WO2023202469A1 - 一种用于遗传性凝血因子缺乏病治疗的核酸构建体及其用途 - Google Patents

一种用于遗传性凝血因子缺乏病治疗的核酸构建体及其用途 Download PDF

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WO2023202469A1
WO2023202469A1 PCT/CN2023/088206 CN2023088206W WO2023202469A1 WO 2023202469 A1 WO2023202469 A1 WO 2023202469A1 CN 2023088206 W CN2023088206 W CN 2023088206W WO 2023202469 A1 WO2023202469 A1 WO 2023202469A1
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nucleic acid
adeno
coagulation factor
acid construct
associated virus
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French (fr)
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吴昊泉
苏玲玲
丁彦甫
傅晓斌
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Kanglin Biotechnology (hangzhou) Co Ltd
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Kanglin Biotechnology (hangzhou) Co Ltd
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Definitions

  • the present invention relates to the technical field of gene therapy, and in particular to nucleic acid constructs for the treatment of hereditary coagulation factor deficiency diseases and their uses.
  • Platelet clotting factors are various protein components involved in the blood clotting process. The joint function of these factors is to maintain normal coagulation physiological function in the bleeding state. If one or more clotting factors are missing, it results in hemophilia, a group of inherited bleeding disorders. Among them, hemophilia A, the most common clinically, is an X-linked recessive genetic disease caused by deficiency of coagulation factor VIII. Patients develop coagulopathy of varying severity and spontaneous bleeding. Hemophilia B is an X-linked recessive hereditary bleeding disorder caused by a mutation in the coagulation factor IX gene that causes a deficiency or qualitative defect in plasma FIX. The incidence rate in men is about 1/30,000.
  • Coagulation factor replacement therapy as the only treatment for hemophilia at present, includes both early plasma-derived (pd) coagulation factors and recombinant human coagulation factor protein products produced by genetic engineering. Not only are these products expensive, but long-term use will produce inhibitory factors.
  • Gene therapy using AAV or lentiviral vectors as delivery methods can permanently integrate coagulation factor protein-coding genes into human cells or implant them into non-dividing cells for a long time, which can achieve full function and continuous expression by human cells themselves. Effects of coagulation factors. Because of its predictable long-term effect and safety, gene therapy has become the most promising treatment method for hemophilia.
  • hemophilia gene therapy In order to avoid liver toxicity and liver cancer risks, hemophilia gene therapy must explore safer and more effective AAV vectors and delivery methods, change the tissue distribution of AAV drugs in the human body, and make it a gene with significant safety advantages. medicine. Moreover, the amount of FIX expressed by existing AAV after delivery into the body is small and cannot meet the blood drug concentration required to treat the disease. Therefore, it is also necessary to develop AAV that can meet the blood drug concentration required to treat the disease.
  • the purpose of the present invention is to provide a nucleic acid construct for the treatment of hereditary coagulation factor deficiency and a new use of muscle-targeted AAV vector serotypes to solve the problems of the prior art. problems in.
  • nucleic acid construct which includes the following elements:
  • the AAV cis-acting elements include one or more of ITR, promoter, kozak sequence, SV40 polyA, UTR or WPRE.
  • the polynucleotide sequence encoding coagulation factor IX is a codon-optimized sequence.
  • the codon-optimized polynucleotide sequence is a coagulation factor IX sequence that includes the R338L mutation; preferably, The sequence of the codon-optimized polynucleotide is as shown in SEQ ID NO:23 or is a sequence that is 95% similar to the sequence shown in SEQ ID NO:23.
  • the promoter is selected from a mammalian constitutive promoter, a mammalian tissue-specific promoter or a mammalian inducible promoter.
  • the structure of the nucleic acid construct is selected from any of the following:
  • the present invention provides the use of muscle-targeted AAV vector serotypes in the preparation of therapeutic products for hereditary coagulation factor deficiency diseases.
  • the muscle-targeting AAV vector serotype is selected from the group consisting of AAV1, AAV2, AAV6, AAV6.2FF, AAV7, AAVrh74, AAV8, AAV9 or their mutants.
  • the product for treating hereditary coagulation factor deficiency is selected from nucleic acid constructs or adeno-associated viruses.
  • the hereditary coagulation factor deficiency disease is selected from the group consisting of hemophilia A, hemophilia B or hemophilia C.
  • the present invention also provides a nucleic acid construct for preparing a treatment product for hereditary coagulation factor deficiency.
  • the adeno-associated virus prepared with the nucleic acid construct has muscle bias and expresses a therapeutically effective dose of coagulation factors; preferably, The nucleic acid construct has muscle bias when delivered by intramuscular injection.
  • the present invention also provides an adeno-associated virus vector system, which includes the two aforementioned nucleic acid constructs.
  • the present invention also provides an adeno-associated virus, which is packaged by the adeno-associated virus vector system.
  • the present invention also provides a cell line, characterized in that the cell line is a cell line infected by the adeno-associated virus.
  • the present invention also provides the use of the adeno-associated virus and the cell line in preparing products for preventing and treating hereditary coagulation factor deficiency diseases.
  • the present invention also provides a method for treating inherited coagulation factor deficiency, the method comprising administering an effective amount of adeno-associated virus to a patient.
  • the present invention provides a new use of muscle-targeting AAV vector serotypes, which has the following beneficial effects: it provides a safer and more effective AAV vector and administration method, changes the tissue distribution of AAV drugs in the human body, making it a gene therapy drug with significant safety advantages.
  • the expression level of FIX is increased, and its expression amount is large enough to meet the blood drug concentration required to treat the disease. Reduce the amount of AAV used while achieving therapeutic effects. It will not cause liver toxicity and is safer and more effective than existing treatment methods.
  • Figure 1 shows a schematic diagram of the design of the coagulation factor IX gene adeno-associated virus vector of the present invention.
  • Figure 2 shows the pAAV-MCS-ITR-130-CAG-SV40 plasmid map of the present invention.
  • Figure 3 shows the expression levels of FIX in C2C12 cell supernatants using different constructs of the present invention.
  • Figure 4 shows a schematic diagram of the design of different promoters of the coagulation factor IX gene adeno-associated virus vector of the present invention.
  • Figure 5 shows the expression levels of FIX in C2C12 cell supernatants using different promoter constructs.
  • Figure 6 shows the effects of different expression vectors on FIX activity in mice.
  • Figure 7 shows a comparison of the expression levels of different promoters in animals.
  • Figure 8-1 to Figure 8-2 show the target base for verifying the biodistribution and vector delivery efficiency of different serotypes of the present invention. Because of the schematic diagram of the construct, Figure 8-1 is a schematic structural diagram of KL-pAAV-HA21, and Figure 8-2 is a schematic structural diagram of KL-pAAV-Luc.
  • Figures 9-1 to 9-4 show in vivo imaging images of Balb/c mice of the present invention on day3, day7, day14, and day21 respectively after injection with different AAVs.
  • Figure 10-1 and Figure 10-2 show the FIX activity and expression amount and blood drug concentration in the hemophilia B model F9-KO3 mice during the 1st, 2nd, 4th, 6th and 8th weeks after AAV injection.
  • Figure 11 shows the APTT test results 8 weeks after administration.
  • Figure 12 shows the bleeding volume detection results of the tail docking test 4 weeks after administration.
  • Figure 13 shows the thromboelastography TEG results four weeks after administration.
  • R (min) represents the start time of the coagulation reaction in the blood of each experimental animal.
  • Figure 14 shows the results of AAV DNA tissue distribution detection at 1, 4, and 8 weeks after administration.
  • the invention provides a nucleic acid construct, which includes the following elements: a polynucleotide encoding coagulation factor IX and an AAV cis-acting element.
  • the polynucleotide sequence encoding coagulation factor IX is a codon-optimized sequence.
  • the AAV Cis-acting elements include expression control elements.
  • the expression control element may be a tissue-specific expression control element.
  • the tissue-specific expression control element is, for example, a muscle tissue-specific expression control element.
  • the tissue-specific expression control element includes any one or more of ITR, promoter, kozak sequence, and SV40 polyA, and the nucleic acid construct is a nucleic acid construct used to prepare adeno-associated virus for intramuscular injection administration.
  • nucleic acid construct refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues.
  • the coagulation factor is selected from coagulation factor VIII, coagulation factor IX or coagulation factor XI and mutants thereof.
  • the coagulation factor IX (or FIX) includes a signal peptide with a length of 28 amino acids and 433 amino acids.
  • the factor IX is an R338L mutated factor IX.
  • the R338L mutated coagulation factor IX mutates the 338th amino acid of FIX from arginine R to leucine L.
  • the mutated coagulation factor IX can be abbreviated as FIX-R338L. After this site mutation, FIX is in mice The potency can be increased by more than 5 times.
  • the sequence of the codon-optimized polynucleotide encoding FIX-R338L (referred to as F9IDTM) is as set forth in SEQ ID NO: 23 or is 95% identical to the sequence set forth in SEQ ID NO: 23 Sequences with the above similarity.
  • the promoter is selected from the group consisting of mammalian constitutive promoters, mammalian tissue-specific promoters, and mammalian inducible promoters.
  • the mammalian constitutive promoter is selected from any of the following: CMV (cytomegalovirus), EF1 ⁇ (elongation factor-1 ⁇ ), EFS, CAG (composed of cytomegalovirus enhancer and chicken ⁇ -actin promoter), CAGG, CBH, SFFV, MSCV, mPGK; hPGK or UBC (ubiquitin C).
  • the mammalian tissue-specific promoter is selected from muscle-specific promoters. Due to the potential risks of hepatotoxicity and liver cancer caused by currently reported AAV targeting the liver, it is necessary to seek other safe and effective delivery methods. The inventor of this application has conducted extensive research on various delivery methods and finally concluded that the delivery method is intramuscular. Injected AAV targeting muscle cells is a good choice for the treatment of hemophilia B, so muscle-specific promoters were selected accordingly in the construction of nucleic acid constructs.
  • the muscle-specific promoter is selected from tMCK, Desmin, SKCRM 4-DES, tMCK2, CK8 or MCK.
  • the ITR sequence can fold into a hairpin structure and is a cis-acting element required for initiation of AAV DNA replication and packaging of recombinant AAV viral particles.
  • the length of the ITR is 130-145bp.
  • the length of the ITR is, for example, 130 to 135 bp, 135 to 140 bp, 141 bp, 142 bp, 143 bp, 144 bp, or 145 bp.
  • the lengths of the upstream ITR and the downstream ITR in the ITR may be the same or different.
  • the upstream ITR and downstream ITR have the same length, both 141 bp.
  • the AAV cis-acting element in the nucleic acid construct further includes a UTR.
  • the UTR Any UTR from any gene known in the art can be introduced into the nucleic acid construct.
  • the UTR is located behind a promoter.
  • Untranslated regions are transcribed but not translated. Typically, the 5'UTR begins at the transcription start site and terminates at the initiation codon, and the 3'UTR begins immediately after the stop codon and continues until the transcription termination signal.
  • the 5'UTR in the viral genome contains kozak sequences. In other embodiments, the 5'UTR in the viral genome does not contain kozak sequences.
  • the AAV cis-acting element in the nucleic acid construct further includes WPRE.
  • the WPRE is located between the F9IDTM gene expression cassette and SV40 polyA.
  • the WPRE element can enhance the expression of the vector and the stability of post-transcriptional mRNA, and improve the efficiency of mRNA splicing.
  • the structure of the nucleic acid construct is selected from any of the following:
  • the nucleic acid construct is an adeno-associated viral vector.
  • the glandular phase Viral vectors also include vector backbones.
  • the carrier framework can be homemade or selected from suitable carrier frameworks available on the market.
  • the vector backbone is pAAV-MCS-ITR-130-CAG-SV40, and the nucleotide sequence is as shown in SEQ ID NO: 1.
