT promoter, a modified SV40 intron, an SV40 poly(A), and a gene encoding FIX.
  • the polynucleotide comprises a modified modified APO-HCR-hAAT promoter, a modified SV40 intron, an SV40 poly(A), and an optimized FIX-encoding gene.
  • the polynucleotide comprises an APO-HCR-hAAT promoter, a modified SV40 intron, an SV40 poly(A), and a gene encoding FIX. In some embodiments, the polynucleotide comprises an APO-HCR-hAAT promoter, a modified SV40 intron, an SV40 poly(A), and an optimized FIX-encoding gene. In some embodiments, the polynucleotide comprises an APO-HCR-hAAT promoter, a modified SV40 intron, bGH poly(A), and a gene encoding FIX. In some embodiments, the polynucleotide comprises an APO-HCR-hAAT promoter, a modified SV40 intron, bGH poly(A), and an optimized FIX-encoding gene.
  • the polynucleotide comprises SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8. In some embodiments, the polynucleotide comprises SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:9. In some embodiments, the polynucleotide comprises SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and a gene encoding FIX. In some embodiments, the polynucleotide comprises SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and an optimized FIX-encoding gene. In some embodiments, the polynucleotide comprises SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:3.
  • the polynucleotide comprises SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:9, and a gene encoding FIX. In some embodiments, the polynucleotide comprises SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:9, and an optimized FIX-encoding gene. In some embodiments, the polynucleotide comprises SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:3.
  • the polynucleotide comprises SEQ ID NO: 10. In some embodiments, the polynucleotide comprises SEQ ID NO: 11. In some embodiments, the polynucleotide comprises SEQ ID NO: 12. In some embodiments, the polynucleotide comprises SEQ ID NO: 13. In some embodiments, the polynucleotide comprises SEQ ID NO: 14. In some embodiments, the polynucleotide comprises SEQ ID NO: 15. In some embodiments, the polynucleotide consists of SEQ ID NO: 10. In some embodiments, the polynucleotide consists of SEQ ID NO: 11. In some embodiments, the polynucleotide consists of SEQ ID NO: 12.
  • the polynucleotide consists of SEQ ID NO: 13. In some embodiments, the polynucleotide consists of SEQ ID NO: 14. In some embodiments, the polynucleotide consists of SEQ ID NO: 15.
  • the present invention provides a cell comprising the composition of the present invention.
  • the cells are insect cells.
  • the insect cells are Sf9 cells.
  • the cells are mammalian cells.
  • the mammalian cells are HEK293 cells and derivatives thereof. Examples of HEK293 derivatives include HEK293A, HEK293AAV, HEK293S, HEK293F, Expi293F, HEK293SG, HEK293SGGD, HEK293FTM, and HEK293T. These derivatives may also be referred to as variants of HEK293.
  • the derivatives are HEK293T cells.
  • cells are transfected with a composition described herein.
  • the transfection includes, but is not limited to, electroporation, calcium phosphate precipitation, lipofection.
  • the composition is stably transfected into the cell.
  • the composition is transiently transfected into the cell.
  • the present invention further provides a cell population comprising the composition of the present invention.
  • the population of cells comprises a plurality of cells described herein.
  • the present invention provides a recombinant adeno-associated virus (rAAV) particle prepared by transfecting the above-mentioned composition of the present invention into insect cells.
  • the insect cells are Sf9 cells.
  • the recombinant adeno-associated virus (rAAV) particle comprises a polynucleotide described herein.
  • the recombinant adeno-associated virus (rAAV) particles may be derived from AAV virus serotypes known in the art to be suitable for use in the present invention.
  • the AAV virus serotypes include, but are not limited to, AAV1, AAV2, AAV3, (including AAV3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV - Rh10, AAV-Rh74, AAV-2i8, AAV-DJ, AAVHSC15, KP1, NP59 and LK03.
  • the AAV virus serotype is AAV2.
  • the AAV virus serotype is AAV8.
  • the AAV virus serotype is AAV5.
  • the cap and the rep can be derived from different AAV serotypes, for example, the cap and the rep can be derived from AAV1, AAV2, AAV3 (including AAV3A and 3B), AAV4, AAV5, respectively , AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-Rh10, AAV-Rh74, AAV-2i8, AAVHSC, DJ, KP1, NP59, LK03 or any other AAV known.
  • the cap is derived from AAV2 and the rep is derived from AAV5.
  • the cap is derived from AAV5 and the rep is derived from AAV2.
  • the compositions of the invention can be delivered into the insect cells by any method known in the art.
  • the methods include, but are not limited to, electroporation, calcium phosphate precipitation, lipofection, and the like.
  • the composition is stably transfected into the insect cell.
  • the composition is transiently transfected into the insect cells.
  • the insect cells are used to produce the rAAV viral particles.
  • the rAAV virus particles can be isolated and purified from the insect cells according to conventional methods known to those skilled in the art.
  • the rAAV can be purified using centrifugation, HPLC, hydrophobic interaction chromatography (HIC), anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, ultrafiltration, gel electrophoresis, affinity chromatography, and/or other purification techniques virus particles.
  • the rAAV virus particles described in the present invention can express high levels of Factor IX in vivo. For example, expressed in vivo in a mouse model of hemophilia B.
  • the expression level of factor IX can be detected by ELISA method. For example, use HYPHEN BioMed's ZYMUTEST FactorIXKit (product number: #RK032A), and use the analysis software SpectraMax software that comes with the multifunctional microplate reader Spectra Max M5 for data analysis.
  • FIX concentration (%) is determined according to the concentration of Plasma FIX Calibrator.
  • the rAAV viral particle expresses greater than about 5% Factor IX in vivo.
  • the rAAV viral particle expresses greater than about 10% Factor IX in vivo. In some embodiments, the rAAV viral particle expresses greater than about 20% Factor IX in vivo. In some embodiments, the rAAV viral particle expresses greater than about 40% Factor IX in vivo. In some embodiments, the rAAV viral particle expresses greater than about 50% Factor IX in vivo. In some embodiments, the rAAV viral particle expresses greater than about 80% Factor IX in vivo. In some embodiments, the rAAV viral particle expresses greater than about 90% Factor IX in vivo.
  • the rAAV viral particles express greater than about 100% Factor IX in vivo. In some embodiments, the rAAV viral particle expresses greater than about 200% Factor IX in vivo. In some embodiments, the rAAV viral particle expresses greater than about 300% Factor IX in vivo. In some embodiments, the rAAV viral particle expresses greater than about 400% Factor IX in vivo. In some embodiments, the rAAV viral particle expresses greater than about 500% Factor IX in vivo. In some embodiments, the rAAV viral particle expresses greater than about 600% Factor IX in vivo.
  • the rAAV viral particle expresses Factor IX in an amount ranging from about 5% to about 600% in vivo. In some embodiments, the rAAV viral particle expresses Factor IX in an amount ranging from about 5% to about 200% in vivo. In some embodiments, the rAAV viral particles express about 5% to about 150% Factor IX in vivo. In some embodiments, the rAAV viral particles express about 10% to about 150% Factor IX in vivo. In some embodiments, the rAAV viral particles express about 5% to about 10% of Factor IX in vivo. In some embodiments, the rAAV viral particles express about 100% to about 600% of Factor IX in vivo.
  • the rAAV viral particle expresses about 200% to about 600% of Factor IX in vivo. In some embodiments, the rAAV viral particle expresses about 300% to about 600% of Factor IX in vivo. In some embodiments, the rAAV viral particle expresses about 400% to about 600% of Factor IX in vivo. In some embodiments, the rAAV viral particle expresses about 450% to about 600% of Factor IX in vivo. In some embodiments, the rAAV viral particle expresses about 500% to about 600% of Factor IX in vivo. In some embodiments, the rAAV virus particles express about 100% Factor IX in vivo.
  • the expression level of factor IX in the rAAV virus particle is about 200% in vivo. In some embodiments, the expression level of factor IX in the rAAV virus particle is about 300% in vivo. In some embodiments, the expression level of factor IX in the rAAV virus particle is about 400% in vivo. In some embodiments, the rAAV virus particles express about 500% Factor IX in vivo. In some embodiments, the rAAV virus particles express about 600% of Factor IX in vivo.
  • the rAAV viral particles express about or at least about 5% of Factor IX in vivo at the fourth week. In some embodiments, the rAAV viral particle expresses Factor IX in vivo at about or at least about 20% at the fourth week. In some embodiments, the rAAV viral particles express about or at least about 40% of Factor IX in vivo at the fourth week. In some embodiments, the rAAV viral particles express about or at least about 100% of Factor IX in vivo at the fourth week. In some embodiments, the rAAV viral particle expresses Factor IX in vivo at about or at least about 400% at the fourth week. In some embodiments, the rAAV viral particle expresses Factor IX in vivo at about or at least about 600% at the fourth week.
  • the rAAV viral particle expresses Factor IX in vivo at about or at least about 5% at week six. In some embodiments, the rAAV viral particles express about or at least about 40% Factor IX in vivo at week six. In some embodiments, the rAAV viral particle expresses Factor IX in vivo at about or at least about 100% at week six. In some embodiments, the rAAV viral particles express about or at least about 400% Factor IX in vivo at week six. In some embodiments, the rAAV viral particles express about or at least about 550% Factor IX in vivo at week six. In some embodiments, the rAAV viral particles express about or at least about 600% Factor IX in vivo at week six.
  • the rAAV viral particle expresses Factor IX in vivo at about or at least about 5% at week eight. In some embodiments, the rAAV viral particles express about or at least about 40% Factor IX in vivo at week eight. In some embodiments, the rAAV viral particle expresses Factor IX in vivo at about or at least about 100% at eighth week. In some embodiments, the rAAV viral particle expresses Factor IX in vivo at about or at least about 300% at eighth week. In some embodiments, the rAAV viral particle expresses Factor IX in vivo at about or at least about 400% at week eight.
  • the rAAV viral particles express about or at least about 450% Factor IX in vivo at week eight. In some embodiments, the rAAV viral particle expresses Factor IX in vivo at about or at least about 500% at week eight.
  • the rAAV viral particle expresses factor IX stably and continuously at about or at least about 5% in the fourth week, sixth week, and eighth week in vivo. In some embodiments, the rAAV viral particles express in vivo factor IX at a stable and sustained expression level of about or at least about 6% at the fourth week, the sixth week, and the eighth week. In some embodiments, the rAAV viral particle expresses factor IX stably and continuously at about or at least about 20% in the fourth week, the sixth week, and the eighth week in vivo.
  • the rAAV viral particle expresses factor IX stably and continuously at about or at least about 40% in the fourth week, the sixth week, and the eighth week in vivo. In some embodiments, the rAAV viral particle expresses factor IX stably and sustainably at about or at least about 400% in the fourth week, the sixth week, and the eighth week in vivo. In some embodiments, the rAAV viral particle expresses factor IX stably and continuously at about or at least about 500% in the fourth week, the sixth week, and the eighth week in vivo.
  • the expression level of factor IX in the rAAV virus particle in vivo is about or at least about 600% in the fourth week, about or at least about 550% in the sixth week, and about or at least about 550% in the eighth week. About 450%.
  • the rAAV viral particles maintain stable expression of Factor IX in vivo for at least four weeks.
  • the rAAV viral particles maintain stable expression of Factor IX in vivo for at least six weeks.
  • the rAAV viral particle maintains stable expression of Factor IX in vivo for at least eight weeks.
  • the rAAV virus particle maintains stable expression of Factor IX in vivo for at least four weeks and the expression level is about or at least about 600%. In some embodiments, the rAAV virus particle maintains stable expression of Factor IX in vivo for at least six weeks and the expression level is about or at least about 600%. In some embodiments, the rAAV viral particle maintains stable expression of Factor IX in vivo for at least eight weeks and the expression level is about or at least about 600%. In some embodiments, the rAAV viral particle maintains stable expression of Factor IX in vivo for at least four weeks and the expression level is about or at least about 550%.
  • the rAAV viral particle maintains stable expression of Factor IX in vivo for at least six weeks and the expression level is about or at least about 550%. In some embodiments, the rAAV viral particle maintains stable expression of Factor IX in vivo for at least eight weeks and the expression level is about or at least about 550%. In some embodiments, the rAAV virus particle maintains stable expression of Factor IX in vivo for at least four weeks and the expression level is about or at least about 500%. In some embodiments, the rAAV virus particle maintains stable expression of Factor IX in vivo for at least six weeks and the expression level is about or at least about 500%.
  • the rAAV viral particle maintains stable expression of Factor IX in vivo for at least eight weeks and the expression level is about or at least about 500%. In some embodiments, the rAAV viral particle maintains stable expression of Factor IX in vivo for at least four weeks and the expression level is about or at least about 450%. In some embodiments, the rAAV virus particle maintains stable expression of Factor IX in vivo for at least six weeks and the expression level is about or at least about 450%. In some embodiments, the rAAV viral particle maintains stable expression of Factor IX in vivo for at least eight weeks and the expression level is about or at least about 450%.
  • the rAAV virus particle maintains stable expression of Factor IX in vivo for at least four weeks and the expression level is about or at least about 400%. In some embodiments, the rAAV viral particle maintains stable expression of Factor IX in vivo for at least six weeks and the expression level is about or at least about 400%. In some embodiments, the rAAV viral particle maintains stable expression of Factor IX in vivo for at least eight weeks and the expression level is about or at least about 400%.
  • the rAAV viral particle maintains stable expression of Factor IX in vivo for at least four weeks and the expression level is at least about or at least about 600%. In some embodiments, the rAAV viral particle maintains stable expression of Factor IX in vivo for at least six weeks and the expression level is at least about or at least about 550%. In some embodiments, the rAAV viral particle maintains stable expression of Factor IX in vivo for at least eight weeks and the expression level is at least about or at least about 500%. In some embodiments, the rAAV viral particle maintains stable expression of Factor IX in vivo for at least eight weeks and the expression level is at least about or at least about 450%.
  • the rAAV viral particle maintains stable expression of Factor IX in vivo for at least eight weeks and the expression level is at least about or at least about 400%. In some embodiments, the rAAV virus particles maintain stable expression of Factor IX in vivo for at least eight weeks, and the expression level lasts from about 400% to about 600%.
  • the rAAV virus particle continuously and stably expresses Factor IX in vivo and the Factor IX is biologically active.
  • Factor IX activity can be detected by two methods: coagulation method and chromogenic substrate method. Coagulation method to detect FIX activity was analyzed and detected by CA-530 coagulation analyzer. The chromogenic substrate method was used to detect FIX activity using HYPHEN BioMed’s BIOPHENTM FIX kit (Cat. No.: REF 221801-RUO) to detect FIX activity. The analysis software SpectraMax software that comes with the multifunctional microplate reader Spectra Max M5 was used for data analysis.
  • the rAAV virus particles continuously and stably express factor IX in vivo and the factor IX has biological activity detected by coagulation method.
  • the rAAV virus particle expresses in vivo factor IX with about or at least about 50% biological activity as detected by a coagulation method.
  • the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 70% as detected by a coagulation method.
  • the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 80% as detected by coagulation.
  • the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about or at least about 200% as detected by a coagulation method. In some embodiments, the rAAV virus particles express in vivo a factor IX biological activity of about or at least about 250% as determined by a coagulation method. In some embodiments, the rAAV virion expresses in vivo a factor IX biological activity of about or at least about 300% as measured by coagulation. In some embodiments, the rAAV virus particle expresses in vivo factor IX with a biological activity of about or at least about 350% as determined by a coagulation method.
  • the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 400% as detected by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 500% as detected by coagulation. In some embodiments, the rAAV virus particle expresses in vivo a factor IX biological activity of about or at least about 550% as determined by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 700% as detected by a coagulation method.
  • the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 1000% as determined by a coagulation method. In some embodiments, the rAAV virus particle expresses in vivo a factor IX biological activity of about or at least about 1500% as determined by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 1700% as determined by coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 1800% as determined by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 2000% as determined by a coagulation method.
  • the rAAV virion expresses in vivo factor IX with greater than about 50% biological activity as measured by a coagulation assay. In some embodiments, the rAAV virion expresses Factor IX in vivo with a biological activity greater than about 70% as measured by coagulation. In some embodiments, the rAAV virion expresses Factor IX in vivo with a biological activity greater than about 80% as determined by a coagulation assay. In some embodiments, the rAAV virion expresses Factor IX in vivo with a biological activity of greater than about 200% as measured by a coagulation assay.
  • the rAAV virus particle expresses Factor IX in vivo and has a biological activity of greater than about 250% as determined by a coagulation assay. In some embodiments, the rAAV virus particle expresses Factor IX in vivo and has a biological activity of greater than about 300% as determined by a coagulation assay. In some embodiments, the rAAV virion expresses Factor IX in vivo with a biological activity greater than about 350% as measured by coagulation. In some embodiments, the rAAV viral particle expresses Factor IX in vivo with a biological activity of greater than about 400% as measured by coagulation.
  • the rAAV virus particle expresses Factor IX in vivo and has a biological activity of greater than about 500% as measured by coagulation. In some embodiments, the rAAV virion expresses Factor IX in vivo with a biological activity of greater than about 550% as determined by a coagulation assay. In some embodiments, the rAAV virus particle expresses Factor IX in vivo with a biological activity of greater than about 700% as measured by coagulation. In some embodiments, the rAAV virus particle expresses Factor IX in vivo and has a biological activity of greater than about 1000% as determined by a coagulation assay.
  • the rAAV virus particle expresses Factor IX in vivo with a biological activity of greater than about 1500% as measured by coagulation. In some embodiments, the rAAV viral particle expresses Factor IX in vivo with a biological activity of greater than about 1700% as determined by a coagulation assay. In some embodiments, the rAAV virion expresses Factor IX in vivo with a biological activity of greater than about 1800% as measured by coagulation. In some embodiments, the rAAV virus particle expresses Factor IX in vivo and has a biological activity of greater than about 2000% as measured by a coagulation assay.
  • the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 50% to about 2000% as detected by the coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 70% to about 2000% as detected by the coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 80% to about 2000% as detected by the coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 200% to about 2000% as detected by the coagulation method.
  • the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 250% to about 2000% as detected by the coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 300% to about 2000% as detected by the coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 350% to about 2000% as detected by the coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 400% to about 2000% as detected by the coagulation method.
  • the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 500% to about 2000% as detected by the coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 550% to about 2000% as detected by the coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 700% to about 2000% as detected by coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 1000% to about 2000% as detected by coagulation method.
  • the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 1500% to about 2000% as detected by the coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 1700% to about 2000% as detected by coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 1800% to about 2000% as detected by the coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 2000% to about 2500% as detected by coagulation method.
