Nucleic acid constructs for increasing adeno-associated virus yields and methods for constructing same

Abstract

The present invention relates to a nucleic acid construct for increasing the yield of adeno-associated virus and a method for constructing the same. The nucleic acid construct comprises an adeno-associated virus (AAV) element and a polynucleotide encoding an IE protein, and the AAV element comprises a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis-element. The construction method involves incorporating an AAV element carrying a foreign target gene and a polynucleotide encoding the IE protein into a baculovirus vector backbone. The resulting recombinant adeno-associated virus (rAAV) of the present invention has a low empty capsid rate and simultaneously increases the rAAV yield per single cell and per unit volume of culture, reducing production costs and facilitating scalability.

Description

[Technical Field]

[0001] The present invention relates to the technical field of gene transfer vectors, and in particular to a nucleic acid construct for increasing the yield of adeno-associated virus and a method for constructing the same. [Background technology]

[0002] Recombinant adeno-associated virus (rAAV) is a gene transfer vector widely used in basic research and clinical gene therapy. Due to its high safety profile, broad host cell range, low immunogenicity, and long in vivo exogenous gene expression, rAAV is considered one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. In recent years, an increasing number of researchers have been using rAAV in large-scale animal studies and human clinical trials. The AAV genome contains three core elements: the ITR sequence, the nonstructural protein Rep, and the structural protein Cap.

[0003] The insect baculovirus system is a common method for producing recombinant proteins. Compared to traditional triple-plasmid transient transfection methods, insect cells offer advantages such as high culture density, serum-free suspension culture, and ease of scale-up. Furthermore, the baculovirus-infected cell production method offers better batch stability. Researchers began using it to produce rAAV in 2002. However, due to various reasons, including the difficulty in achieving an ideal ratio of Cap, Rep, and AAV vector genomic DNA in insect cells and the low AAV packaging efficiency, recombinant AAV vectors packaged using traditional methods have a high empty capsid rate (11%-34%) (Non-Patent Document 1). Because the physical and chemical properties of empty capsid AAV and AAV carrying target genes are very similar, making purification extremely difficult, empty capsid AAV is an impurity that must be removed in the production of clinical-grade AAV vectors. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Benskey et al. 2016 [Non-patent document 2] 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. Summary of the Invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a nucleic acid construct and a method for constructing the same for improving the yield and quality of recombinant adeno-associated viruses, in order to solve the problems of the prior art.

[0006] To achieve the above and other related objects, a nucleic acid construct is provided according to a first aspect of the present invention, comprising an AAV element, a polynucleotide encoding an IE protein, and a polynucleotide encoding a baculovirus recombination homology region, wherein the AAV element comprises a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis element.

[0007] In some embodiments, the IE protein is encoded by one or more of the following genes: Acie0, Acie01, Acie02, and / or the baculovirus recombination homology region is selected from one or more of hr1, hr2, hr3, hr4, or hr5.

[0008] In some embodiments, the nucleic acid construct further comprises a promoter of an IE protein gene, hi some embodiments, the promoter of the IE protein gene is selected from one or more of Gp64, pH, p6.9, or p10.

[0009] In some embodiments, the nucleic acid construct further comprises a baculovirus promoter. In some embodiments, the baculovirus promoter is linked to a baculovirus recombination homology region. In some embodiments, the baculovirus promoter is preferably one or more of pH, Gp64, p6.9, or p10.

[0010] In some embodiments, the AAV cis elements are selected from ITR sequences.

[0011] In some embodiments, the nucleic acid construct further comprises an exogenous target gene, hi some embodiments, the exogenous target gene is embedded in an AAV element.

[0012] In some embodiments, the structure of the nucleic acid construct comprises IE gene expression cassette-Cap gene expression cassette-ITR-foreign 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-foreign target gene expression cassette-ITR-Rep gene expression cassette.

[0013] In some embodiments, the nucleotide sequence of the nucleic acid construct comprises SEQ ID NO: 1 or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 1. In some embodiments, the nucleotide sequence of the nucleic acid construct is as set forth in SEQ ID NO: 1.

[0014] In some embodiments, the nucleic acid construct is an adeno-associated virus vector or a recombinant baculovirus vector. In some embodiments, the nucleic acid construct is an adeno-associated virus 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.

[0015] A second aspect of the present invention provides a recombinant baculovirus, which is obtainable by constructing any of the above-mentioned nucleic acid constructs using a baculovirus system, or by co-constructing a nucleic acid construct containing any element of any of the above-mentioned nucleic acid constructs using a baculovirus system.

[0016] A third aspect of the present invention provides an adeno-associated virus, which is obtained by infecting a cell with any of the above-mentioned recombinant baculoviruses and then packaging the virus.

[0017] A fourth aspect of the present invention provides a cell line, which is a cell line infected with any of the above-mentioned recombinant baculoviruses.

[0018] A fifth aspect of the present invention provides an adeno-associated virus vector system, which comprises a baculovirus system and the nucleic acid construct described above.

[0019] A sixth aspect of the present invention provides a method for constructing the above-mentioned nucleic acid construct, which method comprises incorporating an AAV element carrying a foreign target gene, a polynucleotide encoding an IE protein, and a polynucleotide encoding a baculovirus recombination homology region into a baculovirus vector backbone.

[0020] The method includes one or more (eg, two or three) of the following features. 1) The AAV elements include a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis element, for example, the AAV cis element is preferably an ITR sequence. 2) The polynucleotide encoding the IE protein is selected from one or more of Acie0, Acie01, or Acie02. 3) The baculovirus vector backbone is selected from one of pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB or pFastBacHTC. 4) The baculovirus recombination homology region is selected from one or more of hr1, hr2, hr3, hr4, or hr5.

[0021] A seventh aspect of the present invention provides a production method for increasing the yield of adeno-associated virus, the production method comprising the step of infecting an insect cell line with a recombinant baculovirus.

