WO2022037710A1 - 一种提高腺相关病毒产量的核酸构建体及其构建方法 - Google Patents

一种提高腺相关病毒产量的核酸构建体及其构建方法 Download PDF

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WO2022037710A1
WO2022037710A1 PCT/CN2021/121350 CN2021121350W WO2022037710A1 WO 2022037710 A1 WO2022037710 A1 WO 2022037710A1 CN 2021121350 W CN2021121350 W CN 2021121350W WO 2022037710 A1 WO2022037710 A1 WO 2022037710A1
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nucleic acid
acid construct
baculovirus
aav
protein
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PCT/CN2021/121350
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English (en)
French (fr)
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陶宇
吴昊泉
苏玲玲
党颖
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康霖生物科技（杭州）有限公司
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Priority to CA3190059A priority Critical patent/CA3190059A1/en
Priority to US18/022,001 priority patent/US20230365990A1/en
Priority to JP2023512424A priority patent/JP2023538925A/ja
Priority to CN202180055469.3A priority patent/CN116194576A/zh
Priority to EP21857797.1A priority patent/EP4198135A1/en
Priority to AU2021327911A priority patent/AU2021327911A1/en
Publication of WO2022037710A1 publication Critical patent/WO2022037710A1/zh

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14144—Chimeric viral vector comprising heterologous viral elements for production of another viral vector
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material

Definitions

  • the present invention relates to the technical field of gene transfer vectors, in particular to a nucleic acid construct for improving the yield of adeno-associated virus and a construction method thereof.
  • Recombinant adeno-associated virus is a gene transfer vector widely used in basic research and clinical gene therapy. Due to its good safety, wide range of host cells, low immunogenicity, and long time to express foreign genes in vivo, rAAV is regarded as one of the most promising gene transfer vectors in gene therapy and vaccines worldwide. widely used in research. In recent years, more and more researchers have used rAAV for large animal studies and human clinical trials.
  • the AAV genome contains three core elements: ITR sequence, non-structural protein Rep, and structural protein Cap.
  • the insect baculovirus system is a common method for producing recombinant proteins. Compared with the traditional three-plasmid transient production method, insect cells have the advantages of high culture density, serum-free suspension culture, and easy scale-up, and the baculovirus infects cells.
  • the production method is more batch stable. Although researchers began to use it for the production of rAAV since 2002, the packaging efficiency of AAV is not high due to various reasons, such as the ratio of Cap, Rep and AAV vector genomic DNA is not easy to reach the ideal ratio in insect cells. , and the empty shell rate of the recombinant AAV vector packaged by the traditional method is relatively high (11%-34%) (Benskey et al. 2016).
  • the empty-shell AAV is an impurity that must be removed in the preparation of clinical-grade AAV vectors, because the physicochemical properties of empty-shell AAV and AAV carrying the target gene are very similar, and the purification is very difficult.
  • the object of the present invention is to provide a nucleic acid construct and a construction method for improving the yield and quality of recombinant adeno-associated virus, which are used to solve the problems in the prior art.
  • the nucleic acid construct provided by the first aspect of the present invention includes: AAV element, a polynucleotide encoding IE protein and a polynucleotide encoding a baculovirus recombination homology region, and the AAV element includes Polynucleotides encoding Cap proteins, polynucleotides encoding Rep proteins, and AAV cis-acting elements.
  • the IE protein is encoded by one or more genes of Acie0, Acie01, and Acie02, and/or, the baculovirus recombination homology region is selected from hr1, hr2, hr3, hr4, hr5 one or more of them.
  • the nucleic acid construct further comprises the promoter of the IE protein gene.
  • the promoter of the IE protein gene is selected from one or more of Gp64, pH, p6.9 or p10.
  • the nucleic acid construct further comprises a baculovirus promoter.
  • the baculovirus promoter is linked to a baculovirus recombination homology region.
  • the baculovirus promoter is preferably one or more of pH, Gp64, p6.9 or p10.
  • the AAV cis-acting element is selected from an ITR sequence.
  • the nucleic acid construct further includes an exogenous gene of interest.
  • the exogenous gene of interest is embedded in an AAV element.
  • the structure of the nucleic acid construct comprises: IE gene expression cassette-Cap gene expression cassette-ITR-exogenous target gene expression cassette-ITR-Rep gene expression cassette. In some embodiments, the structure of the nucleic acid construct is: IE gene expression cassette-Cap gene expression cassette-ITR-exogenous target gene expression cassette-ITR-Rep gene expression cassette.
  • the nucleotide sequence of the nucleic acid construct comprises SEQ ID NO. 1, or comprises at least 75%, 80%, 85%, 90%, 95%, 96% compared to SEQ ID NO. 1 %, 97%, 98%, or 99% homology to nucleotide sequences. In some embodiments, the nucleotide sequence of the nucleic acid construct is shown in SEQ ID NO.1.
  • the nucleic acid construct is an adeno-associated viral vector or a recombinant baculovirus vector. In some embodiments, the nucleic acid construct is an adeno-associated viral vector. In some embodiments, the nucleic acid construct is a recombinant baculovirus vector. In some embodiments, the recombinant baculovirus vector is preferably a recombinant baculovirus shuttle vector.
  • the second aspect of the present invention provides a recombinant baculovirus, the recombinant baculovirus is obtained by constructing any one of the nucleic acid constructs through a baculovirus system, or obtained from any one of the nucleic acid constructs including any one of the nucleic acid constructs. Nucleic acid constructs of the elements are collectively obtained by construction of the baculovirus system.
  • a third aspect of the present invention provides an adeno-associated virus, which is obtained by packaging any one of the recombinant baculoviruses after infecting cells.
  • the fourth aspect of the present invention provides a cell line, which is a cell line infected with any one of the recombinant baculoviruses.
  • the fifth aspect of the present invention provides an adeno-associated virus vector system, the adeno-associated virus vector system includes a baculovirus system and the nucleic acid construct.
  • the sixth aspect of the present invention provides a method for constructing the nucleic acid construct, the construction method comprising combining an AAV element carrying an exogenous target gene, a polynucleotide encoding an IE protein, and a polynucleotide encoding a baculovirus recombination homology region Integrated into the baculovirus vector backbone.
  • the method includes one or more (eg, two or three) of the following features:
  • the AAV element includes a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis-acting element, for example, the AAV cis-acting element is preferably an ITR sequence;
  • the polynucleotide encoding the IE protein is selected from one or more of Acie0, Acie01 or Acie02;
  • the baculovirus vector backbone is selected from one of pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB or pFastBacHTC; or
  • the baculovirus recombinant homology region is selected from one or more of hr1, hr2, hr3, hr4 or hr5.
  • a seventh aspect of the present invention provides a production method for adeno-associated virus yield, the production method comprising the following steps: infecting an insect cell line with the recombinant baculovirus.
  • nucleic acid construct for improving the yield of adeno-associated virus and its construction method of the present invention have at least the following beneficial effects:
  • the present invention obviously reduces the expression of the target gene inserted into the baculovirus vector in insect cells, and reduces the influence of the target gene expression on the insect cells; the growth index of the production cell is improved to make it more conducive to the packaging production of rAAV.
  • the optimized vector construction method significantly improves the rAAV yield of single cells and unit volume cultures, and reduces production costs, and can be scaled up.
  • Figure 1 shows a schematic diagram of the structure of the pFBd-Cap-ITR-Rep vector of the present invention.
  • Figure 2 shows a schematic diagram of the pFBd-Cap-ITR-Rep vector map of the present invention.
  • Figure 3 shows a schematic diagram of the structure of the pFBd-IE-hr1Cap-ITR-Rep vector of the present invention.
  • Figure 4 shows a schematic diagram of the pFBd-IE-hr1Cap-ITR-Rep vector map of the present invention.
  • Figure 5 shows the comparison of EGFP expression levels 2-4d after infection of SF9 cells with BV-Cap-ITR-Rep and BV-IE-hr1Cap-ITR-Rep of the present invention.
  • Figure 6 shows the WB detection of the AAV Cap monoclonal antibody of the present invention on two BV-infected cell lysates.
  • Figure 7 shows the electron microscope detection of rAAV2 of the present invention (titer 2.0E+13VG/mL).
  • FIG. 8 shows the fluorescence observation of 293T cells after 2d infection of rAAV2 packaged with BV-Cap-ITR-Rep and BV-IE-hr1Cap-ITR-Rep of the present invention.
  • the present invention is based, at least in part, on the discovery that nucleic acid constructs comprising an AAV element, a polynucleotide encoding an IE protein, and a polynucleotide encoding a baculovirus recombination homology region can significantly control the insertion of a gene of interest in baculovirus hosts
  • the expression in insect cells can improve the cell state and improve the packaging efficiency of adeno-associated virus, thereby greatly increasing the production of adeno-associated virus.