  • the factor IX expressed by the rAAV virus particles at week 6 in vivo has a biological activity of about or at least about 50% as detected by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle at week 6 in vivo has a biological activity of about or at least about 70% as detected by coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle at week 6 in vivo has a biological activity of about or at least about 80% as detected by coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle at week 6 in vivo has a biological activity of about or at least about 100% as detected by a coagulation method.
  • the factor IX expressed by the rAAV virus particle at week 6 in vivo has a biological activity of about or at least about 250% as detected by coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle at week 6 in vivo has a biological activity of about or at least about 300% as detected by the coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle at week 6 in vivo has a biological activity of about or at least about 350% as detected by the coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle at week 6 in vivo has a biological activity of about or at least about 400% as detected by a coagulation method.
  • the factor IX expressed by the rAAV virus particles at week 6 in vivo has a biological activity of about or at least about 600% as detected by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle at week 6 in vivo has a biological activity of about or at least about 700% as detected by coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles at week 6 in vivo has a biological activity of about or at least about 1500% as detected by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles at week 6 in vivo has a biological activity of about or at least about 1700% as detected by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles at week 6 in vivo has a biological activity of about or at least about 2000% as detected by a coagulation method.
  • the factor IX expressed by the rAAV virus particle at week 8 in vivo has a biological activity of about or at least about 50% as detected by coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle at week 8 in vivo has a biological activity of about or at least about 70% as detected by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles at week 8 in vivo has a biological activity of about or at least about 80% as detected by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle at week 8 in vivo has a biological activity of about or at least about 100% as detected by coagulation method.
  • the factor IX expressed by the rAAV virus particles at week 8 in vivo has a biological activity of about or at least about 250% as detected by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles at week 8 in vivo has a biological activity of about or at least about 300% as detected by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles at week 8 in vivo has a biological activity of about or at least about 350% as detected by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles at week 8 in vivo has a biological activity of about or at least about 400% as detected by a coagulation method.
  • the factor IX expressed by the rAAV virus particle at week 8 in vivo has a biological activity of about or at least about 600% as detected by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles at week 8 in vivo has a biological activity of about or at least about 700% as detected by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle at week 8 in vivo has a biological activity of about or at least about 1500% as detected by coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle at week 8 in vivo has a biological activity of about or at least about 1700% as detected by a coagulation method.
  • the factor IX expressed by the rAAV virus particle at week 8 in vivo has a biological activity of about or at least about 1800% as detected by coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particles at week 8 in vivo has a biological activity of about or at least about 2000% as detected by a coagulation method.
  • the factor IX expressed by the rAAV virus particle in vivo remains stable for at least 6 weeks. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo remains stably active for at least 6 weeks and its activity is about or at least about 50% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo remains stably active for at least 6 weeks and its activity is about or at least about 80% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 6 weeks and its activity is about or at least about 200% as measured by a coagulation method.
  • the factor IX expressed by the rAAV virus particle in vivo remains stable for at least 6 weeks and its activity is about or at least about 250% as measured by coagulation. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 6 weeks and its activity is about or at least about 300% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 6 weeks and its activity is about or at least about 350% as measured by a coagulation method.
  • the factor IX expressed by the rAAV virus particle in vivo remains stably active for at least 6 weeks and its activity is about or at least about 400% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 6 weeks and its activity is about or at least about 600% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 6 weeks and its activity is about or at least about 700% as measured by a coagulation method.
  • the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 6 weeks and its activity is about or at least about 1700% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 6 weeks and its activity is about or at least about 1800% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo remains stable for at least 6 weeks and its activity is about or at least about 1900% as measured by coagulation.
  • the factor IX expressed by the rAAV virus particle in vivo remains stable for at least 6 weeks and its activity is about or at least about 2000% as measured by coagulation. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 6 weeks and its activity is about or at least about 2200% as measured by a coagulation method.
  • the factor IX expressed by the rAAV virus particle in vivo remains stable and active for at least 8 weeks. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo remains stably active for at least 8 weeks and its activity is about or at least about 50% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is about or at least about 70% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo remains stably active for at least 8 weeks and its activity is about or at least about 200% as measured by a coagulation method.
  • the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is about or at least about 250% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 300% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is about or at least about 500% as measured by a coagulation method.
  • the factor IX expressed by the rAAV virus particle in vivo remains stable for at least 8 weeks and its activity is about or at least about 550% as measured by coagulation. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo remains stably active for at least 8 weeks and its activity is about or at least about 600% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is about or at least about 700% as measured by a coagulation method.
  • the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is about or at least about 1700% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo remains stable for at least 8 weeks and its activity is about or at least about 1800% as measured by coagulation. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo remains stable for at least 8 weeks and its activity is about or at least about 1900% as determined by coagulation assay.
  • the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is about or at least about 2000% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is about or at least about 2200% as measured by a coagulation method.
  • the factor IX expressed by the rAAV virus particles in vivo maintains its activity stably for at least 8 weeks, and its activity is about or at least about 80% at the 6th week and about or at least about 80% at the 8th week as detected by the coagulation method. At least about 70%. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 300% at the 6th week and about or at least about 300% at the 8th week by the coagulation method. At least about 200%.
  • the factor IX expressed by the rAAV virus particle in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 250% at week 6 and about or at least about 250% at week 8 by coagulation method. At least about 250%. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains its activity stably for at least 8 weeks, and its activity is about or at least about 700% at week 6 and about or at least about 700% at week 8 by coagulation method. At least about 550%.
  • the factor IX expressed by the rAAV virus particles in vivo maintains its activity stably for at least 8 weeks, and its activity is about or at least about 350% at the 6th week and about or at least about 350% at the 8th week by coagulation method. At least about 250%. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains its activity stably for at least 8 weeks, and its activity is about or at least about 1700% at the 6th week and about or at least about 1700% at the 8th week by coagulation method. At least about 1800%.
  • the factor IX expressed by the rAAV virus particles in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 2000% at week 6 and about or at least about 2000% at week 8 by coagulation method. At least about 1800%.
  • the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is at least about or at least about 70% as measured by a coagulation method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is at least about or at least about 200% as measured by coagulation assay. In some embodiments, the factor IX expressed by the rAAV virion in vivo is stably active for at least 8 weeks and has an activity of at least about or at least about 250% as measured by a coagulation assay.
  • the factor IX expressed by the rAAV virus particle in vivo remains stable for at least 8 weeks and its activity is at least about or at least about 300% as measured by coagulation. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is at least about or at least about 350% as measured by coagulation assay. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is at least about or at least about 400% as measured by coagulation assay.
  • the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is at least about or at least about 500% as measured by coagulation. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is at least about or at least about 550% as measured by coagulation. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is at least about or at least about 600% as measured by coagulation assay.
  • the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is at least about or at least about 700% as measured by coagulation assay. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is at least about or at least about 1000% as measured by coagulation assay. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is at least about or at least about 1500% as measured by coagulation assay.
  • the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is at least about or at least about 1700% as measured by coagulation assay. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and its activity is at least about or at least about 1800% as measured by coagulation assay. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo is stably active for at least 8 weeks and has an activity of at least about or at least about 1900% as measured by coagulation. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo remains stable for at least 8 weeks and its activity is at least about or at least about 2000% as measured by coagulation.
  • the rAAV virus particles continuously and stably express factor IX in vivo and the factor IX has biological activity detected by a chromogenic substrate method.
  • the rAAV virus particle expresses in vivo factor IX with a biological activity of about or at least about 10% as detected by a chromogenic substrate method.
  • the rAAV virus particles express in vivo a factor IX biological activity of about or at least about 15% as detected by a chromogenic substrate method.
  • the rAAV virus particle expresses Factor IX in vivo and has a biological activity of about or at least about 50% as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 100% as detected by a chromogenic substrate method. In some embodiments, the rAAV virion expresses in vivo a factor IX biological activity of about or at least about 125% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 200% as detected by a chromogenic substrate method. In some embodiments, the rAAV virus particle expresses in vivo a factor IX biological activity of about or at least about 250% as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 300% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 700% as detected by a chromogenic substrate method. In some embodiments, the rAAV virus particle expresses Factor IX in vivo and has a biological activity of about or at least about 750% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 800% as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 900% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 1000% as detected by a chromogenic substrate method. In some embodiments, the rAAV virus particle expresses Factor IX in vivo and has a biological activity of about or at least about 1100% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about or at least about 1200% as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particle in vivo has a biological activity of about 10% to about 1200% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 15% to about 1200% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 50% to about 1200% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 100% to about 1200% as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 125% to about 1200% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 200% to about 1200% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 250% to about 1200% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 300% to about 1200% as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 700% to about 1200% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 750% to about 1200% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 800% to about 1200% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 900% to about 1200% as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 1000% to about 1200% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo has a biological activity of about 1100% to about 1200% as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 10% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 15% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 100% as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 125% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 200% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 250% as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 300% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 700% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 800% as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 1000% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 1100% as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 1200% as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particle in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 10% at week 8 as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 15% at week 8 as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 100% at week 8 as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particle in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 125% at week 8 as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 250% at week 8 as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 300% at the 8th week as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particles in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 700% at week 8 as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 800% at the 8th week as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particles in vivo maintains stable activity for at least 8 weeks and its activity is about or at least about 1000% at week 8 as detected by a chromogenic substrate method.
  • the factor IX expressed by the rAAV virus particle in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 1100% at week 8 as detected by a chromogenic substrate method. In some embodiments, the factor IX expressed by the rAAV virus particle in vivo maintains its activity stably for at least 8 weeks and its activity is about or at least about 1200% at week 8 as detected by a chromogenic substrate method.
  • the factor IX expression level of the rAAV virus particles in vivo is at least about 5% as detected by ELISA method, and the activity is at least about 50% as detected by coagulation method. In some embodiments, the expression level of factor IX in the rAAV virus particle in vivo is at least about 6% as detected by ELISA method, and the activity is at least about 70% as detected by coagulation method. In some embodiments, the expression level of factor IX in the rAAV virus particle in vivo is at least about 20% as detected by ELISA method, and the activity is at least about 200% as detected by coagulation method.
  • the expression level of factor IX in the rAAV virus particle in vivo is at least about 80% as detected by ELISA method, and the activity is at least about 550% as detected by coagulation method. In some embodiments, the expression level of factor IX in the rAAV virus particle in vivo is at least about 30% as detected by ELISA method, and the activity is at least about 200% as detected by coagulation method. In some embodiments, the expression level of factor IX in the rAAV virus particle in vivo is at least about 300% as detected by ELISA method, and the activity is at least about 1500% as detected by coagulation method.
  • the expression level of factor IX in the rAAV virus particle in vivo is at least about 400% as detected by ELISA method, and the activity is at least about 1500% as detected by coagulation method. In some embodiments, the expression level of factor IX in the rAAV virus particle in vivo is at least about 400% as detected by ELISA method, and the activity is at least about 1600% as detected by coagulation method. In some embodiments, the expression level of factor IX in the rAAV virus particle in vivo is at least about 400% as detected by ELISA method, and the activity is at least about 1700% as detected by coagulation method.
  • the expression level of factor IX in the rAAV virus particle in vivo is at least about 400% as detected by ELISA method, and the activity is at least about 1800% as detected by coagulation method. In some embodiments, the factor IX expression level of the rAAV virus particle in vivo is at least about 400% as detected by ELISA method, and the activity is at least about 1900% as detected by coagulation method. In some embodiments, the factor IX expression level of the rAAV virus particle in vivo is at least about 400% as detected by ELISA method, and the activity is at least about 2000% as detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 4 weeks, and the expression level is at least about 500% detected by ELISA method, and the activity is at least about 1800% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 4 weeks, and the expression level is at least about 500% detected by ELISA method, and the activity is at least about 1900% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 4 weeks, and the expression level is at least about 500% detected by ELISA method, and the activity is at least about 2000% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 4 weeks, and the expression level is at least about 500% detected by ELISA method, and the activity is at least about 2100% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 4 weeks, and the expression level is at least about 550% detected by ELISA method, and the activity is at least about 1800% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 4 weeks, and the expression level is at least about 550% detected by ELISA method, and the activity is at least about 1900% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 4 weeks, and the expression level is at least about 550% detected by ELISA method, and the activity is at least about 2000% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 4 weeks, and the expression level is at least about 550% detected by ELISA method, and the activity is at least about 2100% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 4 weeks, and the expression level is at least about 600% detected by ELISA method, and the activity is at least about 1800% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 4 weeks, and the expression level is at least about 600% detected by ELISA method, and the activity is at least about 1900% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 4 weeks, and the expression level is at least about 600% detected by ELISA method, and the activity is at least about 2000% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 4 weeks, and the expression level is at least about 600% detected by ELISA method, and the activity is at least about 2100% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 6 weeks, and the expression level is at least about 500% detected by ELISA method, and the activity is at least about 1800% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 6 weeks, and the expression level is at least about 500% detected by ELISA method, and the activity is at least about 1900% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 6 weeks, and the expression level is at least about 500% detected by ELISA method, and the activity is at least about 2000% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 6 weeks, and the expression level is at least about 500% detected by ELISA method, and the activity is at least about 2100% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 6 weeks, and the expression level is at least about 550% detected by ELISA method, and the activity is at least about 1800% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 6 weeks, and the expression level is at least about 550% detected by ELISA method, and the activity is at least about 1900% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 6 weeks, and the expression level is at least about 550% detected by ELISA method, and the activity is at least about 2000% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 6 weeks, and the expression level is at least about 550% detected by ELISA method, and the activity is at least about 2100% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 8 weeks, and the expression level is at least about 400% detected by ELISA method, and the activity is at least about 1800% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 8 weeks, and the expression level is at least about 400% detected by ELISA method, and the activity is at least about 1900% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 8 weeks, and the expression level is at least about 400% detected by ELISA method, and the activity is at least about 2000% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 8 weeks, and the expression level is at least about 400% detected by ELISA method, and the activity is at least about 2100% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 8 weeks, and the expression level is at least about 450% detected by ELISA method, and the activity is at least about 1800% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 8 weeks, and the expression level is at least about 450% detected by ELISA method, and the activity is at least about 1900% detected by coagulation method.
  • the factor IX of the rAAV virus particles is stably expressed in vivo for at least 8 weeks, and the expression level is at least about 450% detected by ELISA method, and the activity is at least about 2000% detected by coagulation method. In some embodiments, the factor IX of the rAAV virus particles is stably expressed in vivo for at least 8 weeks, and the expression level is at least about 450% detected by ELISA method, and the activity is at least about 2100% detected by coagulation method.
  • the present invention provides a pharmaceutical composition for treating hemophilia B in a subject in need, comprising the polynucleotide or rAAV particle of the present invention, and pharmaceutically acceptable carrier or excipient.
  • pharmaceutically or therapeutically acceptable carrier or excipient refers to a carrier medium that does not interfere with the effectiveness of the biological activity of the active ingredient and is nontoxic to the host or patient.
  • the type of carrier used in the pharmaceutical formulation will depend on the method of administering the therapeutic compound. Numerous methods of preparing pharmaceutical compositions for various routes of administration are known in the art.
  • the pharmaceutical composition is prepared by dissolving the polynucleotide or rAAV virion of the invention in a suitable solvent.
  • suitable solvents include, but are not limited to, water, saline solutions (eg, NaCl), buffer solutions (eg, Phosphate-buffered saline (PBS)), or other solvents.
  • the solvent is sterile.
  • compositions of the present invention may be prepared by aseptic manipulation, or alternatively sterilized at a suitable stage of manufacture.
  • sterile pharmaceutical compositions can be prepared by aseptically admixing sterile ingredients.
  • the sterile pharmaceutical composition can be prepared by admixing the ingredients followed by sterilization of the final formulation. Sterilization methods may include, but are not limited to, heat sterilization, radiation, and filtration.
  • the present application provides a method for treating hemophilia B, which comprises administering a therapeutically effective amount of the polynucleotide, AAV virus particle or pharmaceutical composition of the present invention to a subject in need thereof.
  • a transgene sequence encoding Factor IX according to the invention is expressed in the subject, thereby treating hemophilia B sick.
  • a transgene sequence encoding Factor IX according to the invention is expressed in the subject's liver, thereby treating type B hemophilia.
  • the polynucleotide, AAV viral particle, or pharmaceutical composition can be administered to the subject by any suitable method known in the art.
  • the administering includes local administration or systemic administration.
  • the administration includes, but is not limited to, oral, nasal, inhalation, intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, topical, or rectal administration.
  • the polynucleotide, AAV viral particle or pharmaceutical composition is administered intravenously.
  • the polynucleotide, AAV viral particle, or pharmaceutical composition is provided in a therapeutically effective amount that achieves the desired biological effect at a medically acceptable level of toxicity.
  • the dosage will vary according to the route of administration and the severity of the disease. The dose can also be adjusted according to the weight, age, sex and/or degree of symptoms of each patient to be treated. The precise dosage and route of administration will ultimately be at the discretion of the treating physician or veterinarian. It will be appreciated that routine changes in dosage may be required depending on the age and weight of the patient and the severity of the condition being treated.
  • the AAV virosomes are administered at a dose of 1010 vg/kg to 1015 vg /kg. In some embodiments of the methods, the AAV virosomes are administered at a dose of 1010 vg/kg to 1013 vg/kg. In some embodiments of the methods, the AAV virosomes are administered at a dose of 1011 vg/kg to 1015 vg/kg. In some embodiments of the methods, the AAV virosomes are administered at a dose of 1011 vg/kg to 1013 vg/kg.
  • the AAV virosomes are administered at a dose of 1012 vg/kg to 1015 vg/kg. In some embodiments of the methods, the AAV virosomes are administered at a dose of 1011 vg/kg to 1012 vg/kg. In some embodiments of the methods, the AAV virosomes are administered at a dose of 1010 vg/kg to 1012 vg/kg.
  • the polynucleotide, AAV viral particle, or pharmaceutical composition is delivered in a volume of about 0.01 mL to 1 mL. In some embodiments, the composition is delivered in a volume of about 0.05 mL to 1 mL. In some embodiments, the composition is delivered in a volume of about 0.1 mL to 1 mL. In some embodiments, the composition is delivered in a volume of about 0.5 mL to 1 mL. In some embodiments, the composition is delivered in a volume of about 0.1 mL to about 0.5 mL. In some embodiments, the composition is delivered in a volume of about 0.01 mL to about 0.5 mL. In some embodiments, the composition is delivered in a volume of about 0.05 mL to about 0.5 mL. In some embodiments, the composition is delivered in a volume of about 0.05 mL to 1 mL.