[0022] As described above, the nucleic acid construct for increasing the yield of adeno-associated virus and the method for constructing the same of the present invention have at least the following beneficial effects: 1) The present invention significantly reduces the expression of target genes inserted into baculovirus vectors in insect cells, reduces the impact of target gene expression on insect cells, improves the growth index of producer cells, and is more advantageous for rAAV packaging. 2) Compared with conventional adherent 293 and 293T cell production and conventional baculovirus production methods, this optimized vector construction method clearly increases rAAV yield per single cell and per unit volume of culture, reduces production costs, and is amenable to scalability. 3) It can be widely applied, such as the production of various scales and types of adeno-associated virus gene therapy vectors. [Brief explanation of the drawings]

[0023] [Figure 1]FIG. 1 is a schematic diagram showing the structure of the pFBd-Cap-ITR-Rep vector of the present invention. [Figure 2] FIG. 1 is a schematic diagram showing a map of the pFBd-Cap-ITR-Rep vector of the present invention. [Figure 3] FIG. 1 is a schematic diagram showing the structure of the pFBd-IE-hr1Cap-ITR-Rep vector of the present invention. [Figure 4] FIG. 1 is a schematic diagram showing a map of the pFBd-IE-hr1Cap-ITR-Rep vector of the present invention. [Figure 5] FIG. 1 shows a comparison of the levels of EGFP expression 2 to 4 days after infection of SF9 cells with BV-Cap-ITR-Rep of the present invention and BV-IE-hr1Cap-ITR-Rep. [Figure 6] FIG. 1 shows WB detection of the AAV Cap monoclonal antibody of the present invention on two BV-infected cell lysates. [Figure 7] FIG. 1 shows electron microscopy detection of the rAAV2 of the present invention (titer 2.0E+13VG / mL). [Figure 8] FIG. 1 shows fluorescence observation two days after infection of 293T cells with rAAV2 packaged with BV-Cap-ITR-Rep and BV-IE-hr1Cap-ITR-Rep of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention is based, at least in part, on the discovery that nucleic acid constructs comprising AAV elements, a polynucleotide encoding an IE protein, and a polynucleotide encoding a baculovirus recombination homology region can significantly control the expression of inserted target genes in insect cells, which are hosts for baculovirus, improve cellular conditions, and increase the packaging efficiency of adeno-associated viruses, thereby significantly increasing the yield of adeno-associated viruses. The present invention unexpectedly reduces the rate of empty capsids in rAAV (e.g., increased Cap expression does not lead to an increase in empty capsids), produces rAAV with superior infectivity, and significantly increases the rAAV yield per single cell and per unit volume of culture, making it suitable for large-scale production of various adeno-associated virus gene therapy vectors.

[0025] Unless otherwise defined below, all technical and scientific terms referred to herein have the meanings commonly understood by one of ordinary skill in the art.

[0026] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into a target cell or tissue, and the nucleic acid construct may be a lentiviral vector or an adeno-associated viral vector, which comprises a vector backbone, i.e., an empty vector, and an expression cassette.

[0027] The term "vector" refers to a nucleic acid or polynucleotide fragment used to introduce or transfer one or more nucleic acids or one or more polynucleotides into a target cell or tissue. Typically, a vector is used to introduce foreign DNA into another cell or tissue. The vector may contain a bacterial resistance gene for propagation in bacteria and a promoter for expression of the target protein in the organism. DNA may be generated in vitro by PCR or any other suitable technique or techniques known to those skilled in the art.

[0028] A first aspect of the present invention provides a nucleic acid construct, which comprises a polynucleotide encoding an AAV element and an IE protein, wherein the AAV element comprises a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis element.

[0029] In one embodiment, the nucleic acid construct further comprises a polynucleotide encoding a baculovirus recombination homology region.

[0030] In some embodiments, the nucleotide sequence of the nucleic acid construct comprises SEQ ID NO: 1 or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO: 1. In some embodiments, the nucleotide sequence of the nucleic acid construct is as set forth in SEQ ID NO: 1.

[0031] SEQ ID NO: 1 TTCTCTGTCACAGAATGAAAATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAACGCTTATTTGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGC ATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCG TCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTC

[0032] In some embodiments, 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. Because the capsid proteins of different serotypes of AAV recognize different receptors on the cell surface, the infection efficiency of different tissue cells can vary greatly, exhibiting specific organ targeting specificity. Depending on the specific experimental purpose, an appropriate serotype of AAV can be selected to achieve specific, highly efficient transduction of a particular serotype of AAV into a particular type of cell tissue.

[0033] In some embodiments, the AAV elements are derived from AAV1. In some embodiments, the AAV elements are derived from AAV2. In some embodiments, the AAV elements are derived from AAV3. In some embodiments, the AAV elements are derived from AAV4. In some embodiments, the AAV elements are derived from AAV5. In some embodiments, the AAV elements are derived from AAV6. In some embodiments, the AAV elements are derived from AAV7. In some embodiments, the AAV elements are derived from AAV8. In some embodiments, the AAV elements are derived from AAV9. In some embodiments, the AAV elements are derived from AAV DJ. In some embodiments, the AAV elements are derived from AAV DJ / 8. In some embodiments, the AAV elements are derived from AAV rh10. In some embodiments, the AAV elements are derived from AAV Retro. In some embodiments, the AAV elements are derived from AAV PHP.eB / PHP.B / PHP.S.

[0034] Cap proteins are structural proteins and typically include VP1, VP2, and VP3 structural proteins. In some embodiments, it is believed that three structural proteins can be assembled into the capsid protein of AAV.

[0035] Rep proteins are non-structural proteins and generally include Rep78 and 52 proteins. In some embodiments, it is believed that Rep proteins can regulate DNA replication and packaging.

[0036] In one embodiment, the AAV cis elements are selected from inverted terminal repeat (ITR) sequences. In some embodiments, the ITR sequences can fold into a hairpin structure, and the AAV It is believed to be the only known cis element required for the initiation of DNA replication and packaging of recombinant AAV virions.

[0037] In some embodiments, the nucleic acid construct further comprises an exogenous target gene. In some embodiments, the exogenous target gene is carried by an AAV element, i.e., the exogenous target gene is embedded in an AAV element.

[0038] Specifically, in some embodiments, the exogenous target gene is embedded between the ITR sequences on both ends.

[0039] Preferably, in some embodiments, the size of the exogenous target gene is ≦3.5 kb. In some embodiments, due to the size restriction between the ITR sequences at both ends, the size of the exogenous target gene generally should not be too large, otherwise the packaging efficiency may be reduced.

[0040] The type of exogenous gene varies depending on the purpose of the experiment. The exogenous gene may be a gene related to the treatment of various diseases, such as AADC, FVIII, or FIX, or it may be a tool gene commonly used in laboratories, such as EGFP or mCherry.

[0041] In some embodiments, the nucleic acid construct will further comprise a promoter of the IE protein gene. In some embodiments, the promoter of the IE protein gene will be 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 comprises 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.

[0042] Preferably, in some embodiments, the promoter of the IE protein gene is selected from the pH strong promoters. In some embodiments, adding an additional copy of ie to the nucleic acid construct and expressing it under the control of the strong promoter pH is believed to exceed the endogenous level of the virus and increase survival of cells at later stages after baculovirus infection.