  • the present invention can unexpectedly reduce the empty shell rate of rAAV (for example, even increasing the expression of Cap does not lead to an increase in empty shell), generate rAAV with better infectivity, and significantly improve the single cell and unit volume culture. rAAV yield, which is suitable for large-scale production of various adeno-associated virus gene therapy vectors.
  • nucleic acid construct refers to an artificially constructed nucleic acid segment that can be introduced into a target cell or tissue, the nucleic acid construct can be a lentiviral vector or an adeno-associated viral vector, including a vector
  • the backbone is an empty vector and an expression framework.
  • vector refers to a nucleic acid fragment or polynucleotide fragment used to introduce or transfer one or more nucleic acids or one or more polynucleotides into a target cell or tissue.
  • a vector is used to introduce foreign DNA into another cell or tissue.
  • the vector may contain a bacterial resistance gene for growth in bacteria and a promoter for expression of the protein of interest in the organism.
  • DNA can be produced in vitro by PCR or any other suitable technique or techniques known to those skilled in the art.
  • a first aspect of the present invention provides a nucleic acid construct comprising: an AAV element and a polynucleotide of a polynucleotide encoding an IE protein, the AAV element comprising a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein polynucleotides and AAV cis-acting elements.
  • the nucleic acid construct further comprises a polynucleotide encoding a baculovirus recombination homology region.
  • the nucleotide sequence of the nucleic acid construct comprises SEQ ID NO. 1, or comprises at least 75%, 80%, 85%, 90%, 95%, 96% compared to SEQ ID NO. 1 %, 97%, 98%, or 99% homology to nucleotide sequences.
  • the nucleotide sequence of the nucleic acid construct is shown in SEQ ID NO. 1 below.
  • the AAV elements are derived from different serotypes of AAV, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV DJ, AAV DJ/8, AAV rh10, AAV Retro , AAV PHP.eB, AAV PHP.B, or AAV PHP.S.
  • the capsid proteins of different serotypes of AAV recognize different receptors on the cell surface, so the infection efficiency of different tissue cells may vary greatly, showing a certain organ targeting specificity.
  • AAV of suitable serotype can be selected according to the specific experimental purpose, so as to achieve efficient transduction of specific serotype AAV to specific types of cells and tissues.
  • the AAV element is derived from AAV1. In some embodiments, the AAV element is derived from AAV2. In some embodiments, the AAV element is derived from AAV3. In some embodiments, the AAV element is derived from AAV4. In some embodiments, the AAV element is derived from AAV5. In some embodiments, the AAV element is derived from AAV6. In some embodiments, the AAV element is derived from AAV7. In some embodiments, the AAV element is derived from AAV8. In some embodiments, the AAV element is derived from AAV9. In some embodiments, the AAV element is derived from AAV DJ. In some embodiments, the AAV element is derived from AAV DJ/8.
  • the AAV element is derived from AAV rh10. In some embodiments, the AAV element is derived from AAV Retro. In some embodiments, the AAV element is derived from AAV PHP.eB/PHP.B/PHP.S.
  • Cap proteins are structural proteins, usually including VP1, VP2 and VP3 structural proteins. It is believed that in some embodiments, the three structural proteins can be assembled into the capsid protein of AAV.
  • the Rep proteins are non-structural proteins, usually including Rep78 and 52 proteins. In some embodiments, Rep proteins are believed to regulate DNA replication and packaging.
  • the AAV cis-acting element is selected from inverted terminal repeats (ITR sequences). It is believed that in some embodiments, the ITR sequence folds into a hairpin structure, the only known cis-acting element required for AAV DNA replication initiation and packaging of recombinant AAV viral particles.
  • ITR sequences inverted terminal repeats
  • the nucleic acid construct further includes an exogenous gene of interest.
  • the exogenous gene of interest is carried by an AAV element, ie, the exogenous gene of interest is embedded in the AAV element.
  • the exogenous gene of interest is inserted between the ITR sequences at both ends.
  • the size of the exogenous target gene is less than or equal to 3.5kb. It is believed that in some embodiments, due to the size limitation between the two ITR sequences, the size of the exogenous target gene should not be too large, otherwise it may lead to poor packaging effect.
  • the types of the exogenous genes vary according to the purpose of the experiment.
  • the exogenous genes can be genes related to the treatment of various diseases, such as AADC, FVIII, FIX, and can also be tool genes commonly used in laboratories, such as EGFP, mCherry gene, and the like.
  • the nucleic acid construct further comprises the promoter of the IE protein gene. It is believed that in some embodiments, the promoter of the IE protein gene is selected from one or more of Gp64, pH, p6.9 or p10. In some embodiments, the promoter of the IE protein gene comprises Gp64. In some embodiments, the promoter of the IE protein gene includes pH. In some embodiments, the promoter of the IE protein gene comprises p6.9. In some embodiments, the promoter of the IE protein gene comprises p10.
  • the promoter of the IE protein gene is selected from a pH strong promoter. It is believed that in some embodiments, the addition of additional copies of ie to the nucleic acid construct and expression under the control of a strong promoter pH above viral endogenous levels increases the survival of cells at a later stage after baculovirus infection .
  • the IE protein is selected from the proteins encoded by one or more (eg, two or three) genes of Acie0, Acie01, or Acie02. In some embodiments, the IE protein is selected from the protein encoded by Acie0, ie, IE0. The nucleotide sequence of the Acie0 is shown in SEQ ID NO.10, and the amino acid sequence of the IEO protein is shown in SEQ ID NO.11. In some embodiments, the IE protein is selected from the protein encoded by Acie01, ie IE1. The nucleotide sequence of the Acie01 is shown in SEQ ID NO.12, and the amino acid sequence of the IE1 protein is shown in SEQ ID NO.13.
  • the IE protein is selected from the protein encoded by Acie02, ie the IE2 protein.
  • the nucleotide sequence of the Acie02 is shown in SEQ ID NO.14
  • the amino acid sequence of the IE2 protein is shown in SEQ ID NO.15.
  • the IE1 protein is the product of the baculovirus immediate early gene 1 (ie1), which may also be referred to as a baculovirus transcriptional regulator protein. It is a multifunctional protein with the ability to activate early and late viral genes and participate in viral genome replication, and is involved in the regulation of the viral cycle.
  • ie1 baculovirus immediate early gene 1
  • the baculovirus recombinant homology region is selected from hr1 (nucleotide sequence shown in SEQ ID NO. 16), hr2 (nucleotide sequence shown in SEQ ID NO. 17) ), hr3 (the nucleotide sequence is shown in SEQ ID NO.18), hr4 (the nucleotide sequence of hr4left is shown in SEQ ID NO.19, and the nucleotide sequence of hr4right is shown in SEQ ID NO.20) , hr5 (nucleotide sequence as shown in SEQ ID NO. 21) in one or more (for example two, three, four or five).
  • the baculovirus recombination homology region is selected from hr1. It is believed that in some embodiments, the hr1 is a repetitive sequence interspersed in the baculovirus genome, and hr1 is both the baculovirus origin of replication and functions as an enhancer. Although the enhancement effect is not obvious in the early stage of infection, it becomes more significant in the later stage of infection.
  • the baculovirus recombination homology region (hr) comprises hr2.
  • the baculovirus recombination homology region (hr) comprises hr3.
  • the baculovirus recombination homology region (hr) comprises hr4.
  • the baculovirus recombination homology region (hr) comprises hr5.
  • the nucleic acid construct further comprises a baculovirus promoter.
  • the baculovirus promoter is selected from one or more (eg, two, three, or four) of polyhedrin promoter (pH), Gp64, p6.9, or p10. It is believed that in some embodiments, the hr is cis-linked to the baculovirus promoter, which can promote IE-mediated transactivation, and at the same time, IE will bind to hr1 in a dimer form to increase the expression of downstream proteins.
  • the structure of the nucleic acid construct comprises: IE gene expression cassette-Cap gene expression cassette-hr1-ITR-exogenous target gene expression cassette-ITR-Rep gene expression cassette. In some embodiments, the structure of the nucleic acid construct is: IE gene expression cassette-Cap gene expression cassette-ITR-exogenous target gene expression cassette-ITR-Rep gene expression cassette.
  • the gene expression cassette includes the gene and its promoter.
  • the structure of the nucleic acid construct includes: pA-IE1-pH-pA-Cap-p6.9p10-hr1-ITR-exogenous target gene and its promoter-pA-ITR-pH- Rep-pA.
  • the structure of the nucleic acid construct is: pA-IE1-pH-pA-Cap-p6.9p10-hr1-ITR-exogenous target gene and its promoter-pA-ITR-pH- Rep-pA.