  • the frequency of administration of the polynucleotide, AAV viral particle or pharmaceutical composition may be at least once per day, including 2, 3, 4 or 5 times per day.
  • the treatment can last for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days , 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days days, 48 days, 49 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 150 days, 200 days, 250 days, 300 days, 400 days, 500 days, 750 days, 1000 days or More than 1000 days.
  • the present invention provides a kit for treating hemophilia B, which comprises the polynucleotide, AAV virus particle or pharmaceutical composition of the present invention, and instructions.
  • the instructions are for a method of administering the polynucleotide, AAV viral particle, or pharmaceutical composition to treat hemophilia B.
  • the kit further comprises a container.
  • the container is configured to deliver a polynucleotide, AAV viral particle, or pharmaceutical composition described herein.
  • containers include vials, droppers, bottles, tubes and syringes.
  • the container is a syringe for administering the polynucleotide, AAV viral particle, or pharmaceutical composition.
  • Embodiment 1 Design and cloning of recombinant AAV vector
  • rep coding sequences derived from AAV2 and the cap coding sequences of AAV5 together with their corresponding promoters were respectively cloned into bacmid vectors to obtain the first polynucleotide comprising the coding sequences of cap and rep proteins in this application.
  • a nucleotide sequence comprising factor IX encoding SEQ ID NO: 2 or SEQ ID NO: 3 together with a liver-specific promoter (SEQ ID NO: 4 or 5), an intron (SEQ ID NO: 6 or 7), the poly(A) signal (SEQ ID NO: 8 or 9) and other linking sequences are combined into the second polynucleotide cloned into the bacmid vector.
  • SEQ ID NO: 4 or 5 an intron
  • SEQ ID NO: 8 or 9 the poly(A) signal
  • other linking sequences are combined into the second polynucleotide cloned into the bacmid vector.
  • 6 different second polynucleotides expressing factor IX were tested, that is, the second polynucleotide sequences 1-6 of the test, the sequences of which are shown in SEQ ID NO.10-15.
  • the first polynucleotide and the second polynucleotide bacmid obtained in Example 1 were respectively transfected into Sf9 cells to obtain baculovirus particles. Then the baculovirus particles were combined to infect Sf9 cells to produce recombinant AAV virus. Finally, the recombinant AAV5/factor IX virus particles can be isolated and purified from Sf9 cells.
  • the (F9-KO) hemophilia B model mice knocked out by factor IX were divided into experimental group and control group, and the purified rAAV5/factor IX (a dose of 2x10 ⁇ 13vg/kg) and PBS were intravenously injected into the experimental group and the control group mice.
  • blood was taken at various time points and plasma was extracted to detect the concentration and activity of Factor IX (FIX) in plasma.
  • the concentration of FIX was detected by ELISA method.
  • the ZYMUTEST Factor IX Kit (article number: #RK032A) of HYPHEN BioMed was used for data analysis using the analysis software SpectraMax software that comes with the multifunctional microplate reader Spectra Max M5.
  • FIX concentration (%) is determined according to the concentration of Plasma FIX Calibrator. .
  • Coagulation method to detect FIX activity was analyzed and detected by CA-530 coagulation analyzer.
  • the chromogenic substrate method was used to detect FIX activity using HYPHEN BioMed’s BIOPHENTM FIX kit (Cat. No.: REF 221801-RUO) to detect FIX activity.
  • the analysis software SpectraMax software that comes with the multifunctional microplate reader Spectra Max M5 was used for data analysis. The results showed that compared with the control group, the delivered AAV could significantly express factor IX in mice ( Figure 1 and Table 1), and the expressed factor IX was active ( Figure 2-3 and Table 2-3). In addition, different expression combinations had different levels of expression, and the expression and activity of FIX remained stable at each time point detected.
  • Embodiment 4 The effectiveness of the composition of the present application in vivo

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Abstract

提供一种组合物，包含：(1)第一多核苷酸序列，所述第一多核苷酸序列包含与第一启动子可操作连接的第一细小病毒衣壳基因第一开放阅读框(ORF)和与第二启动子可操作连接的第二细小病毒衣壳基因第二开放阅读框，其中所述第一ORF包含含有所述第二启动子的内含子序列；和(2)第二多核苷酸序列，所述第二多核苷酸序列包含编码人因子IX(FIX)蛋白的转基因序列。

Description

用于治疗B型血友病的组合物和方法
优先权信息
本申请请求2021年6月23日向中国国家知识产权局提交的、专利申请号为PCT/CN2021/101845的专利申请的优先权和权益，并且通过参照将其全文并入此处。
背景技术
B型血友病(Hemophilia B)是一种罕见的遗传性出血性疾病，其通常由个体内因子IX(Factor IX，也称为FIX蛋白)基因的改变(突变)引起。根据患者体内因子IX的活性水平，B型血友病可进一步分为轻度，中度或重度。在轻度情况下，只有在手术、受伤或牙科手术之后才出现出血症状。在某些中度和最严重的情况下，轻伤后甚至在没任何外因触发下即可能会出现出血症状。
凝血因子是血液凝结所需的一类蛋白质。因子IX(Factor IX，或Christmas因子，FIX蛋白)是凝血系统中的一种丝氨酸蛋白酶，其属于肽酶家族S1。由于因子IX的基因位于X染色体(Xq27.1-q27.2)上，因此B型血友病在男性中发生频率更高。
发明内容
目前，本领域对开发能够有效治疗B型血友病的药物和方法存在需求，尤其在于寻找并开发一种可以在病人体内高表达因子IX并且该表达的因子IX在病人体内具有并维持高活性的药物和方法。本发明所提供的组合物和方法解决了上述需求。
一方面，本发明提供一种组合物，其包含：(1)第一多核苷酸序列，所述第一多核苷酸序列包含与第一启动子可操作连接的第一细小病毒衣壳基因第一开放阅读框(ORF)和与第二启动子可操作连接的第二细小病毒衣壳基因第二开放阅读框，其中所述第一ORF包含含有所述第二启动子的内含子序列；和(2)第二多核苷酸序列，所述第二多核苷 酸序列包含编码人因子IX(FIX)蛋白的转基因序列。
在一些实施方案中，所述第一启动子和所述第二启动子适于在昆虫细胞或哺乳动物细胞中表达中表达。在一些实施方案中，所述第一ORF、第一启动子、第二ORF和第二启动子自3’至5’为如下顺序：第一启动子，包含第二启动子的第一ORF，第二ORF，其中所述第二ORF包含至少一个翻译初始密码子并与所述第一ORF的3’部分重叠。
在一些实施方案中，所述昆虫细胞是Sf9细胞。在一些实施方案中，所述哺乳动物细胞是HEK293细胞或其衍生物。在一些实施方案中，所述衍生物为HEK293T细胞。在一些实施方案中，所述第一启动子或所述第二启动子是Polh启动子。在一些实施方案中，所述第一启动子或所述第二启动子是p10启动子。在一些实施方案中，所述第一多核苷酸序列进一步在3’末端包含多聚A序列。
在一些实施方案中，所述细小病毒为腺相关病毒(AAV)。在一些实施方案中，所述第一ORF编码腺相关病毒(AAV)cap蛋白。在一些实施方案中，所述第二ORF编码腺相关病毒(AAV)rep蛋白。在一些实施方案中，所述AAV cap蛋白是AAV血清型5蛋白。在一些实施方案中，所述AAV cap蛋白是VP1、VP2和/或VP3蛋白。在一些实施方案中，所述AAV rep蛋白是AAV血清型2蛋白。在一些实施方案中，所述AAV rep蛋白是Rep78和/或Rep52蛋白。
在一些实施方案中，所述第二多核苷酸序列包含启动子。在一些实施方案中，所述第二多核苷酸的启动子是肝特异性启动子。在一些实施方案中，所述启动子是APO-HCR-hATT启动子。在一些实施方案中，所述启动子包含SEQ ID NO:4的序列。在一些实施方案中，所述第二多核苷酸序列进一步包含第二启动子。在一些实施方案中，所述第二多核苷酸的第二启动子是修饰的APO-HCR-hAAT启动子。在一些实施方案中，所述第二多核苷酸的第二启动子包含SEQ ID NO:5的序列。在一些实施方案中，所述第二多核苷酸序列包含内含子。在一些实施方案中，所述内含子为SV40内含子或修饰后的人因子IX第一内含子。在一些实施方案中，所述SV40内含子为修饰后的SV40内含子。在一些实施方 案中，所述修饰后的SV40内含子包含SEQ ID NO:6的序列。在一些实施方案中，所述修饰后的人因子IX第一内含子包含SEQ ID NO:7的序列。
在一些实施方案中，编码所述人因子IX的所述转基因序列是密码子优化的。在一些实施方案中，所述转基因序列包含少于5个CG二核苷酸。在一些实施方案中，所述人因子IX相比野生型人因子IX包含一个或多个氨基酸取代。在一些实施方案中，所述人因子IX相比SEQ ID NO:1的序列包含氨基酸取代R338X，其中X可以是除精氨酸外的任意氨基酸。在一些实施方案中，所述人因子IX相比SEQ ID NO:1的序列包含氨基酸取代R338L、R338D或R338Q。在一些实施方案中，所述人因子IX相比SEQ ID NO:1的序列包含氨基酸取代R338L。在一些实施方案中，所述转基因序列与SEQ ID NO:3具有至少50％、60％、70％、80％、85％、90％、95％或99％的同一性。在一些实施方案中，所述转基因序列包含SEQ ID NO:3的序列。在一些实施方案中，所述转第二多核苷酸序列进一步包含填充序列。
另一方面，本发明提供一种细胞，其包含本发明所述的组合物。在一些实施方案中，所诉细胞是昆虫细胞。在一些实施方案中，所述昆虫细胞是Sf9细胞。另一方面，本发明提供一种哺乳动物细胞，其包含本发明所述的组合物。在一些实施方案中，所述哺乳动物细胞是HEK293细胞或其衍生物。在一些实施方案中，所述衍生物为HEK293T细胞。
另一方面，本发明提供多核苷酸序列，其包含编码人因子IX的转基因序列，其中所述转基因序列与SEQ ID NO:3具有至少50％、60％、70％、80％、85％、90％或95％的同一性。在一些实施方案中，所述转基因序列与SEQ ID NO:3具有至少96％、97％、98％或99％的同一性。在一些实施方案中，所述转基因序列包含少于5个CG二核苷酸。在一些实施方案中，所述转基因序列不包含CG二核苷酸。在一些实施方案中，所述转基因序列包含SEQ ID NO:3的序列。在一些实施方案中，所述多核苷酸序列包含启动子。在一些实施方案中，所述启动子是肝特异性启动子。在一些实施方案中，所述启动子是APO-HCR-hATT启动子。 在一些实施方案中，所述启动子包含SEQ ID NO:4的序列。在一些实施方案中，所述多核苷酸序列进一步包含修饰的APO-HCR-hATT启动子。在一些实施方案中，所述修饰的APO-HCR-hATT启动子包含SEQ ID NO:5的序列。
另一方面，本发明提供一种腺相关病毒(AAV)病毒颗粒，其包含本发明所述的多核苷酸。在一些实施方案中，所述AAV病毒颗粒源自AAV血清型5。
另一方面，本发明提供一种药物组合物，其包含本发明所述的多核苷酸或AAV病毒颗粒。在一些实施方案中，所述药物组合物进一步包含药学上可接受的载体或赋形剂。
另一方面，本发明提供一种用于治疗B型血友病的方法，其包括对有需要的受试者施用本发明所述的多核苷酸、AAV病毒颗粒，或药物组合物，其中所述转基因序列在所述受试者中表达，由此治疗B型血友病。在一些实施方案中，所述受试者是人。所述转基因序列在所述受试者的肝脏中表达。所述施用经由静脉注射进行。所述施用的剂量为10 10vg/kg to 10 15vg/kg。
另一方面，本发明提供一种用于治疗B型血友病的试剂盒，包含本发明所述的组合物、所述的多核苷酸、所述的AAV病毒颗粒、或所述的药物组合物，以及说明书。在一些实施方案中，所述说明书用于指示施用所述组合物、所述多核苷酸、所述AAV病毒颗粒、或所述药物组合物以治疗B型血友病的方法。
在一些实施方案中，本发明所述的组合物、所述的多核苷酸、所述的AAV病毒颗粒、所述的药物组合物、所述的方法、或所述的试剂盒，其中的所述转基因序列在体内稳定表达所述FIX蛋白至少4周、6周、或8周，且表达的所述FIX蛋白具有生物活性。在一些实施方案中，本发明所述的组合物、所述的多核苷酸、所述的AAV病毒颗粒、所述的药物组合物、所述的方法、或所述的试剂盒，其中所述转基因序列为SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、或SEQ ID NO:15。
在一些实施方案中，本发明所述的组合物、所述的多核苷酸、所述的AAV病毒颗粒、所述的药物组合物、所述的方法、或所述的试剂盒，其中所述转基因序列为SEQ ID NO:15。
附图说明
图1显示rAAV5-FIX在B型血友病小鼠模型体内的FIX的表达。各个编码FIX的多核苷酸序列(即试验第二多核苷酸序列1-6，见SEQ ID NOs:10-15)均有显著的FIX的表达，并且远高于对照组(Control)的基础水平。不同多核苷酸序列之间有着不同水平的表达且在不同时间的表达稳定。其中，与其他序列相比，试验第二多核苷酸序列6(SEQ ID NO:15)在第29天、第43天、和第57天均持续并且稳定高表达FIX。实验显示第二多核苷酸6(SEQ ID NO:15)为最佳的表达载体设计。
图2显示rAAV5-FIX在B型血友病小鼠模型体内表达的FIX具有稳定的活性。该活性由凝固法检测得到。其中，与其他序列相比，试验第二多核苷酸序列6(SEQ ID NO:15)在第43天和第57天表达的FIX持续具有高活性。实验显示第二多核苷酸6(SEQ ID NO:15)为最佳的表达载体设计。
图3显示rAAV5-FIX在B型血友病小鼠体内表达的FIX具有活性。FIX活性由发色底物法(chromogenic activity assay)检测。该方法得到的FIX活性与凝固法检测得到的活性趋势一致。
发明详述
虽然本文已经显示和描述了本发明的各种实施方案，但是对于本领域技术人员显而易见的是，这些实施方案仅以示例性的方式提供。在不脱离本发明的情况下，本领域技术人员可以想到许多变化、改变和替换。应该理解，可以采用本文所述的本发明实施方案的各种替代方案。
除非另有说明，本文公开的一些实施方案的实践采用免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA的常规技术。参见例如Sambrook和Green,Molecular Cloning:A Laboratory Manual,4th Edition(2012)；the series Current  Protocols in Molecular Biology(F.M.Ausubel,et al.eds.)；the series Methods In Enzymology(Academic Press,Inc.),PC 2:A Practical Approach(M.J.MacPherson,B.D.Hames and G.R.Taylor eds.(1995)),Harlow and Lane,eds.(1988)Antibodies,A Laboratory Manual,and Culture of Animal Cells:A Manual of Basic Technique and Specialized Applications,6th Edition(R.I.Freshney,ed.(2010))。
定义
如说明书和权利要求书中所用，单数形式“一”，“一个”和“所述”包括复数指代，除非上下文另有明确说明。例如，术语“rAAV颗粒”包括一个或多个rAAV颗粒。
术语“约”或“近似”指在本领域普通技术人员确定的特定值的可接受误差范围内，这将部分取决于如何测量或确定该值，即，测量系统的局限性。例如，根据本领域的实践，“约”可以表示在1或大于1的标准偏差内。或者，“约”可表示给定值的最多20％，最多10％，最多5％或最多1％的范围。或者，特别是对于生物系统或过程，该术语可以表示数值的一个数量级，优选地在5倍内，更优选地在2倍内。在申请和权利要求中描述了特定值的情况下，除非另有说明，否则应当假定术语“约”意味着在特定值的可接受误差范围内。
如本文中所使用的，术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，以指任何长度的氨基酸聚合物。聚合物可以是直链、环状或支链的，它可以包含修饰的氨基酸，并且可以被非氨基酸中断。该术语还包括已经被修饰的氨基酸聚合物，该修饰例如是通过硫酸化、糖基化、脂化、乙酰化、磷酸化、碘化、甲基化、氧化、蛋白水解处理、磷酸化、异戊烯化、外消旋化、硒化、转移RNA介导的氨基酸向蛋白质的添加(如精氨酸化)、遍在蛋白化，或其它任何操作，如与标记组分的缀合。如本文所用的，术语“氨基酸”是指天然和/或非天然或合成的氨基酸，包括甘氨酸和D或L光学异构体，以及氨基酸类似物和拟肽。由指定蛋白质“衍生”的多肽或氨基酸序列是指多肽的起 源。优选地，多肽具有与序列中编码的多肽的氨基酸序列基本相同的氨基酸序列，或其部分，其中该部分由至少10-20个氨基酸或至少20-30氨基酸或至少30-50个氨基酸组成，或者其可用序列中编码的多肽免疫学地标识。该术语还包括从指定核酸序列表达的多肽。如本文中所使用的，术语“结构域”是指蛋白质的一部分，它在物理上或功能上与该蛋白质或肽的其它部分区分开。物理上定义的结构域包括极具疏水性或亲水性的氨基酸序列，如那些膜结合的或细胞质结合的序列。结构域还可以通过例如由基因复制引起的内部同源性来定义。功能上定义的结构域具有不同的生物学功能。例如，抗原结合域是指抗原结合单元或抗体中与抗原结合的部分。功能上定义的结构域不需要由连续的氨基酸序列编码，且功能上定义的结构域可以含有一个或多个物理上定义的结构域。
如本文中所使用的，术语“氨基酸”是指天然的和/或非天然的或合成的氨基酸，包括但不限于D或L旋光异构体，以及氨基酸类似物和拟肽。标准的单字母或三字母代码用来指称氨基酸。在本文中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，在多肽的情况下，“序列”是多肽中的氨基酸在从氨基末端到羧基末端方向上的顺序，其中该序列中彼此相邻的残基在该多肽的一级结构中是连续的。序列也可以是已知在一个或两个方向上包含额外残基的多肽的一部分的线性序列。
如本文中所使用的，“同一性”、“同源性”或“序列同一性”是指在两个或更多个多核苷酸序列之间或在两个或更多个多肽序列之间的序列相似性或可互换性。当使用诸如Emboss Needle或BestFit等程序确定两个不同氨基酸序列之间的序列同一性、相似性或同源性时，可以使用默认设置，或者可以选择适当的打分矩阵，例如blosum45或blosum80，来优化同一性、相似性或同源性得分。优选地，同源的多核苷酸是那些在如本文所定义的严格条件下杂交并且与这些序列相比具有至少70％、优选至少80％、更优选至少90％、更优选95％、 更优选97％、更优选98％并且甚至更优选99％的序列同一性的多核苷酸。当对可比长度的序列进行最佳比对时，同源的多肽优选具有至少80％，或至少90％，或至少95％，或至少97％，或至少98％的序列同一性，或具有至少99％的序列同一性。
就本文所确定的抗原结合单元而言，“序列同一性百分比(％)”被定义为在比对序列并在必要情况下引入缺口以获得最大序列同一性百分比后，并且不把任何保守置换视为序列同一性的一部分，查询序列中与第二、参考多肽序列或其部分的氨基酸残基相同的氨基酸残基的百分比。可以以本领域技术内的各种方式，例如使用可公开获得的计算机软件，如BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件，来实现旨在确定氨基酸序列同一性百分比的比对。本领域技术人员可以确定用于测量比对的合适的参数，包括在所比较的序列的全长上获得最大比对所需的任何算法。同一性百分比可以在整个定义的多肽序列的长度上进行测量，或者可以在较短的长度上，例如，在从较大的、定义的多肽序列取得的片段的长度上进行测量，该片段例如是至少5、至少10、至少15、至少20、至少50、至少100或至少200个连续残基的片段。这些长度只是示例性的，并且应该理解，由本文的表格、附图或序列表中所示的序列支持的任何片段长度可以用来描述在其上可以测量同一性百分比的长度。
本文所述的蛋白可具有相对于参考序列的一个或多个修饰。所述修饰可以是缺失、插入或添加，或氨基酸残基的取代或置换。“缺失”是指由于缺少一个或多个氨基酸残基而导致的氨基酸序列变化。“插入”或“添加”是指导致与参考序列相比添加一个或多个氨基酸残基的氨基酸序列变化。“取代”或“置换”是指一个或多个氨基酸被不同的氨基酸所取代。在本文中，可以通过将抗原结合单元与参考序列进行比较来确定抗原结合单元相对于参考序列的突变。用于比较的序列的最佳比对可以根据本领域的任何已知方法进行。
如本文中所使用的，术语“分离的”是指与细胞的和其它方面的成分分离的，其中在自然界中，多核苷酸、肽、多肽、蛋白质、抗体 或其片段在正常情况下与之相关联。本领域技术人员知晓，非天然存在的多核苷酸、肽、多肽、蛋白质、抗体或其片段不需要“分离”来与其天然存在的对应物区分开来。另外，“浓缩的”、“分离的”或“稀释的”多核苷酸、肽、多肽、蛋白质、抗体或其片段与其天然存在的对应物是可区分的，因为每单位体积的分子浓度或数目大于(“浓缩的”)或小于从其天然存在的对应物(“分离的”)。富集可基于绝对量来测量，例如每单位体积的溶液的重量，或者其可相对于来源混合物中存在的第二、可能干扰性的物质来测量。
术语“多核苷酸”、“核酸”、“核苷酸”和“寡核苷酸”可互换使用。它们是指任何长度的核苷酸(无论是脱氧核糖核苷酸还是核糖核苷酸)或其类似物的聚合形式。多核苷酸可具有任何三维结构，并且可以执行任何已知或未知的功能。以下是多核苷酸的非限制性实例：基因或基因片段的编码区或非编码区、从连锁分析确定的基因座、外显子、内含子、信使RNA(mRNA)、转移RNA、核糖体RNA、核酶、cDNA、重组多核苷酸、支链多核苷酸、质粒、载体、任意序列的分离的DNA、任意序列的分离的RNA、核酸探针、引物、寡核苷酸或合成的DNA。多核苷酸可以包含修饰的核苷酸，如甲基化核苷酸和核苷酸类似物。如果存在的话，对核苷酸结构的修饰可以在聚合物装配之前或之后赋予。核苷酸的序列可以被非核苷酸组分中断。多核苷酸可以在聚合后进一步修饰，例如通过与标记组分缀合。
当应用于多核苷酸时，“重组的”意味着该多核苷酸是克隆、限制酶切消化和/或连接步骤以及产生与自然界中发现的多核苷酸不同的构建体的其它程序的各种组合的产物。
术语“基因”或“基因片段”在本文中可互换使用。它们是指含有至少一个开放阅读框的多核苷酸，该开放阅读框能够在转录和翻译后编码特定蛋白质。基因或基因片段可以是基因组的、cDNA或合成的，只要该多核苷酸含有至少一个开放阅读框，该开放阅读框可以覆盖整个编码区或其区段。
术语“可操作地连接”或“有效连接”指并置，其中这样描述的组 件所处的关系允许它们以其预期方式发挥作用。例如，如果启动子序列促进编码序列的转录，则该启动子序列与该编码序列可操作地连接。
如本文所用的，“表达”是指多核苷酸被转录为mRNA的过程，和/或转录的mRNA(也称为“转录物”)随后被翻译成肽、多肽或蛋白质的过程。转录物和被编码的多肽统称为基因产物。如果多核苷酸衍生自基因组DNA，则表达可以包括真核细胞中mRNA的剪接。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文使用的术语“密码子优化”指利用遗传密码中的冗余来改变核苷酸序列同时保持其所编码的蛋白质序列相同。在一些情况下，进行密码子优化可促进编码的蛋白质的表达增加或降低，这是通过针对特定细胞类型对密码子使用的偏好如tRNA在细胞类型中的相对丰度，使核苷酸序列中的密码子适应该偏好来实现的。在一些情况中，也可以通过选择已知在特定细胞类型中罕见的tRNA的密码子来减少表达。在一些情况下，通过密码子优化还能够增加序列复制的保真度，即在多核苷酸复制循环期间如克隆过程中较少发生突变。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的 细胞，其包括但不限于，如大肠杆菌或枯草芽孢杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK293细胞或人细胞等的动物细胞。
如本文中使用的“有效量”指至少实现特定病症的可测量改善或预防所需要的最小量。本文中的有效量可以随患者的疾病状态、年龄、性别和体重等因素变化。有效量也是治疗有益效果超过治疗的任何毒性或不利效果的量。在癌症或肿瘤的治疗中，药物的有效量可以具有以下效果：减少癌细胞的数目、降低肿瘤大小、抑制癌细胞浸润入外周器官、抑制肿瘤转移、在一定程度上抑制肿瘤生长和/或在一定程度上减轻一种或多种与病症有关的症状。可以在一次或多次施用中施用有效量。