[0043] In some embodiments, the IE protein is selected from proteins encoded by one or more (e.g., two or three) of the following genes: Acie0, Acie01, or Acie02. In some embodiments, the IE protein is selected from the protein encoded by Acie0, i.e., IE0. The nucleotide sequence of Acie0 is as set forth in SEQ ID NO:10, and the amino acid sequence of the IE0 protein is as set forth in SEQ ID NO:11. In some embodiments, the IE protein is selected from the protein encoded by Acie01, i.e., IE1. The nucleotide sequence of Acie01 is as set forth in SEQ ID NO:12, and the amino acid sequence of the IE1 protein is as set forth in SEQ ID NO:13. In some embodiments, the IE protein is selected from the protein encoded by Acie02, i.e., the IE2 protein. The nucleotide sequence of Acie02 is as set forth in SEQ ID NO:14, and the amino acid sequence of the IE2 protein is as set forth in SEQ ID NO:15.

[0044] The sequences of sequence numbers 10 to 15 are as follows:

[0045] Sequence number 10 ATGATAAGAACCAGCAGTCACGTGCTGAACGTCCAGGAAAATATAATGACGTCAAACTGTGCGTCATCGCCATATTCGTGCGAGGCAACGTCCGCTTGCGCAGAAGCTCAGCAGGTAATGATCGATAACTTTGTTTTCTTTCACATGTACAACGCCGACATACAAATTGACGCAAAGCTGCAATGCGGCGTGCGCTCGGCCGCGTTTGCAATGATCGACGATAAACATTTGGAAATGTACAAGCATAGAATAGAGAATAAATTTTTTTATTACTATGATCAATGTGCCGACATTGCCAAACCCGACCGTCTGCCCGATGACGACGGCGCGTGCTGTCACCATTTTATTTTTGATGCCCAACGTATTATTCAATGTATTAAAGAGATTGAAAGCGCGTACGGCGTGCGTGATCGCGGCAATGTAATAGTGTTTTATCCGTACTTGAAACAGTTGCGAGACGCGTTGAAGCTAATTAAAAACTCTTTTGCGTGTTGTTTTAAAATTATAAATTCTATGCAAATGTACGTGAACGAGTTAATATCAAATTGCCTGTTGTTTATTGAAAAGCTGGAAACTATTAATAAAACTGTTAAAGTTATGAATTTGTTTGTAGACAATTTGGTTTTGTACGAATGCAATGTTTGTAAAGAAATATCTACGGATGAAAGATTTTTAAAGCCAAAAGAATGTTGCGAATACGCTATATGCAACGCGTGCTGCGTTAACATGTGGAAGACGGCCACCACGCACGCAAAATGTCCAGCGTGCAGGACATCGTATAAATAA

[0046] Sequence number 11 MIRTSSHVLNVQENIMTSNCASSPYSCEATSACAEAQQVMIDNFVFFHMYNADIQIDAKLQCGVRSAAFAMIDDKHLEMYKHRIENKFFYYYDQCADIAKPDRLPDDDGACCHHFIFDAQRIIQCIKEIE SAYGVRDRGNVIVFYPYLKQLRDALKLIKNSFACCFKIINSMQMYVNELISNCLLFIEKLETINKTVKVMNLFVDNLVLYECNVCKEISTDERFLKPKECCEYAICNACCVNMWKTATTHAKCPACRTSYK

[0047] SEQ ID NO: 12

[0048] SEQ ID NO: 13 MIRTSSHVLNVQENIMTSNCASSPYSCEATSACAEAQQLQVDTGGDKIVNNQVTMTQINFNASYTSASTPSRASFDNSYSEFCDKQPNDYLSYYNHPTPDGADTVISDSETAAASNFLASVNSLTDNDLVECLLKTTDNLEEAVSSAYYSESLEQPVVEQPSPSSAYHAESFEHSAGVNQPSATGTKRKLDEYLDNSQGV VGQFNKIKLRPKYKKSTIQSCATLEQTINHNTNICTVASTQEITHYFTNDFAPYLMRFDDNDYNSNRFSDHMSETGYYMFVVKKSEVKPFEIIFAKYVSN VVYEYTNNYYMVDNRVFVVTFDKIRFMISYNLVKETGIEIPHSQDVCNDETAAQNCKKCHFVDVHHTFKAALTSYFNLDMYYAQTTFVTLLQSLGERKCG FLLSKLYEMYQDKNLFTLPIMLSRKESNEIETASNNFFVSPYVSQILKYSESVQFPDNPNKYVVDNLNLIVNKKSTLTYKYSSVANLLFNNYKYHDNIASNNNAENLKKVKKEDGSM HIVEQYLTQNVDNVKGHNFIVLSFKNEERLTIAKKNKEFYWISGEIKDVDVSQVIQKYNRFKHHMFVIGKVNRRESTLHNNLLKLLALILQGLVPLSDAITFAEQKLNCKYKKFEFN

[0049] SEQ ID NO: 14

[0050] SEQ ID NO: 15 MSRQINAATPSSSRRHRLSLSRRRINFTTSPEAQPSSSSRSQPSSSSRSHRRQERRQEQRVSEENVQIIGNVNEPLTRTYHRQGVTYYVHGQVNISNDDPLL SQEDDVILINSENVDRERFPDITAQQYQDNIASETAAQRALQRGLDLEAQLMNEIAPRSPTYSPSYSPNYVIPQSPDLFASPQSPQPQQQQQQSEPEEEVE VSCNICFTTFKDTKNVNSSFVTSIHCNHAVCFKCYVKIIMDNSVYKCFCSATSSDCRVYNKHGYVEFMPINVTRNQDSIKQHWRELLENNTVNNHTTDLNYVEQLQKELSELRAKTSQVEHKMTMLNSDYIMLKHKHAVAELDQKANYDLQESTKKSEELQSTVNNLQEQLRKQVAESQAKFSEFERSNSDLVSKLQTVMSRR

[0051] In some embodiments, the IE1 protein is believed to be the product of baculovirus immediate early gene 1 (ie1), also referred to as baculovirus transcriptional regulator protein, which is a multifunctional protein with the ability to activate early and late viral genes and participate in viral genome replication, and is involved in regulating the viral cycle.

[0052] In some embodiments, the baculovirus recombinant homology regions (hr) are selected from one or more (e.g., two, three, four, or five) of hr1 (nucleotide sequence set forth in SEQ ID NO: 16), hr2 (nucleotide sequence set forth in SEQ ID NO: 17), hr3 (nucleotide sequence set forth in SEQ ID NO: 18), hr4 (nucleotide sequence of hr4 left set forth in SEQ ID NO: 19 and nucleotide sequence of hr4 right set forth in SEQ ID NO: 20), and hr5 (nucleotide sequence set forth in SEQ ID NO: 21).

