A nucleic acid construct for improving adeno-associated virus production and a method for constructing the same

Abstract

Provided are a nucleic acid construct for improving production of adeno-associated virus and a method for constructing the same, the nucleic acid construct comprising: an adeno-associated virus (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 an AAV cis-acting element. The method for constructing comprises integrating the AAV element carrying an exogenous target gene and the polynucleotide encoding the IE protein into a baculovirus vector skeleton. The recombinant adeno-associated virus (rAAV) obtained by the method has a lower empty shell rate, while improving the rAAV production of a single cell and a unit volume of culture, reducing the production cost, and being easy to scale up.

Description

Technical Field

[0001] This invention relates to the field of gene transfer vector technology, and in particular to a nucleic acid construct for increasing adeno-associated virus yield and its construction method. Background Technology

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

[0003] Baculovirus systems are a common method for producing recombinant proteins. Compared to traditional three-plasmid transient conversion, insect cells offer advantages such as high culture density, serum-free suspension culture, and ease of scaling up. Furthermore, baculovirus-infected cell production provides greater batch stability. Although researchers began using them for rAAV production in 2002, various factors, such as the difficulty in achieving ideal ratios of Cap, Rep, and AAV vector genomic DNA in insect cells, have resulted in low AAV packaging efficiency. Moreover, traditional methods yield recombinant AAV vectors with a high empty shell rate (11%-34%) (Benskey et al. 2016). Empty shell AAV is a crucial impurity that must be removed in the preparation of clinical-grade AAV vectors because its physicochemical properties are very similar to those of AAV carrying the target gene, making purification extremely difficult. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a nucleic acid construct and its construction method for improving the yield and quality of recombinant adeno-associated virus, thereby solving the problems in the prior art.

[0005] To achieve the above and other related objectives, the nucleic acid construct provided in the first aspect of the present invention includes: an AAV element, a polynucleotide encoding an IE protein, and a polynucleotide encoding a baculovirus recombinant homologous region, wherein the AAV element includes a polynucleotide encoding a Cap protein, a polynucleotide encoding a Rep protein, and an AAV cis-acting element.

[0006] In some embodiments, the IE protein is encoded by one or more genes selected from Acie0, Acie01, and Acie02, and / or the baculovirus recombinant homologous region is selected from one or more genes selected from hr1, hr2, hr3, hr4, and hr5.

[0007] In some embodiments, the nucleic acid construct further includes a promoter for the IE protein gene. In some embodiments, the promoter for the IE protein gene is selected from one or more of Gp64, pH, p6.9, or p10.

[0008] In some embodiments, the nucleic acid construct further includes a baculovirus promoter. In some embodiments, the baculovirus promoter is linked to a baculovirus recombinant homologous region. In some embodiments, the baculovirus promoter is preferably one or more of pH, Gp64, p6.9, or p10.

[0009] In some embodiments, the AAV cis-acting element is selected from the ITR sequence.

[0010] In some embodiments, the nucleic acid construct further includes a foreign target gene. In some embodiments, the foreign target gene is embedded in an AAV element.

[0011] In some embodiments, the structure of the nucleic acid construct includes: IE gene expression box - Cap gene expression box - ITR - exogenous target gene expression box - ITR - Rep gene expression box. In some embodiments, the structure of the nucleic acid construct is: IE gene expression box - Cap gene expression box - ITR - exogenous target gene expression box - ITR - Rep gene expression box.

[0012] In some embodiments, the nucleotide sequence of the nucleic acid construct includes SEQ ID NO.1, or includes 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 shown in SEQ ID NO.1.

[0013] 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.

[0014] A second aspect of the present invention provides a recombinant baculovirus, wherein the recombinant baculovirus is obtained by constructing any of the described nucleic acid constructs using a baculovirus system, or by constructing nucleic acid constructs comprising any element of any of the described nucleic acid constructs using a baculovirus system.

[0015] A third aspect of the present invention provides an adeno-associated virus, wherein the adeno-associated virus is obtained by infecting cells with any of the recombinant baculoviruses described herein and then packaging the cells.

[0016] A fourth aspect of the present invention provides a cell line, said cell line being a cell line infected with any of the recombinant baculoviruses described above.