如本文中所使用的，术语“接受者”、“个体”、“受试者”、“宿主”和“患者”在本文中可互换使用，并且是指希望对其进行诊断、处理或治疗的任何哺乳动物受试者，特别是人类。
如本文中所使用的，术语“治疗”、“处理”指获得所需的药理学和/或生理学效果。该效果就完全或部分防止疾病或其症状而言可以是预防性的，和/或就部分或完全稳定或治愈疾病和/或归因于该疾病的不良反应而言可以是治疗性的。如本文所用的“治疗”涵盖在哺乳动物中对疾病的任何治疗，该哺乳动物例如是小鼠、大鼠、兔、猪、灵长类动物，包括人类和其它猿类，特别是人，并且该术语包括：(a)防止疾病或症状在可能易患该疾病或症状但尚未发生诊断的受试者中发生；(b)抑制疾病症状；(c)阻止疾病的发展；(d)缓解疾病症状；(e)引起疾病或症状消退；或其任意组合。如本文所用的术语“试剂盒”是指经包装以共同使用或市售的组合。例如，本公开的试剂盒可包含本公开的组合物，以及使用组合物或试剂盒的说明。术语“说明书”指治疗产品的商业化包装中通常含有的说明性插页，其含有关于适应症、使用、剂量、施用、组合疗法、禁忌症的信息和/或关于使用这类治疗产品的警告。
如本文中所使用的，术语“生物活性”或“活性”是指通常将被本领域技术人员归因于该蛋白的任何活性。例如，因子XI的活性指本领域技术人员理解的天然或天然存在的因子XI蛋白的能够导致生物功能的活性。活性可以由凝固法或底物发色法检测。
B型血友病
B型血友病也被称为乙型血友病或Christmas病，其是由凝血因子IX的遗传突变而引起的一种遗传性疾病。B型血友病于1952年首次在患者Stephen Christmas体内确认，因此其也被称为Christmas病。
B型血友病的症状和严重程度可因人而异。范围包括轻度、中度以及重度。轻度血友病患者的IX因子水平在正常水平的5％至40％；中度血友病患者的因子水平为正常水平的1％至5％；重度血友病患者的因子水平低于正常水平的1％。在B型血友病的轻度病例中，个体在手术、牙科手术、受伤或外伤后可能会出现瘀伤和出血。尽管在受伤或外伤后正常个体中也会发生一些出血，但患有血友病B的个体通常会出现较长的出血事件。许多患有轻度B型血友病的人可能直到接受外科手术或受伤后才被诊断出来。
患有中度B型血友病的人则可能偶尔会从关节和肌肉等深部组织自然出血。这些发作通常与一些伤害诱发事件有关。患有中度B型血友病的个体在手术或外伤后有长时间出血的危险。在严重的B型血友病病例中，频繁的自发性出血是最常见的症状。自发性出血可能包括肌肉和关节的出血，并引起疼痛和肿胀并限制关节的运动。如果不及时治疗，可能会导致长期损害，包括关节内膜发炎(滑膜炎)和关节疾病(关节炎)、肌肉无力和/或肿胀、紧绷以及受累关节活动受限，并造成永久性关节损伤。自发性关节出血是严重血友病B的最常见症状。影响患有严重B型血友病的个体的其他症状包括频繁和严重的瘀伤和肌肉出血、胃肠道和中枢神经系统出血。
患有中度或重度血友病个体出血后还可能会渗入其他器官，包括肾脏、胃、肠和脑。肾脏或胃和肠内的出血可能会导致尿液中的血 液(称为血尿)和大便中的血液(分别称为黑便或便血)。脑内出血可能会导致头痛、脖子僵硬、呕吐、癫痫发作和精神状态改变，包括过度嗜睡和易唤醒性，如不及时治疗可能会导致死亡。
B型血友病的常用治疗方法是施用替代性因子IX，以实现足够的血液凝结并防止与疾病相关的并发症。当前，通常使用重组产物或衍生自人血液或血浆的产物来完成因子IX的置换以达到足够的水平。
因子IX
因子IX(也称为Christmas因子或FIX蛋白)是凝血系统的丝氨酸蛋白酶之一，属于肽酶家族S1。这种蛋白的缺乏会导致B型血友病。由于因子IX的基因位于X染色体(Xq27.1-q27.2)上，因此B型血友病在男性中发生频率更高。
已经描述了100多种因子IX的突变，有些不会引起症状，但是很多会导致严重的出血性疾病。目前已商品化的用于治疗B型血友病的因子IX重组产物制剂包括：BeneFix、Idelvion、Alprolix和Refixia。
重组AAV载体
腺相关病毒(Adeno-associated virus，AAV)属于细小病毒科，是一种单链DNA(ssDNA)病毒。AAV的基因组全长约为4.7千个碱基，包含位于DNA链两端的反向末端重复(ITR)以及被称为rep和cap的两个开放阅读框(ORF)。
“AAV反向末端重复(ITR)”序列是在天然单链AAV基因组两末端存在的约145个核苷酸的序列。ITR是腺相关病毒基因组中用于有效复制的对称核酸序列，其可用作病毒DNA合成的复制起点，并且是重组AAV载体所必需的结构组分。
“rep”包含编码AAV生命周期所需的四个rep蛋白rep78、rep68、rep52和rep40的多核苷酸序列。“cap”则包含编码AAV cap蛋白VP1、VP2和VP3的多核苷酸序列，其中AAV的cap蛋白VP1、VP2和VP3能够相互作用以形成二十四面对称的AAV衣壳。
AAV能够有效感染分裂的以及非分裂的人类细胞，其基因组可整合到宿主细胞基因组中的单一染色体位点。最为重要的是，尽管 AAV存在于许多人的体内，但目前的研究认为AAV并不与任何疾病相关。基于其安全性高、免疫原性低、宿主范围广、能介导外源基因在动物体内长期稳定表达等特点，AAV已成为基因治疗中最具前景的载体系统。
迄今为止，根据AAV血清型或感染的组织或细胞不同，已鉴定出13种不同的AAV，分别为AAV1-AAV13。并且，如下表1所示，不同AAV已开发为针对特定细胞类型转染的有利的载体系统。在众多AAV血清型中，血清型2(AAV2)是被研究和使用最为广泛的一种，其能够感染视网膜上皮、感光细胞、骨骼肌、中枢神经和肝细胞等，已作为载体被用于多项临床研究中。
表1 AAV血清型及其在基因治疗中作为载体用于递送的组织
AAV血清型 递送组织
AAV1,AAV2,AAV4,AAV5,AAV8,AAV9 中枢神经
AAV1,AAV8,AAV9 心脏
AAV2 肾脏
AAV2,AAV5,AAV7,AAV8,AAV9 肝脏
AAV4,AAV5,AAV6,AAV9 肺
AAV8 胰腺
AAV2,AAV5,AAV8 感光细胞
AAV1,AAV2,AAV4,AAV5,AAV8 视网膜上皮
AAV1,AAV6,AAV7,AAV8,AAV9 骨骼肌
如本文使用的术语“重组AAV载体(rAAV载体)”指含有一个或多个异源序列(即非AAV来源的核酸序列)的多核苷酸载体，其侧翼为两个AAV反向末端重复序列(ITR)。当存在于表达AAV rep和cap蛋白的宿主细胞中时，该rAAV载体可复制并包装入AAV病毒颗粒中。
“重组AAV(rAAV)病毒”或“rAAV病毒颗粒”指由至少一种AAVcap蛋白包封rAAV载体所组成的AAV病毒颗粒。rAAV病毒颗粒可以通过宿主细胞(HEK293，SF9，Hela，和A549)或HSV系统 生产。目前常用于rAAV病毒颗粒生产的宿主细胞是来自哺乳动物的细胞类型，如HEK293细胞、COS细胞、HeLa细胞、KB细胞和其他哺乳动物细胞系。通过提供rAAV质粒在所述哺乳动物细胞培养系统中可产生rAAV病毒颗粒。然而，大多数的上述哺乳动物细胞培养系统的产量均难以达到临床试验和商业规模生产所需。为此，最近开发了使用昆虫细胞如Sf9细胞的rAAV病毒颗粒生产系统。然而，在昆虫细胞中产生AAV，必须进行一些修饰以获得AAVcap蛋白的正确化学计量比。
杆状病毒(baculovirus)属于杆状病毒科，是双链环状DNA病毒，其基因组大小在90kb-230kb之间。杆状病毒专一性寄生于节肢动物体内，已知可感染600多种昆虫。1983年，Smith等人利用苜蓿银纹夜蛾核型多角体病毒(Autographa Californica Multicapsid Nuclear Polyhedrosis Virus，AcMNPV)在草地贪夜蛾细胞系Sf9中成功表达了人β-干扰素，首创了杆状病毒表达系统(Mol Cell Biol,1983,3:2156-2165)。此后，杆状病毒表达系统被不断完善和发展，已经成为应用非常广泛的一种真核表达系统。2002年，Urabe等人证实了感染杆状病毒的Sf9昆虫细胞可以支持AAV的复制，其利用分别携带有AAV的rep基因、Cap基因以及ITR核心表达元件的三种重组杆状病毒共同感染Sf9细胞并成功制备了rAAV病毒颗粒。在此基础上，研究者相继开发出了更适合大规模制备rAAV病毒颗粒的系统。
目前利用杆状病毒表达系统大规模制备rAAV病毒颗粒的方法主要有以下两种：两杆状病毒系统(Two Bac system)和依赖包装细胞系的一杆状病毒系统(One Bac system)。使用两杆状病毒系统制备rAAV病毒颗粒的主要流程是，将AAV的rep基因和Cap基因整合在一个杆状病毒基因组中，将ITR核心表达元件和感兴趣的目的基因整合到另外一个杆状病毒基因组中，然后使用上述两种重组杆状病毒共同感染宿主细胞，产生出携带目的基因的rAAV病毒颗粒。使用依赖包装细胞系的一杆状病毒系统制备rAAV病毒颗粒的主要流程是，先 建立诱导表达rep基因和Cap基因的包装细胞系，这种包装细胞系整合了rep基因和Cap基因表达元件，其中rep基因和Cap基因分别置于杆状病毒晚期基因表达强启动子PH调控下，在PH启动子的上游进一步加入hr2增强子序列和AAV的rep蛋白结合序列。在感染包含AAV ITR和目的基因的重组杆状病毒后，包装细胞系中的rep基因和Cap基因被诱导表达，进而产生整合了目的基因的rAAV病毒颗粒。
在一些实施方案中，rAAV病毒颗粒中用于携带目的基因的rAAV载体还可包括一个或多个“表达调控元件”。如本文使用的术语“表达调控元件”指影响可操作地连接的多核苷酸的表达的核酸序列，包括促进异源多核苷酸转录和翻译的多核苷酸序列。可用于本发明中的表达调控元件包括但不限于启动子、增强子、内含子剪接信号、多聚A(polyA)、反向末端重复序列(ITR)等。
“启动子”是位于编码目标产物的异源多核苷酸序列相邻处的DNA序列，其通常可操作地连接在相邻的序列例如异源多核苷酸上。与不存在启动子的情况下所表达的量相比较，启动子通常增加异源多核苷酸所表达的量。
“增强子”是一段增强启动子活性的序列。与启动子不同的是，增强子并不具有启动子活性，并且通常可不依赖于其相对启动子的位置(即启动子的上游或下游)而起作用。可用于本发明的增强子元件(或其部分)的非限制性实例包括杆状病毒增强子和在昆虫细胞中发现的增强子元件。
“填充序列”是指包含在较大核酸分子(诸如载体)中的核苷酸序列，通常用于在两个核酸特征之间(诸如在启动子和编码序列之间)产生所需间隔，或延伸核酸分子以使其具有期望长度。填充序列不含有蛋白质编码信息，并且可以具有未知/合成来源和/或与较大核酸分子内的其他核酸序列不相关。
组合物
一方面，本发明提供一种组合物，其包含：(1)第一多核苷酸序列，所述第一多核苷酸序列包含与第一启动子可操作连接的第一细 小病毒衣壳基因第一开放阅读框(ORF)和与第二启动子可操作连接的第二细小病毒衣壳基因第二开放阅读框，其中所述第一ORF包含含有所述第二启动子的内含子序列；和(2)第二多核苷酸序列，所述第二多核苷酸序列包含编码人因子IX(FIX)蛋白的转基因序列。
在本发明的组合物的一些实施方案中，所述所述第一ORF、第一启动子、第二ORF和第二启动子可以任何适于分别表达第一ORF和第二ORF所编码的基因的顺序排列。在一些实施方案中，所述第一ORF、第一启动子、第二ORF和第二启动子自3’至5’为如下顺序：第一启动子，包含第二启动子的第一ORF，第二ORF。在一些实施方案中，其中所述第二ORF包含至少一个翻译初始密码子并与所述第一ORF的3’部分重叠。在一些实施方案中，所述第二ORF包含至少一个翻译初始密码子并不与所述第一ORF的3’部分重叠。在一些实施方案中，所述第一ORF、第一启动子、第二ORF和第二启动子自3’至5’为如下顺序：第一启动子，包含第二启动子的第一ORF，第二ORF，其中所述第二ORF包含至少一个翻译初始密码子并与所述第一ORF的3’部分重叠。
在本发明的组合物的一些实施方案中，所述细小病毒为腺相关病毒(AAV)。在本发明的组合物的一些实施方案中，所述第一ORF编码腺相关病毒(AAV)cap蛋白。在一些实施方案中，所述cap蛋白可以是本领域已知的任何能够形成功能性AAV衣壳(即能够包装DNA并感染靶细胞)的结构蛋白。在一些实施方案中，所述cap蛋白包括VP1、VP2和VP3。在一些实施方案中，所述cap蛋白无需包括VP1、VP2、VP3中的全部，只要其能够产生功能性AAV衣壳。在一些实施方案中，所述cap蛋白包括VP1和VP2。在一些实施方案中，所述cap蛋白包括VP1和VP3。在一些实施方案中，所述cap蛋白包括VP2和VP3。在一些实施方案中，所述cap蛋白包括VP1。在一些实施方案中，所述cap蛋白包括VP2。在一些实施方案中，所述cap蛋白包括VP3。
所述VP1、VP2、VP3可衍生自任何AAV血清型。在一些实施方案中，所述VP1可衍生自AAV血清型1(AAV1)、AAV血清型 2(AAV2)、AAV血清型3(AAV3，包括血清型3A和3B)、AAV血清型4(AAV4)、AAV血清型5(AAV5)、AAV血清型6(AAV6)、AAV血清型7(AAV7)、AAV血清型8(AAV8)、AAV血清型9(AAV9)、AAV血清型10(AAV10)、AAV血清型11(AAV11)、AAV血清型12(AAV12)、AAV血清型13(AAV13)、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03和已知的任何其他AAV。在一些实施方案中，所述VP1衍生自AAV血清型2(AAV2)。在一些实施方案中，所述VP1衍生自AAV血清型5(AAV5)。
在一些实施方案中，所述VP1与源自AAV1、AAV2、AAV3，(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59或LK03中的野生型VP1具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述VP1与源自AAV2或AAV5中的野生型VP1具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述VP1相比源自AAV1、AAV2、AAV3，(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59或LK03中的野生型VP1具有一个或多个氨基酸的取代、缺失和/或添加。在一些实施方案中，所述VP1相比源自AAV2或AAV5中的野生型VP1具有一个或多个氨基酸的取代、缺失和/或添加。
在一些实施方案中，所述VP2可衍生自AAV1、AAV2、AAV3，(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03和已知的任何其他AAV。在一些实施方案中，所述VP2衍生自AAV血清型2(AAV2)。在一些实施方案中，所述VP2衍生自AAV血清型5(AAV5)。在一些实施方案中，所述VP2与源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、 AAV12、AAV13、AAV-Rh10、AAV-Rh74或AAV-2i8中的野生型VP2具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述VP2与源自AAV2或AAV5中的野生型VP1具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述VP2相比源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74或AAV-2i8中的野生型VP2具有一个或多个氨基酸的取代、缺失和/或添加。在一些实施方案中，所述VP2相比源自AAV2或AAV5中的野生型VP1具有一个或多个氨基酸的取代、缺失和/或添加。
所述VP3可衍生自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03和已知的任何其他AAV。在一些实施方案中，所述VP3衍生自AAV血清型2(AAV2)。在一些实施方案中，所述VP3与源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03中的野生型VP3具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述VP3与源自AAV2或AAV5中的野生型VP1具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述VP3相比源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03中的野生型VP3具有一个或多个氨基酸的取代、缺失和/或添加。在一些实施方案中，所述VP3相比源自AAV2或AAV5中的野生型VP1具有一个或多个氨基酸的取代、缺失和/或添加。
在一些实施方案中，所述cap蛋白包含衍生自相同血清型的 AAV的VP1、VP2和/或VP3。在一些实施方案中，所述cap蛋白可以包含衍生自AAV2的VP1、VP2和/或VP3。在一些实施方案中，所述cap蛋白可以包含衍生自AAV5的VP1、VP2和/或VP3。在一些实施方案中，所述cap包含衍生自不同血清型的AAV的VP1、VP2和/或VP3，例如所述cap可以包含衍生自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03中任何一种或多种的VP1、VP2和/或VP3。
在本发明的组合物的一些实施方案中，所述第一ORF与第一启动子可操作连接。所述第一启动子可以是本领域已知的任何能够驱动所述cap表达的适当的启动子。在一些实施方案中，所述第一启动子可以是组织特异性启动子、组成型启动子、可调控启动子。在一些实施方案中，所述第一启动子可以选自不同来源，例如所述第一启动子可以是病毒启动子、植物启动子和哺乳动物启动子。
所述第一启动子的实例包括但不限于，人巨细胞病毒(CMV)即刻早期增强子/启动子、SV40早期增强子/启动子、JC多瘤病毒启动子、髓磷脂碱性蛋白(MBP)或神经胶质原纤维酸性蛋白(GFAP)启动子、单纯疱疹病毒(HSV-1)潜伏期相关启动子(LAP)、劳斯肉瘤病毒(RSV)长末端重复序列(LTR)启动子、神经元特异性启动子(NSE)、血小板源性生长因子(PDGF)启动子、hSYN、黑色素聚集激素(MCH)启动子、CBA、基质金属蛋白质启动子(MPP)、鸡β-肌动蛋白启动子、CAG、MNDU3、PGK和EF1a启动子。
在一些实施方案中，所述第一启动子是适于在昆虫细胞中表达的启动子。在一些实施方案中，所述适于在昆虫细胞中表达的启动子包括但不限于PolH启动子、p10启动子、碱性启动子、诱导型启动子、E1启动子或ΔE1启动子。在一些实施方案中，所述第一启动子是PolH启动子。在一些实施方案中，所述第一启动子是p10启动子。
在一些实施方案中，所述第一ORF的3’末端进一步包含聚腺苷 酸化序列或“多聚A序列”。在一些实施方案中，所述聚腺苷酸化序列或“多聚A序列”的长度范围可以为约1-500bp。在一些实施方案中，所述聚腺苷酸化序列或“多聚A序列”的长度可以是但不限于1、2、3、4、5、6、7、8、9、10、20、30、50、10、200或500个核苷酸。
在本发明的组合物的一些实施方案中，所述第二ORF编码AAV rep蛋白，其中所述rep蛋白可以是任何rAAV载体复制和包装成rAAV病毒颗粒所必需的复制蛋白。在一些实施方案中，所述rep蛋白包括rep78、rep68、rep52和rep40。在一些实施方案中，所述rep蛋白无需包括rep78、rep68、rep52和rep40中的全部，只要其能够允许rAAV载体复制和包装成rAAV病毒颗粒。在一些实施方案中，所述rep蛋白包括rep78、rep68、rep52和rep40中的任意三种。在一些实施方案中，所述rep蛋白包括rep78、rep68、rep52和rep40中的任意两种。在一些实施方案中，所述rep蛋白包括rep78、rep68、rep52和rep40中的任意一种。在一些实施方案中，所述rep蛋白包括rep78和rep52。在一些实施方案中，所述rep蛋白包括rep78和rep40。在一些实施方案中，所述rep蛋白包括rep68和rep52。在一些实施方案中，所述rep蛋白包括rep68和rep40。
所述rep78、rep68、rep52和rep40可以衍生自任何AAV血清型。在一些实施方案中，所述rep78可衍生自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8和已知的任何其他AAV。在一些实施方案中，所述rep78可衍生自AAV2或AAV5。在一些实施方案中，所述rep78与源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74或AAV-2i8中的野生型rep78具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述rep78与源自AAV2或AAV5中的野生型rep78具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述rep78相比源自AAV1、 AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74或AAV-2i8中的野生型rep78具有一个或多个氨基酸的取代、缺失和/或添加。在一些实施方案中，所述rep78相比源自AAV2或AAV5中的野生型rep78具有一个或多个氨基酸的取代、缺失和/或添加。
在一些实施方案中，所述rep68可衍生自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03和已知的任何其他AAV。在一些实施方案中，所述rep68可衍生自AAV2或AAV5。在一些实施方案中，所述rep68与源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、DJ、KP1、NP59、LK03中的野生型rep68具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述rep68与源自AAV2或AAV5中的野生型rep68具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述rep68相比源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03中的野生型rep68具有一个或多个氨基酸的取代、缺失和/或添加。在一些实施方案中，所述rep68相比源自AAV2或AAV5中的野生型rep68具有一个或多个氨基酸的取代、缺失和/或添加。
在一些实施方案中，所述rep52可衍生自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03和已知的任何其他AAV。在一些实施方案中，所述rep52可衍生自AAV2或AAV5。在一些实 施方案中，所述rep52与源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03中的野生型rep52具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述rep52与源自AAV2或AAV5中的野生型rep52具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述rep52相比源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03中的野生型rep52具有一个或多个氨基酸的取代、缺失和/或添加。在一些实施方案中，所述rep52相比源自AAV2或AAV5中的野生型rep52具有一个或多个氨基酸的取代、缺失和/或添加。
在一些实施方案中，所述rep40可衍生自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03和已知的任何其他AAV。在一些实施方案中，所述rep40可衍生自AAV2或AAV5。在一些实施方案中，所述rep40与源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03中的野生型rep40具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述rep40与源自AAV2或AAV5中的野生型rep40具有至少75％、80％、85％、90％、95％或更高的同一性。在一些实施方案中，所述rep40相比源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03中的野生型rep40具有一个或多个氨基酸的取代、缺失和/或添 加。在一些实施方案中，所述rep40相比源自AAV2或AAV5中的野生型rep40具有一个或多个氨基酸的取代、缺失和/或添加。
在一些实施方案中，所述rep包含衍生自相同血清型的AAV的rep78、rep68、rep52和/或rep40。在一些实施方案中，所述rep可以包含衍生自AAV2的rep78、rep68、rep52和/或rep40。在一些实施方案中，所述rep可以包含衍生自AAV2的rep78和/或rep52。在一些实施方案中，所述rep可以包含衍生自AAV5的rep78、rep68、rep52和/或rep40。在一些实施方案中，所述rep可以包含衍生自AAV5的rep78和/或rep52。在一些实施方案中，所述rep包含衍生自不同血清型的AAV的rep78、rep68、rep52和/或rep40，例如所述rep可以包含衍生自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03中的野生型和已知的任何其他AAV中任何一种或多种的rep78、rep68、rep52和/或rep40。
在一些实施方案中，编码所述rep蛋白的第二ORF与第二启动子可操作连接。所述第二启动子可以是本领域已知的任何能够驱动所述cap表达的适当的启动子。在一些实施方案中，所述第二启动子可以是组织特异性启动子、组成型启动子、可调控启动子。在一些实施方案中，所述第二启动子可以选自不同来源，例如所述第二启动子可以是病毒启动子、植物启动子和哺乳动物启动子。
所述第二启动子的实例包括但不限于，人巨细胞病毒(CMV)即刻早期增强子/启动子、SV40早期增强子/启动子、JC多瘤病毒启动子、髓磷脂碱性蛋白(MBP)或神经胶质原纤维酸性蛋白(GFAP)启动子、单纯疱疹病毒(HSV-1)潜伏期相关启动子(LAP)、劳斯肉瘤病毒(RSV)长末端重复序列(LTR)启动子、神经元特异性启动子(NSE)、血小板源性生长因子(PDGF)启动子、hSYN、黑色素聚集激素(MCH)启动子、CBA、基质金属蛋白质启动子(MPP)、鸡β-肌动蛋白启动子、CAG、MNDU3、PGK和EF1a启动子。
在一些实施方案中，所述第二启动子是适于在昆虫细胞中表达的启动子。在一些实施方案中，所述适于在昆虫细胞中表达的启动子包括但不限于PolH启动子、p10启动子、碱性启动子、诱导型启动子、E1启动子或ΔE1启动子。在一些实施方案中，所述第二启动子是PolH启动子。在一些实施方案中，所述第二启动子是p10启动子。
在一些实施方案中，所述第二ORF的3’末端进一步包含聚腺苷酸化序列或“多聚A序列”。在一些实施方案中，所述聚腺苷酸化序列或“多聚A序列”的长度范围可以为约1-500bp。在一些实施方案中，所述聚腺苷酸化序列或“多聚A序列”的长度可以是但不限于1、2、3、4、5、6、7、8、9、10、20、30、50、10、200或500个核苷酸。
在本发明的组合物的一些实施方案中，所述cap和所述rep可以衍生自相同的AAV血清型。例如，所述cap和rep可以衍生自相同的AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03或已知的任何其他AAV。
在一些实施方案中，所述cap和所述rep可以衍生自不同的AAV血清型，例如，所述cap和所述rep可以分别衍生自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03或已知的任何其他AAV。举例来说，在一些实施方案中，所述cap可以衍生自AAV2，而所述rep则衍生自AAV5。
在本发明的组合物的一些实施方案中，所述第一启动子和第二启动子可以是相同的启动子。例如，所述第一启动子和第二启动子均为选自PolH启动子、p10启动子、碱性启动子、诱导型启动子、E1启动子或ΔE1启动子中的任何一种。举例而言，在一些实施方案中，所述所述第一启动子和第二启动子均为PolH启动子。在一些实施方案中，所述所述第一启动子和第二启动子均为p10启动子。
在本发明的组合物的一些实施方案中，所述第一启动子和第二启动子可以是不同的启动子。例如，所述第一启动子和第二启动子可以是分别选自PolH启动子、p10启动子、碱性启动子、诱导型启动子、E1启动子或ΔE1启动子中的任何二种启动子。举例而言，在一些实施方案中，所述所述第一启动子为PolH启动子，而所述第二启动子为p10启动子。在一些实施方案中，所述所述第一启动子为p10启动子，而所述第二启动子为PolH启动子。
在本发明的组合物的一些实施方案中，第二ORF包含至少一个翻译初始密码子并与所述第一ORF的3’部分重叠。在一些实施方案中，所述第一ORF包含含有所述第二启动子的内含子序列。在一些实施方案中，所述第一ORF与所述第二ORF通过编码接头的序列连接。在一些实施方案中，所述接头为可裂解接头。在一些实施方案中，所述可裂解接头为包含2A肽的序列。在一些实施方案中，所述2A肽可以是选自源自口疮病毒属或心病毒属的2A肽，例如源自口蹄疫病毒(FMDV)、马鼻炎A病毒(ERAV)、Thoseaasigna病毒(TaV)或猪捷申病毒(PTV-1)的2A肽。