[0053] The sequences of SEQ ID NOs: 16 to 21 are as follows:

[0054] SEQ ID NO: 16 ATCGATGTTGACCCCAACAAAAGATTTATAATTAATCATAATCACGAACAACAACAAGTCAATGAAACAAATAAACAAGTTGTCGATAAAACATTCATAAATGACACAGCAACATACAATTCTTGCATAATAAAAATTTAAATGACATCATATTTGAGAATAACAAATGACATTATCCCTCGATTGTGTTTTACAAGTAGAATTCTACCCGTAAAGCGAGTTTAGTTTTGAAAAACAAATGACATCATTTGTATAATGACATCATCCCCTGATTGTGTTTTACAAGTAGAATTCTATCCGTAAAGCGAGTTCAGTTTTGA AAACAAATGAGTCATACCTAAACACGTTAATAATCTTCTGATATCAGCTTATGACTCAAGTTATGAGCCGTGTGCAAAACATGAGATAAGTTTATGACATCATCCACTGATCGTGCGTTACAAGTAGAATTCTACTCGTAAAGCCAGTTCGGTTATGAGCCGTGTGCAAAACATGACATCAGCTTATGACTCATACTTGATTGTGTTTTACGCGTAGAATTCTACTCGTAAAGCGAGTTCGGTTATGAGCCGTGTGCAAAACATGACATCAGCTTATGAGTCATAATTAATCGTGCGTTACAAGTAGAATTCTACTCGTAAAGCGAGTTGAAGGATCATATTTAGTTGCGTTTATGAGATAAGATTGAAAGCACGTGTAAAATGTTTCCCGCGCGTTGGCACAACTATTTACAATGCGGCCAAGTTATAAAAGATTCTAATCTGATATGTTTTAAAACACCTTTGCGGCCCGAGTTGTTTGCGTACGTGACTAGCGAAGAAGATGTGTGGACCGCAGAACAGATAGTAAAACAAAACCCTAGTATTGGAGCAATAATCGAT

[0055] SEQ ID NO: 17 TGAGCAAAACACAACCGGCAAATTCTCGGCGGCCGTTTGGGAATGCGGAATAATTGCCATATGTAAATGATGTCATCGGTTCTAACTCGCTTTACGAGTAGAATTCTACGTGTAAAACATAATCAAGAGATGATGTCATTTGTTTTTCAAAACTGAACTCAAGAAATGATGTCATTTGTTTTTCAAAACTGAACTGGCTTTACGAGTAGAATTCTACTTGTAAAACACAATCGAGAGATGATGTCATATTTTGCACACGGCTCTAATTAAACTCGCTTTACGAGTAAAATTCTACTTGTAACGCATGATCAAAGGGATGATGTCATTGGATGAGTCATTTGTTTTTCAAAACTAAACTCGCTTTACGAGTAGAATTCTACTTGTAAAACAGCAACTCGAGGGGATGAT GTCATCCTTTACTCGATGATTATAAACGTGTTTATGTATGACTCATTTGTTTTTCAAAACTAAACTCGCTTTACGAGTAGAATTCTACTTGTAACGCACGATCAGGGATGATGTCATTTATTTGTGCAAAGCTCGATGTCATCTTTTGCACACGATTATAAACACAATCCAAAATAATGACTCATTTGTTTTCAAAACTGAACTCGCTTTACGAGTAGAATTCTACTTGTAAAACACAATCAAGGGATGATGTCATTTTCAAAATGATGTCATTTGTTTTTCAAAACTAAACTCGCTTTACGAGTAGAATTCTACTTGTAAAACACAATCAAGGGATGATGTCATTTTAAAAATGATCATTTGTTTTTCAAAACTAAACTCGCTTTACGAGTAGAATTCTACGTGTAAAACACAATCAAGGGATGATGTCATTTACTAAATAAAATAATTATTTAAATAAAACTGTTTTTTATTGTCAAATACACATTGATTCAC

[0056] sequence number 18 ACGCGTAGAATTCTACTTGTAAAGCAAGTTAAAATAAGCCGTGTGCAAAAATGACATCAGACAAATGACATCATCTACCTATCATGATCATGTTAATAATCATGTTTTAAAATGACATCAGCTTATGACTAATAATTGATCGTGCGTTACAAGTAGAATTCTACTCGTAAA GCGAGTTTAGTTTTGAAAAACAAATGAGTCATCATTAAAACATGTTAATAATCGTGTATAAAGGATGACATCATCCACTAATCGTGCGTTACAAGTAGAATTCTACTCGTAAAGCGAGTTCGGTTTTGAAAAACAAATGACATCATTTCTTGATTGTGTTTTACACGTAGAAT TCTACTCGTAAAGTATGTTCAGTTTAAAAAACAAATGACATCATTTTACAGATGACATCATTTCTTGATTATGTTTTACAAGTAGAATTCTACTCGTAAAGCGAGTTTTAGTTTTAAAAAACAAATGACATCATCTCTTGATTATGTTTTACAAGTAGAATTCTACTCGTAAAGCGAGTTTAGTTTTGAAAAACAAATGACATCATCTCTTGATTATGTTTTACAAGTAGAATTCTACTCGTAAAGCGAGTTTAGTTTTGAAAAACAAATGACATCCCTTGATCATGCGTTACAAGTAGAATTCTACTCGTAAAGCGAGTTGATTTTGATTACAATATT

[0057] sequence number 19 ATGCATATAATTGTGTACAAAATATGACTCATTAATCGATCGTGCGTTACAAGTAGAATTCTACTGGTAAAGCAAGTTCGGTTGTGAGCCGTGTGCAAAACATGACATCATAACTAATCATGTTTATAATCATGTGCAAAATATGACATCATCCGACGATTGTGTTTT ACAAGTAGAATTCTACTCGTAAAGCGAGTTTAAAAATTTTGTGACGTCAATGAAACAACGTGTAATATTTTTTACAATATTTAAGTGAAACATTATGACTTCCAATAATTTTGTGGATGTGGATACGTTTGCAAGACAATTGATTACAGATAAATGTAGTGCTCTAATC GAAAGATGCGGATCTGTTGCCGGCAAACATTTTAGAGATTTAGAGAAAGGCCAGAGACAAGTATTTTGAGGTGCCAACTCAAAAAAACTATGAATACATTAAAAAATTATTTTTACGACAAAATATATGGACGATTCGATAGATTATAAAGATTTTAACAGACGCATCCTATTGATAGTTTTTAAAATTCGCTTTAAACAAGAGCACCAACTACTTTCCATCGTACTAAAAGAGATCGAGGTGGCCATTAAACGTTTAAACAAAAATTAACCCCGATTTAAAGAGTTCCGCGCAATGCTTCAGCATTACAAATGAATGTTTGGAAAATCTAGA