  • the nucleotide sequence of the nucleic acid construct is selected from any of the following, and has at least 75%, 80%, 85%, 90%, 95%, 96% compared to any of the following sequences, Nucleotide sequences with 97%, 98%, or 99% homology: SEQ ID NO:22, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21.
  • the present invention also provides the use of muscle-targeted AAV vector serotypes in the preparation of products for the prevention or treatment of hereditary coagulation factor deficiency diseases, or the use of muscle-targeted AAV vector serotypes in the prevention or treatment of hereditary coagulation factor deficiency diseases. use.
  • serotype is a distinction used to refer to AVVs that have capsids that are serologically distinct from other AVV serotypes. Serological distinction is based on the absence of cross-reactivity between antibodies to one AAV compared with another AVV. This difference in cross-reactivity is usually due to differences in capsid protein sequence/antigenic determinants (eg due to VP1, VP2 and/or VP3 sequence differences among AAV serotypes). Although AAV variants containing capsid variants may be serologically indistinguishable from reference AAV or other AAV serotypes, they differ from the reference or other AAV serotypes by at least one nucleotide or amino acid residue.
  • a serotype is one in which the target virus has been tested for neutralizing activity by sera specific for all existing and characterized serotypes, and no antibodies neutralizing the target virus have been found.
  • viruses may or may not have serological differences from any existing serotype. Therefore, when a new virus (eg AAV) has no serological differences, the new virus (eg AAV) may be a subtype or variant of the corresponding serotype.
  • serological tests for neutralizing activity need to be performed on mutant viruses with modified capsid sequences to determine whether they are another serotype within the traditional serotype definition.
  • serotype refers broadly to serologically distinct viruses (e.g., AAV) as well as to viruses that are not serologically distinct and are subtypes or variants of a particular serotype (e.g., AAV). .
  • the muscle-targeting AAV vector serotype is selected from the group consisting of AAV1, AAV2, AAV6, AAV6.2FF, AAV7, AAVrh74, AAV8, AAV9, or mutants thereof.
  • the muscle-targeting AAV vector serotype is AAV6.2FF.
  • AAV6.2FF is a recombinant adeno-associated virus triple mutation vector. Using the Firefly-Luciferase reporter gene vector packaged in the capsid of this serotype, in vivo imaging technology was used to find that the AAV6.2FF serotype vector has more muscle bias in wild-type c57 mice. sex, less liver bias.
  • bias refers to the specificity of the AAV capsid protein present in the AAV virion for infecting a specific type of cell or tissue.
  • the "muscle bias” refers to a bias toward muscle cells or tissues.
  • the muscle cells are selected from muscle cells, myotubes, myoblasts or satellite cells.
  • the muscle tissue is selected from cardiac or skeletal muscle tissue. Muscle tissue accounts for 35% to 40% of human body weight and has the potential to serve as a "biofactory" suitable for gene therapy.
  • a treatment product for hereditary coagulation factor deficiency causes severe local adverse reactions, the cost of muscle tissue removal will be much lower than that of the liver, and tumors in muscle tissue are often benign. No malignant spread occurs. Taken together, the safety risk impact of targeted muscle delivery is lower.
  • the product for treating hereditary coagulation factor deficiency is selected from the group consisting of nucleic acid constructs and adeno-associated viruses.
  • the hereditary coagulation factor deficiency disease is selected from the group consisting of hemophilia A, hemophilia B or hemophilia C.
  • the present invention also provides a nucleic acid construct for preparing a treatment product for hereditary coagulation factor deficiency.
  • the adeno-associated virus prepared using the nucleic acid construct has muscle bias and expresses a therapeutically effective dose of coagulation factors.
  • the nucleic acid construct has muscle bias when delivered by intramuscular injection.
  • the therapeutically effective amount refers to an amount effective in treating hereditary coagulation factor deficiency.
  • the dosage to achieve a therapeutic effect e.g., the dose of vector genome per kilogram of body weight (vg/kg) will vary based on several different factors, including but not limited to: route of administration, and the level of expression of the heterologous polynucleotide required to achieve the therapeutic effect. , the specific disease being treated, any host immune response to the viral vector, the host immune response to the heterologous polynucleotide or expression product (protein), and the stability of the expressed protein.
  • route of administration e.g., the level of expression of the heterologous polynucleotide required to achieve the therapeutic effect.
  • the specific disease being treated any host immune response to the viral vector, the host immune response to the heterologous polynucleotide or expression product (protein), and the stability of the expressed protein.
  • protein protein
  • the dosage range is at least 1 ⁇ 10 8 or more, such as 1 ⁇ 10 9 , 1 ⁇ 10 10 , 1 ⁇ 10 11 , 1 ⁇ 10 12 , 1 ⁇ 10 13 or 1 ⁇ 10 14 or more.
  • vector genome per kilogram of subject body weight vg/kg
  • the effective dose range of AAV in mice is 1 ⁇ 10 10 -1 ⁇ 10 11 and in dogs is 1 ⁇ 10 12 -1 ⁇ 10 13 .
  • hemophilia B As an example, generally speaking, it is believed that in order to achieve a therapeutic effect, the blood coagulation factor concentration needs to be greater than 1% of the normal individual factor concentration to change the severe disease phenotype to a mild phenotype. Severe phenotypes are characterized by joint damage and fatal bleeding. In order to convert a mild disease phenotype to a mild phenotype, it is believed that the coagulation factor concentration needs to be greater than 5% of normal concentrations.
  • the usual dose is at least 1 ⁇ 10 vector genome (vg) per kilogram (vg/kg) of host body weight, or about 1 ⁇ 10 to 1 ⁇ 10 vg/kg of host body weight, or About 1 ⁇ 10 11 to 1 ⁇ 10 12 vg/kg host body weight, or about 1 ⁇ 10 12 to 1 ⁇ 10 13 vg/kg host body weight, to achieve the desired therapeutic effect.
  • a dosage that is therapeutically "effective” or “sufficient” is usually in a measurable range. Effectively responsive to one, more or all adverse disease symptoms, consequences or complications, one or more adverse symptoms, disorders, diseases, pathologies or complications, e.g. caused by or related to a disease, but reduced, reduced , inhibiting, stopping, limiting or controlling the development or progression of the disease is a satisfactory result.
  • an effective amount or sufficient amount can be provided in a single administration, but not necessarily, it can require multiple administrations and can be alone or in combination with other components (e.g., therapeutic agents), treatments, regimens, or regimens, but this It's not necessary either.
  • the amount may be proportionally increased as required by the subject, type, state and severity of the disease being treated, or side effects, if any, of treatment.
  • an effective amount or a sufficient amount need not be effective or a sufficient amount if administered in single or multiple doses without a second composition (e.g., another drug or agent), treatment, regimen, or regimen, Because additional doses, amounts, or durations above and beyond this dosage may be included, or additional components (e.g., drugs or agents), treatments, regimens, or regimens may be included that are considered effective in a given subject or sufficient amount.
  • Amounts considered effective also include amounts that result in the reduction of other treatments, regimens, or regimens, such as the administration of a recombinant coagulation factor protein (eg, FVIII) for the treatment of a coagulation disorder (eg, hemophilia A).
  • the nucleic acid construct is an adeno-associated viral vector.
  • the nucleic acid construct includes a vector backbone expressing adeno-associated virus capsid protein; preferably, the adeno-associated virus capsid protein vector backbone expresses adeno-associated virus capsid protein targeting muscle tissue.
  • the carrier framework can be homemade or selected from suitable carrier frameworks available on the market.
  • the carrier scaffold is a muscle-targeted carrier scaffold.
  • the vector skeleton is, for example, KL-pAAV-RC1, KL-pAAV-RC2, KL-pAAV-RC6, KL-pAAV-RC6.2FF, KL-pAAV-RC7, KL-pAAV-RCrh74, KL-pAAV-RC8, KL-pAAV-RC9, the nucleotide sequence is shown in SEQ ID NO.1 ⁇ 8.
  • the present invention also provides an adeno-associated virus vector system, which includes the nucleic acid construct for preparing a treatment product for hereditary coagulation factor deficiency disease.
  • the adeno-associated virus vector expression system is selected from E. coli expression system, yeast expression system, insect expression system, mammalian expression system, plant expression system; preferably, the adeno-associated virus vector expression system is selected from plasmid transient transfection expression Any of the system, baculovirus expression system, stably transformed cell line expression system, adenovirus expression system, and poxvirus expression system.
  • the lentiviral vector system also includes host cells.
  • the host cells carry adeno-associated viral vectors.
  • the host cell can be selected from various applicable host cells in the art, as long as it does not limit the purpose of the present invention. Specific applicable cells may be cells that produce adeno-associated virus, such as 293T cells.
  • the adeno-associated virus vector system also includes a nucleic acid construct carrying the gene of interest body and helper plasmids.
  • the nucleic acid construct carrying the gene of interest includes a polynucleotide encoding a coagulation factor and an AAV cis-acting element.
  • the nucleic acid construct carrying the gene of interest is the aforementioned construct of the present invention including a polynucleotide encoding a coagulation factor and an AAV cis-acting element.
  • helper plasmid includes necessary viral packaging components, and the helper plasmid may include a packaging plasmid.
  • the present invention also provides an adeno-associated virus (AAV), which is packaged by a virus using an adeno-associated virus vector system.
  • AAV adeno-associated virus
  • the adeno-associated virus can be used to treat various diseases, such as hereditary coagulation factor deficiencies.
  • the present invention also provides a cell line, which is a cell line infected with the adeno-associated virus.
  • the cell line is a mammalian cell line.
  • the present invention also provides uses of the adeno-associated virus vector system, adeno-associated virus, and cell lines in preparing products for preventing and treating hereditary coagulation factor deficiency diseases.
  • the inherited coagulation factor deficiency disorder is hemophilia A, B or C.
  • the hereditary coagulation factor deficiency is a mammalian hereditary coagulation factor deficiency.
  • the hereditary coagulation factor deficiency disease is a human hereditary coagulation factor deficiency disease.
  • the present invention also provides a method for treating inherited coagulation factor deficiency, the method comprising administering an effective amount of the adeno-associated virus to a patient.
  • the hereditary coagulation factor deficiency disease is hemophilia type A, type B or type C.
  • the hereditary coagulation factor deficiency is a mammalian hereditary coagulation factor deficiency.
  • the hereditary coagulation factor deficiency disease is a human hereditary coagulation factor deficiency disease.
  • the method includes intramuscularly injecting an effective amount of the adeno-associated virus into the patient.
  • the following examples construct a series of molecular constructs carrying target genes based on codon optimization, ITR sequences, UTR and WPRE, and promoters, and clone them into recombinant adeno-associated viruses.
  • the corresponding adeno-associated virus is packaged and the differentiated muscle cell line is infected with the virus at a certain biological titer. Quantitatively detect and compare the FIX produced in cell supernatants.
  • AAV vectors with better performance in cells were selected and injected into the hind leg muscles of C57 mice for in vivo comparative screening.
  • the expression framework was designed based on the amino acid sequence of human coagulation factor IX on Uniprot ⁇ UniProtKB-P00740(FA9_HUMAN) ⁇ . This sequence contains a 46-amino-acid signal peptide and a 415-amino-acid FIX sequence.
  • the 338th amino acid of mature FIX is composed of Arginine R is mutated into leucine L. This mutation can increase the FIX titer in mice by more than 5 times.
  • IDT is used for online codon optimization to obtain the F9IDTM nucleotide sequence. (For the codon-optimized nucleotide sequence, see SEQ ID NO:23, and for the amino acid sequence, see SEQ ID NO:24)
  • the F9IDTM gene expression framework is designed to be inserted into the adeno-associated virus vector backbone pAAV-MCS-ITR-130-CAG-SV40.