在一些实施方案中，本发明所述的组合物中的第二多核苷酸包含与CMV、CAG、MNDU3、PGK、EF1a启动子或肝部特异性启动子可操作连接的转基因。在一些实施方案中，所述肝部特异性启动子包括如下的一种或多种：最小TTR启动子(minimal TTR promotor，TTRm)、hAAT启动子、白蛋白(ALB)启动子、载脂蛋白(APO)启动子、载脂蛋白A(apolipoprotein A，APOA1)启动子、载脂蛋白C3(apolipoprotein C3，APOC3)启动子、补体因子B(complement factor B，CFB)启动子、酮己糖激酶(ketohexokinase，KHK)启动子、血凝素(hemopexin，HPX)启动子、烟酰胺N-甲基转移酶(icotinamide N-methyltransferase，NNMT)启动子或最小启动子、(肝)羧酸酯酶1(carboxylesterase 1，CES1)启动子、蛋白C(protein C，PROC)启动子、甘露聚糖结合凝集素丝氨酸蛋白酶2(mannan-binding lectin serine protease 2，MASP2)启动子、铁调素抗菌肽(epcidin antimicrobial  peptide，HAMP)启动子，或其经修饰的变体。
在一些实施方案中，所述肝部特异性启动子进一步包括其他调控序列。在一些实施方案中，所述调控序列包括增强子序列。在一些实施方案中，所述增强子序列包括肝特异性肝控制区(liver-specific hepatic control region，HCR)增强子。
在一些实施方案中，所述肝部特异性启动子选自Apo-hAAT、HCR-hAAT、APO-HCR-hAAT、APOA1-HCR-hAAT、APOC3-HCR-hAAT，和其经修饰的变体。
在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:4具有至少75％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:4具有至少80％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:4具有至少85％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:4具有至少90％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:4具有至少95％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:4具有至少96％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:4具有至少97％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:4具有至少98％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:4具有至少99％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含SEQ ID NO:4的多核苷酸序列。
在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:5具有至少75％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:5具有至少80％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:5具有至少85％同一性的多核苷酸序列。在一些实施方案 中，所述第二多核苷酸的启动子包含与SEQ ID NO:5具有至少90％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:5具有至少95％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:5具有至少96％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:5具有至少97％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:5具有至少98％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含与SEQ ID NO:5具有至少99％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的启动子包含SEQ ID NO:5的多核苷酸序列。
在一些实施方案中，所述第二多核苷酸进一步包含第二启动子。所述第二多核苷酸的第二启动子可以是任何本领域已知的适于所述转基因表达的启动子或其经修饰的变体。在一些实施方案中，所述第二多核苷酸的第二启动子包括但不限于CMV、CAG、MNDU3、PGK、EF1a启动子或肝部特异性启动子。在一些实施方案中，所述第二多核苷酸的第二启动子为肝部特异性启动子，且所述肝部特异性启动子包括如下的一种或多种：最小TTR启动子(minimal TTR promotor，TTRm)、人α-1抗胰蛋白酶启动子(human alpha 1-antitrypsin，hAAT)、白蛋白(ALB)启动子、载脂蛋白(APO)启动子、载脂蛋白A(apolipoprotein A，APOA1)启动子、载脂蛋白C3(apolipoprotein C3，APOC3)启动子、补体因子B(complement factor B，CFB)启动子、酮己糖激酶(ketohexokinase，KHK)启动子、血凝素(hemopexin，HPX)启动子、烟酰胺N-甲基转移酶(icotinamide N-methyltransferase，NNMT)启动子或最小启动子、(肝)羧酸酯酶1(carboxylesterase 1，CES1)启动子、蛋白C(protein C，PROC)启动子、甘露聚糖结合凝集素丝氨酸蛋白酶2(mannan-binding lectin serine protease 2，MASP2)启动子、铁调素抗菌肽(epcidin antimicrobial peptide，HAMP)启动子，或其经修饰的变体。
在一些实施方案中，所述第二多核苷酸的第二启动子为肝部特异性启动子，且所述肝部特异性启动子进一步包括其他调控序列。在一些实施方案中，所述调控序列包括增强子序列。在一些实施方案中，所述增强子序列包括载脂蛋白E(apolipoprotein E，ApoE)的肝特异性肝控制区(liver-specific hepatic control region，HCR)增强子。
在一些实施方案中，所述第二多核苷酸的第二启动子为肝部特异性启动子，且所述肝部特异性启动子选自Apo-hAAT、HCR-hAAT、APO-HCR-hAAT、APOA1-HCR-hAAT、APOC3-HCR-hAAT，和其经修饰的变体。
在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:4具有至少75％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:4具有至少80％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:4具有至少85％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:4具有至少90％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:4具有至少95％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:4具有至少96％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:4具有至少97％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:4具有至少98％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:4具有至少99％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含SEQ ID NO:4的多核苷酸序列。
在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:5具有至少75％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:5具有至少 80％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:5具有至少85％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:5具有至少90％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:5具有至少95％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:5具有至少96％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:5具有至少97％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:5具有至少98％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含与SEQ ID NO:5具有至少99％同一性的多核苷酸序列。在一些实施方案中，所述第二多核苷酸的第二启动子包含SEQ ID NO:5的多核苷酸序列。
在一些实施方案中，所述转基因编码人因子IX(FIX)蛋白序列。本文所述的因子IX可以是源自任何哺乳动物的因子IX及其变体。在一些实施方案中，所述哺乳动物包括但不限于如灵长类动物(例如人)、牛、狗、猫、啮齿动物(例如，豚鼠、大鼠或小鼠)。在一些实施方案中，本文所述的因子IX是源自人的因子IX或其变体。
在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少75％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少80％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少85％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少90％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少95％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少96％同一性的 序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少97％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少98％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少99％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含人因子IX的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX相比具有一个或多个氨基酸的突变、取代、缺失或添加的序列。
在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含SEQ ID NO:1的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少75％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少80％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少85％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少90％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少95％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少96％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少97％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少98％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少99％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1相比具有一个或多个氨基酸的突变、取代、缺失或添加的序列。
在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含相比SEQ ID NO:1的序列具有至少一个氨基酸取代的氨基酸序列。在一些实施方案中，所述氨基酸取代包括R338X，其中X可以是除精氨酸外的任意氨基酸。在一些实施方案中，所述X选自H、K、D、E、S、T、N、Q、C、G、P、A、V、I、L、M、F、Y或W。在一些实施方案中，所述X选自L、D或Q。在一些实施方案中，所述氨基酸取代包括R338L、R338D或R338Q。在一些实施方案中，所述氨基酸取代为R338L。
在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于300个CG二核苷酸。在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于250个CG二核苷酸。在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于200个CG二核苷酸。在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于150个CG二核苷酸。在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于100个CG二核苷酸。在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于50个CG二核苷酸。在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于25个CG二核苷酸。在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于20个CG二核苷酸。在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于15个CG二核苷酸。在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于10个CG二核苷酸。在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于5个CG 二核苷酸。在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且其中所述编码人因子IX的序列不包含CG二核苷酸。
在一些实施方案中，所述第二多核苷酸序列包含编码人因子IX的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少50％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少55％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少60％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少65％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少70％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少75％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少80％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少85％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少90％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少95％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少96％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少97％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少98％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2具有至少99％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:2相比具有一个或多个核苷酸突变、取代、缺失或添加的序列。
在一些实施方案中，所述第二多核苷酸包含编码人因子IX的序列，且是密码子优化的。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少50％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少55％同一性的序列。 在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少60％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少65％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少70％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少75％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少80％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少85％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少90％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少95％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少96％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少97％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少98％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3具有至少99％同一性的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3的序列。在一些实施方案中，所述第二多核苷酸包含与SEQ ID NO:3相比具有一个或多个核苷酸突变、取代、缺失或添加的序列。
在一些实施方案中，所述第二多核苷酸进一步包含内含子。在一些实施方案中，所述内含子为SV40内含子或修饰后的人因子IX第一内含子。在一些实施方案中，所述SV40内含子为修饰后的SV40内含子。在一些实施方案中，所述修饰后的SV40内含子含有SEQ ID NO:6的编码序列。
在一些实施方案中，所述第二多核苷酸进一步包含其他调控序列，所述调控序列包括但不限于反向末端重复序列(ITR)、增强子、剪接信号、聚腺苷酸化信号、填充序列、终止子、蛋白降解信号、内部核糖体进入元件(IRES)、2A序列等。
在一些实施方案中，所述第二多核苷酸进一步包含增强子区。 在一些实施方案中，所述增强子区包括SV40增强子、即刻早期巨细胞病毒增强子、IRBP增强子、源于免疫球蛋白基因的增强子或肝特异性肝控制区(liver-specific hepatic control region，HCR)增强子。在一些实施方案中，所述增强子区位于所述第二多核苷酸的启动子的上游。在一些实施方案中，所述增强子区位于所述第二多核苷酸的启动子的下游。在一些实施方案中，所述增强子区位于所述第二多核苷酸的第二启动子的上游。在一些实施方案中，所述增强子区位于所述第二多核苷酸的第二启动子的下游。
在一些实施方案中，所述所述第二多核苷酸进一步包含反向末端重复序列(ITR)。在一些实施方案中，所述所述第二多核苷酸包含至少一个反向末端重复序列(ITR)。在一些实施方案中，所述所述第二多核苷酸包含两个反向末端重复序列(ITR)。在一些实施方案中，所述两个ITR是彼此相同的。在一些实施方案中，所述两个ITR是彼此不同的。在一些实施方案中，所述反向末端重复序列(ITR)是源自AAV的ITR。在一些实施方案中，所述ITR可以源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8和已知的任何其他AAV的ITR。在一些实施方案中，所述ITR相比源自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8和已知的任何其他AAV的野生型ITR具有一个或多个碱基的突变、插入或缺失，只要其保留所期望的末端重复序列功能，如目的基因的复制、病毒颗粒的包装和/或整合等。
在一些实施方案中，所述第二多核苷酸进一步包含一个或多个填充序列。在一些实施方案中，所述填充序列位于所述第二多核苷酸的启动子的上游。在一些实施方案中，所述填充序列位于所述第二多核苷酸的启动子的下游。在一些实施方案中，所述填充序列位于所述第二多核苷酸的第二启动子的上游。在一些实施方案中，所述填充序列位于所述第二多核苷酸的第二启动子的下游。在一些实施方案中， 所述填充序列位于所述5’ITR序列的5’端。在一些实施方案中，所述填充序列位于所述5’ITR序列的3’端。在一些实施方案中，所述填充序列位于所述5’ITR序列的5’端。在一些实施方案中，所述填充序列位于所述3’ITR序列的5’端。在一些实施方案中，所述填充序列位于所述3’ITR序列的3’端。
在一些实施方案中，所述填充序列的长度可以为约0.1kb-5kb，例如但不限于0.1kb、0.2kb、0.3kb、0.4kb、0.5kb、0.6kb、0.7kb、0.8kb、0.9kb、1kb、1.1kb、1.2kb、1.3kb、1.4kb、1.5kb、1.6kb、1.7kb、1.8kb、1.9kb、2kb、2.1kb、2.2kb、2.3kb、2.4kb、2.5kb、2.6kb、2.7kb、2.8kb、2.9kb、3kb、3.1kb、3.2kb、3.3kb、3.4kb、3.5kb、3.6kb、3.7kb、3.8kb、3.9kb、4.0kb、4.1kb、4.2kb、4.3kb、4.4kb、4.5kb、4.6kb、4.7kb、4.8kb、4.9kb或5.0kb。
另一方面，本发明提供多核苷酸序列，其包含编码人因子IX的转基因序列，其中所述转基因序列与SEQ ID NO:2具有至少50％的同一性。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少55％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少60％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少65％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少70％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少75％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少80％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少85％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少90％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少95％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少96％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少97％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至 少98％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少99％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2相比具有一个或多个核苷酸突变、取代、缺失或添加的序列。
在一些实施方案中，被编码的人因子IX的氨基酸序列包含与人因子IX具有至少75％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少80％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少85％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少90％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少95％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少96％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少97％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少98％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX具有至少99％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含人因子IX的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与人因子IX相比具有一个或多个氨基酸的突变、取代、缺失或添加的序列。
在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含SEQ ID NO:1的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX由SEQ ID NO:1的序列组成。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少75％同一性的序列。在一些实施方案中， 所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少80％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少85％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少90％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少95％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少96％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少97％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少98％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1具有至少99％同一性的序列。在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含与SEQ ID NO:1相比具有一个或多个氨基酸的突变、取代、缺失或添加的序列。
在一些实施方案中，所述第二多核苷酸中所述转基因编码的因子IX包含相比SEQ ID NO:1的序列具有至少一个氨基酸取代的氨基酸序列。在一些实施方案中，所述氨基酸取代包括R338X，其中X可以是除精氨酸外的任意氨基酸。在一些实施方案中，所述X选自H、K、D、E、S、T、N、Q、C、G、P、A、V、I、L、M、F、Y或W。在一些实施方案中，所述X选自L、D或Q。在一些实施方案中，所述氨基酸取代包括R338L、R338D或R338Q。在一些实施方案中，所述氨基酸取代为R338L。