[0058] sequence number 20 AACTGGCTTTACGAGTAGAATTCTACTTGTAAAACACAATCAAGAAATGATGTCATTTTTGTACGTGATTATAAACATGTTTAAACATGGTACATTGAACTTAATTTTTGCAAGTTGATAAACTAGATTAATGTATGACTCATTTGTTTGTGCAAGTTGATAAACGTGATTAATATATGACTCATATGTTTGTGCAAAAATGGTGTCCATCGTACAAACTCGCTTTACGATAGAATTCTA CTTGTAAAACACAATCGAGGGATGATGTCATTTGTAGAATGATGTCATTTGTTTTTTCAAACCGAACTCGCTTACGAGTAGAATTCTACTTGTAAAACACAATCGAGGGATGATGTCATTTGTAGAATGATGTCATCGTACAAACTCGCTTTACGAGTAGAATTCTAGTAAAACAC

[0059] sequence number 21 TTGAAAATTATTGCCTAATATTATTTTTGTCAGTTCGTTGTCATTATTAATTTGGATGATGTCCATTTGTTTTTAAAATTGAACTGGCTTTACGAGTAGAATTCTACGCGTAAACACAATCAAGTATGAGTCATAAGCTGATGTCATGTTTTGCACACGGCTCATAACCGAACTGGCT TTACGAGTAGAATTCTACTTGTAACGCACGATCGAGTGGATGATGGTCATTGTTTTTCAAATCGAGATGATGTCATGTTTTGCACAGGGCTCATAAACTGCTTTACGAGTAGAATTCTACGTGTAACGCACGATCGATTGATGAGTCATTGTTTTGCAATATGATATCATACATATG ACTCATTTGTTTTTCAAAACCGAACTTGATTTACGGGTAGAATTCTACTCGTAAAGCACAATCAAAAGATGATGTCATTTGTTTTTCAAAACTGAACTCTCGGCTTTACGAGTAGAATTCTACGTGTAAAACACAATCAAGAAATGATGTCATTTGTTATAAAAATAAAGCTGATGTCA TGTTTTGCACATGGCTCATAACTAAACTCGCTTTACGGGTAGAATTCTACGCGTAAAACATGATTGATAATTAAATAATTCATTTGCAAAGCTATACGTTAAATCAAACGGACGTTATGGAATTGTATAATATTAAATATGCAATTGATCCAACAAAATAAAATTATAATAGAGCAAGTCGAC

[0060] Preferably, in some embodiments, the baculovirus recombinant homology region is selected from hr1. In some embodiments, hr1 is a repeated sequence interspersed in the baculovirus genome, and hr1 is thought to function not only as a baculovirus replication origin but also as an enhancer. Its enhancing effect is not apparent in the early stages of infection but becomes more pronounced in the later stages of infection. In some embodiments, the baculovirus recombinant homology region (hr) comprises hr2. In some embodiments, the baculovirus recombinant homology region (hr) comprises hr3. In some embodiments, the baculovirus recombinant homology region (hr) comprises hr4. In some embodiments, the baculovirus recombinant homology region (hr) comprises hr5.

[0061] In some embodiments, the nucleic acid construct further comprises a baculovirus promoter. In some embodiments, the baculovirus promoter is selected from one or more (e.g., two, three, or four) of the polyhedron promoter (pH), Gp64, p6.9, or p10. In some embodiments, hr can bind in cis to the baculovirus promoter to promote IE-mediated transactivation, and simultaneously, IEs are believed to bind to hr1 in a dimeric form to increase expression of downstream proteins.

[0062] In some embodiments, the structure of the nucleic acid construct comprises IE gene expression cassette-Cap gene expression cassette-hr1-ITR-foreign 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-foreign target gene expression cassette-ITR-Rep gene expression cassette.

[0063] The gene expression cassette comprises a gene and its promoter.

[0064] In a preferred embodiment, the structure of the nucleic acid construct comprises pA-IE1-pH-pA-Cap-p6.9p10-hr1-ITR-foreign target gene and its promoter-pA-ITR-pH-Rep-pA. In a preferred embodiment, the structure of the nucleic acid construct is pA-IE1-pH-pA-Cap-p6.9p10-hr1-ITR-foreign target gene and its promoter-pA-ITR-pH-Rep-pA.

[0065] Specifically, in some embodiments, the nucleic acid construct is an adeno-associated virus vector or a recombinant baculovirus vector. In some embodiments, the nucleic acid construct is an adeno-associated virus vector. In some embodiments, the nucleic acid construct is a recombinant baculovirus vector. In some embodiments, the recombinant baculovirus vector is a recombinant baculovirus shuttle vector.

[0066] In some embodiments, the adeno-associated virus vector or recombinant baculovirus vector further comprises a vector backbone.

[0067] In some embodiments, the vector backbone can be selected from suitable vector backbones available on the market, such as pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB or pFastBacHTC.

[0068] In one embodiment, the vector backbone is pFastBacdual.

[0069] In some embodiments, the titer of rAAV obtained by packaging the nucleic acid construct is at least 1 x 10 12 VG / mL (e.g., at least 1.5x10 12 , 2x10 12 , 2.5x10 12 , 3x10 12 , 3.5x10 12 , 4x10 12 , 4.5x10 12 , 5x1012 , 5.5x10 12 , 6x10 12 , 7x10 12 , 7.5x10 12 , 8x10 12 , 8.5x10 12 , 9x10 12 , 9.5x10 12 , 1x10 13 , 5x10 13 , or 1x10 14 VG / mL), i.e., the number of AAV genome copies contained in 1 ml of virus culture is at least 1 x 10 12 (e.g., at least 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 , or 1x10 14 )

[0070] In some embodiments, the titer of the rAAV obtained by packaging the nucleic acid construct is at least 2.60E+12VG / mL, i.e., the number of copies of the AAV genome contained per ml of virus culture is at least 2.60E+12.

[0071] The yield of rAAV was detected by qPCR, and the detection procedure was as follows:

[0072] The standard used in quantitative PCR is the pAAV-MCS plasmid linearized by PvuI-HF(NEB) single restriction enzyme digestion.

[0073] The primer sequences used in quantitative PCR are as follows: ITR Forward primer 5'-GGAACCCCTAGTGATGGAGTT-3' (SEQ ID NO: 2) ITR Reverse primer 5'-CGGCCTCAGTGAGCGA-3' (SEQ ID NO: 3)

[0074] Quantitative PCR operating program: 95°C for 60 seconds (95°C for 15 seconds, 60°C for 30 seconds, 40 cycles). After drawing a standard curve from the C values obtained by quantitative PCR and the concentrations of the standards, the titers of the samples were calculated.

[0075] A second aspect of the present invention provides a recombinant baculovirus, which is obtainable by constructing any of the above-mentioned nucleic acid constructs using a baculovirus system, or by co-constructing a nucleic acid construct containing any element of any of the above-mentioned nucleic acid constructs using a baculovirus system.