  • the AAV vector contains the CAG promoter, the polyadenylation signal of the SV40 virus (SV40SL), and the inverted terminal repeats Sequence (ITR) and other elements, the upstream ITR of the vector is 130bp and the downstream ITR is 141bp, named HA21-01.
  • adeno-associated virus vector HA21-01 On the basis of adeno-associated virus vector HA21-01, two other vectors, adeno-associated virus vector HA21-02 and HA21-03, were designed respectively.
  • the upstream and downstream ITRs of HA21-02 are both 141bp, and the upstream and downstream ITRs of HA21-03 are both 145bp.
  • the adeno-associated virus vector HA21-04 was designed, adding a UTR sequence after the promoter and a WPRE sequence between the target gene and SV40.
  • the structure of each adeno-associated virus vector is shown in Figure 1.
  • the coagulation factor F9IDTM gene expression framework designed in Example 1 was synthesized by GenScript Nanjing and cloned into the adeno-associated virus vector backbone pAAV-MCS-ITR-130-CAG-SV40 by homologous recombination methods well known in the art. Between AgeI/SalI of the multiple cloning site, it is named HA21-01 (nucleotide sequence SEQ ID NO: 22).
  • the adeno-associated virus vector construct skeleton is derived from the self-prepared skeleton of Kanglin Biotechnology (Hangzhou) Co., Ltd.— —pAAV-MCS-ITR-130-CAG-SV40 (nucleotide sequence SEQ ID NO: 1), see Figure 2.
  • the 141bp ITR sequence (nucleotide sequence SEQ ID NO: 2) was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and cloned into the multiple cloning site NarI/ on HA21-01 through homologous recombination methods well known in the art. Between MluI, after cloning was completed, the sequence information was confirmed by sequencing and named HA21-02 (nucleotide sequence SEQ ID NO: 3).
  • the 145bp ITR sequence (nucleotide sequence SEQ ID NO: 4) was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and cloned into the multiple cloning site NarI/ on HA21-01 through homologous recombination methods well known in the art. Between MluI and PmeI/Pci I, after cloning was completed, the sequence information was confirmed by sequencing and named HA21-03 (nucleotide sequence SEQ ID NO: 5).
  • hepatitis B virus post-transcriptional regulatory element (nucleotide sequence SEQ ID NO: 6) was synthesized by Genscript Nanjing, the UTR sequence (nucleotide sequence) was synthesized using PCR amplification methods well known in the art.
  • the sequence SEQ ID NO: 7 is placed on the primer to amplify the CAG promoter, and at the same time, F9IDTM is amplified, and then cloned into the upstream ITR of HA21-02 and SV40pA signal through the homologous recombination method well known in the art, and the cloning is completed.
  • the sequence information was then confirmed by sequencing and named HA21-04 (nucleotide sequence SEQ ID NO: 8).
  • Example 3 Viral packaging of coagulation factor IX gene expression adeno-associated vector
  • the coagulation factor FIX gene adeno-associated virus expression vector (HA21-01, HA21-02, HA21-03, HA21-04) constructed in Example 2, the capsid plasmid (KL-pAAV-RC6.2FF, its nucleotide The sequence is shown in SEQ ID NO:9) and the packaging plasmid (pHelper, whose nucleotide sequence is shown in SEQ ID NO:10) were simultaneously co-transfected into 293T cells (purchased from the American Type Culture Collection Center (ATCC) and preserved No. CRL-3216), the coagulation factor IX gene therapy adeno-associated virus vector was packaged in this 293T cell line.
  • ATCC American Type Culture Collection Center
  • the transfection method is transient transfection of eukaryotic cells mediated by PEI cationic polymer.
  • the PEI cationic polymer is PEI-Max transfection reagent purchased from Polysciences (purchased from Polysciences, Cat. No.: 24765-1).
  • the transfection operation refers to the production method.
  • the supplier recommends standardized operations, and the transfection scale is 15cm 2 cell culture dishes.
  • adeno-associated virus AAV
  • AAV adeno-associated virus
  • the lysate supernatant and culture medium supernatant were mixed and purified by affinity chromatography, then aliquoted and frozen at -80°C for later use.
  • the physical titer of AAV was calculated based on the quantitative PCR results and the AAV standard (AAV2 purchased from ATCC) as a reference.
  • the primer and probe sequences used in quantitative PCR are:
  • ITR probe 5’-FAM-CACTCCCTCTCTGCGCGC-BHQ1-3’(SEQ ID NO:12)
  • Example 4 Detection of protein expression after transducing C2C12 cells with coagulation factor FIX gene expression adeno-associated virus vector
  • C2C12 cells 24-well cell culture plate, 1E5 cells per well. Cells were differentiated in 2% horse serum medium. Differentiated C2C12 cells were infected with the FIX gene adeno-associated virus packaged and purified in the above Example 3 at an MOI of 1E5, and the supernatant was collected at 96 hours. The supernatant was detected using a human coagulation factor IX (FIXa) kit (ELISA). (Purchased from Ruixin Biotech, product number: SU-BN16070). The test results showed that the ITR length of 141bp was significantly higher than that of 130bp. On this basis, after adding UTR and WPRE, the expression level increased significantly. The results are shown in Figure 3.
  • Example 5 Design of different promoters for coagulation factor IX gene expression AAV vector
  • Example 6 Construction of different promoters for coagulation factor IX gene expression adeno-associated viral vectors
  • the CMV promoter (nucleotide sequence SEQ ID NO: 14) was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and cloned into the space between MluI and AgeI of HA21-04 using homologous recombination methods well known in the art, and replaced CAG promoter, after cloning, the sequence information was confirmed by sequencing and named HA21-05 (nucleotide sequence SEQ ID NO: 15).
  • the CBH promoter (nucleotide sequence SEQ ID NO: 16) was synthesized by Nanjing GenScript Biotechnology Co., Ltd. and cloned into the space between MluI and AgeI of HA21-04 using homologous recombination methods well known in the art, and replaced CAG promoter, after cloning, the sequence information was confirmed by sequencing and named HA21-06 (nucleotide sequence SEQ ID NO: 17).
  • the CAGG promoter (nucleotide sequence SEQ ID NO: 18) was synthesized by Nanjing GenScript Biotechnology Co., Ltd. The well-known method in the art was to clone HA21-04 between MluI and AgeI using homologous recombination, and replace the CAG promoter. After the cloning was completed, sequencing was performed to confirm the sequence information, and it was named HA21-07 (nucleotide sequence SEQ ID NO. :19).
  • the tMCK muscle-specific promoter (nucleotide sequence SEQ ID NO: 20) was synthesized by Nanjing Genscript Biotechnology Co., Ltd. and cloned between MluI and AgeI of HA21-04 through homologous recombination methods well known in the art. , replaced the CAG promoter, and after cloning was completed, the sequence information was confirmed by sequencing and named HA21-08 (nucleotide sequence SEQ ID NO: 21).
  • Example 7 Detection of protein expression after adeno-associated virus transduction of C2C12 cells with coagulation factor FIX gene expression from different promoters
  • Example 5 Use the method in Example 3 to perform AAV packaging, purification, and titer detection on the different promoter constructs (CAG, CBH, CAGG, tMCK,) in Example 5, and then use the same method to detect FIX expression in the cell supernatant.
  • the results showed that CAG, CBH, and CAGG promoters were significantly better than the tMCK promoter. The results are shown in Figure 5.
  • blood was collected from the mice using orbital blood collection. During the blood collection process, 3.2% sodium citrate anticoagulant was added (ratio 1:9). After the blood is centrifuged, the FIX activity in the plasma is detected.
  • the FIX detection kit is Biophen FIX:C (Chromogenic assay for measuring Factor IX:C in plasma, or in concentrates), the product number is 221806, and the standard is recombinant human coagulation factor IX for injection (Befu) produced by Pfizer Pharmaceuticals. 250IU/bottle, registration number S20120053;
  • HA21-04 has added UTR and WPRE elements. These two elements also have a greater promotion effect on FIX expression in vivo. , as shown in Figure 6, and the specific activity values are shown in Table 3.
  • the plasma at different time points was detected according to the method of Example 8.
  • CAG HA21-04
  • CBH HA21-06
  • HA21-04 can greatly improve the expression activity of FIX.
  • the specific activity values are shown in Table 4.
  • Selected serotypes include AAV1, AAV2, AAV6, AAV6.2FF, AAV7, AAVrh74, AAV8, and AAV9.
  • the capsid sequences of the above serotypes were synthesized through commercial methods and cloned into the AAV packaging vector KL-pAAV-Rep through a reasonable molecular biology vector construction strategy.
  • the AAV vector undergoes packaging and purification processes to obtain corresponding AAV products of several different serotypes, including a vector carrying the gene sequence encoding FIX-co-padua protein (AAV-HA21) and a vector carrying the Firefly-Luciferase reporter gene and the same promoter The corresponding version vector (AAV-Luc) of expression regulatory elements.
  • Firefly-Luciferase reporter gene vectors packaged in capsids of different serotypes were used, and in vivo imaging technology was used to explore the biodistribution characteristics of different serotype vectors in wild-type c57 mice, and screen out those with more muscle bias and liver bias. Less sexual serotype (AAV6.2FF).
  • the gene delivery efficiency of the AAV6.2FF vector was then verified in the FIX gene knockout transgenic mouse F9-KO3 (southern model organism, strain name: C57BL/6-F9em3Smoc, catalog number: NM-KO-200607), and passed Proof-of-concept methods such as the tail-clip test have proven that the gene therapy drug KL-HA21, which encodes the target gene of the normal-functioning FIX protein (FIX-co-padua) delivered by the AAV6.2FF system, can target muscles by Gene delivery method can safely, significantly and long-term reduce the symptoms of hemophilia B in F9-KO3 mice.
  • KL-pAAV-Rep After cloning, the sequence information was confirmed by sequencing.
  • the packaging vectors of different serotypes were named: KL-pAAV-RC1, KL-pAAV-RC2, KL-pAAV-RC6, KL-pAAV-RC6.2FF, and KL-pAAV-RC7.
  • KL-pAAV-RCrh74, KL-pAAV-RC8, KL-pAAV-RC9 the nucleotide sequences are shown in SEQ ID NO: 25 ⁇ 27, 9, 28 ⁇ 31.
  • the packaging vector plasmids of different serotypes obtained in Example 10 (KL-pAAV-RC1, KL-pAAV-RC2, KL-pAAV-RC6, KL-pAAV-RC6.2FF, KL-pAAV-RC7, KL-pAAV- RCrh74, KL-pAAV-RC8, KL-pAAV-RC9), helper plasmid (KL-pAAV-Helper, nucleotide sequence SEQ ID NO: 34), target gene expression plasmid (KL containing FIX-co-padua target gene - pAAV-HA21 vector, nucleotide sequence SEQ ID NO: 32 or KL-pAAV-Luc vector containing Firefly-Luciferase target gene, nucleotide sequence SEQ ID NO: 33) co-transfected 293T cells (purchased from American model The Culture Collection Center (ATCC), deposit number CRL-3216) uses the 293T system for AAV packaging.
  • the transfection method is transient transfection of eukaryotic cells mediated by PEI cationic polymer.
  • the PEI cationic polymer is specifically PEI-Max transfection reagent (purchased from Polysciences, product number: 24765-1).
  • the transfection operation was performed according to the standardized operation recommended by the manufacturer, and the transfection scale was 15 cm 2 cell culture dish.
  • AAV final products of different serotypes AAV1 -CBH-Luc, AAV2-CBH-Luc, AAV6-CBH-Luc, AAV6.2FF-CBH-Luc, AAV7-CBH-Luc, AAVrh74-CBH-Luc, AAV8-CBH-Luc, AAV9-CBH-Luc, AAV6 .2FF-CBH-HA21).
  • the physical titer of AAV is calculated based on the quantitative PCR results and the AAV standard as a reference.