在所述多核苷酸序列的一些实施方案中，所述转基因序列包含编码人因子IX的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少50％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少55％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少60％同一性 的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少65％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少70％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少75％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少80％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少85％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少90％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少95％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少96％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少97％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少98％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2具有至少99％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2的序列。在一些实施方案中，所述转基因序列由SEQ ID NO:2的序列组成。在一些实施方案中，所述转基因序列包含与SEQ ID NO:2相比具有一个或多个核苷酸突变、取代、缺失或添加的序列。
在所述多核苷酸序列的一些实施方案中，所述转基因序列包含编码人因子IX的序列，且是密码子优化的。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少50％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少55％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少60％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少65％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少70％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少75％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少80％同一性的序列。在一些实施方案中，所述转基因序列 包含与SEQ ID NO:3具有至少85％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少90％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少95％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少96％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少97％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少98％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3具有至少99％同一性的序列。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3的序列。在一些实施方案中，所述转基因序列由SEQ ID NO:3的序列组成。在一些实施方案中，所述转基因序列包含与SEQ ID NO:3相比具有一个或多个核苷酸突变、取代、缺失或添加的序列。
在所述多核苷酸序列的一些实施方案中，所述转基因序列包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于300个CG二核苷酸。在一些实施方案中，所述转基因序列包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于250个CG二核苷酸。在一些实施方案中，所述转基因序列包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于200个CG二核苷酸。在一些实施方案中，所述转基因序列包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于150个CG二核苷酸。在一些实施方案中，所述转基因序列包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于100个CG二核苷酸。在一些实施方案中，所述转基因序列包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于50个CG二核苷酸。在一些实施方案中，所述转基因序列包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于25个CG二核苷酸。在一些实施方案中，所述转基因序列包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于20个CG二核苷酸。在一些实施方案中，所述转基因序列包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少 于15个CG二核苷酸。在一些实施方案中，所述转基因序列包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于10个CG二核苷酸。在一些实施方案中，所述转基因序列包含编码人因子IX的序列，且其中所述编码人因子IX的序列包含少于5个CG二核苷酸。在一些实施方案中，所述转基因序列包含编码人因子IX的序列，且其中所述编码人因子IX的序列不包含CG二核苷酸。
在所述多核苷酸序列的一些实施方案中，所述多核苷酸进一步包含启动子。所述多核苷酸的启动子可以是任何本领域已知的适于所述转基因表达的启动子或其经修饰的变体。在一些实施方案中，所述多核苷酸的启动子包括但不限于CMV、CAG、MNDU3、PGK、EF1a启动子或肝部特异性启动子。在一些实施方案中，所述多核苷酸的启动子为肝部特异性启动子，且所述肝部特异性启动子包括如下的一种或多种：最小TTR启动子(minimal TTR promotor，TTRm)、人α-1抗胰蛋白酶启动子(human alpha 1-antitrypsin，hAAT)、白蛋白(ALB)启动子、载脂蛋白(APO)启动子、载脂蛋白A(apolipoprotein A，APOA1)启动子、载脂蛋白C3(apolipoprotein C3，APOC3)启动子、补体因子B(complement factor B，CFB)启动子、酮己糖激酶(ketohexokinase，KHK)启动子、血凝素(hemopexin，HPX)启动子、烟酰胺N-甲基转移酶(icotinamide N-methyltransferase，NNMT)启动子或最小启动子、(肝)羧酸酯酶1(carboxylesterase 1，CES1)启动子、蛋白C(protein C，PROC)启动子、甘露聚糖结合凝集素丝氨酸蛋白酶2(mannan-binding lectin serine protease 2，MASP2)启动子、铁调素抗菌肽(epcidin antimicrobial peptide，HAMP)启动子，或其经修饰的变体。
在所述多核苷酸序列的一些实施方案中，所述多核苷酸的启动子为肝部特异性启动子，且所述肝部特异性启动子进一步包括其他调控序列。在一些实施方案中，所述调控序列包括增强子序列。在一些实施方案中，所述增强子序列包括肝特异性肝控制区(liver-specific hepatic control region，HCR)增强子。
在所述多核苷酸序列的一些实施方案中，所述多核苷酸的启动子为肝部特异性启动子，且所述肝部特异性启动子选自Apo-hAAT、HCR-hAAT、APO-HCR-hAAT、APOA1-HCR-hAAT、APOC3-HCR-hAAT，和其经修饰的变体。
在所述多核苷酸序列的一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:4具有至少75％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子与SEQ ID NO:4具有至少80％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:4具有至少85％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:4具有至少90％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:4具有至少95％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:4具有至少96％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:4具有至少97％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:4具有至少98％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:4具有至少99％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含SEQ ID NO:4的多核苷酸序列。
在所述多核苷酸序列的一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:5具有至少75％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子与SEQ ID NO:5具有至少80％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:5具有至少85％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:5具有至少90％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:5具有至少95％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:5具有至少96％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含 与SEQ ID NO:5具有至少97％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:5具有至少98％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含与SEQ ID NO:5具有至少99％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的启动子包含SEQ ID NO:5的多核苷酸序列。
在所述多核苷酸序列的一些实施方案中，所述多核苷酸进一步包含第二启动子。所述多核苷酸的第二启动子可以是任何本领域已知的适于所述转基因表达的启动子或其经修饰的变体。在一些实施方案中，所述多核苷酸的第二启动子包括但不限于CMV、CAG、MNDU3、PGK、EF1a启动子或肝部特异性启动子。在一些实施方案中，所述多核苷酸的第二启动子为肝部特异性启动子，且所述肝部特异性启动子包括如下的一种或多种：最小TTR启动子(minimal TTR promotor，TTRm)、人α-1抗胰蛋白酶启动子(human alpha 1-antitrypsin，hAAT)、白蛋白(ALB)启动子、载脂蛋白(APO)启动子、载脂蛋白A(apolipoprotein A，APOA1)启动子、载脂蛋白C3(apolipoprotein C3，APOC3)启动子、补体因子B(complement factor B，CFB)启动子、酮己糖激酶(ketohexokinase，KHK)启动子、血凝素(hemopexin，HPX)启动子、烟酰胺N-甲基转移酶(icotinamide N-methyltransferase，NNMT)启动子或最小启动子、(肝)羧酸酯酶1(carboxylesterase 1，CES1)启动子、蛋白C(protein C，PROC)启动子、甘露聚糖结合凝集素丝氨酸蛋白酶2(mannan-binding lectin serine protease 2，MASP2)启动子、铁调素抗菌肽(epcidin antimicrobial peptide，HAMP)启动子，或其经修饰的变体。
在所述多核苷酸序列的一些实施方案中，所述多核苷酸的第二启动子为肝部特异性启动子，且所述肝部特异性启动子进一步包括其他调控序列。在一些实施方案中，所述调控序列包括增强子序列。在一些实施方案中，所述增强子序列包括肝特异性肝控制区(liver-specific hepatic control region，HCR)增强子。
在所述多核苷酸序列的一些实施方案中，所述多核苷酸的第二启 动子为肝部特异性启动子，且所述肝部特异性启动子选自Apo-hAAT、HCR-hAAT、APO-HCR-hAAT、APOA1-HCR-hAAT、APOC3-HCR-hAAT，和其经修饰的变体。
在所述多核苷酸序列的一些实施方案中，所述多核苷酸的第二启动子包含与SEQ ID NO:5具有至少75％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的第二启动子与SEQ ID NO:5具有至少80％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的第二启动子包含与SEQ ID NO:5具有至少85％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的第二启动子包含与SEQ ID NO:5具有至少90％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的第二启动子包含与SEQ ID NO:5具有至少95％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的第二启动子包含与SEQ ID NO:5具有至少96％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的第二启动子包含与SEQ ID NO:5具有至少97％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的第二启动子包含与SEQ ID NO:5具有至少98％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的第二启动子包含与SEQ ID NO:5具有至少99％同一性的多核苷酸序列。在一些实施方案中，所述多核苷酸的第二启动子包含SEQ ID NO:5的多核苷酸序列。
在所述多核苷酸序列的一些实施方案中，所述多核苷酸包含APO-HCR-hAAT启动子、SV40内含子、SV40 poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、SV40内含子、SV40 poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含APO-HCR-hAAT启动子、修饰的SV40内含子、SV40 poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含APO-HCR-hAAT启动子、SV40内含子、bGH poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含APO-HCR-hAAT启动子、SV40内含子、SV40poly(A)、以及优化的FIX编码基因。在一些实施方案中，所述多核苷 酸包含修饰的APO-HCR-hAAT启动子、SV40内含子、SV40 poly(A)、以及优化的FIX编码基因。在一些实施方案中，所述多核苷酸包含APO-HCR-hAAT启动子、修饰的SV40内含子、SV40 poly(A)、以及优化的FIX编码基因。在一些实施方案中，所述多核苷酸包含APO-HCR-hAAT启动子、SV40内含子、bGH poly(A)、以及优化的FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、SV40 poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、SV40 poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、bGH poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、SV40 poly(A)、以及优化的FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、SV40 poly(A)、以及优化的FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、bGH poly(A)、以及优化的FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、bGH poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、bGH poly(A)、以及优化的FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、SV40内含子、bGH poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、bGH poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、bGH poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、SV40内含子、bGH poly(A)、以及优化的 FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、bGH poly(A)、以及优化的FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、bGH poly(A)、以及优化的FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、SV40 poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含修饰的修饰的APO-HCR-hAAT启动子、修饰的SV40内含子、SV40 poly(A)、以及优化的FIX编码基因。
在一些实施方案中，所述多核苷酸包含APO-HCR-hAAT启动子、修饰的SV40内含子、SV40 poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含APO-HCR-hAAT启动子、修饰的SV40内含子、SV40 poly(A)、以及优化的FIX编码基因。在一些实施方案中，所述多核苷酸包含APO-HCR-hAAT启动子、修饰的SV40内含子、bGH poly(A)、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含APO-HCR-hAAT启动子、修饰的SV40内含子、bGH poly(A)、以及优化的FIX编码基因。
在一些实施方案中，所述多核苷酸包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8。在一些实施方案中，所述多核苷酸包含SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:9。在一些实施方案中，所述多核苷酸包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、以及优化的FIX编码基因。在一些实施方案中，所述多核苷酸包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、以及SEQ ID NO:3。在一些实施方案中，所述多核苷酸包含SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:9、以及FIX编码基因。在一些实施方案中，所述多核苷酸包含SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:9、以及优化的FIX编码基因。在一些实施方案中，所述多核苷酸包含SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:9、以及SEQ ID  NO:3。
在一些实施方案中，所述多核苷酸包含SEQ ID NO:10。在一些实施方案中，所述多核苷酸包含SEQ ID NO:11。在一些实施方案中，所述多核苷酸包含SEQ ID NO:12。在一些实施方案中，所述多核苷酸包含SEQ ID NO:13。在一些实施方案中，所述多核苷酸包含SEQ ID NO:14。在一些实施方案中，所述多核苷酸包含SEQ ID NO:15。在一些实施方案中，所述多核苷酸由SEQ ID NO:10组成。在一些实施方案中，所述多核苷酸由SEQ ID NO:11组成。在一些实施方案中，所述多核苷酸由SEQ ID NO:12组成。在一些实施方案中，所述多核苷酸由SEQ ID NO:13组成。在一些实施方案中，所述多核苷酸由SEQ ID NO:14组成。在一些实施方案中，所述多核苷酸由SEQ ID NO:15组成。
另一方面，本发明提供一种细胞，其包含本发明所述的组合物。在一些实施方案中，所述细胞是昆虫细胞。在一些实施方案中，所述昆虫细胞为Sf9细胞。在一些实施方案中，所述细胞是哺乳动物细胞。在一些实施方案中，所述哺乳细胞是HEK293细胞及其衍生物。HEK293衍生物的实例包括HEK293A、HEK293AAV、HEK293S、HEK293F、Expi293F、HEK293SG、HEK293SGGD、HEK293FTM和HEK293T。也可以将这些衍生物称为HEK293的变体。在一些实施方案中，所述衍生物是HEK293T细胞。在一些实施方案中，细胞经转染包含本发明所述的组合物。在一些实施方案中，所述转染包括但不限于电穿孔、磷酸钙沉淀、脂质体转染。在一些实施方案中，所述组合物被稳定转染入所述细胞。在一些实施方案中，所述组合物被瞬时转染入所述细胞中。
所述另一方面，本发明进一步提供细胞群，其包含其包含本发明所述的组合物。在一些实施方案中，所述细胞群包含本文所述的多个细胞。
重组AAV病毒颗粒
另一方面，本发明提供一种重组腺相关病毒(rAAV)颗粒，其通过将本发明上述的组合物转染至昆虫细胞制备。在一些实施方案中，所述昆虫细胞为Sf9细胞。
在一些实施方案中，所述重组腺相关病毒(rAAV)颗粒包含本申请所述的多核苷酸。所述重组腺相关病毒(rAAV)颗粒可以衍生自本领域已知的适合适用于本发明的AAV病毒血清型。在一些实施方案中，所述AAV病毒血清型包括但不限于AAV1、AAV2、AAV3，(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAV-DJ、AAVHSC15、KP1、NP59和LK03。在一些实施方案中，所述AAV病毒血清型为AAV2。在一些实施方案中，所述AAV病毒血清型为AAV8。在一些实施方案中，所述AAV病毒血清型为AAV5。
在一些实施方案中，所述cap和所述rep可以衍生自不同的AAV血清型，例如，所述cap和所述rep可以分别衍生自AAV1、AAV2、AAV3(包括AAV3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV-Rh10、AAV-Rh74、AAV-2i8、AAVHSC、DJ、KP1、NP59、LK03或已知的任何其他AAV。举例来说，在一些实施方案中，所述cap衍生自AAV2，而所述rep则衍生自AAV5。在一些实施方案中，所述cap衍生自AAV5，而所述rep衍生自AAV2。
在一些实施方案中，本发明的组合物可通过本领域任何已知的方法递送入所述昆虫细胞。在一些实施方案中，所述方法包括但不限于电穿孔、磷酸钙沉淀、脂质体转染等。在一些实施方案中，所述组合物被稳定转染入所述昆虫细胞。在一些实施方案中，所述组合物被瞬时转染入所述昆虫细胞中。在一些实施方案中，所述昆虫细胞用于生产所述rAAV病毒颗粒。
如果有需要，可根据本领域技术人员已知的常规方法从所述昆虫细胞分离并纯化所述rAAV病毒颗粒。例如，可使用离心、HPLC、 疏水相互作用色谱(HIC)、阴离子交换色谱、阳离子交换色谱、尺寸排阻色谱、超滤、凝胶电泳、亲和色谱、和/或其他纯化技术纯化所述rAAV病毒颗粒。
本发明中所述rAAV病毒颗粒可以在体内表达高水平的因子IX。例如，在B型血友病小鼠模型体内表达。因子IX的表达水平可以用ELISA法检测。例如，采用HYPHEN BioMed的ZYMUTEST FactorⅨKit(货号：#RK032A)，使用多功能酶标仪Spectra Max M5自带分析软件SpectraMax软件进行数据分析。FIX浓度(％)依据Plasma FIX Calibrator的浓度来决定。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约5％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约10％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约20％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约40％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约50％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约80％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约90％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约100％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约200％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约300％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约400％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约500％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量大于约600％。
在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约5％到约600％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约5％到约200％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约5％到约150％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约10％到约 150％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约5％到约10％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约100％到约600％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约200％到约600％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约300％到约600％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约400％到约600％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约450％到约600％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约500％到约600％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约100％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约200％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约300％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约400％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约500％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量为约600％。
在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周为约或至少约5％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周为约或至少约20％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周为约或至少约40％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周为约或至少约100％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周为约或至少约400％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周为约或至少约600％。