[0076] A nucleic acid construct comprising any element in any of the above nucleic acid constructs is primarily an AAV gene. The term "nucleic acid construct" refers to a nucleic acid construct comprising a polynucleotide encoding an IE protein, a nucleic acid construct comprising a polynucleotide encoding a baculovirus recombination homology region, or a nucleic acid construct comprising a polynucleotide encoding a baculovirus recombination homology region. The nucleic acid construct comprising an AAV element can also be selected from a nucleic acid construct comprising a polynucleotide encoding a Cap protein, a nucleic acid construct comprising a polynucleotide encoding a Rep protein, or a nucleic acid construct comprising a polynucleotide encoding an AAV cis element. Recombinant baculovirus can be obtained by co-constructing the above nucleic acid constructs using a baculovirus system.

[0077] The baculovirus system is selected from the Bac-to-Bac system (from ThermoFisher / Invitrogen), the flashBac / BacMagic system (from Mirus / EMD / OET / Nextgen), the BaculoDirect system (from ThermoFisher / Invitrogen) or the BacPAK6 / Baculogold system (from BD Biosciences / Clonetech).

[0078] Specifically, a recombinant baculovirus is obtained by transforming competent cells with the above nucleic acid construct, extracting the Bacmid, and transfecting SF9 insect cells.

[0079] The competent cells can be selected from any competent cells available in the art, as long as they do not limit the scope of the present invention. For example, the competent cells can be DH10Bac.

[0080] A third aspect of the present invention provides an adeno-associated virus (AAV), which can be obtained by packaging any of the above-mentioned recombinant baculoviruses. The adeno-associated virus can be used to treat various diseases, such as hemophilia, spinal muscular atrophy, Duchenne muscular dystrophy, Parkinson's disease, and age-related macular degeneration.

[0081] A fourth aspect of the present invention provides a cell line, which is a cell line infected with any of the above-mentioned recombinant baculoviruses.

[0082] In some embodiments, the cell line is an insect cell line such as SF9 cells, SF21 cells, or High5 cells.

[0083] A fifth aspect of the present invention provides an adeno-associated virus vector system, which comprises a baculovirus system and the nucleic acid construct described above.

[0084] A sixth aspect of the present invention provides a method for constructing the above-mentioned nucleic acid construct, which method comprises incorporating an AAV element carrying a foreign target gene and a polynucleotide encoding an IE protein into a baculovirus vector backbone.

[0085] The method of construction further comprises incorporating a polynucleotide encoding a baculovirus recombinant homology region into a baculovirus vector backbone.

[0086] In some embodiments, the AAV elements include a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis element.

[0087] In some embodiments, the AAV cis elements are from inverted terminal repeat (ITR) sequences to be selected.

[0088] Specifically, in some embodiments, the polynucleotide encoding the IE protein is linked to the strong promoter pH.

[0089] In some embodiments, the polynucleotide encoding the baculovirus recombinant homology region protein is linked in cis to a baculovirus promoter. In some embodiments, the baculovirus promoter is p6.9 or p10.

[0090] In some embodiments, the baculovirus vector backbone is selected from pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB, or pFastBacHTC, or the like.

[0091] In some embodiments, the baculovirus recombination homology regions are selected from one or more (eg, two, three, four, or five) of hr1, hr2, hr3, hr4, or hr5.

[0092] A seventh aspect of the present invention provides a method for producing an adeno-associated virus, the method comprising the step of infecting an insect cell line with a recombinant baculovirus.

[0093] This method for producing adeno-associated virus can increase the yield of adeno-associated virus.

[0094] In some embodiments, the cell line is selected from an insect cell line, hi one embodiment, the insect cell line is selected from SF9 cells.

[0095] An eighth aspect of the invention provides a method of treating a disease, the method comprising administering to a patient an effective amount of any of the adeno-associated viruses described above.

[0096] In some embodiments, the disease includes, for example, hemophilia, spinal muscular atrophy, Duchenne muscular dystrophy, Parkinson's disease, age-related macular degeneration, and the like.

[0097] The present invention also includes any of the following numbered paragraphs:

[0098] 1. A nucleic acid construct comprising an AAV element and a polynucleotide encoding an IE protein, wherein the AAV element comprises a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis element.

[0099] 2. The nucleic acid construct of paragraph 1, further comprising a polynucleotide encoding a baculovirus recombination homology region.

[0100] 3. A nucleic acid construct according to paragraph 2, characterized in that the IE protein is selected from proteins encoded by one or more of Acie0, Acie01 or Acie02, and / or the baculovirus recombination homology region is selected from one or more of hr1, hr2, hr3, hr4 or hr5.

[0101] 4. A nucleic acid construct described in any one of paragraphs 1 to 3, characterized in that the nucleic acid construct further comprises a promoter of an IE protein gene, and the promoter of the IE protein gene is selected from one or more of Gp64, pH, p6.9 or p10.

[0102] 5. A nucleic acid construct according to any one of paragraphs 1 to 4, characterized in that the nucleic acid construct further comprises a baculovirus promoter, which is linked to a baculovirus recombinant homology region, and the baculovirus promoter is preferably one or more of pH, Gp64, p6.9 or p10.

[0103] 6. A nucleic acid construct according to any one of paragraphs 1 to 5, characterized in that the AAV cis elements are selected from ITR sequences.

[0104] 7. A nucleic acid construct according to any one of paragraphs 1 to 6, characterized in that the nucleic acid construct further comprises an exogenous target gene, the exogenous target gene being embedded in an AAV element.

[0105] 8. A nucleic acid construct described in any one of paragraphs 1 to 7, characterized in that the structure of the nucleic acid construct is IE gene expression cassette-Cap gene expression cassette-ITR-foreign target gene expression cassette-ITR-Rep gene expression cassette.

[0106] 9. A nucleic acid construct according to any one of paragraphs 1 to 8, characterized in that the nucleotide sequence of the nucleic acid construct is SEQ ID NO:1.

[0107] 10. A nucleic acid construct according to any one of paragraphs 1 to 9, characterized in that the nucleic acid construct is an adeno-associated virus vector or a recombinant baculovirus vector, the recombinant baculovirus vector preferably being a recombinant baculovirus shuttle vector.

[0108] 11. A recombinant baculovirus, characterized in that it is obtained by constructing the nucleic acid construct described in any one of paragraphs 1 to 10 using a baculovirus system, or by co-constructing a nucleic acid construct containing any element of the nucleic acid construct described in any one of paragraphs 1 to 10 using a baculovirus system.

[0109] 12. An adeno-associated virus, characterized in that it is obtained by infecting a cell with the recombinant baculovirus described in paragraph 11 and then packaging the virus.

[0110] 13. A cell line, characterized in that it is a cell line infected with a recombinant baculovirus according to paragraph 11.