  • the primer and probe sequences used in quantitative PCR are:
  • CMV probe 5’-CAAGTGTATCATATGCCAAGTACGCCCCC-3’(SEQ ID NO:36)
  • the CMV probe has a FAM fluorescent group on the 5' end and a BHQ1 fluorescent group on the 3' end;
  • Example 12 Experiment to explore tissue distribution characteristics of AAV-Luc of different serotypes
  • mice Inject 10 ⁇ l into the unilateral gastrocnemius muscle, 10 ⁇ l into the tibialis anterior muscle, and 10 ⁇ l into the front and rear quadriceps muscles. The day of injection is defined as day 0 of the experiment (day0). There are 3 mice in each group. 8-week-old male mice. See Table 5 for detailed grouping information.
  • mice underwent in vivo imaging on day3, day7, day14, and day21 respectively.
  • mice were intraperitoneally injected with the substrate D-luciferin (Beyotime, product number: ST196) at a concentration of 15 ⁇ g/ml at a dose of 200 ⁇ l/mouse; 5 minutes after substrate injection, the mice were anesthetized with isoflurane gas; Twelve minutes after substrate injection, the small animal in vivo imager (PerkinElmer, IVIS Spectrum) was placed in the supine and prone positions for in vivo imaging data collection.
  • substrate D-luciferin Beyotime, product number: ST196
  • AAV6.2FF can deliver therapeutic levels of FIX protein through muscle targeting
  • a nucleic acid construct KL-pAAV-HA21, nucleotide sequence SEQ ID NO. 32 carrying the gene sequence encoding FIX-co-padua protein was used.
  • packaging vector plasmid and helper plasmid to prepare AAV.
  • the AAV carrying FIX-co-padua protein was named AAV6.2FF-HA21 (or HA21), and it was proved that AAV6.2FF-HA21 (HA21) can indeed partially or even completely restore hemophilia.
  • the present invention selected a hemophilia B model mouse (C57BL/6-F9 em3Smoc , F9-KO3) that uses gene editing technology to knock out the F9 gene exon and is deficient in coagulation factor IX. , verifying that AAV6.2FF-HA21 can repair the coagulation function of mice by delivering drugs by targeting muscle tissue, which is the final proof of concept of the present invention.
  • mice 240 male 8-week-old F9-KO mice were put into cages at a scale of ⁇ 5/cage, and the animals had free access to adult mouse feed and drinking water. Raising conditions: corncob bedding, room temperature 16 ⁇ 26°C, relative humidity 40% ⁇ 70%, artificial lighting 12/12 hours alternating light and dark day and night.
  • mice After 1 week of animal adaptive rearing, mice were injected with bilateral gastrocnemius and quadriceps muscles, 50 ⁇ l for each point, and anesthetized with isoflurane gas during injection. The day of injection was defined as experimental day 0 (day0). Participants were randomly divided into groups according to body weight. The detailed grouping information is shown in Table 6.
  • tissue distribution detection was set up. Three animals in each group were culled for testing 1 and 4 weeks after administration. Two animals in each group were culled for testing at week 8. Eight more animals were raised in each group for testing. The genome was extracted by dissection and sampling according to time points, and CMV primers and probes were used for qPCR detection. First, AAV DNA copies/ug DNA was calculated. According to the fact that 1ng DNA is approximately equal to 172 cells, it was converted to AAV DNA copies/cell.
  • FIX-KO mice were injected with HA21 IM or placebo IM at 1 week, 2 weeks, 4 weeks, and 6 weeks respectively. Weeks and 8 weeks were used to collect plasma and detect the content and activity of FIX in the plasma (FIX activity assay kit 221806, BIOPHEN; FIX content assay kit SU-BN16070, Ruixin Biotech).
  • FIX-KO mice were injected IV with BeneFIX at 4 weeks and plasma was collected one hour after administration to detect the content and activity of FIX in the plasma. The changes in FIX content and activity in plasma are shown in the figure ( Figure 10-1, Figure 10-2).
  • FIX content in each HA21 administration group and the BeneFIX administration group increased significantly compared with the placebo group, and the FIX content was positively correlated with the dose of the HA21 injection group.
  • FIX activity in plasma showed an upward trend as the concentration increased.
  • activated partial thromboplastin time was measured at the eighth week ( Figure 11).
  • the experimental results show that as the concentration of HA21 increases (G1-G4), the APTT time of the test mice decreases.
  • the HA21 administration groups are significantly lower than the placebo group.
  • the lowest dose group has the effect of the drug.
  • G3, G4 and even The G6 time is similar to that of the positive control group.
  • the tail of the mouse has one central artery, two lateral veins, and one dorsal vein, arranged regularly.
  • the mice were vertically cut at the cross-sectional area of the tail of the same size (diameter 2.0 mm), and an open wound was introduced into the above-mentioned blood vessels.
  • the wound surface was kept moist in a warm saline bath while the outflowing blood was collected.
  • Mice with normal coagulation ability will initiate a coagulation reaction in a shorter period of time to prevent further bleeding from the wound.
  • AAV DNA copy number detection showed that compared with other tissues, the copy number was highest in the gastrocnemius and quadriceps muscles.