在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第六周为约或至少约5％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第六周为约或至少约40％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第六周为 约或至少约100％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第六周为约或至少约400％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第六周为约或至少约550％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第六周为约或至少约600％。
在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第八周为约或至少约5％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第八周为约或至少约40％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第八周为约或至少约100％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第八周为约或至少约300％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第八周为约或至少约400％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第八周为约或至少约450％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第八周为约或至少约500％。
在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周、第六周、和第八周稳定持续表达为约或至少约5％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周、第六周、和第八周稳定持续表达为约或至少约6％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周、第六周、和第八周稳定持续表达为约或至少约20％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周、第六周、和第八周稳定持续表达为约或至少约40％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周、第六周、和第八周稳定持续表达为约或至少约400％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周、第六周、和第八周稳定持续表达为约或至少约500％。
在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量在第四周为约或至少约600％、第六周为约或至少约550％、和第八 周为约或至少约450％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少四周。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少六周。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少八周。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少四周且表达量为约或至少约600％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少六周且表达量为约或至少约600％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少八周且表达量为约或至少约600％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少四周且表达量为约或至少约550％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少六周且表达量为约或至少约550％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少八周且表达量为约或至少约550％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少四周且表达量为约或至少约500％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少六周且表达量为约或至少约500％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少八周且表达量为约或至少约500％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少四周且表达量为约或至少约450％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少六周且表达量为约或至少约450％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少八周且表达量为约或至少约450％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少四周且表达量为约或至少约400％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少六周且表达量为约或至少约400％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少八周且表达量为约或至少约400％。
在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少四周且表达量至少为约或至少约600％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少六周且表达量至少为约或至少约550％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少八周且表达量至少为约或至少约500％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少八周且表达量至少为约或至少约450％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少八周且表达量至少为约或至少约400％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少八周且表达量持续为约400％到约600％。
在一些实施例中，所述rAAV病毒颗粒在体内持续稳定表达因子IX并且所述因子IX具有生物活性。例如，在B型血友病小鼠模型体内表达。因子IX的活性可以由两种方法来检测：凝固法和发色底物法。凝固法检测FIX活性是由CA-530凝血分析仪分析检测。发色底物法检测FIX活性采用HYPHEN BioMed的BIOPHENTM FIX kit(货号：REF 221801-RUO)检测FIX活性。使用多功能酶标仪Spectra Max M5自带分析软件SpectraMax软件进行数据分析。
在一些实施例中，所述rAAV病毒颗粒在体内持续稳定表达因子IX并且所述因子IX经凝固法检测具有生物活性。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约50％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约70％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约80％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约250％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法 检测具有的生物活性约为或至少约300％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约350％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约400％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约500％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约550％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约1000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约1500％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约1700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约1800％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性约为或至少约2000％。
在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约50％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约70％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约80％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约250％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约300％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约350％。在一些实施 例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约400％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约500％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约550％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约1000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约1500％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约1700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约1800％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性大于约2000％。
在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约50％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约70％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约80％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约200％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约250％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约300％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约350％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约400％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约500％ 到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约550％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约700％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约1000％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约1500％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约1700％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约1800％到约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经凝固法检测具有的生物活性为约2000％到约2500％。
在一些实施例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约50％。在一些实施例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约70％。在一些实施例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约80％。在一些实施例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约100％。在一些实施例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约250％。在一些实施例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约300％。在一些实施例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约350％。在一些实施例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约400％。在一些实施例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约600％。在一些实施 例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约700％。在一些实施例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约1500％。在一些实施例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约1700％。在一些实施例中，所述rAAV病毒颗粒在体内第6周表达的因子IX经凝固法检测具有的生物活性为约或至少约2000％。
一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约50％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约70％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约80％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约100％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约250％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约300％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约350％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约400％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约600％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约700％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约1500％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约1700％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX 经凝固法检测具有的生物活性为约或至少约1800％。在一些实施例中，所述rAAV病毒颗粒在体内第8周表达的因子IX经凝固法检测具有的生物活性为约或至少约2000％。
在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约50％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约80％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约250％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约300％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约350％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约400％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约600％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约1700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约1800％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约1900％。在一些实施例中，所述rAAV病毒颗粒在体内 表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少6周并且其活性经凝固法检测为约或至少约2200％。
在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约50％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约70％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约250％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约300％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约500％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约550％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约600％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约1700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约1800％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测 为约或至少约1900％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约2000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测为约或至少约2200％。
在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测在第6周为约或至少约80％、在第8周为约或至少约70％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测在第6周为约或至少约300％、在第8周为约或至少约200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测在第6周为约或至少约250％、在第8周为约或至少约250％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测在第6周为约或至少约700％、在第8周为约或至少约550％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测在第6周为约或至少约350％、在第8周为约或至少约250％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测在第6周为约或至少约1700％、在第8周为约或至少约1800％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测在第6周为约或至少约2000％、在第8周为约或至少约1800％。
在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约70％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子 IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约250％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约300％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约350％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约400％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约500％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约550％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约600％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约1000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约1500％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约1700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约1800％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约1900％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经凝固法检测至少为约或至少约2000％。
在一些实施例中，所述rAAV病毒颗粒在体内持续稳定表达因子IX并且所述因子IX经发色底物法检测具有生物活性。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约10％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约15％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约50％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约100％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约125％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约250％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约300％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约750％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约800％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约900％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约1000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约1100％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性约为或至少约1200％。
在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经 发色底物法检测具有的生物活性为约10％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约15％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约50％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约100％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约125％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约200％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约250％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约300％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约700％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约750％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约800％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约900％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约1000％到约1200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX经发色底物法检测具有的生物活性为约1100％到约1200％。
在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测为约或至少约10％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测为约或至少约15％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX 持续稳定保持活性至少8周并且其活性经发色底物法检测为约或至少约100％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测为约或至少约125％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测为约或至少约200％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测为约或至少约250％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测为约或至少约300％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测为约或至少约700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测为约或至少约800％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测为约或至少约1000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测为约或至少约1100％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测为约或至少约1200％。