[0111] 14. An adeno-associated virus vector system, characterized in that it comprises a baculovirus system and a nucleic acid construct according to any one of paragraphs 1 to 10.

[0112] 15. A method for constructing a nucleic acid construct according to any one of paragraphs 1 to 10, characterized in that it comprises incorporating an AAV element carrying a foreign target gene and a polynucleotide encoding an IE protein into a baculovirus vector backbone.

[0113] 16. A method of construction according to paragraph 15, characterized in that the method of construction comprises one or more of the following features: 1) The AAV elements include a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis element, and the AAV cis element is preferably an ITR sequence. 2) The IE protein is encoded by one or more of the Acie0, Acie01, or Acie02 genes. 3) The baculovirus vector backbone is selected from one of pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB or pFastBacHTC. 4) The baculovirus recombination homology region is selected from one or more of hr1, hr2, hr3, hr4, or hr5. 5) Incorporating a polynucleotide encoding a baculovirus recombinant homology region into the baculovirus vector backbone.

[0114] 17. A method for producing an adeno-associated virus, comprising the step of infecting an insect cell line with the recombinant baculovirus described in paragraph 11.

[0115] Hereinafter, embodiments of the present invention will be described through specific examples, but those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied by other different specific embodiments, and various modifications or changes can be made to the details of the specification based on different viewpoints and applications without departing from the spirit of the present invention.

[0116] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the following specific specific embodiments. It should also be understood that the terms used in the examples of the present invention are intended to describe specific specific embodiments, and do not limit the scope of protection of the present invention. In the description and claims of the present invention, the singular forms "one", "one" and "this" include the plural forms unless the context clearly dictates otherwise.

[0117] When an example provides a range of values, it should be understood that the two endpoints of each range and any value between the two endpoints can be selected unless otherwise specified in the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, devices, and materials used in the examples, those skilled in the art can also realize the present invention using any methods, devices, and materials in the prior art that are similar or equivalent to the methods, devices, and materials described in the examples of the present invention, based on an understanding of the prior art and the description of the present invention.

[0118] In the following examples, EGFP is used as an example of a foreign target gene.

[0119] Example 1 Construction of a recombinant baculovirus shuttle vector and acquisition of a recombinant baculovirus Identification of related genes: The genes encoding the Cap and Rep proteins were both derived from the AAV2 genome (GenBank: AF043303.1). Point mutations were performed based on the ribosome leaky scanning mechanism, with reference to the literature (Non-Patent Document 2): Acie01 (GenBank: NC_001623.1), and the baculovirus recombination homology region hr1 (GenBank: M14313.1).

[0120] The synthesis of the corresponding sequences was outsourced to a gene company: the Cap gene sequence (nucleotide sequence of SEQ ID NO: 4), the Rep gene sequence (nucleotide sequence of SEQ ID NO: 5), the combination of hr1 with p6.9 and p10 promoters (hr1p6.9p10, nucleotide sequence of SEQ ID NO: 6), and the combination of pH promoter and Acie01 (pH-Acie01, nucleotide sequence of SEQ ID NO: 7). Using a homologous recombination method well known in the art, the above sequences and the ITR-CMV-EGFP sequence in the pAAV-EGFP vector (transformed by Konglin Biotechnology (Hangzhou) Co., Ltd.) were cloned into pFastBacdual (Invitrogen). After sequencing and identification, two recombinant baculovirus shuttle vectors were created: pFBd-Cap-ITR-Rep (Figures 1 and 2, nucleotide sequence of SEQ ID NO: 8) and pFBd-IE-hr1Cap-ITR-Rep (Figures 3 and 4, SEQ ID NO: 9). The nucleotide sequences of the respective genes (nucleotide sequence no. 9) were obtained.

[0121] The sequences of SEQ ID NOs: 4 to 9 are as follows:

[0122] SEQ ID NO:4

[0123] SEQ ID NO:5 GTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCCAAATTTGACTCACCGCGCGTCTGGATCATGACTTGGGAAGGTCACCAAGCAGGAAGTCCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAAGACCCCCCAGTGACGCAGTATAAGTGAGCCCAAACGGTGCGCGAGTC GTTGCGCAGCCATCGACGTCAGACGCGGAAGCATCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAG ACTGTTTAGAGTGCTTTCCCGTGTCCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAA

[0124] அக்க்கு நுர்க்கு 6

[0125] SEQ ID NO:7 CCAGGACAAGAACCTCTTCACTTTGCCCATCATGCTGAGCCGCAAGGAATCAAACGAGATCGAAACCGCCTCTAACACTTCTTCGTCTCGCCATACGTTTCCCAGATCCTCAAGTACTCGGAGTCCGTCCAATTCCCGGACAACCCTCCCAACAAGTACGTCGTTGATA ACCTGAACCTCATCGTGAACAAGAAGAGCACTCTGACATACAAGTACTCGTCCGTCGCTAACCTGCTCTTCAACAACTACAAGTACCACGACAACATCGCTTCTAACAACAACGCCGAGAACCTCAAGAAGGTCAAGAAGGAAGACGGAAGCATGCACATCGTTGAGCAGT ACTTGACTCAAAACGTCGATAACGTTAAGGGTCCAACTTCATCGTGTTGTCCTTCAAGAACGAGGAAAGGCTGACCATCGCTAAGAAGAACAAGGAGTTCTACTGGATCTCTGGCGAAATCAAGGACGTTGATGTGAGCCAGGTCATCCAAAGTACAACAGATTCAAG CACCACATGTTCGTGATCGGCAAGGTCAACCGTCGGAGTCAACTACACTGCACAACAACTTGCTGAAGCTCTTGGCCTTGATCCTGCAGGGACTGGTGCCACTCTCCGACGCCATCACATTCGCCGAGCAAAAGCTCAACTGCAAGTACAAGAAGTTCGAGTTCAACTAA

[0126] sequence number 8 CTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCACAGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACTCCATGTGACAAACCGTCATCTTCGGCTACTTT

[0127] SEQ ID NO: 9

[0128] The two recombinant vectors were each transformed into DH10Bac competent cells (Invitrogen) and subjected to blue-white selection. White-positive clones were collected and cultured, and bacmids were extracted using PureLink™ HiPure Plasmid DNA Purification Kits (Invitrogen). The two types of bacmids were each transfected into adherent SF9 cells (Gbico). Three days later, primary recombinant baculoviruses were collected and amplified up to the P2 passage. Finally, the resulting P2-passage recombinant baculoviruses were named BV-Cap-ITR-Rep and BV-IE-hr1Cap-ITR-Rep, respectively, and the viral titers were measured using the TCID50 method. For details on this section, please refer to the Bac to Bac Expression System operation manual (Invitrogen). stomach.