  • the average copy number in the gastrocnemius muscle of the highest dose group G4C was 273 copies/cell, and in the quadriceps muscles, the average copy number was 273 copies/cell.
  • the average copy number in muscle is 737 copies/cell.
  • the administration method in this experiment was a total of four injections into the gastrocnemius and quadriceps muscles on both sides. The extremely high copy number suggests a correlation with the administration method.

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Abstract

涉及基因治疗技术领域，特别是涉及用于遗传性凝血因子缺乏病治疗的核酸构建体及其新用途。所述构建体包括以下元件：编码凝血因子IX的多核苷酸；AAV顺式作用元件，所述AAV顺式作用元件包括ITR、启动子、kozak序列、SV40 polyA、UTR或WPRE中的一种或多种。优选的，所述编码凝血因子IX的多核苷酸序列为经过密码子优化的序列，优选的，所述经过密码子优化的多核苷酸序列为包括R338L突变的凝血因子IX的序列。所述新用途为靶向肌肉的AAV载体血清型在制备遗传性凝血因子缺乏病治疗产品中的用途。以该核酸构建体和携带凝血因子目的基因的核酸构建体制备的腺相关病毒在肌肉注射的递送方式下，具有肌肉偏向性，并表达治疗有效剂量的凝血因子。

Description

一种用于遗传性凝血因子缺乏病治疗的核酸构建体及其用途 技术领域
本发明涉及基因治疗技术领域，特别是涉及用于遗传性凝血因子缺乏病治疗的核酸构建体及其用途。
背景技术
血小板凝血因子是参与血液凝固过程的各种蛋白质组分。这些因子的共同作用是维持在出血状态下正常的凝血生理功能。如果一种或多种凝血因子缺失，会导致一组遗传性出血性疾病——血友病。其中临床上最常见的A型血友病是一种由凝血因子VIII缺乏所引起的X连锁隐性遗传病。患者出现严重程度不等的凝血功能障碍及自发性出血。血友病B是凝血因子Ⅸ基因突变引起血浆FIX量的缺乏或质的缺陷所导致的一种X连锁隐性遗传性出血性疾病，在男性中的发病率约为1/30000。
凝血因子替代疗法，作为目前血友病唯一的治疗手段，既包括早期的血浆衍生(plasma-derived,pd)凝血因子，也包括由基因工程生产的重组人凝血因子蛋白产品。这些产品不仅价格昂贵，且长期应用会产生抑制因子。以AAV或者慢病毒载体作为递送手段的基因治疗通过将凝血因子蛋白编码基因永久性地整合到人体细胞中或长期植入到非分裂细胞中，可以起到由人体细胞自体持续表达具有完全功能的凝血因子的效果。因其可预见的长效性及安全性，基因治疗成为血友病治疗最具前景的治疗手段。
近年来，在血友病临床应用上，AAV载体递送以肝脏为靶器官逐渐成为主流趋势，比如BioMarin的BMN 270、UniQure的AMT-061等明星产品，均已开展至临床III期试验。然而这种技术路线无法避免药物产生肝脏毒性的风险。2020年11月16日，国际顶尖学术期刊Nature子刊Nature Biotechnology杂志发表了一篇题为：A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells的研究论文，该研究对患有A型血友病的狗进行的AAV病毒基因治疗试验，而在治疗后长达十年的观测中，研究团队发现AAV病毒携带的治疗性基因片段有些被整合到了狗的染色体上控制生长的基因附近，有诱发癌症的可能性。另外目前血友病B主要治疗方式还是凝血因子替代疗法，凝血因子经过血源→重组→长效的发展历程，但是患者需要终生用药，虽然这可在一定程度上控制疾病，但仍有出血风险，也给患者带来许多副作用和很大的经济负担。因此，全球有很多基因治疗公司试图以一次性方式，利用病毒载体递送FIX基因的功能性拷贝至患者体内，使肝脏表达缺失的凝血因子，从而消除患者的出血风险，达到治疗益处。目前临床 上的几款基因治疗的药也已进展到临床阶段，如Pfizer(Spark)的SPK-9001-LTFU101、Uniqure的AMT-061等，尤其是Uniqure的AMT-061已进入到3期临床，在2020年12月22日，uniQure公司宣布暂停其治疗B型血友病的III期临床，原因是AMT-061的临床实验中报告了一例肝癌患者病例。
为避免肝脏毒性和肝癌隐患，血友病基因治疗必须探索更为安全且有效的AAV载体及给药方式，改变AAV药物在人体内的组织分布，使其成为一种具有显著安全性优势的基因治疗药物。而且现有的AAV递送至体内后表达的FIX量较小，不能满足治疗疾病所需的血药浓度，因此同时需要开发能满足治疗疾病所需血药浓度的AAV。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种用于遗传性凝血因子缺乏病治疗的核酸构建体及靶向肌肉的AAV载体血清型的新用途，用于解决现有技术中的问题。
为实现上述目的及其他相关目的，本发明提供一种核酸构建体，所述核酸构建体包括以下元件：
编码凝血因子IX的多核苷酸；
AAV顺式作用元件，所述AAV顺式作用元件包括ITR、启动子、kozak序列、SV40 polyA、UTR或WPRE中的一种或多种。
优选的，所述编码凝血因子IX的多核苷酸序列为经过密码子优化的序列，优选的，所述经过密码子优化的多核苷酸序列为包括R338L突变的凝血因子IX的序列；优选的，所述经过密码子优化的多核苷酸的序列如SEQ ID NO:23所示或为与SEQ ID NO:23所示的序列具有95％相似性的序列。
所述启动子选自哺乳动物组成型启动子、哺乳动物组织特异性启动子或哺乳动物诱导型启动子。
所述核酸构建体的结构选自以下任一：
1)ITR-启动子-kozak序列-F9IDTM基因表达框-SV40 polyA序列-ITR；
2)ITR-启动子-kozak序列-F9IDTM基因表达框-WPRE-SV40 polyA序列-ITR；
3)ITR-启动子-UTR序列-kozak序列-F9IDTM基因表达框-WPRE-SV40 polyA序列-ITR。
本发明提供靶向肌肉的AAV载体血清型在制备遗传性凝血因子缺乏病治疗产品中的用途。
所述靶向肌肉的AAV载体血清型选自AAV1、AAV2、AAV6、AAV6.2FF、AAV7、AAVrh74、AAV8、AAV9或它们的突变体。
所述遗传性凝血因子缺乏病治疗产品选自核酸构建体或腺相关病毒。
所述遗传性凝血因子缺乏病选自A型血友病、B型血友病或C型血友病。
本发明还提供一种用于制备遗传性凝血因子缺乏病治疗产品的核酸构建体，以该核酸构建体制备的腺相关病毒具有肌肉偏向性，并表达治疗有效剂量的凝血因子；优选的，所述核酸构建体在肌肉注射的递送方式下，具有肌肉偏向性。
本发明还提供一种腺相关病毒载体系统，所述腺相关病毒载体系统包括前述的两种核酸构建体。
本发明还提供一种腺相关病毒，所述腺相关病毒所述腺相关病毒载体系统包装而成。
本发明还提供一种细胞系，其特征在于，所述细胞系为经所述腺相关病毒感染的细胞系。
本发明还提供所述腺相关病毒、所述细胞系在制备预防、治疗遗传性凝血因子缺乏病的产品中的用途。
本发明还提供一种治疗遗传性凝血因子缺乏病的方法，所述方法包括向患者给予有效量的腺相关病毒。
如上所述，本发明的提供靶向肌肉的AAV载体血清型的新用途，具有以下有益效果：提供了更为安全且有效的AAV载体及给药方式，改变AAV药物在人体内的组织分布，使其成为一种具有显著安全性优势的基因治疗药物。提高了FIX的表达量，其表达的量足够大，能满足治疗疾病所需的血药浓度。在达到治疗效果的同时减少AAV的使用量。不会导致肝毒性，相比现有的治疗方法更加安全有效。
附图说明
图1显示为本发明的凝血因子IX基因腺相关病毒载体设计示意图。
图2显示为本发明的pAAV-MCS-ITR-130-CAG-SV40质粒图谱。
图3显示为本发明的不同构建体在C2C12细胞上清中FIX的表达量。
图4显示为本发明的凝血因子IX基因腺相关病毒载体不同启动子设计示意图。
图5显示为不同启动子的构建体在C2C12细胞上清中FIX的表达量。
图6显示为不同表达载体对小鼠体内FIX活性的影响。
图7显示为不同启动子在动物体内的表达水平比较。
图8-1至图8-2显示为本发明的用于验证不同血清型生物分布和载体递送效率的目的基 因构建体示意图，其中图8-1为KL-pAAV-HA21结构示意图，图8-2为KL-pAAV-Luc结构示意图。
图9-1至图9-4显示为本发明的Balb/c小鼠分别在注射不同AAV后day3、day7、day14、day21的活体成像图。
图10-1和图10-2显示为乙型血友病模型F9-KO3小鼠注射AAV后第1、2、4、6、8周内的FIX活性和表达量血药浓度。
图11显示为给药后8周的APTT检测结果。
图12显示为给药后4周断尾实验的出血量检测结果。
图13显示为给药后四周血栓弹力图TEG结果，R(min)代表着各实验动物血液的凝血反应开始时间。
图14为给药后1、4、8周AAV DNA组织分布检测结果。
具体实施方式
本发明提供一种核酸构建体，包括以下元件：编码凝血因子IX的多核苷酸和AAV顺式作用元件，所述编码凝血因子IX的多核苷酸序列为经过密码子优化的序列，所述AAV顺式作用元件包括表达控制元件。在一种实施方式中，所述表达控制元件可以是组织特异性表达控制元件。所述组织特异性表达控制元件例如为肌肉组织特异性表达控制元件。所述组织特异性表达控制元件包括ITR、启动子、kozak序列、SV40 polyA中的任一种或多种，所述核酸构建体为用于制备肌肉注射给药的腺相关病毒的核酸构建体。
本发明中，术语“核酸构建体”是指可以被引入靶细胞或组织中的人工构建的核酸区段。
所述凝血因子选自凝血因子VIII、凝血因子IX或凝血因子XI以及它们的突变体。
所述凝血因子IX(或称为FIX)包括长度为28个氨基酸的信号肽和433个氨基酸。
在一种实施方式中，凝血因子IX为经过R338L突变的凝血因子IX。所述R338L突变的凝血因子IX即将FIX的第338个氨基酸由精氨酸R突变为亮氨酸L，经过该突变的凝血因子IX可简写为FIX-R338L，该位点突变后FIX在小鼠上的效价可提高5倍以上。
在一种实施方式中，经过密码子优化的编码FIX-R338L的多核苷酸(称为F9IDTM)的序列如SEQ ID NO:23所示或为与SEQ ID NO:23所示的序列具有95％以上相似性的序列。
在一种实施方式中，所述启动子选自哺乳动物组成型启动子、哺乳动物组织特异性启动子、哺乳动物诱导型启动子。
所述哺乳动物组成型启动子选自以下任意：CMV(巨细胞病毒)、EF1α(延伸因子-1α)、 EFS、CAG(巨细胞病毒增强子和鸡β-肌动蛋白启动子组成)、CAGG、CBH、SFFV、MSCV、mPGK；hPGK或UBC(泛素C)。
在一较佳实施方式中，所述哺乳动物组织特异性启动子选自肌肉特异性启动子。由于目前报道的AAV靶向肝脏造成的肝脏毒性和肝癌隐患，因此需要寻求其他的安全有效的给药方式，本申请的发明人经过大量研究多种给药方式，最终得出给药方式是肌肉注射的靶向肌肉细胞的AAV是治疗血友病B很好的选择，因此在核酸构建体的构建中相应的选择肌肉特异性启动子。
所述肌肉特异性启动子选自tMCK、Desmin、SKCRM 4-DES、tMCK2、CK8或MCK。
据信在一些实施例中，所述ITR序列可折叠成发夹结构，是AAV DNA复制起始和包装重组AAV病毒颗粒所需的顺式作用元件。
所述ITR的长度为130～145bp。所述ITR的长度例如为130～135bp、135～140bp、141bp、142bp、143bp、144bp、145bp。
所述ITR中上游ITR和下游ITR的长度可以相同或不同。
在一较佳实施方式中，所述上游ITR和下游ITR的长度相同，均为141bp。
在一种实施方式中，所述核酸构建体中AAV顺式作用元件还包括UTR。
所述UTR可将来自本领域已知的任何基因的任何UTR引入到所述核酸构建体中。
在一种实施方式中，所述UTR位于启动子后。
非翻译区(UTR)被转录但不被翻译。通常，5'UTR在转录起始位点开始，并在起始密码子处终止，3'UTR在终止密码子后立即开始，并一直持续到转录终止信号为止。
在某些实施方案中，病毒基因组中的5'UTR包含kozak序列。在另一些实施方案中，病毒基因组中的5'UTR不包含kozak序列。
在一种实施方式中，所述核酸构建体中AAV顺式作用元件还包括WPRE。在一种实施方式中，所述WPRE位于F9IDTM基因表达框和SV40 polyA之间。