  • the nucleic acid construct is an adeno-associated virus vector or a recombinant baculovirus vector.
  • the nucleic acid construct is an adeno-associated viral vector.
  • the nucleic acid construct is a recombinant baculovirus vector.
  • the recombinant baculovirus vector is a recombinant baculovirus shuttle vector.
  • the adeno-associated virus vector or recombinant baculovirus vector further includes a vector backbone.
  • the vector backbone may be selected from suitable commercially available vector backbones, such as pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB, or pFastBacHTC, among others.
  • suitable commercially available vector backbones such as pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB, or pFastBacHTC, among others.
  • the vector backbone is pFastBacdual.
  • the rAAV titer obtained by packaging the nucleic acid construct is at least 1 ⁇ 10 12 VG/mL (e.g., at least 1.5 ⁇ 10 12 , 2 ⁇ 10 12 , 2.5 ⁇ 10 12 , 3 ⁇ 10 12 , 3.5 ⁇ 10 12 , 4 ⁇ 10 12 , 4.5x1012,5x1012,5.5x1012,6x1012,7x1012,7.5x1012,8x1012,8.5x1012,9x1012,9.5x1012,1x1013,5x1013 , or 1x1014 VG / mL ) _ _ _ _ _
  • the number of copies of the AAV genome contained in each ml of viral broth is at least 1x10 12 (eg at least 1.5x10 12 , 2x10 12 , 2.5x10 12 , 3x10 12 , 3.5x10 12 , 4x10 12 , 4.5x10 12 , 5x10 12 , 5.5 x10 12 , 6x10 12 , 7x10 12 ,
  • the rAAV titer obtained by packaging the nucleic acid construct is at least 2.60E+12VG/mL, that is, the number of copies of the AAV genome contained in each ml of viral culture fluid is at least 2.60E+12.
  • the output of the rAAV is detected by qPCR method, and the detection steps are as follows:
  • the standard used in quantitative PCR was pAAV-MCS plasmid linearized by single enzyme digestion with Pvu I-HF (NEB).
  • the primer sequences used for quantitative PCR are:
  • the quantitative PCR running program is: 95°C for 60s, (95°C for 15s, 60°C for 30s, 40cycles). After the standard curve was drawn according to the CT value obtained by quantitative PCR and the standard concentration, the sample titer was converted.
  • the second aspect of the present invention provides a recombinant baculovirus
  • the recombinant baculovirus is obtained by constructing any one of the nucleic acid constructs through a baculovirus system, or by including any element in any of the nucleic acid constructs
  • the nucleic acid constructs were obtained through baculovirus system construction.
  • nucleic acid constructs comprising any element in any of the nucleic acid constructs mainly refer to: nucleic acid constructs comprising AAV elements, nucleic acid constructs comprising polynucleotides encoding IE proteins, including those encoding baculoviruses. polynucleotides of the source region.
  • the nucleic acid construct comprising an AAV element can also be selected from nucleic acid constructs comprising a polynucleotide encoding a Cap protein, nucleic acid constructs comprising a polynucleotide encoding a Rep protein, nucleic acid constructs comprising a cis-acting element encoding an AAV .
  • the recombinant baculovirus can be obtained by jointly constructing the above nucleic acid constructs through the baculovirus system.
  • the baculovirus system is selected from Bac-to-Bac system (derived from ThermoFisher/Invitrogen), flashBac/BacMagic system (derived from Mirus/EMD/OET/Nextgen), BaculoDirect system (derived from ThermoFisher/Invitrogen) or BacPAK6/ Baculogold system (from BD Biosciences/Clonetech).
  • Bac-to-Bac system derived from ThermoFisher/Invitrogen
  • flashBac/BacMagic system derived from Mirus/EMD/OET/Nextgen
  • BaculoDirect system derived from ThermoFisher/Invitrogen
  • BacPAK6/ Baculogold system from BD Biosciences/Clonetech.
  • the recombinant baculovirus is obtained by transforming competent cells with the nucleic acid construct, extracting Bacmid, and transfecting SF9 insect cells.
  • the competent cells can be selected from any applicable competent cells in the art, as long as the purpose of the present invention is not limited.
  • the competent cells can be DH10Bac.
  • the third aspect of the present invention provides an adeno-associated virus (AAV) obtained by packaging any one of the recombinant baculoviruses.
  • AAV adeno-associated virus
  • the adeno-associated virus can be used to treat various diseases, such as hemophilia, spinal muscular atrophy, Duchenne muscular dystrophy, Parkinson's disease, age-related macular degeneration and the like.
  • a fourth aspect of the present invention provides a cell line, which is a cell line infected with any one of the recombinant baculoviruses.
  • the cell line is an insect cell line, eg, SF9 cells, SF21 cells, High5 cells.
  • a fifth aspect of the present invention provides an adeno-associated virus vector system, the adeno-associated virus vector system includes a baculovirus system and the nucleic acid construct.
  • the sixth aspect of the present invention provides a method for constructing the nucleic acid construct, which comprises integrating an AAV element carrying an exogenous target gene and a polynucleotide encoding an IE protein into a baculovirus vector backbone.
  • the construction method also includes integrating a polynucleotide encoding a baculovirus recombination homology region into a baculovirus vector backbone.
  • the AAV elements include a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis-acting element.
  • the AAV cis-acting element is selected from inverted terminal repeats (ITR sequences).
  • the polynucleotide encoding the IE protein is linked to a strong promoter pH.
  • the polynucleotide encoding a baculovirus recombinant homology region protein is linked in cis to a baculovirus promoter.
  • the baculovirus promoter is p6.9 or p10.
  • the baculovirus vector backbone is selected from the group consisting of pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB, or pFastBacHTC, among others.
  • the baculovirus recombination homology region is selected from one or more (eg, two, three, four or five) of hr1, hr2, hr3, hr4, or hr5.
  • a seventh aspect of the present invention provides a method for producing adeno-associated virus, the production method comprising the following steps: infecting an insect cell line with the recombinant baculovirus.
  • the production method of adeno-associated virus can improve the yield of adeno-associated virus.
  • the cell line is selected from insect cell lines. In one embodiment, the insect cell line is selected from SF9 cells.
  • An eighth aspect of the present invention provides a method of treating a disease, said method comprising administering to a patient an effective amount of any one of said adeno-associated viruses.
  • the disease includes, for example, hemophilia, spinal muscular atrophy, Duchenne muscular dystrophy, Parkinson's disease, age-related macular degeneration, and the like.
  • the present invention also includes any of the following numbered paragraphs:
  • a nucleic acid construct characterized in that the nucleic acid construct comprises: an AAV element, a polynucleotide encoding an IE protein, the AAV element comprising a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and AAV cis-acting element.
  • nucleic acid construct according to paragraph 1, wherein the nucleic acid construct further comprises a polynucleotide encoding a baculovirus recombinant homology region.
  • nucleic acid construct according to paragraph 2 wherein the IE protein is selected from one or more encoded proteins of Acie0, Acie01 or Acie02, and/or the baculovirus recombinant homology
  • the region is selected from one or more of hr1, hr2, hr3, hr4 or hr5.
  • nucleic acid construct according to any of paragraphs 1-3, wherein the nucleic acid construct also comprises a promoter of an IE protein gene, and the promoter of the IE protein gene is selected from Gp64, pH, p6. One or more of 9 or p10.
  • nucleic acid construct according to any one of paragraphs 1-4, wherein the nucleic acid construct further comprises a baculovirus promoter, and the baculovirus promoter is linked to the baculovirus recombination homology region,
  • the baculovirus promoter is preferably one or more of pH, Gp64, p6.9 or p10.
  • nucleic acid construct according to any of paragraphs 1-5, wherein the AAV cis-acting element is selected from ITR sequences.
  • nucleic acid construct according to any one of paragraphs 1-6, wherein the nucleic acid construct further comprises an exogenous gene of interest embedded in an AAV element.
  • nucleic acid construct according to any one of paragraphs 1-7, wherein the nucleic acid construct has the following structure: IE gene expression box-Cap gene expression box-ITR-exogenous target gene expression box-ITR- Rep gene expression box.
  • nucleic acid construct according to any of paragraphs 1-8, wherein the nucleic acid construct has a nucleotide sequence such as SEQ ID NO.1.
  • nucleic acid construct according to any of paragraphs 1-9, wherein the nucleic acid construct is an adeno-associated virus vector or a recombinant baculovirus vector, and the recombinant baculovirus vector is preferably a recombinant baculovirus Shuttle vector.