[0129] Example 2 Packaging and Titering of rAAV2 Two types of P2-passaged recombinant baculoviruses were each infected into 25 mL of suspension SF9 cells (density 3.0E+06 cells / mL) at an MOI of 0.1. Four days later, the cells were harvested and lysed in 5 mL of TNT lysis solution (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton® X-100, 10 mmol / L MgCl2) by incubation at room temperature for 1 hour. Nuclease was added to a final concentration of 50 U / mL and digested at 37°C for 2 hours. Samples were then taken and the outer shells were digested with proteinase K (TIANGEN) at 56°C for 1 hour. Titers were measured by qPCR. The detection procedure was as follows:

[0130] The standard used in quantitative PCR was the pAAV-EGFP plasmid linearized by PvuI-HF(NEB) single restriction enzyme digestion (plasmid sequence shown below in SEQ ID NO: 22).

[0131] SEQ ID NO: 22

[0132] The primer sequences used in quantitative PCR were as follows: ITR Forward primer 5'-GGAACCCCTAGTGATGGAGTT-3' (SEQ ID NO: 2) ITR Reverse primer 5'-CGGCCTCAGTGAGCGA-3' (SEQ ID NO: 3)

[0133] Quantitative PCR operating program: 95°C for 60 seconds (95°C for 15 seconds, 60°C for 30 seconds, 40 cycles).

[0134] A standard curve was constructed from the C values obtained by quantitative PCR and the concentrations of the standard samples, and the titers of the samples were calculated. The rAAV yields packaged with BV-Cap-ITR-Rep and BV-IE-hr1Cap-ITR-Rep were 2.39E+04 VG / cell (equivalent to 7.18E+10 VG / mL) and 8.67E+05 VG / cell (equivalent to 2.60E+12 VG / mL), respectively.

[0135] Example 3 Comparison of EGFP expression during cell infection process and Cap protein expression level in cell lysate At 48, 72, and 96 hours after infection, cells infected with the two types of baculoviruses were observed under an inverted fluorescence microscope. The optimized recombinant baculovirus significantly reduced the expression of the target gene (in this example, the target gene was EGFP) (Figure 5). The reduced expression of the target gene (EGFP) appears to reduce interference with rAAV packaging in the cells, favoring the expression of rAAV packaging-related structural and functional proteins.

[0136] WB detection was performed on the same amount of cell lysate using a Cap protein antibody (Progen) (Figure 6). The results showed that the expression level of Cap protein in cells significantly increased after infection with the optimized recombinant baculovirus.

[0137] Example 4 Electron Microscopy Detection and Infectivity Measurement of Purified rAAV2 Cell lysates were purified using POROS™ CaptureSelect® affinity chromatography media, and titers were measured by fluorescent quantitative PCR (as in Example 2). The purified rAAV2 was negatively stained and observed under a transmission electron microscope (Figure 7). Genome-packaged rAAV2 particles were solid, while the centers of defective rAAV particles containing no nucleic acid were darkly stained. Overall, the shape was intact, and the empty capsid rate was significantly reduced (approximately 3%) compared to conventional methods (Non-Patent Document 1). This indicates that the use of optimized baculovirus vector constructs significantly reduces the rate of empty capsid virus in packaged products, significantly easing the burden of downstream processes such as empty capsid virus removal.

[0138] The purified rAAV2 was serially diluted and infected into 293T cells (48-well plates, 5.0E+04 cells / well) cultured at MOIs of 10,000, 2,000, 400, and 80, respectively. Two days after infection, EGFP expression was observed under a fluorescence microscope. The experimental results demonstrated that the rAAV2 produced by this system had high in vitro infection activity (Figure 8).

[0139] The above examples are intended to illustrate the disclosed embodiments of the present invention and should not be construed as limiting the present invention. Moreover, various modifications and variations of the methods of the present invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the present invention is not limited to these specific embodiments. Indeed, various modifications such as those described above that would be apparent to those skilled in the art for achieving the present invention are intended to be included within the scope of the present invention.

Claims

1. A nucleic acid construct, comprising: AAV elements, a polynucleotide encoding an IE protein, and a polynucleotide encoding a baculovirus recombination homology region; the AAV element comprises a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis element; the IE protein is encoded by Acie01; A nucleic acid construct, characterized in that the baculovirus recombination homology region is hr1.

2. The nucleic acid construct of claim 1, further comprising a promoter of an IE protein gene, wherein the promoter of the IE protein gene is selected from one or more of Gp64, pH, p6.9 or p10.

3. 2. The nucleic acid construct of claim 1, further comprising a baculovirus promoter, wherein the baculovirus promoter is linked to a baculovirus recombination homology region.

4. 4. The nucleic acid construct of claim 3, wherein the baculovirus promoter is one or more of pH, Gp64, p6.9 or p10.

5. The nucleic acid construct of claim 1, wherein the AAV cis elements are selected from ITR sequences.

6. The nucleic acid construct of claim 1, further comprising an exogenous target gene, wherein the exogenous target gene is embedded in an AAV element.

7. The nucleic acid construct has a structure of IE gene expression cassette-Cap gene expression cassette-ITR-foreign target gene expression cassette-ITR-Rep gene expression cassette. The nucleic acid construct according to claim 6 .

8. 2. The nucleic acid construct of claim 1, wherein the nucleotide sequence of the nucleic acid construct is as set forth in SEQ ID NO:

1.

9. The nucleic acid construct according to claim 1, characterized in that the nucleic acid construct is an adeno-associated virus vector or a recombinant baculovirus vector.

10. 10. The nucleic acid construct of claim 9, wherein the recombinant baculovirus vector is a recombinant baculovirus shuttle vector.

11. A recombinant baculovirus obtained by constructing the nucleic acid construct according to any one of claims 1 to 10 using a baculovirus vector.

12. An adeno-associated virus obtained by infecting a cell with the recombinant baculovirus of claim 11 and then packaging the virus.

13. A cell line, characterized in that it is a cell line infected with the recombinant baculovirus of claim 11.

14. An adeno-associated virus vector, comprising a baculovirus vector and the nucleic acid construct according to any one of claims 1 to 10.

15. 11. A method for constructing a nucleic acid construct according to any one of claims 1 to 10, comprising incorporating the AAV element carrying a foreign target gene, the polynucleotide encoding the IE protein, and the polynucleotide encoding the baculovirus recombination homology region into a baculovirus vector backbone.

16. 16. The method of claim 15, wherein the method comprises the following features: The baculovirus vector backbone is selected from one of pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB or pFastBacHTC.

17. The method of claim 16, wherein the AAV cis element is an ITR sequence.

18. A method for producing an adeno-associated virus, comprising the step of infecting an insect cell line with the recombinant baculovirus of claim 11.

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