WPRE元件能增强载体的表达及转录后mRNA的稳定性，提高mRNA剪接效率。
在一种实施方式中，所述核酸构建体的结构选自以下任一：
1)ITR-启动子-kozak序列-F9IDTM基因表达框-SV40 polyA序列-ITR；
2)ITR-启动子-kozak序列-F9IDTM基因表达框-WPRE-SV40 polyA序列-ITR；
3)ITR-启动子-UTR序列-kozak序列-F9IDTM基因表达框-WPRE-SV40 polyA序列-ITR。
在一些实施方式中，所述核酸构建体为腺相关病毒载体。在一些实施方式中，所述腺相 关病毒载体还包括载体骨架。
在一些实施方式中，所述载体骨架可以自制或选自市场上出售的合适载体骨架。
在一种实施方式中，所述载体骨架为pAAV-MCS-ITR-130-CAG-SV40，核苷酸序列如SEQ ID NO：1所示。
在一种实施方式中，所述核酸构建体的核苷酸序列选自以下任一，以及与以下任一序列比较至少具有75％，80％，85％，90％，95％，96％，97％，98％，或99％同源性的核苷酸序列：SEQ ID NO:22、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21。
本发明还提供靶向肌肉的AAV载体血清型在制备遗传性凝血因子缺乏病预防或治疗产品中的用途，或提供靶向肌肉的AAV载体血清型在预防或治疗遗传性凝血因子缺乏病中的用途。
术语“血清型”是一区别，用于指具有血清学上不同于其他AVV血清型的衣壳的AVV。血清学区别性基于与另一种AVV相比，一种AAV的抗体之间没有交叉反应。这种交叉反应区别通常由于衣壳蛋白序列/抗原决定簇的不同引起(例如由于AAV血清型的VP1、VP2和/或VP3序列差异)。尽管包含衣壳变体的AAV变体可能在血清学上无法与参考AAV或其他AAV血清型区分，他们至少与参考或其他AAV血清型具有一个核苷酸或氨基酸残基的差异。
在传统定义下，血清型是指目标病毒已经过对所有现存的和特征化的血清型具有特异性的血清测试中和活性，没有发现中和目标病毒的抗体。因为更多自然产生的病毒分离物是被发现的和/或衣壳突变产生的，他们与任意现存的血清型可具有也可没有血清学差异。因此，当新病毒(例如AAV)没有血清学差异，该新病毒(例如AAV)可能是相应血清型的亚型或变体。在许多情形下，用于中和活性的血清学测试需要在衣壳序列修饰的突变病毒上进行，以确定其是否是传统血清型定义中的另一种血清型。相应的，为了便利和避免重复，术语“血清型”广泛的指血清学上不同的病毒(例如AAV)以及血清学上并非不同的属于特定血清型的亚型或变体的病毒(例如AAV)。
在一种实施方式中，所述靶向肌肉的AAV载体血清型选自AAV1、AAV2、AAV6、AAV6.2FF、AAV7、AAVrh74、AAV8、AAV9或它们的突变体。
在一较佳实施方式中，所述靶向肌肉的AAV载体血清型为AAV6.2FF。AAV6.2FF为重组腺相关病毒三突变载体，使用该血清型衣壳包装的Firefly-Luciferase报告基因载体，在野生型c57小鼠上利用活体成像技术发现AAV6.2FF血清型载体具有更多肌肉偏向性、较少肝脏偏向性。
所述“偏向性”是指存在于AAV病毒颗粒中的AAV衣壳蛋白用于感染特定类型的细胞或组织的特异性。
本发明中，所述“肌肉偏向性”是指对肌肉细胞或组织具有偏向性。
所述肌肉细胞选自肌细胞、肌管、成肌细胞或卫星细胞。
所述肌肉组织选自心脏或骨骼肌组织。肌肉组织占人体重量的35％～40％，具有作为适用于基因治疗的“生物工厂”的潜能。另一方面，从风险管理的角度，即使遗传性凝血因子缺乏病治疗产品引起了局部的严重不良反应，肌肉组织切除的代价也将远低于肝脏，而且肌肉组织的肿瘤也往往是良性的，不发生恶性扩散。综合来看，靶向肌肉给药的安全性的风险影响更低。
所述遗传性凝血因子缺乏病治疗产品选自核酸构建体、腺相关病毒。
所述遗传性凝血因子缺乏病选自A型血友病、B型血友病或C型血友病。
本发明还提供一种用于制备遗传性凝血因子缺乏病治疗产品的核酸构建体，以该核酸构建体制备的腺相关病毒具有肌肉偏向性，并表达治疗有效剂量的凝血因子。
所述核酸构建体在肌肉注射的递送方式下，具有肌肉偏向性。
所述治疗有效量是指在治疗遗传性凝血因子缺乏病中有效果的量。
实现治疗效果的剂量，例如载体基因组的剂量/kg体重(vg/kg)，基于几种不同因素而变化，包括但不限于：给药途径、实现治疗效果所需的异源多核苷酸表达水平，治疗的特异性疾病，针对病毒载体的任何宿主免疫响应，针对异源多核苷酸或表达产物(蛋白)的宿主免疫响应，以及表达的蛋白的稳定性。本领域技术人员能确定rAAV/载体基因组剂量范围，以基于上述因素和其他因素治疗具有特定疾病或失调的患者。通常而言，剂量范围是至少1×108或更多，例如1×109、1×1010、1×1011、1×1012、1×1013或1×1014或更多的载体基因组每千克主体体重(vg/kg)，以实现治疗效果。AAV在小鼠的有效剂量范围是1×1010-1×1011，在狗中是1×1012-1×1013。
以B型血友病为例，通常而言，认为为了实现治疗效果，需要血凝因子浓度大于正常个体因子浓度的1％，以改变严重疾病表型为温和表型。严重表型特征为关节损伤和致命流血。为了将温和疾病表型转变成轻微表型，认为血凝因子浓度需要大于正常浓度的5％。关于治疗这类血友病主体，通常剂量至少为1×1010载体基因组(vg)每千克(vg/kg)宿主体重，或者约1×1010至1×1011vg/kg宿主体重，或者约1×1011至1×1012vg/kg宿主体重，或者约1×1012至1×1013vg/kg宿主体重，以实现所需的治疗效果。
治疗“有效量”或“足够量”(例如减轻或提供治疗益处或改善)的剂量通常以可测量程 度有效响应于一种、多种或全部不利疾病症状、后果或并发症，一种或多种不利症状、失调、疾病、病理学或并发症，例如由疾病引起或与其相关，但降低、减少、抑制、制止、限制或控制疾病发展或恶化是满意的结果。
有效量或足够量能够以单次给药提供，但不是必须的，其可需要多次给药以及能够单独或与其他组分(例如治疗剂)、治疗物、方案或治疗方案组合，但这也不是必须的。例如，数量可随着主体、类型、治疗疾病的状态和严重性或治疗副反应(如果有)的需要按比例增加。另外，有效量或足够量如果以单或多剂量给药而没有第二种组合物(例如另一种药物或试剂)、治疗物、方案或治疗方案，其无需是有效的或足量的，因为可包括另外在该剂量之上和超过该剂量的剂量、数量或持续时间，或者可包括另外的组分(例如药物或试剂)、治疗物、方案或治疗方案，以考虑对给定主体有效或足够量。认为有效的量也包括导致其他治疗物、治疗方案或方案减少的量，例如给予用于治疗凝血疾病(例如A型血友病)的重组凝血因子蛋白(例如FVIII)。
在一些实施方式中，所述核酸构建体为腺相关病毒载体。在一些实施方式中，所述核酸构建体包括表达腺相关病毒衣壳蛋白的载体骨架；优选的，腺相关病毒衣壳蛋白载体骨架表达靶向肌肉组织的腺相关病毒衣壳蛋白。
在一些实施方式中，所述载体骨架可以自制或选自市场上出售的合适载体骨架。
在一些实施方式中，所述载体骨架为靶向肌肉的载体骨架。所述载体骨架例如为KL-pAAV-RC1、KL-pAAV-RC2、KL-pAAV-RC6、KL-pAAV-RC6.2FF、KL-pAAV-RC7、KL-pAAV-RCrh74、KL-pAAV-RC8、KL-pAAV-RC9，核苷酸序列依次如SEQ ID NO.1～8所示。
本发明还提供一种腺相关病毒载体系统，所述腺相关病毒载体系统包括所述的用于制备遗传性凝血因子缺乏病治疗产品的核酸构建体。
所述腺相关病毒载体表达系统选自大肠杆菌表达系统、酵母表达系统、昆虫表达系统、哺乳动物表达系统、植物表达系统；优选的，所述腺相关病毒载体表达系统选自质粒瞬时转染表达系统、杆状病毒表达系统、稳转细胞系表达系统、腺病毒表达系统、痘病毒表达系统中的任一种。
进一步的，所述慢病毒载体系统还包括宿主细胞。所述宿主细胞携带腺相关病毒载体。所述宿主细胞可以选自本领域各种可适用的宿主细胞，只要不对本发明的发明目的产生限制即可。具体的可适用的细胞可以为生产腺相关病毒的细胞，例如可以为293T细胞。
在质粒瞬时转染表达系统中，所述腺相关病毒载体系统还包括携带目的基因的核酸构建 体和辅助质粒。在本发明的一种实施方式中，所述携带目的基因核酸构建体中包括编码凝血因子的多核苷酸和AAV顺式作用元件。具体的，所述携带目的基因的核酸构建体为本发明前述的包括编码凝血因子的多核苷酸和AAV顺式作用元件的构建体。
更进一步的，所述辅助质粒中包括必需的病毒包装组件，所述辅助质粒可以包括包装质粒。
本发明还提供一种腺相关病毒(AAV)，所述腺相关病毒由腺相关病毒载体系统经病毒包装而成。所述腺相关病毒可以用来治疗各种疾病，例如遗传性凝血因子缺乏病。
本发明还提供一种细胞系，所述细胞系为经所述腺相关病毒感染的细胞系。
在一种实施方式中，所述细胞系为哺乳动物细胞系。
本发明还提供所述腺相关病毒载体系统、腺相关病毒、细胞系在制备预防、治疗遗传性凝血因子缺乏病的产品中的用途。
在一种实施方式中，所述遗传性凝血因子缺乏病为A型、B型或C型血友病。在一种实施方式中，所述遗传性凝血因子缺乏病为哺乳动物遗传性凝血因子缺乏病。更进一步的，所述遗传性凝血因子缺乏病为人遗传性凝血因子缺乏病。
本发明还提供一种治疗遗传性凝血因子缺乏病的方法，所述方法包括向患者给予有效量的所述腺相关病毒。
所述遗传性凝血因子缺乏病为A型、B型或C型血友病。在一种实施方式中，所述遗传性凝血因子缺乏病为哺乳动物遗传性凝血因子缺乏病。更进一步的，所述遗传性凝血因子缺乏病为人遗传性凝血因子缺乏病。
在一种实施方式中，所述方法包括向患者肌肉注射有效量的所述腺相关病毒。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术 语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
以下实施例根据密码子优化、ITR序列、UTR及WPRE、启动子、构建了一系列搭载目的基因的分子构建体，并将其克隆到重组腺相关病毒中。在293T细胞中，包装相应的腺相关病毒，以一定生物学滴度病毒感染分化肌肉细胞系。对细胞上清中产生的FIX定量检测、比较。选取细胞中表现较好的AAV载体在C57小鼠后腿肌肉进行注射，进行体内比较筛选。
主要的构建体如下表1：
实施例1凝血因子IX基因表达AAV载体设计
表达框架根据Uniprot{UniProtKB-P00740(FA9_HUMAN)}上人凝血因子IX的氨基酸序列为基础设计，该序列包含46个氨基酸的信号肽和415个氨基酸的FIX序列，将成熟FIX的第338个氨基酸由精氨酸R突变为亮氨酸L，该突变在小鼠上FIX效价可提高5倍以上，氨基酸序列确定后，再用IDT在线进行密码子优化，得到F9IDTM核苷酸序列。(密码子优化的核苷酸序列见SEQ ID NO:23，氨基酸序列见SEQ ID NO:24)
该F9IDTM基因表达框架被设计插入腺相关病毒载体骨架pAAV-MCS-ITR-130-CAG-SV40中，该AAV载体包含CAG启动子、SV40病毒的多腺苷酸化信号(SV40SL)、反向末端重复序列(ITR)等元件，该载体上游ITR为130bp，下游ITR为141bp，命名为HA21-01。
在腺相关病毒载体HA21-01的基础上，分别设计腺相关病毒载体HA21-02、HA21-03另外2个载体。HA21-02的上下游ITR均为141bp，HA21-03的上下游ITR均为145bp。
在腺相关病毒载体HA21-02的基础上，设计腺相关病毒载体HA21-04，在启动子后添加UTR序列，在目的基因和SV40之间添加WPRE序列。各腺相关病毒载体的结构如图1所示。
实施例2凝血因子IX基因表达腺相关病毒载体的构建
将实施例1设计的凝血因子F9IDTM基因表达框架经南京金斯瑞合成后，通过本领域熟知的以同源重组的方法克隆至腺相关病毒载体骨架pAAV-MCS-ITR-130-CAG-SV40中多克隆位点的AgeI/SalI之间，命名为HA21-01(核苷酸序列SEQ ID NO:22)，腺相关病毒载体构建体骨架来源于康霖生物科技(杭州)有限公司自备骨架——pAAV-MCS-ITR-130-CAG-SV40(核苷酸序列SEQ ID NO：1)，见图2。
由南京金斯瑞生物技术有限公司合成141bp的ITR序列(核苷酸序列SEQ ID NO:2)，通过本领域熟知的同源重组的方法分别克隆至HA21-01上的多克隆位点NarI/MluI之间之间，克隆完成后以测序确认序列信息，命名为HA21-02(核苷酸序列SEQ ID NO:3)。