  • a recombinant baculovirus characterized in that the recombinant baculovirus is obtained by constructing the nucleic acid construct described in any of paragraphs 1-10 through a baculovirus system, or obtained from any of paragraphs 1-10 The nucleic acid constructs of any element in the nucleic acid constructs are collectively obtained by constructing the baculovirus system.
  • An adeno-associated virus wherein the adeno-associated virus is obtained by infecting cells with the recombinant baculovirus described in paragraph 11 after packaging.
  • a cell line wherein the cell line is a cell line infected with the recombinant baculovirus of paragraph 11.
  • adeno-associated virus vector system wherein the adeno-associated virus vector system comprises a baculovirus system and the nucleic acid construct described in any of paragraphs 1-10.
  • the AAV element includes a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis-acting element, and the AAV cis-acting element is preferably an ITR sequence;
  • the IE protein is encoded by one or more of the Acie0, Acie01 or Acie02 genes;
  • the baculovirus vector backbone is selected from one of pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB or pFastBacHTC;
  • the baculovirus recombination homology region is selected from one or more of hr1, hr2, hr3, hr4 or hr5;
  • a production method for adeno-associated virus characterized in that, the production method comprises the following steps: infecting an insect cell line with the recombinant baculovirus described in paragraph 11.
  • the following examples take EGFP as an exogenous target gene as an example.
  • Example 1 Construction of recombinant baculovirus shuttle vector and acquisition of recombinant baculovirus
  • Cap and Rep proteins are all derived from the AAV2 genome (GenBank: AF043303.1), according to the ribosome leak scanning mechanism reference (Smith RH, Levy JR, Kotin RM: A simplified baculovirus-AAV expression vector system coupledd with one-step affinity purification yields high-titer rAAV stocks from insect cells.Mol Ther 2009,17(11):1888-1896.) point mutation of the bases; Acie01(GenBank:NC_001623.1), baculovirus Recombinant homology region hr1 (GenBank: M14313.1).
  • Cap gene sequence (nucleotide sequence SEQ ID NO: 4), Rep gene sequence (nucleotide sequence SEQ ID NO: 5), hr1 and p6.9, p10 promoter combination (hr1p6 .9p10, nucleotide sequence SEQ ID NO:6), pH promoter and Acie01 combination (pH-Acie01, nucleotide sequence SEQ ID NO:7).
  • the two recombinant vectors were transformed into DH10Bac competent cells (Invitrogen), respectively, and screened by blue and white spots. White positive clones were picked and cultured, and the baculovirus plasmid (Bacmid) was extracted using PureLink TM HiPure Plasmid DNA Purification Kits (Invitrogen). The two kinds of Bacmid were transfected into adherent SF9 cells (Gbico) respectively, and the primary recombinant baculovirus was harvested 3 days later and amplified to the P2 generation.
  • the P2 generation recombinant baculoviruses were named BV-Cap-ITR-Rep and BV-IE-hr1Cap-ITR-Rep, respectively, and the virus titers were determined by TCID50 method. Please refer to the Bac to Bac Expression System Operation Manual (Invitrogen) for details.
  • the standard used in quantitative PCR is the pAAV-EGFP plasmid linearized by Pvu I-HF (NEB) single enzyme digestion (the plasmid sequence is shown in the following SEQ ID NO: 22).
  • the primer sequences used for quantitative PCR are:
  • the quantitative PCR running program is: 95°C for 60s, (95°C for 15s, 60°C for 30s, 40cycles).
  • the calculated rAAV yields of BV-Cap-ITR-Rep and BV-IE-hr1Cap-ITR-Rep packaging were 2.39E+04VG/cell (equivalent to 7.18E+10VG/mL) and 8.67E+05VG/cell (equivalent to at 2.60E+12VG/mL).
  • the cells infected with the two baculoviruses were observed under an inverted fluorescence microscope.
  • the optimized recombinant baculovirus can greatly reduce the expression of the target gene (in this example, the target gene is EGFP).
  • Figure 5 Decreased expression of the gene of interest (EGFP) appears to attenuate the interference with intracellular rAAV packaging, making it more favorable for the expression of structural and functional proteins associated with rAAV packaging.