在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测在第8周为约或至少约10％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测在第8周为约或至少约15％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测在第8周为约或至少约100％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测在第8周为约或至少约125％。在一些实施例中， 所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测在第8周为约或至少约250％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测在第8周为约或至少约300％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测在第8周为约或至少约700％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测在第8周为约或至少约800％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测在第8周为约或至少约1000％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测在第8周为约或至少约1100％。在一些实施例中，所述rAAV病毒颗粒在体内表达的因子IX持续稳定保持活性至少8周并且其活性经发色底物法检测在第8周为约或至少约1200％。
在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量经ELISA法检测至少为约5％，且活性经凝固法检测至少为约50％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量经ELISA法检测至少为约6％，且活性经凝固法检测至少为约70％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量经ELISA法检测至少为约20％，且活性经凝固法检测至少为约200％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量经ELISA法检测至少为约80％，且活性经凝固法检测至少为约550％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量经ELISA法检测至少为约30％，且活性经凝固法检测至少为约200％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量经ELISA法检测至少为约300％，且活性经凝固法检测至少为约1500％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量经 ELISA法检测至少为约400％，且活性经凝固法检测至少为约1500％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量经ELISA法检测至少为约400％，且活性经凝固法检测至少为约1600％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量经ELISA法检测至少为约400％，且活性经凝固法检测至少为约1700％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量经ELISA法检测至少为约400％，且活性经凝固法检测至少为约1800％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量经ELISA法检测至少为约400％，且活性经凝固法检测至少为约1900％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX的表达量经ELISA法检测至少为约400％，且活性经凝固法检测至少为约2000％。
在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少4周，且表达量经ELISA法检测至少为约500％，且活性经凝固法检测至少为约1800％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少4周，且表达量经ELISA法检测至少为约500％，且活性经凝固法检测至少为约1900％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少4周，且表达量经ELISA法检测至少为约500％，且活性经凝固法检测至少为约2000％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少4周，且表达量经ELISA法检测至少为约500％，且活性经凝固法检测至少为约2100％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少4周，且表达量经ELISA法检测至少为约550％，且活性经凝固法检测至少为约1800％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少4周，且表达量经ELISA法检测至少为约550％，且活性经凝固法检测至少为约1900％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少4周，且表达量经ELISA法检测至少为约550％，且活性经凝固法检测至少为约2000％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至 少4周，且表达量经ELISA法检测至少为约550％，且活性经凝固法检测至少为约2100％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少4周，且表达量经ELISA法检测至少为约600％，且活性经凝固法检测至少为约1800％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少4周，且表达量经ELISA法检测至少为约600％，且活性经凝固法检测至少为约1900％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少4周，且表达量经ELISA法检测至少为约600％，且活性经凝固法检测至少为约2000％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少4周，且表达量经ELISA法检测至少为约600％，且活性经凝固法检测至少为约2100％。
在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少6周，且表达量经ELISA法检测至少为约500％，且活性经凝固法检测至少为约1800％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少6周，且表达量经ELISA法检测至少为约500％，且活性经凝固法检测至少为约1900％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少6周，且表达量经ELISA法检测至少为约500％，且活性经凝固法检测至少为约2000％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少6周，且表达量经ELISA法检测至少为约500％，且活性经凝固法检测至少为约2100％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少6周，且表达量经ELISA法检测至少为约550％，且活性经凝固法检测至少为约1800％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少6周，且表达量经ELISA法检测至少为约550％，且活性经凝固法检测至少为约1900％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少6周，且表达量经ELISA法检测至少为约550％，且活性经凝固法检测至少为约2000％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至 少6周，且表达量经ELISA法检测至少为约550％，且活性经凝固法检测至少为约2100％。
在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少8周，且表达量经ELISA法检测至少为约400％，且活性经凝固法检测至少为约1800％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少8周，且表达量经ELISA法检测至少为约400％，且活性经凝固法检测至少为约1900％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少8周，且表达量经ELISA法检测至少为约400％，且活性经凝固法检测至少为约2000％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少8周，且表达量经ELISA法检测至少为约400％，且活性经凝固法检测至少为约2100％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少8周，且表达量经ELISA法检测至少为约450％，且活性经凝固法检测至少为约1800％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少8周，且表达量经ELISA法检测至少为约450％，且活性经凝固法检测至少为约1900％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少8周，且表达量经ELISA法检测至少为约450％，且活性经凝固法检测至少为约2000％。在一些实施例中，所述rAAV病毒颗粒在体内的因子IX持续稳定表达至少8周，且表达量经ELISA法检测至少为约450％，且活性经凝固法检测至少为约2100％。
药物组合物
另一方面，本发明提供一种用于在有需要的受试者中治疗B型血友病的药物组合物，其包含本发明所述的多核苷酸或rAAV颗粒，以及药物上可接受的载体或赋形剂。
如本文所用的，“药学上或治疗上可接受的载体或赋形剂”是指不干扰活性成分的生物活性的有效性并且对宿主或患者无毒的载体 介质。药物制剂中使用的载体类型将取决于施用该治疗性化合物的方法。制备用于多种给药途径的药物组合物的许多方法是本领域公知的。
在本发明的药物组合物的一些实施方案中，通过将本发明的多核苷酸或rAAV病毒颗粒溶解在适当的溶剂中来制备所述药物组合物。适当的溶剂包括但不限于水、盐溶液(例如，NaCl)、缓冲溶液(例如，Phosphate-buffered saline(PBS))或其他溶剂。在某些实施方案中，该溶剂为无菌的。
本发明的药物组合物可通过无菌操作来制备，或者可替代地在制备的合适阶段进行灭菌。例如，无菌药物组合物可以通过无菌地混合无菌成分来制备。或者，该无菌药物组合物可通过先将成分混合随后将最终制剂灭菌来制备。灭菌方法可包括但不限于热灭菌、辐射和过滤。
方法
另一方面，本申请提供用于治疗B型血友病的方法，其包括将治疗上有效量的本发明的多核苷酸、AAV病毒颗粒或药物组合物施用至有需要的受试者。在一些实施方案中，在施用所述多核苷酸、AAV病毒颗粒或药物组合物后，编码本发明所述的因子IX的转基因序列在所述受试者中表达，由此治疗B型血友病。在一些实施方案中，在施用所述多核苷酸、AAV病毒颗粒或药物组合物后，编码本发明所述的因子IX的转基因序列在所述受试者的肝脏中表达，由此治疗B型血友病。
在一些实施方案中，所述多核苷酸、AAV病毒颗粒或药物组合物可以通过本领域已知的任何适当的方法施用至所述受试者。在一些实施方案中，所述施用包括局部施用或全身性施用。在一些实施方案中，所述施用包括但不限于口服、经鼻、吸入、静脉内、腹膜内、皮下、肌肉内、皮内、局部或直肠途径的施用。在一些实施方案中，所述多核苷酸、AAV病毒颗粒或药物组合物通过静脉注射施用。
在一些实施方案中，所述多核苷酸、AAV病毒颗粒或药物组合物以治疗上有效的量提供，该治疗有效量在医学上可接受的毒性水平下达到所需的生物效果。该剂量可根据给药途径和疾病的严重程度而变化。该剂量还可根据待治疗的每个患者的体重、年龄、性别和/或症状程度而调整。精确的剂量和给药途径将最终由经治医生或兽医来决定。可以理解，可能需要根据患者的年龄和体重以及待治疗的状况的严重程度对剂量作出常规改变。
在所述方法的一些实施方案中，所述AAV病毒颗粒施用剂量为10 10vg/kg至10 15vg/kg。在所述方法的一些实施方案中，所述AAV病毒颗粒施用剂量为10 10vg/kg至10 13vg/kg。在所述方法的一些实施方案中，所述AAV病毒颗粒施用剂量为10 11vg/kg至10 15vg/kg。在所述方法的一些实施方案中，所述AAV病毒颗粒施用剂量为10 11vg/kg至10 13vg/kg。在所述方法的一些实施方案中，所述AAV病毒颗粒施用剂量为10 12vg/kg至10 15vg/kg。在所述方法的一些实施方案中，所述AAV病毒颗粒施用剂量为10 11vg/kg至10 12vg/kg。在所述方法的一些实施方案中，所述AAV病毒颗粒施用剂量为10 10vg/kg至10 12vg/kg。
在所述方法的一些实施方案中，所述多核苷酸、AAV病毒颗粒或药物组合物的递送体积约为0.01mL-1mL。在一些实施方案中，所述组合物的递送体积约为0.05mL-1mL。在一些实施方案中，所述组合物的递送体积约为0.1mL-1mL。在一些实施方案中，所述组合物的递送体积约为0.5mL-1mL。在一些实施方案中，所述组合物的递送体积约为0.1mL-0.5mL。在一些实施方案中，所述组合物的递送体积约为0.01mL-0.5mL。在一些实施方案中，所述组合物的递送体积约为0.05mL-0.5mL。在一些实施方案中，所述组合物的递送体积约为0.05mL-1mL。
在所述方法的一些实施方案中，所述多核苷酸、AAV病毒颗粒或药物组合物的给药频率可以是每天施加至少一次，包括每天2、3、4或5次。在一些实施方案中，所述治疗可持续1天、2天、3天、4 天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天、22天、23天、24天、25天、26天、27天、28天、29天、30天、31天、32天、33天、34天、35天、36天、37天、38天、39天、40天、41天、42天、43天、44天、45天、46天、47天、48天、49天、50天、60天、70天、80天、90天、100天、150天、200天、250天、300天、400天、500天、750天、1000天或多于1000天。
试剂盒
另一方面，本发明提供用于治疗B型血友病的试剂盒，其包含本发明所述的多核苷酸、AAV病毒颗粒或药物组合物，以及说明书。在一些实施方案中，所述说明书用于指示施用所述多核苷酸、AAV病毒颗粒或药物组合物以治疗B型血友病的方法。
在一些实施方案中，所述试剂盒进一步包含容器。在一些实施方案中，所述容器被配置为递送本文所述的多核苷酸、AAV病毒颗粒或药物组合物。在一些实施例中，容器包括小瓶，滴管，瓶，管和注射器。在一些实施方式中，容器是用于施用所述多核苷酸、AAV病毒颗粒或药物组合物的注射器。
本发明的一些实施方案通过以下实施例进一步阐明，该实施例不应被解释为限制性的。本领域技术人员将会理解，下面的实施例中公开的技术代表本发明人发现在本文描述的本发明实施方案的实施中运作良好的技术，并因此可被认为构成用于实施这些实施方案的优选方式。然而，根据本公开内容，本领域技术人员将理解，在不脱离本发明的精神和范围的情况下，可以在本文公开的具体实施方案中作出许多改变，并且仍可获得同样或相似的结果。
实施例
下列实施例进一步阐述了本发明。这些实施例仅旨在说明本发明，而不应被解释为对本发明的限制。
实施例1 重组AAV载体的设计和克隆
将源自AAV2的rep和AAV5的cap编码序列连同其相应的启动子分别克隆至杆状病毒质粒载体中以获得本申请中包含cap和rep蛋白的编码序列的第一多核苷酸。
将包含编码SEQ ID NO:2或SEQ ID NO:3所示的因子IX的核苷酸序列连同肝脏特异性启动子(SEQ ID NO:4或5)、内含子(SEQ ID NO:6或7)、poly(A)信号(SEQ ID NO:8或9)及其他连接序列进行组合成为的第二多核苷酸克隆至杆状病毒质粒载体中。本实验测试了6个不同的表达因子IX的第二多核苷酸，即试验第二多核苷酸序列1-6，其序列见SEQ ID NO.10-15。
实施例2 重组AAV病毒颗粒的制备
将实施例1中获得的第一多核苷酸和第二多核苷酸杆状病毒质粒分别转染Sf9细胞，以获得杆状病毒颗粒。再将杆状病毒颗粒进行组合感染Sf9细胞以生产重组AAV病毒。最后从Sf9细胞中就可以分离和纯化所述重组AAV5/因子IX病毒颗粒。
实施例3 因子IX在B型血友病小鼠模型中的表达和活性
在本实施例中，将因子IX敲除的(F9-KO)B型血友病模型小鼠分为实验组和对照组，分别将实施例2中获得的纯化的rAAV5/因子IX(剂量为2x10^13vg/kg)和PBS静脉注射至实验组和对照组小鼠。一段时间后，在各时间点取血并提取血浆，检测因子IX(FIX)在血浆中的浓度和活性。FIX浓度用ELISA法检测。采用HYPHEN BioMed的ZYMUTEST Factor Ⅸ Kit(货号：#RK032A)，使用多功能酶标仪Spectra Max M5自带分析软件SpectraMax软件进行数据分析。FIX浓度(％)依据Plasma FIX Calibrator的浓度来决定。。凝固法检测FIX活性是由CA-530凝血分析仪分析检测。发色底物法检测FIX活性采用HYPHEN BioMed的BIOPHENTM FIX kit(货号：REF 221801-RUO)检测FIX活性。使用多功能酶标仪Spectra Max M5自带分析软件SpectraMax软件进行数据分析。结果显示，相比对照组，递送的AAV均能够在小鼠体内显著表达因子IX(图1和表1)， 且表达的因子IX具有活性(图2-3和表2-3)。另外，不同的表达组合有着不同水平的表达，并且FIX的表达和活性在检测的各时间点持续稳定。
表1.因子IX(FIX)的体内表达浓度(MEAN(％),SD(％),N)
Figure PCTCN2022100382-appb-000001
表2.因子IX(FIX)的活性检测：凝固法(MEAN(％),SD(％),N)
Figure PCTCN2022100382-appb-000002
表3.因子IX(FIX)的活性检测：发色底物法(MEAN(％),SD(％),N)
Figure PCTCN2022100382-appb-000003
实施例4 本申请组合物在体内的效力
使用对照和包含rAAV5/因子IX病毒颗粒的药物组合物进行临床实验以验证本申请中所述药物组合物的有效性。
序列表
Figure PCTCN2022100382-appb-000004
Figure PCTCN2022100382-appb-000005
Figure PCTCN2022100382-appb-000006
Figure PCTCN2022100382-appb-000007
Figure PCTCN2022100382-appb-000008
Figure PCTCN2022100382-appb-000009
Figure PCTCN2022100382-appb-000010
Figure PCTCN2022100382-appb-000011
Figure PCTCN2022100382-appb-000012
Figure PCTCN2022100382-appb-000013
Figure PCTCN2022100382-appb-000014
Figure PCTCN2022100382-appb-000015
Figure PCTCN2022100382-appb-000016
Figure PCTCN2022100382-appb-000017
Figure PCTCN2022100382-appb-000018
Figure PCTCN2022100382-appb-000019
Figure PCTCN2022100382-appb-000020

Claims (65)

  1. 一种组合物，其包含：
    (1)第一多核苷酸序列，所述第一多核苷酸序列包含与第一启动子可操作连接的第一细小病毒衣壳基因第一开放阅读框(ORF)和与第二启动子可操作连接的第二细小病毒衣壳基因第二开放阅读框，其中所述第一ORF包含含有所述第二启动子的内含子序列；和
    (2)第二多核苷酸序列，所述第二多核苷酸序列包含编码人因子IX(FIX)蛋白的转基因序列。
  2. 如权利要求1所述的组合物，其中所述第一启动子和所述第二启动子适于在昆虫细胞或哺乳动物细胞中表达。
  3. 如权利要求1所述的组合物，其中所述第一ORF、第一启动子、第二ORF和第二启动子自3’至5’为如下顺序：第一启动子，包含第二启动子的第一ORF，第二ORF，其中所述第二ORF包含至少一个翻译初始密码子并与所述第一ORF的3’部分重叠。
  4. 如权利要求2所述的组合物，其中所述昆虫细胞是Sf9细胞，所述哺乳动物细胞为HEK293细胞或其衍生物。
  5. 如权利要求4所述的组合物，其中所述衍生物为HEK293T细胞。
  6. 如权利要求1-5任一项所述的组合物，其中所述第一启动子或所述第二启动子是Polh启动子。
  7. 如权利要求1-5任一项所述的组合物，其中所述第一启动子或所述第二启动子是p10启动子。
  8. 如权利要求1-7任一项所述的组合物，其中所述第一多核苷酸序列进一步在3’末端包含多聚A序列。
  9. 如权利要求1-8任一项所述的组合物，其中所述细小病毒为腺相关病毒(AAV)。
  10. 如权利要求9所述的组合物，其中所述第一ORF编码腺相关病毒(AAV)cap蛋白。
  11. 如权利要求9所述的组合物，其中所述第二ORF编码腺相关病毒(AAV)rep蛋白。
  12. 如权利要求或9-11任一项所述的组合物，其中所述AAV cap蛋白优选AAV血清型5蛋白。
  13. 如权利要求12所述的组合物，其中所述AAV cap蛋白是VP1、VP2和/或VP3蛋白。
  14. 如权利要求10-13任一项所述的组合物，其中所述AAV rep蛋白是AAV血清型2蛋白。
  15. 如权利要求14所述的组合物，其中所述AAV rep蛋白是Rep78和/或Rep52蛋白。
  16. 如权利要求1-15任一项所述的组合物，其中所述第二多核苷酸序列包含启动子。
  17. 如权利要求16所述的组合物，其中所述第二多核苷酸的启动子是肝特异性启动子。
  18. 如权利要求16所述的组合物，其中所述启动子是APO-HCR-hATT启动子。
  19. 如权利要求18所述的组合物，其中所述启动子包含SEQ ID NO:4的序列。
  20. 如权利要求16-19任一项所述的组合物，其中所述第二多核苷酸序列进一步包含第二启动子。
  21. 如权利要求18所述的组合物，其中所述第二多核苷酸的第二启动子是修饰的APO-HCR-hAAT启动子。
  22. 如权利要求21所述的组合物，其中所述第二多核苷酸的第二启动子包含SEQ ID NO:5的序列。
  23. 如权利要求1-22任一项所述的序列，其中所述第二多核苷酸序列包含内含子。
  24. 如权利要求23所述的组合物，其中所述内含子为SV40内含子或修饰后的人因子IX第一内含子。
  25. 如权力要求24所述的组合物，其中所述SV40内含子为修饰后的SV40内含子。
  26. 如权力要求25所述的组合物，其中所述修饰后的SV40内含子包含SEQ ID NO:6的序列。
  27. 如权力要求24所述的组合物，其中所述修饰后的人因子IX第一内含子包含SEQ ID NO:7的序列。
  28. 如权利要求1-27任一项所述的组合物，其中编码所述人因子IX的所述转基因序列是密码子优化的。
  29. 如权利要求28所述的组合物，其中所述转基因序列包含少于5个CG二核苷酸。
  30. 如权利要求1-29任一项所述的组合物，其中所述人因子IX相比野生型人因子IX包含一个或多个氨基酸取代。
  31. 如权利要求30所述的组合物，其中所述人因子IX相比SEQ ID NO:1的序列包含氨基酸取代R338X，其中X可以是除精氨酸外的任意氨基酸。
  32. 如权利要求31所述的组合物，其中所述人因子IX相比SEQ ID NO:1的序列包含氨基酸取代R338L、R338D或R338Q。
  33. 如权利要求32所述的组合物，其中所述人因子IX相比SEQ ID NO:1的序列包含氨基酸取代R338L。
  34. 如权利要求1-33任一项所述的组合物，其中所述转基因序列与SEQ ID NO:3具有至少50％、60％、70％、80％、85％、90％、95％或99％的同一性。
  35. 如权利要求34所述的组合物，其中所述转基因序列包含SEQ ID NO:3的序列。
  36. 如权利要求1-35任一项所述的组合物，其中所述转第二多核苷酸序列进一步包含填充序列。
  37. 一种细胞，其包含权利要求1-36任一项所述的组合物。
  38. 如权利要求37所述的细胞，其中所述细胞为昆虫细胞或哺乳动物细胞。
  39. 如权利要求38所述的细胞，其中所述昆虫细胞是Sf9细胞，其中所述哺乳动物细胞是HEK293细胞或其衍生物。
  40. 如权利要求39所述的细胞，其中所述衍生物是HEK293T细胞。
  41. 多核苷酸序列，其包含编码人因子IX的转基因序列，其中所述转基因序列与SEQ ID NO:3具有至少50％、60％、70％、80％、85％、90％或95％的同一性。
  42. 如权利要求41所述的多核苷酸，其中所述转基因序列与SEQ ID NO:3具有至少96％、97％、98％或99％的同一性。
  43. 如权利要求41或42所述的多核苷酸，其中所述转基因序列包含少于5个CG二核苷酸。
  44. 如权利要求43所述的多核苷酸，其中所述转基因序列不包含CG二核苷酸。
  45. 如权利要求41-44任一项所述的多核苷酸，其中所述转基因序列包含SEQ ID NO:3的序列。
  46. 如权利要求41-45任一项所述的多核苷酸，其进一步包含启动子。
  47. 如权利要求46所述的多核苷酸，其中所述启动子是肝特异性启动子。
  48. 如权利要求47所述的多核苷酸，其中所述启动子是APO-HCR-hATT启动子。
  49. 如权利要求48所述的多核苷酸，其中所述启动子包含SEQ ID NO:4的序列。
  50. 如权利要求47或48所述的多核苷酸，其进一步包含修饰的APO-HCR-hATT启动子。
  51. 如权利要求50所述的多核苷酸，其中所述修饰的APO-HCR-hATT启动子包含SEQ ID NO:5的序列。
  52. 一种腺相关病毒(AAV)病毒颗粒，其包含权利要求41-51任一项所述的多核苷酸。
  53. 如权利要求52所述的AAV病毒颗粒，其中所述AAV病毒颗粒源自AAV血清型5。
  54. 一种药物组合物，其包含如权利要求41-51任一项所述的多核苷酸或如权利要求52或53所述的AAV病毒颗粒。
  55. 如权利要求54所述的药物组合物，其进一步包含药学上可接受的载体或赋形剂。
  56. 一种用于治疗B型血友病的方法，其包括对有需要的受试者施用如权利要求41-51任一项所述的多核苷酸，如权利要求52或53任一项所述的AAV病毒颗粒，或如权利要求54-55任一项所述的药物组合物，其中所述转基因序列在所述受试者中表达，由此治疗B型血友病。
  57. 如权利要求56所述的方法，其中所述受试者是人。
  58. 如权利要求56或57所述的方法，其中所述转基因序列在所述受试者的肝脏中表达。
  59. 如权利要求56-58任一项所述的方法，其中所述施用经由静脉注射进行。
  60. 如权利要求56-59任一项所述的方法，其中所述施用的剂量为10 10vg/kg-10 15vg/kg。
  61. 一种用于治疗B型血友病的试剂盒，包含权利要求1-36任一项所述的组合物、权利要求41-51任一项所述的多核苷酸、权利要求52-53任一项所述的AAV病毒颗粒、或权利要求54-55任一项所述的药物组合物，以及说明书。
  62. 如权利要求61所述的试剂盒，所述说明书用于指示施用所述组合物、所述多核苷酸、所述AAV病毒颗粒、或所述药物组合物以治疗B型血友病的方法。
  63. 如权利要求1-33任一项所述的组合物、权利要求41-51任一项所述的多核苷酸、权利要求52-53任一项所述的AAV病毒颗粒、权利要求54-55任一项所述的药物组合物、权利要求56-60任一项所述的方法、或权利要求61-62任一项所述的试剂盒，其中所述转基因序列在体内稳 定表达所述FIX蛋白至少4周、6周、或8周，且表达的所述FIX蛋白具有生物活性。
  64. 如权力要求1-33任一项所述的组合物、权利要求41-51任一项所述的多核苷酸、权利要求52-53任一项所述的AAV病毒颗粒、权利要求54-55任一项所述的药物组合物、权利要求56-60任一项所述的方法、或权利要求61-62任一项所述的试剂盒，其中所述转基因序列为SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、或SEQ ID NO:15。
  65. 如权力要求1-33任一项所述的组合物、权利要求41-51任一项所述的多核苷酸、权利要求52-53任一项所述的AAV病毒颗粒、权利要求54-55任一项所述的药物组合物、权利要求56-60任一项所述的方法、或权利要求61-62任一项所述的试剂盒，其中所述转基因序列为SEQ ID NO:15。
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