由南京金斯瑞生物技术有限公司合成145bp的ITR序列(核苷酸序列SEQ ID NO:4)，通过本领域熟知的同源重组的方法分别克隆至HA21-01上的多克隆位点NarI/MluI之间和PmeI/Pci I之间，克隆完成后以测序确认序列信息，命名为HA21-03(核苷酸序列SEQ ID NO：5)。
将土拨鼠乙肝病毒转录后调控元件(WPRE)(核苷酸序列SEQ ID NO:6)经南京金斯瑞合成后，以本领域熟知的PCR扩增的方法，将UTR序列(核苷酸序列SEQ ID NO:7)放在引物上扩增CAG启动子，同时再扩增F9IDTM，再通过本领域熟知的同源重组的方法克隆至HA21-02的上游ITR与SV40pA signal之间，克隆完成后以测序确认序列信息，命名为HA21-04(核苷酸序列SEQ ID NO:8)。
实施例3：凝血因子IX基因表达腺相关载体的病毒包装
将实施例2中构建的凝血因子FIX基因腺相关病毒表达载体(HA21-01、HA21-02、HA21-03、HA21-04)，衣壳质粒(KL-pAAV-RC6.2FF，其核苷酸序列如SEQ ID NO:9所示)和包装质粒(pHelper，其核苷酸序列如SEQ ID NO:10所示)同时共转染293T细胞(购自美国模式菌种收集中心(ATCC)，保藏号为CRL-3216)，在该293T细胞系中进行凝血因子IX基因治疗腺相关病毒载体的包装。
转染方法为PEI阳离子聚合物介导的真核细胞瞬时转染，PEI阳离子聚合物为购自Polysciences的PEI-Max转染试剂(购自Polysciences，货号：24765-1)，转染操作参照生产商推荐标准化操作进行，转染规模为15cm2细胞培养皿。
转染完成72小时后，收获重组腺相关病毒(AAV)，收集上清和细胞，4200rpm离心10min，离心完成后分离上清跟细胞，细胞加裂解液跟核酶，裂解消化1h，10000g离心10min取裂解液上清。将裂解液上清及培养基上清混合后经亲和层析纯化后分装冻存于-80℃备用。
取纯化后的AAV 5μL加入到45μL QE裂解液中，(QuickExtractTM DNA Extraction  Solutionr)(购自Lucigen，货号QE09050)并用PCR仪运行以下表2程序来裂解AAV。
表2 PCR程序
通过本领域熟知的方法，根据定量PCR结果以及AAV标准品(AAV2购自ATCC)为参照计算出AAV的物理滴度。
定量PCR使用的引物探针序列为：
ITR-For primer：GGAACCCCTAGTGATGGAGTT(SEQ ID NO:11)
ITR probe：5’-FAM-CACTCCCTCTCTGCGCGC-BHQ1-3’(SEQ ID NO:12)
ITR-Rev primer：CGGCCTCAGTGAGCGA(SEQ ID NO:13)
实施例4：凝血因子FIX基因表达腺相关病毒载体转导C2C12细胞后蛋白表达检测
C2C12细胞24孔细胞培养板，每孔1E5cells。用2％马血清培养基分化细胞。将上述实施例3中包装纯化的各凝血因子FIX基因腺相关病毒按照1E5的MOI感染分化好的C2C12细胞，96小时时收集上清，上清用人凝血因子IX(FIXa)试剂盒(ELISA)检测(购自睿信生物，货号：SU-BN16070)。检测结果发现ITR长度为141bp时相比130bp有明显，在此基础上添加UTR和WPRE后，表达量显著上升，结果如图3所示。
实施例5：凝血因子IX基因表达AAV载体不同启动子设计
根据前期研究肌肉注射的效果较好，因此进一步考虑到肌肉注射是否选择肌肉特异性启动子比较好，于是本实施例又设计了几种通用启动子和肌肉特异性启动子进行系统比较，见图4。
实施例6：凝血因子IX基因表达腺相关病毒载体的不同启动子构建
由南京金斯瑞生物技术有限公司合成CMV启动子(核苷酸序列SEQ ID NO:14)，通过本领域熟知的以同源重组的方法克隆至HA21-04的MluI和AgeI之间，替换掉CAG启动子，克隆完成后以测序确认序列信息，命名为HA21-05(核苷酸序列SEQ ID NO:15)。
由南京金斯瑞生物技术有限公司合成CBH启动子(核苷酸序列SEQ ID NO:16)，通过本领域熟知的以同源重组的方法克隆至HA21-04的MluI和AgeI之间，替换掉CAG启动子，克隆完成后以测序确认序列信息，命名为HA21-06(核苷酸序列SEQ ID NO:17)。
由南京金斯瑞生物技术有限公司合成CAGG启动子(核苷酸序列SEQ ID NO:18)，通过 本领域熟知的以同源重组的方法克隆至HA21-04的MluI和AgeI之间，替换掉CAG启动子，克隆完成后以测序确认序列信息，命名为HA21-07(核苷酸序列SEQ ID NO:19)。
由南京金斯瑞生物技术有限公司合成tMCK肌肉特异性启动子(核苷酸序列SEQ ID NO:20)，通过本领域熟知的以同源重组的方法克隆至HA21-04的MluI和AgeI之间，替换掉CAG启动子，克隆完成后以测序确认序列信息，命名为HA21-08(核苷酸序列SEQ ID NO:21)。
实施例7：不同启动子凝血因子FIX基因表达腺相关病毒转导C2C12细胞后蛋白表达检测
以实施例3中方法对实施例5中的不同启动子构建体(CAG、CBH、CAGG、tMCK、)进行AAV包装、纯化、滴度检测后以相同的方法进行细胞上清FIX表达量检测，结果显示CAG、CBH、CAGG启动子明显优于tMCK启动子。结果如图5所示。
实施例8：不同AAV在动物体内的表达水平比较
选取HA21-02、HA21-04腺相关病毒以相同剂量2.5×1011VG通过小鼠大腿外侧肌肉注射方式注射到8周龄的C57小鼠体内(n＝5)。注射后不同时间点，采用眼眶取血的方式对小鼠进行采血，采血过程中需要添加3.2％柠檬酸钠抗凝剂(比例为1：9)。血液经过离心后，对血浆中的FIX活性进行检测。FIX检测试剂盒为Biophen FIX:C(Chromogenic assay for measuring Factor IX:C in plasma,or in concentrates)，货号为221806，标准品为辉瑞制药公司生产的注射用重组人凝血因子IX(贝赋)，250IU/瓶，注册证号S20120053；
数据表明以肌肉注射的方式进行给药能发挥较好的效果，HA21-04相比于HA21-02增加了UTR和WPRE元件，这两个元件对在体内同样对FIX表达有较大的促进作用，如图6所示，具体活性数值见表3。
表3两种构建体C57BL/6J体内水平比较

实施例9：不同启动子的AAV在动物体内的表达水平比较
选取HA21-04、HA21-06、H21-08腺相关病毒以相同剂量2.5×1011VG通过小鼠大腿外侧肌肉注射方式注射到8周龄的C57小鼠体内(n＝5)。按照实施例8的方法对不同时间点的血浆进行检测。
数据表明CAG(HA21-04)和CBH(HA21-06)相对肌肉特异性启动子在体内有明显优势，如图7所示。
综合比较后HA21-04能够很大程度的提高FIX的表达活性，具体活性数值见表4。
表4不同启动子构建体C57BL/6J体内水平比较
以下实施例首先筛选出几种可能合适的载体类型作为待选用的血友病基因治疗递送系统。选择的血清型包括AAV1、AAV2、AAV6、AAV6.2FF、AAV7、AAVrh74、AAV8、AAV9。以上血清型的衣壳的序列经过商业化途径合成后，经过合理的分子生物学载体构建策略克隆到AAV包装载体KL-pAAV-Rep上。AAV载体经过包装、纯化工艺，得到相应的几种不同血清型的AAV产品，包括搭载编码FIX-co-padua蛋白的基因序列的载体(AAV-HA21)和携带Firefly-Luciferase报告基因及相同启动子等表达调控元件的对应版本载体(AAV-Luc)。
接着使用不同血清型衣壳包装的Firefly-Luciferase报告基因载体，利用活体成像技术，在野生型c57小鼠上探索不同血清型载体的生物分布特征，筛选出具有更多肌肉偏向性、同时肝脏偏向性较少的血清型类型(AAV6.2FF)。
再在FIX基因敲除的转基因小鼠F9-KO3(南方模式生物，品系名：C57BL/6-F9em3Smoc，目录号：NM-KO-200607)中验证了AAV6.2FF载体的基因递送效率，并通过断尾实验(tail-clip test)等概念验证的方式证明了由AAV6.2FF系统递送编码功能正常的FIX蛋白(FIX-co-padua)目的基因的基因治疗药物KL-HA21可以通过靶向肌肉的基因递送方式，安全、显著且长效地减轻F9-KO3小鼠的乙型血友病症状。
实施例10不同血清型AAV的选择、载体设计与构建
通过美国临床试验注册中心(https://clinicaltrials.gov/)和美国国家生物信息中心(https://www.ncbi.nlm.nih.gov)的检索，着重筛选出与肌肉递送和肌肉靶向性相关的载体血清型子集，其中包括AAV1、AAV2、AAV6、AAV6.2FF、AAV7、AAVrh74、AAV8、AAV9。通过美国国家生物信息中心(https://www.ncbi.nlm.nih.gov)和全球科学家质粒共享非盈利组织Addgene(http://www.addgene.org)的数据库检索，结合多篇新型血清型改造策略相关文献的分析，整理出上述提及的8种血清型载体的核苷酸序列。使用分子生物学工具SnapGene设计构建相应血清型的包装载体序列，经南京金斯瑞生物技术有限公司合成DNA片段后，通过SmaI+AgeI双酶切、T4DNA连接酶连接的方法分别克隆至AAV包装载体KL-pAAV-Rep上。克隆完成后经测序确认序列信息，不同血清型的包装载体分别命名为：KL-pAAV-RC1、KL-pAAV-RC2、KL-pAAV-RC6、KL-pAAV-RC6.2FF、KL-pAAV-RC7、KL-pAAV-RCrh74、KL-pAAV-RC8、KL-pAAV-RC9，核苷酸序列依次如SEQ ID NO:25～27、9、28～31所示。
实施例11不同血清型的AAV-HA21和AAV-Luc生产
将实施例10得到的不同血清型的包装载体质粒(KL-pAAV-RC1、KL-pAAV-RC2、KL-pAAV-RC6、KL-pAAV-RC6.2FF、KL-pAAV-RC7、KL-pAAV-RCrh74、KL-pAAV-RC8、KL-pAAV-RC9)、辅助质粒(KL-pAAV-Helper,核苷酸序列SEQ ID NO:34)、目的基因表达质粒(含FIX-co-padua目的基因的KL-pAAV-HA21载体，核苷酸序列SEQ ID NO:32或者含Firefly-Luciferase目的基因的KL-pAAV-Luc载体，核苷酸序列SEQ ID NO:33)共同转染293T细胞(购自美国模式菌种收集中心(ATCC)，保藏号为CRL-3216)，利用293T系统进行AAV的包装。转染方法为PEI阳离子聚合物介导的真核细胞瞬时转染，PEI阳离子聚合物具体为PEI-Max转染试剂(购自Polysciences，货号：24765-1)， 转染操作参照生产商推荐的标准化操作进行，转染规模为15cm2细胞培养皿。
转染完成72小时后，将细胞上清及细胞一起收集至50mL离心管中。使用台式离心机室温4200rpm离心10分钟分离细胞及上清。将上清转移至新的50mL离心管中，添加MgCl2及核酶，混匀待用。剩余细胞添加核酶和细胞裂解液，室温静置1h，随后室温10000g离心10min转移上清至原先细胞上清中，混匀，最后进行亲和纯化浓缩，得到不同血清型的AAV终产品(AAV1-CBH-Luc、AAV2-CBH-Luc、AAV6-CBH-Luc、AAV6.2FF-CBH-Luc、AAV7-CBH-Luc、AAVrh74-CBH-Luc、AAV8-CBH-Luc、AAV9-CBH-Luc、AAV6.2FF-CBH-HA21)。
通过本领域熟知的方法，根据定量PCR结果以及AAV标准品为参照计算出AAV的物理滴度。定量PCR使用的引物探针序列为：
CMV-For primer：ACTTTCCATTGACGTCAATGGGTGG(SEQ ID NO:35)
CMV probe：5’-CAAGTGTATCATATGCCAAGTACGCCCCC-3’(SEQ ID NO:36)
CMV-Rev primer：AGGTCATGTACTGGGCATAATGC(SEQ ID NO:37)
其中CMV probe中5’端带有FAM荧光基团，3’带有BHQ1荧光基团；
实施例12：不同血清型AAV-Luc组织分布特征摸索试验
根据载体构建的时间顺序差异，选择上述实施例2中部分优先包装纯化得到的数种血清型AAV-Luc载体(AAV6.2FF-CBH-LucAAV8-CBH-Luc、AAV9-CBH-Luc)，在Balb/c和C57BL/6小鼠中先行探索该部分血清型载体的生物分布规律。