  • the cell lysate was purified with POROS TM CaptureSelect TM affinity chromatography packing (Thermo), and the titer was determined by fluorescence quantitative PCR (the same as in Example 2), and the purified rAAV2 was subjected to negative staining, transmission electron microscopy. observed below ( Figure 7). Genome-packaged rAAV2 appears as a solid particle, and the nucleic acid-free defective rAAV particle is stained dark in the middle. Overall, the morphology was intact, and the empty shell rate was significantly reduced ( ⁇ 3%) compared with the traditional method (Benskey et al. 2016), indicating that the use of the optimized baculovirus vector construct significantly reduced the proportion of empty shell viruses in the packaging product. Significantly reduces the pressure on downstream steps such as empty virus removal.

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Abstract

提供一种提高腺相关病毒产量的核酸构建体及其构建方法，核酸构建体包括：腺相关病毒(AAV)元件、编码IE蛋白的多核苷酸；AAV元件包括编码Cap蛋白的多核苷酸、编码Rep蛋白的多核苷酸和AAV顺式作用元件。构建方法包括将携带外源目的基因的AAV元件和编码IE蛋白的多核苷酸的多核苷酸整合入杆状病毒载体骨架中。该方法获得的重组腺相关病毒(rAAV)具有较低的空壳率，同时提高了单个细胞以及单位体积培养物的rAAV产量，降低了生产成本，并且易于放大规模。

Description

一种提高腺相关病毒产量的核酸构建体及其构建方法 技术领域
本发明涉及基因转移载体技术领域，特别是涉及一种提高腺相关病毒产量的核酸构建体及其构建方法。
背景技术
重组腺相关病毒(rAAV)是一种在基础研究和临床基因治疗中被广泛应用的基因转移载体。由于其安全性好、宿主细胞范围广、免疫原性低、在体内表达外源基因时间长等特点，rAAV被视为最有前途的基因转移载体之一，在世界范围内的基因治疗和疫苗研究中得到广泛应用。近年来，越来越多的研究者使用rAAV进行大型动物研究和人类临床试验。AAV基因组包含三个核心元件：ITR序列、非结构蛋白Rep、结构蛋白Cap。
昆虫杆状病毒系统是生产重组蛋白的常用手段，相比于传统三质粒瞬转生产的方式，昆虫细胞具有培养密度高、可无血清悬浮培养、易于放大规模等优点，且杆状病毒感染细胞的生产方式更具有批次稳定性。尽管从2002年开始，研究者们开始将其用于rAAV的生产，但是昆虫细胞中由于各种原因，例如Cap、Rep和AAV载体基因组DNA的比例不容易达到理想比例，AAV的包装效率不高，而且传统的方法包装完成的重组AAV载体空壳率较高(11％-34％)(Benskeyetal.2016)。而空壳AAV是制备临床级AAV载体中必须要去除的杂质，因为空壳AAV和携带目的基因的AAV理化性质非常接近，纯化难度很高。
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提高重组腺相关病毒产量及其质量的核酸构建体及其构建方法，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，本发明第一方面提供的核酸构建体包括：AAV元件、编码IE蛋白的多核苷酸和编码杆状病毒重组同源区的多核苷酸，所述AAV元件包括编码Cap蛋白的多核苷酸、编码Rep蛋白的多核苷酸和AAV顺式作用元件。
在一些实施方式中，所述IE蛋白由Acie0、Acie01、Acie02中的一种或几种基因编码，和/或，所述杆状病毒重组同源区选自hr1、hr2、hr3、hr4、hr5中的一种或几种。
在一些实施方式中，所述核酸构建体还包括IE蛋白基因的启动子。在一些实施方式中，所述IE蛋白基因的启动子选自Gp64、pH、p6.9或p10中的一种或几种。
在一些实施方式中，所述核酸构建体还包括杆状病毒启动子。在一些实施方式中，所述 杆状病毒启动子与杆状病毒重组同源区连接。在一些实施方式中，所述杆状病毒启动子优选为pH、Gp64、p6.9或p10中的一种或几种。
在一些实施方式中，所述AAV顺式作用元件选自ITR序列。
在一些实施方式中，所述核酸构建体还包括外源目的基因。在一些实施方式中，所述外源目的基因嵌入AAV元件中。
在一些实施方式中，所述核酸构建体的结构包括：IE基因表达框-Cap基因表达框-ITR-外源目的基因表达框-ITR-Rep基因表达框。在一些实施方式中，所述核酸构建体的结构为：IE基因表达框-Cap基因表达框-ITR-外源目的基因表达框-ITR-Rep基因表达框。
在一些实施方式中，所述核酸构建体的核苷酸序列包括SEQ ID NO.1，或者包括跟SEQ ID NO.1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。在一些实施方式中，所述核酸构建体的核苷酸序列如SEQ ID NO.1所示。
在一些实施方式中，所述核酸构建体为腺相关病毒载体或重组杆状病毒载体。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，所述核酸构建体为重组杆状病毒载体。在一些实施方式中，所述重组杆状病毒载体优选为重组杆状病毒穿梭载体。
本发明第二方面提供一种重组杆状病毒，所述重组杆状病毒为由任何一种所述的核酸构建体经杆状病毒系统构建获得，或由包括任一所述核酸构建体中任意元件的核酸构建体共同经杆状病毒系统构建获得。
本发明第三方面提供一种腺相关病毒，所述腺相关病毒为由任何一种所述的重组杆状病毒感染细胞后包装获得。
本发明第四方面提供一种细胞系，所述细胞系为经任何一种所述的重组杆状病毒感染的细胞系。
本发明第五方面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杆状病毒系统和所述的核酸构建体。
本发明第六方面提供所述核酸构建体的构建方法，所述构建方法包括将携带外源目的基因的AAV元件、编码IE蛋白的多核苷酸和编码杆状病毒重组同源区的多核苷酸整合入杆状病毒载体骨架中。
所述方法包括以下特征中的一项或几项(例如两项或三项)：
1)所述AAV元件包括编码Cap蛋白的多核苷酸、编码Rep蛋白的多核苷酸和AAV顺式作用元件，例如所述AAV顺式作用元件优选为ITR序列；
2)所述编码IE蛋白的多核苷酸选自Acie0、Acie01或Acie02中的一种或几种；
3)所述杆状病毒载体骨架选自pFastBacdual、pFastBac1、pFastBacHTA、pFastBacHTB或pFastBacHTC中的一种；或
4)所述杆状病毒重组同源区选自hr1、hr2、hr3、hr4或hr5中的一种或几种。
本发明第七方面提供一种腺相关病毒产量的生产方法，所述生产方法包括以下步骤：将所述的重组杆状病毒感染昆虫细胞系。
如上所述，本发明的一种提高腺相关病毒产量的核酸构建体及其构建方法，至少具有以下有益效果：
1)本发明明显降低了杆状病毒载体中插入目标基因在昆虫细胞中的表达，减少了目标基因表达对昆虫细胞的影响；改善生产细胞生长指标使其更有利于rAAV的包装生产。
2)相比传统的贴壁293和293T细胞的生产，以及常规的杆状病毒生产方式，本优化后的载体构建方法明显提高了单个细胞以及单位体积培养物的rAAV产量，降低了生产成本，并且可规模放大。
3)应用范围广，包括各种规模和类型的腺相关病毒基因治疗载体生产。
附图说明
图1显示为本发明的pFBd-Cap-ITR-Rep载体结构示意图。
图2显示为本发明的pFBd-Cap-ITR-Rep载体图谱示意图。
图3显示为本发明的pFBd-IE-hr1Cap-ITR-Rep载体结构示意图。
图4显示为本发明的pFBd-IE-hr1Cap-ITR-Rep载体图谱示意图。
图5显示为本发明的BV-Cap-ITR-Rep和BV-IE-hr1Cap-ITR-Rep感染SF9细胞后2-4d EGFP表达量比较。
图6显示为本发明的AAV Cap单抗对两种BV感染细胞裂解液的WB检测。
图7显示为本发明的rAAV2电镜检测(滴度2.0E+13VG/mL)。
图8显示为本发明的BV-Cap-ITR-Rep和BV-IE-hr1Cap-ITR-Rep包装的rAAV2感染293T细胞2d后荧光观察。
具体实施方式
本发明至少部分基于以下发现，即包含AAV元件，编码IE蛋白的多核苷酸，以及编码杆状病毒重组同源区的多核苷酸的核酸构建体可以明显控制插入目标基因在作为杆状病毒宿主的昆虫细胞内的表达，改善细胞状态，提高腺相关病毒的包装效率，从而大幅增加腺相关 病毒的产量。本发明能够出人意料地降低rAAV的空壳率(比如，即使提高Cap的表达量也不会导致空壳的增加)，产生具有更好感染性的rAAV，以及明显提高单个细胞和单位体积培养物的rAAV产量，从而适合大规模的生产各种腺相关病毒基因治疗载体。
除非下文另外定义，本发明所提及的所有技术和科学用语具有本发明所属领域的技术人员通常理解的意义。
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，所述核酸构建体可以为慢病毒载体或腺相关病毒载体，慢病毒载体或腺相关病毒载体包括载体骨架即空载体与表达框架。
术语“载体”是指用于将一个或多个核酸或一个或多个多核苷酸引入或转移到靶细胞或组织中的核酸片段或多核苷酸片段。典型地，载体用于将外源DNA引入另一个细胞或组织中。载体可以包含用于在细菌中生长的细菌抗性基因和用于在生物体中表达目的蛋白质的启动子。DNA可以通过PCR或任何其他本领域技术人员已知的一种或多种合适的技术在体外产生。
本发明第一方面提供一种核酸构建体，所述核酸构建体包括：AAV元件和编码IE蛋白的多核苷酸的多核苷酸，所述AAV元件包括编码Cap蛋白的多核苷酸、编码Rep蛋白的多核苷酸和AAV顺式作用元件。
在一种实施方式中，所述核酸构建体还包括编码杆状病毒重组同源区的多核苷酸。
在一些实施方式中，所述核酸构建体的核苷酸序列包括SEQ ID NO.1，或者包括跟SEQ ID NO.1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。在一些实施方式中，所述核酸构建体的核苷酸序列如以下SEQ ID NO.1所示。
SEQ ID NO:1：
Figure PCTCN2021121350-appb-000001
Figure PCTCN2021121350-appb-000002
Figure PCTCN2021121350-appb-000003
Figure PCTCN2021121350-appb-000004
Figure PCTCN2021121350-appb-000005
Figure PCTCN2021121350-appb-000006
Figure PCTCN2021121350-appb-000007
Figure PCTCN2021121350-appb-000008
在一些实施方式中，所述AAV元件来源于不同血清型的AAV，例如AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV DJ、AAV DJ/8、AAV rh10、AAV Retro、AAV PHP.eB、AAV PHP.B或AAV PHP.S。不同血清型AAV的衣壳蛋白识别细胞表面的受体不同，因而对不同组织细胞的侵染效率差异可能很大，表现出一定的器官靶向特异性。可以根据具体的实验目的选择合适血清型的AAV，从而实现特定血清型AAV对特定类型细胞组织特异性的高效转导。
在一些实施方式中，所述AAV元件来源于AAV1。在一些实施方式中，所述AAV元件来源于AAV2。在一些实施方式中，所述AAV元件来源于AAV3。在一些实施方式中，所述AAV元件来源于AAV4。在一些实施方式中，所述AAV元件来源于AAV5。在一些实施方 式中，所述AAV元件来源于AAV6。在一些实施方式中，所述AAV元件来源于AAV7。在一些实施方式中，所述AAV元件来源于AAV8。在一些实施方式中，所述AAV元件来源于AAV9。在一些实施方式中，所述AAV元件来源于AAV DJ。在一些实施方式中，所述AAV元件来源于AAV DJ/8。在一些实施方式中，所述AAV元件来源于AAV rh10。在一些实施方式中，所述AAV元件来源于AAV Retro。在一些实施方式中，所述AAV元件来源于AAV PHP.eB/PHP.B/PHP.S。
所述Cap蛋白为结构蛋白，通常包括VP1、VP2和VP3结构蛋白。据信在一些实施方式中，三种结构蛋白可组装成AAV的衣壳蛋白。
所述Rep蛋白为非结构蛋白，通常包括Rep78和52蛋白。据信在一些实施方式中，Rep蛋白可调控DNA的复制和包装。
在一种实施方式中，所述AAV顺式作用元件选自反向末端重复序列(ITR序列)。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病毒颗粒所需的唯一已知顺式作用元件。
在一些实施方式中，所述核酸构建体还包括外源目的基因。在一些实施方式中，所述外源目的基因由AAV元件携带，即外源目的基因嵌入AAV元件中。
具体的，在一些实施方式中，所述外源目的基因嵌入两端ITR序列之间。
较佳的，在一些实施方式中，所述外源目的基因的大小≤3.5kb。据信在一些实施方式中，由于受到两端ITR序列之间的大小限制，因此外源目的基因的大小一般不可过大，否则可能会导致包装效果较差。
所述外源基因的种类根据实验目的不同而不同。所述外源基因可以为各种疾病治疗的相关基因，例如AADC、FVIII、FIX，还可以为实验室常用的工具基因如EGFP、mCherry基因等等。
据信在一些实施方式中，所述核酸构建体还包括IE蛋白基因的启动子。据信在一些实施方式中，所述IE蛋白基因的启动子选自Gp64、pH、p6.9或p10中的一种或几种。在一些实施方式中，所述IE蛋白基因的启动子包括Gp64。在一些实施方式中，所述IE蛋白基因的启动子包括pH。在一些实施方式中，所述IE蛋白基因的启动子包括p6.9。在一些实施方式中，所述IE蛋白基因的启动子包括p10。
较佳的，在一些实施方式中，所述IE蛋白基因的启动子选自pH强启动子。据信在一些实施方式中，在核酸构建体中加入ie的额外拷贝并在强启动子pH的控制下表达，使其高于病毒内源性水平，增加了杆状病毒感染后晚期细胞的存活。
在一些实施方式中，所述IE蛋白选自由Acie0、Acie01或Acie02中的一种或几种(例如两种或三种)基因编码的蛋白。在一些实施方式中，所述IE蛋白选自Acie0编码的蛋白，即IE0。所述Acie0的核苷酸序列如SEQ ID NO.10所示，所述IE0蛋白的氨基酸序列如SEQ ID NO.11所示。在一些实施方式中，所述IE蛋白选自Acie01编码的蛋白，即IE1。所述Acie01的核苷酸序列如SEQ ID NO.12所示，所述IE1蛋白的氨基酸序列如SEQ ID NO.13所示。在一些实施方式中，所述IE蛋白选自Acie02编码的蛋白，即IE2蛋白。所述Acie02的核苷酸序列如SEQ ID NO.14所示，所述IE2蛋白的氨基酸序列如SEQ ID NO.15所示。
SEQ ID NO.10-15的序列如下所示：
SEQ ID NO.10：
Figure PCTCN2021121350-appb-000009
SEQ ID NO.11：
Figure PCTCN2021121350-appb-000010
SEQ ID NO.12：
Figure PCTCN2021121350-appb-000011
Figure PCTCN2021121350-appb-000012
SEQ ID NO.13：
Figure PCTCN2021121350-appb-000013
SEQ ID NO.14：
Figure PCTCN2021121350-appb-000014
SEQ ID NO.15：
Figure PCTCN2021121350-appb-000015
据信在一些实施方式中，所述IE1蛋白为杆状病毒立即早期基因1(ie1)的产物，所述IE蛋白亦可称为杆状病毒转录调控因子蛋白。它是一种多功能蛋白，具有激活早期和晚期病毒基因和参与病毒基因组复制的能力，参与病毒周期的调控。
在一些实施方式中，所述杆状病毒重组同源区(hr)选自hr1(核苷酸序列如SEQ ID NO.16所示)、hr2(核苷酸序列如SEQ ID NO.17所示)、hr3(核苷酸序列如SEQ ID NO.18所示)、hr4(hr4left的核苷酸序列如SEQ ID NO.19所示，hr4right的核苷酸序列如SEQ ID NO.20所示)、hr5(核苷酸序列如SEQ ID NO.21所示)中的一种或几种(例如两种，三种，四种或五种)。
SEQ ID NO.16-21的序列如下所示：
SEQ ID NO.16：
Figure PCTCN2021121350-appb-000016
Figure PCTCN2021121350-appb-000017
SEQ ID NO.17：
Figure PCTCN2021121350-appb-000018
SEQ ID NO.18：
Figure PCTCN2021121350-appb-000019
SEQ ID NO.19：
Figure PCTCN2021121350-appb-000020
SEQ ID NO.20：
Figure PCTCN2021121350-appb-000021
SEQ ID NO.21：
Figure PCTCN2021121350-appb-000022
Figure PCTCN2021121350-appb-000023
较佳的，在一些实施方式中，所述杆状病毒重组同源区选自hr1。据信在一些实施方式中，所述hr1为分散在杆状病毒基因组中的重复序列，hrl既是杆状病毒复制原点，又具有增强子的作用。增强作用尽管在感染初期不明显，但在感染后期，变得比较显著。在一些实施方式中，所述杆状病毒重组同源区(hr)包括hr2。在一些实施方式中，所述杆状病毒重组同源区(hr)包括hr3。在一些实施方式中，所述杆状病毒重组同源区(hr)包括hr4。在一些实施方式中，所述杆状病毒重组同源区(hr)包括hr5。
在一些实施方式中，所述核酸构建体还包括杆状病毒启动子。在一些实施方式中，所述杆状病毒启动子选自多角体启动子(pH)、Gp64、p6.9或p10中的一种或几种(例如两种，三种，或四种)。据信在一些实施方式中，所述hr与杆状病毒启动子顺式连接，可以促进IE介导的反式激活，同时IE会以二聚体形式与hr1结合，提高下游蛋白的表达量。
在一些实施方式中，所述核酸构建体的结构包括：IE基因表达框-Cap基因表达框-hr1-ITR-外源目的基因表达框-ITR-Rep基因表达框。在一些实施方式中，所述核酸构建体的结构为：IE基因表达框-Cap基因表达框-ITR-外源目的基因表达框-ITR-Rep基因表达框。
所述基因表达框包括基因及其启动子。
在一较佳实施方式中，所述核酸构建体的结构包括：pA-IE1-pH-pA-Cap-p6.9p10-hr1-ITR-外源目的基因及其启动子-pA-ITR-pH-Rep-pA。在一较佳实施方式中，所述核酸构建体的结构为：pA-IE1-pH-pA-Cap-p6.9p10-hr1-ITR-外源目的基因及其启动子-pA-ITR-pH-Rep-pA。
具体的，在一些实施方式中，所述核酸构建体为腺相关病毒载体或重组杆状病毒载体。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，所述核酸构建体为重组杆状病毒载体。在一些实施方式中，所述重组杆状病毒载体为重组杆状病毒穿梭载体。
在一些实施方式中，所述腺相关病毒载体或重组杆状病毒载体还包括载体骨架。
在一些实施方式中，所述载体骨架可以选自市场上出售的合适载体骨架，例如pFastBacdual、pFastBac1、pFastBacHTA、pFastBacHTB或pFastBacHTC等。
在一种实施方式中，所述载体骨架为pFastBacdual。