Balb/c小鼠实验方案：以单侧腓肠肌10μl、胫骨前肌10μl、股四头肌前后各10μl的方案进行注射给药，注射当天定义为实验第0天(day0)，每组3只，8周龄雄鼠。详细分组情况见表5。
表5 Balb/c小鼠不同血清型AAV-Luc组织分布特征摸索实验分组
动物分别在day3、day7、day14、day21、进行活体成像。活体成像时，小鼠按照200μl/只的剂量腹腔注射浓度为15μg/ml的底物D-luciferin(碧云天，货号：ST196)；底物注射后5分钟，利用异氟烷气体麻醉小鼠；底物注射后12分钟，放入小动物活体成像仪(PerkinElmer，IVIS Spectrum)，分别以仰卧位和俯卧位摆放，进行活体成像数据收集。
如图9所示，每组3只动物，每组动物腹侧与背侧各进行一次成像拍照。Day3～day14观察可见，AAV8-CBH-Luc、AAV9-CBH-Luc均有显著的肝脏载体表达信号分布，而AAV6.2FF-Luc的注射部位肌肉靶向性非常专一，没有产生明显的肝脏或其他组织器官的非特异性分布。
实施例13.AAV6.2FF-HA21在F9-KO3模型小鼠体内的药效学试验
为验证AAV6.2FF能够通过肌肉靶向递送具有治疗水平的FIX蛋白，利用搭载编码FIX-co-padua蛋白的基因序列的核酸构建体(KL-pAAV-HA21，核苷酸序列SEQ ID NO.32)与包装载体质粒与辅助质粒制备AAV，携带FIX-co-padua蛋白的AAV命名为AAV6.2FF-HA21(或HA21)，并证明AAV6.2FF-HA21(HA21)的确可以部分乃至彻底恢复血友病引起的凝血功能障碍，本发明选择了一种利用基因编辑技术敲除了F9基因外显子、凝血因子IX缺陷的乙型血友病模型小鼠(C57BL/6-F9em3Smoc，F9-KO3)，验证AAV6.2FF-HA21通过靶向肌肉组织的方式递送药物，能够产生修复该小鼠凝血功能的效果，即本发明的最终概念验证。
将240只雄性8周龄F9-KO小鼠以≤5只/笼的规模入笼，动物自由摄取大小鼠育成饲料和饮用水。饲养条件：玉米芯垫料，室温16～26℃，相对湿度40％～70％，人工照明12/12小时昼夜明暗交替。
注射方案：动物适应性饲养1周后，小鼠双侧腓肠肌和股四头肌注射，每个点50μl，注射时异氟烷气体麻醉。注射当天定义为实验第0天(day0)。根据体重随机分组，详细分组情况见表6。
表6实验分组及剂量设计
在1、2、4、6、8周采集3.2％枸橼酸钠1:9抗凝血检测FIX活性和表达量的检测，在给药后第4周分别进行断尾实验、血栓弹力图TEG检测和APTT检测。
同时设置组织分布检测，给药后1、4周每组各扑杀3只检测，第8周每组各扑杀2只检测，该动物为每组另饲养8只进行。按时间点解剖取样提取基因组，用CMV引物探针进行qPCR检测，先算出AAV DNA copies/ug DNA，根据1ng DNA约等于172个细胞换算到AAV DNA copies/cell。
血药浓度药代动力学结果分析
FIX-KO小鼠在IM注射HA21HA21或安慰剂或IV注射BeneFIX(单次给药)后，IM注射HA21或IM注射安慰剂的FIX-KO小鼠分别于1周，2周，4周，6周，8周收集血浆并检测血浆中FIX的含量与活性(FIX活性测定试剂盒221806，BIOPHEN；FIX含量测定试剂盒SU-BN16070，睿信生物)。IV注射BeneFIX的FIX-KO小鼠在4周进行给药并在给药一小时后收集血浆，检测血浆中FIX的含量与活性。FIX含量与活性在血浆中的变化如图所示(图10-1，图10-2)。HA21各给药组和BeneFIX给药组中FIX含量与安慰剂组相比均有显著上升，FIX含量与HA21注射组剂量成正相关。在HA21给药组中，血浆中FIX活性随着浓度升高有上升趋势。
活化部分凝血活酶时间(APTT)检测结果分析
为了检测不同给药组给药后内源性凝血系统凝血因子活性，在第八周(图11)进行活化部分凝血活酶时间(APTT)检测。实验结果可见随着HA21给药浓度的提升(G1-G4)，受试小鼠APTT时间减少，HA21给药组均显著低于安慰剂组，最低剂量组就起到药效，G3、G4甚至与阳性对照组G6时间相近。
断尾实验结果分析
小鼠的尾部有中央动脉一条，侧静脉两条，背部静脉一条，排布规则。在断尾实验中，通过在小鼠同样大小面积尾截面面积处(直径2.0mm)垂直切断，向上述血管引入开放创面，在生理盐水温水浴中保持创面湿润同时收集流出血液。具备正常凝血能力的小鼠会在较短时间内启动凝血反应，阻止创面进一步出血。通过断尾实验，我们可以评价HA21各剂量给药组分别与安慰剂组或重组F IX阳性对照组对FIX-KO小鼠凝血能力的功能性改善。
如图12所示，大部分FIX-KO小鼠断尾在安慰剂量组中无法自行停止尾部断面流血，同时出血量平均值在各组中最高，而在重组F IX阳性对照组中，出血量平均值为32.92±12.72μL，表明一次性外源性补充FIX可以短时显著改善小鼠凝血功能。而HA21各剂量组均显著减少断尾后出血量。
血栓弹力图TEG检测结果分析
如图13所示，FIX-KO小鼠血栓弹力图中，安慰剂量组中除1只小鼠外其他凝血因子作 用时间均超上限，即没有凝血功能。而在重组FIX阳性对照组中，凝血因子作用时间平均值为16.34±5.84min，表明外源性补充F IX可以显著缩短凝血时间。而HA21剂量组1.25E+12vg/kg也能缩短60％小鼠的凝血时间，随着剂量递增，平均凝血时间也随之缩短，趋于重组FIX阳性对照组的凝血时间。
组织分布检测结果分析
AAV DNA拷贝数检测显示，相比其他组织腓肠肌和股四头肌中的拷贝数最高，在给药后1周时，最高剂量组G4C的腓肠肌中拷贝数平均值为273copies/cell，股四头肌中的拷贝数平均值为737copies/cell。本实验的给药方式为双侧腓肠肌、股四头肌共四点注射，极高的拷贝数提示了与给药方式的相关性。
在本实验中，除肌肉外，肝和脾中的拷贝数较高，但与肌肉相差大于1个数量级，心和肾中的拷贝数较低，肺、脑和睾丸中的拷贝数极低。且组织分布也有剂量效应。相比于给药后1周，给药后4周和8周的拷贝数降低，具体数据见表7和图14。
表7不同时间点组织分布数据
本次实验通过血药浓度检测、活化部分凝血活酶时间、断尾实验、血栓弹力图检测等手段，发现各剂量组与安慰剂组均有显著性差异，可显著减轻F9-KO3小鼠的乙型血友病症状，组织分布检测明确了肌肉部位的分布绝对优势，在F9-KO3乙型血友病模型小鼠上验证了AAV6.2FF-HA21药物抗乙型血友病的效果，验证了一种靶向肌肉组织的血清型--AAV6.2FF用于血友病基因治疗的有效性。
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。

Claims (27)

  1. 一种核酸构建体，其特征在于，所述核酸构建体包括以下元件：
    编码凝血因子IX的多核苷酸；
    AAV顺式作用元件，所述AAV顺式作用元件包括ITR、启动子、kozak序列、SV40 polyA、UTR或WPRE中的一种或多种。
  2. 根据权利要求1所述的核酸构建体，其特征在于，所述编码凝血因子IX的多核苷酸序列为经过密码子优化的序列，优选的，所述经过密码子优化的多核苷酸序列为包括R338L突变的凝血因子IX的序列；优选的，所述经过密码子优化的多核苷酸的序列如SEQ ID NO:23所示或为与SEQ ID NO:23所示的序列具有95％以上相似性的序列。
  3. 根据权利要求1所述的核酸构建体，其特征在于，所述启动子选自哺乳动物组成型启动子、哺乳动物组织特异性启动子或哺乳动物诱导型启动子。
  4. 根据权利要求1所述的核酸构建体，其特征在于，所述启动子选自肌肉组织特异性启动子；优选的，所述肌肉组织特异性启动子选自tMCK、Desmin、SKCRM 4-DES、tMCK2、CK8或MCK。
  5. 根据权利要求1所述的核酸构建体，其特征在于，所述ITR的长度为130～145bp；优选的，所述ITR的长度为130bp、141bp或145bp；更优选的，所述上游ITR和下游ITR的长度相同，均为141bp。
  6. 根据权利要求1所述的核酸构建体，其特征在于，所述核酸构建体的结构选自以下任一：
    1)ITR-启动子-kozak序列-F9IDTM基因表达框-SV40 polyA序列-ITR；
    2)ITR-启动子-kozak序列-F9IDTM基因表达框-WPRE-SV40 polyA序列-ITR；
    3)ITR-启动子-UTR序列-kozak序列-F9IDTM基因表达框-WPRE-SV40 polyA序列-ITR。
  7. 根据权利要求1所述的核酸构建体，其特征在于，所述核酸构建体为腺相关病毒载体。
  8. 根据权利要求1所述的核酸构建体，其特征在于，所述腺相关病毒载体还包括载体骨架；优选的，所述载体骨架为pAAV-MCS-ITR-130-CAG-SV40，核苷酸序列如SEQ ID NO：1所示。
  9. 根据权利要求1所述的核酸构建体，其特征在于，所述核酸构建体的核苷酸序列选自以下任一：SEQ ID NO:22、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21。
  10. 靶向肌肉组织的AAV载体血清型在制备遗传性凝血因子缺乏病治疗产品中的用途。
  11. 根据权利要求10所述的用途，其特征在于，所述靶向肌肉的AAV载体血清型选自 AAV1、AAV2、AAV6、AAV6.2FF、AAV7、AAVrh74、AAV8、AAV9或它们的突变体。
  12. 根据权利要求10所述的用途，其特征在于，所述遗传性凝血因子缺乏病治疗产品选自核酸构建体或腺相关病毒。
  13. 根据权利要求10所述的用途，其特征在于，所述遗传性凝血因子缺乏病选自A型血友病、B型血友病或C型血友病。
  14. 一种用于制备遗传性凝血因子缺乏病治疗产品的核酸构建体，其特征在于，以该核酸构建体制备的腺相关病毒具有肌肉偏向性，并表达治疗有效剂量的凝血因子；优选的，所述核酸构建体在肌肉注射的递送方式下，具有肌肉偏向性。
  15. 根据权利要求14所述的核酸构建体，其特征在于，所述核酸构建体包括表达腺相关病毒衣壳蛋白的载体骨架；优选的，腺相关病毒衣壳蛋白载体骨架表达靶向肌肉组织的腺相关病毒衣壳蛋白。
  16. 根据权利要求15所述的核酸构建体，其特征在于，所述腺相关病毒衣壳蛋白载体骨架为KL-pAAV-RC1、KL-pAAV-RC2、KL-pAAV-RC6、KL-pAAV-RC6.2FF、KL-pAAV-RC7、KL-pAAV-RCrh74、KL-pAAV-RC8、KL-pAAV-RC9，核苷酸序列依次如SEQ ID NO.25～27、9、28～31所示。
  17. 一种腺相关病毒载体表达系统，其特征在于，所述腺相关病毒载体系统包括权利要求1～9任一所述的核酸构建体和权利要求14～16任一所述的核酸构建体。
  18. 根据权利要求17所述的腺相关病毒载体系统，其特征在于，所述腺相关病毒载体系统还包括宿主细胞。
  19. 根据权利要求17所述的腺相关病毒载体系统，其特征在于，所述腺相关病毒载体表达系统选自大肠杆菌表达系统、酵母表达系统、昆虫表达系统、哺乳动物表达系统、植物表达系统；优选的，所述腺相关病毒载体表达系统选自质粒瞬时转染表达系统、杆状病毒表达系统、稳转细胞系表达系统、腺病毒表达系统、痘病毒表达系统中的任一种。
  20. 一种腺相关病毒，其特征在于，所述腺相关病毒由权利要求17-19任一所述的腺相关病毒载体系统包装而成。
  21. 一种细胞系，其特征在于，所述细胞系为经权利要求20所述的腺相关病毒感染的细胞系。
  22. 权利要求20所述的腺相关病毒、权利要求21所述的细胞系在制备预防、治疗遗传性凝血因子缺乏病的产品中的用途。
  23. 根据权利要求22所述的用途，其特征在于，所述遗传性凝血因子缺乏病为A型、B型 或C型血友病。
  24. 一种治疗遗传性凝血因子缺乏病的方法，其特征在于，所述方法包括向患者给予有效量的权利要求20所述的腺相关病毒。
  25. 根据权利要求24所述的方法，其特征在于，所述遗传性凝血因子缺乏病为A型、B型或C型血友病。
  26. 根据权利要求24所述的方法，其特征在于，所述遗传性凝血因子缺乏病为哺乳动物遗传性凝血因子缺乏病；优选的，所述遗传性凝血因子缺乏病为人遗传性凝血因子缺乏病。
  27. 根据权利要求24所述的方法，其特征在于，向患者肌肉注射有效量的权利要求20所述的腺相关病毒。
PCT/CN2023/088206 2022-04-19 2023-04-13 一种用于遗传性凝血因子缺乏病治疗的核酸构建体及其用途 Ceased WO2023202469A1 (zh)

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