在一些实施方式中，利用所述核酸构建体包装得到的rAAV滴度至少为1x10 12VG/mL (例如至少为1.5x10 12,2x10 12,2.5x10 12,3x10 12,3.5x10 12,4x10 12,4.5x10 12,5x10 12,5.5x10 12,6x10 12,7x10 12,7.5x10 12,8x10 12,8.5x10 12,9x10 12,9.5x10 12,1x10 13,5x10 13,或1x10 14VG/mL)，即每ml病毒培养液中含有的AAV基因组的拷贝数至少为1x10 12(例如至少为1.5x10 12,2x10 12,2.5x10 12,3x10 12,3.5x10 12,4x10 12,4.5x10 12,5x10 12,5.5x10 12,6x10 12,7x10 12,7.5x10 12,8x10 12,8.5x10 12,9x10 12,9.5x10 12,1x10 13,5x10 13,或1x10 14)。
在一些实施方式中，利用所述核酸构建体包装得到的rAAV滴度至少为2.60E+12VG/mL，即每ml病毒培养液中含有的AAV基因组的拷贝数至少为2.60E+12。
所述rAAV的产量通过qPCR方法检测，检测步骤如下：
定量PCR使用的标准品为Pvu Ⅰ-HF(NEB)单酶切线性化的pAAV-MCS质粒。
定量PCR使用的引物序列为：
ITR Forward primer 5’-GGAACCCCTAGTGATGGAGTT-3’(SEQ ID NO:2)
ITR Reverse primer 5’-CGGCCTCAGTGAGCGA-3’(SEQ ID NO:3)
定量PCR运行程序为：95℃ 60s，(95℃ 15s，60℃ 30s，40cycles)。根据定量PCR所得CT值和标准品浓度绘制标准品曲线后，换算出样品滴度。
本发明第二方面提供一种重组杆状病毒，所述重组杆状病毒为由任何一种所述核酸构建体经杆状病毒系统构建获得，或由包括任一所述核酸构建体中任意元件的核酸构建体共同经杆状病毒系统构建获得。
所述的包括任一所述核酸构建体中的任意元件的核酸构建体主要是指：包括AAV元件的核酸构建体、包括编码IE蛋白的多核苷酸的核酸构建体、包括编码杆状病毒同源区的多核苷酸。所述包括AAV元件的核酸构建体还可以选自包括编码Cap蛋白的多核苷酸的核酸构建体、包括编码Rep蛋白的多核苷酸的核酸构建体、包括编码AAV顺式作用元件的核酸构建体。所述重组杆状病毒能由以上核酸构建体共同经杆状病毒系统构建获得。
所述杆状病毒系统选自Bac-to-Bac系统(来源于ThermoFisher/Invitrogen)、flashBac/BacMagic系统(来源于Mirus/EMD/OET/Nextgen)、BaculoDirect系统(来源于ThermoFisher/Invitrogen)或BacPAK6/Baculogold系统(来源于BD Biosciences/Clonetech)。
具体的，所述重组杆状病毒为由所述核酸构建体转化感受态细胞，提取Bacmid后转染SF9昆虫细胞获得。
所述感受态细胞可以选自本领域各种可适用的任何感受态细胞，只要不对本发明的发明目的产生限制即可。例如所述感受态细胞可以为DH10Bac。
本发明第三方面提供一种腺相关病毒(AAV)，所述腺相关病毒为由任何一种所述重组 杆状病毒包装获得。所述腺相关病毒可以用来治疗各种疾病，例如血友病，脊髓性肌萎缩，杜氏肌营养不良症，帕金森病，年龄相关性黄斑病变等。
本发明第四方面提供一种细胞系，所述细胞系为经任何一种所述重组杆状病毒感染的细胞系。
在一些实施方式中，所述细胞系为昆虫细胞系，例如为SF9细胞、SF21细胞、High5细胞。
本发明第五方面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杆状病毒系统和所述核酸构建体。
本发明第六方面提供所述核酸构建体的构建方法，所述构建方法包括将携带外源目的基因的AAV元件、编码IE蛋白的多核苷酸整合入杆状病毒载体骨架中。
所述构建方法还包括将编码杆状病毒重组同源区的多核苷酸整合入杆状病毒载体骨架中。
在一些实施方式中，所述AAV元件包括编码Cap蛋白的多核苷酸、编码Rep蛋白的多核苷酸和AAV顺式作用元件。
在一些实施方式中，所述AAV顺式作用元件选自反向末端重复序列(ITR序列)。
具体的，在一些实施方式中，所述编码IE蛋白的多核苷酸与强启动子pH连接。
在一些实施方式中，所述编码杆状病毒重组同源区蛋白的多核苷酸与杆状病毒启动子顺式连接。在一些实施方式中，所述杆状病毒启动子为p6.9或p10。
在一些实施方式中，所述杆状病毒载体骨架选自pFastBacdual、pFastBac1、pFastBacHTA、pFastBacHTB或pFastBacHTC等。
在一些实施方式中，所述杆状病毒重组同源区选自hr1、hr2、hr3、hr4或hr5中的一种或几种(例如两种，三种，四种或五种)。
本发明第七方面提供一种腺相关病毒的生产方法，所述生产方法包括以下步骤：将所述的重组杆状病毒感染昆虫细胞系。
所述腺相关病毒的生产方法能提高腺相关病毒的产量。
在一些实施方式中，所述细胞系选自昆虫细胞系。在一种实施方式中，所述昆虫细胞系选自SF9细胞。
本发明第八方面提供用一种治疗疾病的方法，所述方法包括向患者给予有效量的任何一种所述的腺相关病毒。
在一些实施方式中，所述疾病包括例如血友病、脊髓性肌萎缩、杜氏肌营养不良症、帕 金森病、年龄相关性黄斑病变等。
本发明还包括以下任何编号的段落：
1、核酸构建体，其特征在于，所述核酸构建体包括：AAV元件、编码IE蛋白的多核苷酸，所述AAV元件包括编码Cap蛋白的多核苷酸、编码Rep蛋白的多核苷酸和AAV顺式作用元件。
2、根据段落1所述的核酸构建体，其特征在于，所述核酸构建体还包括编码杆状病毒重组同源区的多核苷酸。
3、根据段落2所述的核酸构建体，其特征在于，所述IE蛋白选自Acie0、Acie01或Acie02中的一种或几种编码的蛋白，和/或，所述杆状病毒重组同源区选自hr1、hr2、hr3、hr4或hr5中的一种或几种。
4、根据段落1-3任一所述的核酸构建体，其特征在于，所述核酸构建体还包括IE蛋白基因的启动子，所述IE蛋白基因的启动子选自Gp64、pH、p6.9或p10中的一种或几种。
5、根据段落1-4任一所述的核酸构建体，其特征在于，所述核酸构建体还包括杆状病毒启动子，所述杆状病毒启动子与杆状病毒重组同源区连接，所述杆状病毒启动子优选为pH、Gp64、p6.9或p10中的一种或几种。
6、根据段落1-5任一所述的核酸构建体，其特征在于，所述AAV顺式作用元件选自ITR序列。
7、根据段落1-6任一所述的核酸构建体，其特征在于，所述核酸构建体还包括外源目的基因，所述外源目的基因嵌入AAV元件中。
8、根据段落1-7任一所述的核酸构建体，其特征在于，所述核酸构建体的结构为：IE基因表达框-Cap基因表达框-ITR-外源目的基因表达框-ITR-Rep基因表达框。
9、根据段落1-8任一所述的核酸构建体，其特征在于，所述核酸构建体的核苷酸序列如SEQ ID NO.1。
10、根据段落1-9任一所述的核酸构建体，其特征在于，所述核酸构建体为腺相关病毒载体或重组杆状病毒载体，所述重组杆状病毒载体优选为重组杆状病毒穿梭载体。
11、一种重组杆状病毒，其特征在于，所述重组杆状病毒为由段落1-10任一所述的核酸构建体经杆状病毒系统构建获得，或由包括段落1-10任一所述核酸构建体中任意元件的核酸构建体共同经杆状病毒系统构建获得。
12、一种腺相关病毒，其特征在于，所述腺相关病毒为由段落11所述的重组杆状病毒感染细胞后包装获得。
13、一种细胞系，其特征在于，所述细胞系为经段落11所述重组杆状病毒感染的细胞系。
14、一种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包括杆状病毒系统和段落1-10任一所述的核酸构建体。
15、一种构建段落1-10任一所述核酸构建体的方法，其特征在于，所述构建方法包括将携带外源目的基因的AAV元件、编码IE蛋白的多核苷酸整合入杆状病毒载体骨架中。
16、根据段落15所述的构建方法，其特征在于，所述构建方法包括以下特征中的一项或几项：
1)所述AAV元件包括编码Cap蛋白的多核苷酸、编码Rep蛋白的多核苷酸和AAV顺式作用元件，所述AAV顺式作用元件优选为ITR序列；
2)所述IE蛋白由Acie0、Acie01或Acie02基因中的一种或几种编码；
3)所述杆状病毒载体骨架选自pFastBacdual、pFastBac1、pFastBacHTA、pFastBacHTB或pFastBacHTC中的一种；
4)所述杆状病毒重组同源区选自hr1、hr2、hr3、hr4或hr5中的一种或几种；
5)将编码杆状病毒重组同源区的多核苷酸整合入杆状病毒载体骨架中。
17、一种腺相关病毒的生产方法，其特征在于，所述生产方法包括以下步骤：将段落11所述的重组杆状病毒感染昆虫细胞系。
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。
以下实施例以EGFP为外源目的基因为例。
实施例1重组杆状病毒穿梭载体的构建及重组杆状病毒的获得
确定相关基因：Cap和Rep蛋白的基因均来源于AAV2基因组(GenBank:AF043303.1)，根据核糖体泄露扫描机制参考文献(Smith RH,Levy JR,Kotin RM:A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells.Mol Ther 2009,17(11):1888-1896.)对其中碱基进行点突变；Acie01(GenBank:NC_001623.1)，杆状病毒重组同源区hr1(GenBank:M14313.1)。
委托基因公司分别合成相应序列：Cap基因序列(核苷酸序列SEQ ID NO:4)，Rep基因序列(核苷酸序列SEQ ID NO:5)，hr1和p6.9、p10启动子组合(hr1p6.9p10，核苷酸序列SEQ ID NO:6)，pH启动子和Acie01组合(pH-Acie01，核苷酸序列SEQ ID NO:7)。使用本领域熟知的同源重组方法将上述序列以及pAAV-EGFP载体(康霖生物科技(杭州)有限公司改造)中的ITR-CMV-EGFP序列克隆至pFastBacdual(Invitrogen)上，测序鉴定后分别得到pFBd-Cap-ITR-Rep(图1和图2，核苷酸序列SEQ ID NO:8)和pFBd-IE-hr1Cap-ITR-Rep(图3和图4，核苷酸序列SEQ ID NO:9)两个重组杆状病毒穿梭载体。
SEQ ID NO.4-9的序列如下所示：
SEQ ID NO:4：
Figure PCTCN2021121350-appb-000024
Figure PCTCN2021121350-appb-000025
SEQ ID NO:5：
Figure PCTCN2021121350-appb-000026
Figure PCTCN2021121350-appb-000027
SEQ ID NO:6：
Figure PCTCN2021121350-appb-000028
Figure PCTCN2021121350-appb-000029
SEQ ID NO:7：
Figure PCTCN2021121350-appb-000030
Figure PCTCN2021121350-appb-000031
SEQ ID NO:8：
Figure PCTCN2021121350-appb-000032
Figure PCTCN2021121350-appb-000033
Figure PCTCN2021121350-appb-000034
Figure PCTCN2021121350-appb-000035
Figure PCTCN2021121350-appb-000036
Figure PCTCN2021121350-appb-000037
Figure PCTCN2021121350-appb-000038
SEQ ID NO:9：
Figure PCTCN2021121350-appb-000039
Figure PCTCN2021121350-appb-000040
Figure PCTCN2021121350-appb-000041
Figure PCTCN2021121350-appb-000042
Figure PCTCN2021121350-appb-000043
Figure PCTCN2021121350-appb-000044
Figure PCTCN2021121350-appb-000045
Figure PCTCN2021121350-appb-000046
将两个重组载体分别转化至DH10Bac感受态细胞(Invitrogen)，进行蓝白斑筛选，挑取白色阳性克隆培养，使用PureLink TM HiPure Plasmid DNA Purification Kits(Invitrogen)提取杆状病毒质粒(Bacmid)。将两种Bacmid分别转染至贴壁SF9细胞(Gbico)，3d后收获初代重组杆状病毒，并进行扩增至P2代。最终得到P2代重组杆状病毒分别命名为BV-Cap-ITR-Rep和BV-IE-hr1Cap-ITR-Rep，使用TCID50法分别测定病毒滴度。此部分内容具体参照Bac to Bac Expression System操作手册(Invitrogen)。
实施例2 rAAV2的包装及滴度测定
将两种P2代重组杆状病毒分别以MOI＝0.1感染25mL悬浮SF9细胞(密度3.0E+06cell/mL)，4d后收获细胞，使用5mL TNT裂解液(20mM Tris–HCl(pH 7.5)，150mM NaCl，1％Triton X-100，10mmol/l MgCl 2)室温孵育1h裂解细胞。以50U/mL终浓度加入核酸酶37℃消化2h，取样使用蛋白酶K(天根)56℃消化外壳1h，qPCR测定滴度，检测步骤如下：
定量PCR使用的标准品为Pvu Ⅰ-HF(NEB)单酶切线性化的pAAV-EGFP质粒，(质粒序列如以下SEQ ID NO:22所示)。
SEQ ID NO.22：
Figure PCTCN2021121350-appb-000047
Figure PCTCN2021121350-appb-000048
Figure PCTCN2021121350-appb-000049
定量PCR使用的引物序列为：
ITR Forward primer 5’-GGAACCCCTAGTGATGGAGTT-3’(SEQ ID NO:2)
ITR Reverse primer 5’-CGGCCTCAGTGAGCGA-3’(SEQ ID NO:3)
定量PCR运行程序为：95℃ 60s，(95℃ 15s，60℃ 30s，40cycles)。
根据定量PCR所得Ct值和标准品浓度绘制标准品曲线后，换算出样品滴度。计算出BV-Cap-ITR-Rep和BV-IE-hr1Cap-ITR-Rep包装的rAAV产量分别为2.39E+04VG/cell(相当于7.18E+10VG/mL)和8.67E+05VG/cell(相当于2.60E+12VG/mL)。
实施例3细胞感染过程中EGFP的表达和细胞裂解液中Cap蛋白表达量比较
在感染48h、72h、96h后分别于倒置荧光显微镜下观察被两种杆状病毒感染的细胞，优化后的重组杆状病毒可大幅度降低目的基因(本实施例中目的基因为EGFP)的表达(图5)。目的基因(EGFP)表达量的减少似乎减弱了对细胞内rAAV包装的干扰，使其更有利于rAAV包装相关的结构与功能蛋白的表达。
对等量细胞裂解液使用Cap蛋白抗体(Progen)进行WB检测(图6)。结果表明优化后的重组杆状病毒感染细胞后，细胞内的Cap蛋白表达水平显著提高。
实施例4纯化后rAAV2电镜检测，感染性测定
使用POROS TM CaptureSelect TM亲和层析填料(Thermo)纯化细胞裂解液，用荧光定量PCR方法测定滴度(测定方法同实施例2)，并对纯化后的rAAV2进行负染色处理，透射式电子显微镜下观察(图7)。包装了基因组的rAAV2呈实心颗粒，无核酸的缺陷rAAV颗粒中间被染成深色。总体来看形态完好，与传统方法(Benskeyetal.2016)相比空壳率显著降低(～3％)，表明采用优化后的杆状病毒载体构建体明显降低了包装产物中空壳病毒的比例，显著减少了下游的空壳病毒去除等步骤的压力。
将纯化的rAAV2梯度稀释，分别以MOI＝10000、2000、400、80感染培养的293T细胞(48孔板，5.0E+04cells/well)。感染2天后，用荧光显微镜观察EGFP的表达。实验结果 表明，该系统制备出的rAAV2具有较高的体外感染活性(图8)。
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 (17)

  1. 核酸构建体，其特征在于，所述核酸构建体包括以下元件：AAV元件和编码IE蛋白的多核苷酸，所述AAV元件包括编码Cap蛋白的多核苷酸、编码Rep蛋白的多核苷酸和AAV顺式作用元件。
  2. 根据权利要求1所述的核酸构建体，其特征在于，所述核酸构建体还包括编码杆状病毒重组同源区的多核苷酸。
  3. 根据权利要求2所述的核酸构建体，其特征在于，所述IE蛋白由Acie0、Acie01或Acie02基因中的一种或几种编码，和/或，所述杆状病毒重组同源区选自hr1、hr2、hr3、hr4或hr5中的一种或几种。
  4. 根据权利要求1所述的核酸构建体，其特征在于，所述核酸构建体还包括IE蛋白基因的启动子，所述IE蛋白基因的启动子选自Gp64、pH、p6.9或p10中的一种或几种。
  5. 根据权利要求1所述的核酸构建体，其特征在于，所述核酸构建体还包括杆状病毒启动子，所述杆状病毒启动子与杆状病毒重组同源区连接，所述杆状病毒启动子优选为pH、Gp64、p6.9或p10中的一种或几种。
  6. 根据权利要求1所述的核酸构建体，其特征在于，所述AAV顺式作用元件选自ITR序列。
  7. 根据权利要求1所述的核酸构建体，其特征在于，所述核酸构建体还包括外源目的基因，所述外源目的基因嵌入AAV元件中。
  8. 根据权利要求7所述的核酸构建体，其特征在于，所述核酸构建体的结构为：IE基因表达框-Cap基因表达框-ITR-外源目的基因表达框-ITR-Rep基因表达框。
  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如SEQ ID NO.1。
  10. 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或重组杆状病毒载体，所述重组杆状病毒载体优选为重组杆状病毒穿梭载体。
  11. 一种重组杆状病毒，其特征在于，所述重组杆状病毒为由权利要求1-10任一所述的核酸构建体经杆状病毒系统构建获得，或由包括权利要求1-10任意所述核酸构建体中任意元件的核酸构建体共同经杆状病毒系统构建获得。
  12. 一种腺相关病毒，其特征在于，所述腺相关病毒为由权利要求11所述的重组杆状病毒感染细胞后包装获得。
  13. 一种细胞系，其特征在于，所述细胞系为经权利要求11所述的重组杆状病毒感染的细胞系。
  14. 一种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包括杆状病毒系统和权利要求1-10任一所述的核酸构建体。
  15. 一种构建权利要求1-10任一所述核酸构建体的方法，其特征在于，所述构建方法包括将携带外源目的基因的AAV元件、编码IE蛋白的多核苷酸和编码杆状病毒重组同源区的多核苷酸整合入杆状病毒载体骨架中。
  16. 根据权利要求15所述的构建方法，其特征在于，所述构建方法包括以下特征中的一项或几项：
    1)所述AAV元件包括编码Cap蛋白的多核苷酸、编码Rep蛋白的多核苷酸和AAV顺式作用元件，所述AAV顺式作用元件优选为ITR序列；
    2)所述编码IE蛋白的多核苷酸选自Acie0、Acie01或Acie02中的一种或几种；
    3)所述杆状病毒载体骨架选自pFastBacdual、pFastBac1、pFastBacHTA、pFastBacHTB或pFastBacHTC中的一种；
    4)所述杆状病毒重组同源区选自hr1、hr2、hr3、hr4或hr5中的一种或几种。
  17. 一种腺相关病毒的生产方法，其特征在于，所述生产方法包括以下步骤：将权利要求11所述的重组杆状病毒感染昆虫细胞系。
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