[0017] A fifth aspect of the present invention provides an adeno-associated virus vector system, the adeno-associated virus vector system comprising a baculovirus system and the aforementioned nucleic acid construct.

[0018] The sixth aspect of the present invention provides a method for constructing the nucleic acid construct, the method comprising integrating an AAV element carrying a foreign target gene, a polynucleotide encoding an IE protein, and a polynucleotide encoding a baculovirus recombinant homologous region into a baculovirus vector backbone.

[0019] The method includes one or more (e.g., two or three) of the following features:

[0020] 1) The AAV element includes a polynucleotide encoding the Cap protein, a polynucleotide encoding the Rep protein, and an AAV cis-acting element, for example, the AAV cis-acting element is preferably an ITR sequence;

[0021] 2) The polynucleotide encoding the IE protein is selected from one or more of Acie0, Acie01, or Acie02;

[0022] 3) The baculovirus vector backbone is selected from one of pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB, or pFastBacHTC; or

[0023] 4) The baculovirus recombinant homologous region is selected from one or more of hr1, hr2, hr3, hr4 or hr5.

[0024] The seventh aspect of the present invention provides a method for producing adeno-associated virus, the method comprising the following steps: infecting an insect cell line with the recombinant baculovirus.

[0025] As described above, the nucleic acid construct and its construction method for increasing adeno-associated virus yield according to the present invention have at least the following beneficial effects:

[0026] 1) This invention significantly reduces the expression of the target gene inserted in the baculovirus vector in insect cells, reduces the impact of target gene expression on insect cells, and improves the growth indicators of production cells to make them more conducive to the packaging and production of rAAV.

[0027] 2) Compared with the traditional production of adherent 293 and 293T cells and conventional baculovirus production methods, the optimized vector construction method significantly increases the rAAV yield per cell and per unit volume of culture, reduces production costs, and can be scaled up.

[0028] 3) It has a wide range of applications, including the production of adeno-associated virus gene therapy vectors of various scales and types. Attached Figure Description

[0029] Figure 1 The diagram shown is a schematic diagram of the pFBd-Cap-ITR-Rep carrier structure of the present invention.

[0030] Figure 2 The image shown is a schematic diagram of the pFBd-Cap-ITR-Rep vector of the present invention.

[0031] Figure 3 The diagram shown is a schematic diagram of the pFBd-IE-hr1Cap-ITR-Rep carrier structure of the present invention.

[0032] Figure 4 The image shown is a schematic diagram of the pFBd-IE-hr1Cap-ITR-Rep vector of the present invention.

[0033] Figure 5 The image shows a comparison of EGFP expression levels in SF9 cells 2-4 days after infection with BV-Cap-ITR-Rep and BV-IE-hr1Cap-ITR-Rep of the present invention.

[0034] Figure 6 The image shows the Western blot (WB) assay of two types of BV-infected cell lysates using the AAV Cap monoclonal antibody of this invention.

[0035] Figure 7 The image shows the electron microscopy findings of rAAV2 (titer 2.0E+13VG / mL) of this invention.

[0036] Figure 8 The image shows fluorescence observation of 293T cells infected with rAAV2 packaged with BV-Cap-ITR-Rep and BV-IE-hr1Cap-ITR-Rep according to the present invention, 2 days later. Detailed Implementation

[0037] This invention is at least partly based on the discovery that nucleic acid constructs containing AAV elements, polynucleotides encoding IE proteins, and polynucleotides encoding baculovirus recombinant homologous regions can significantly control the expression of inserted target genes in insect cells serving as baculovirus hosts, improving cell state, increasing adeno-associated virus (aAAV) packaging efficiency, and thus significantly increasing aAAV yield. This invention unexpectedly reduces the empty shell rate of rAAV (e.g., even increasing Cap expression does not lead to an increase in empty shells), produces rAAV with better infectivity, and significantly increases rAAV yield per single cell and per unit volume of culture, thus making it suitable for large-scale production of various aAAV gene therapy vectors.

[0038] Unless otherwise defined below, all technical and scientific terms used in this invention shall have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0039] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. The nucleic acid construct can be a lentiviral vector or an adeno-associated virus vector, which includes a vector backbone, i.e., an empty vector and an expression framework.

[0040] The term "vector" refers to a nucleic acid or polynucleotide fragment used to introduce or transfer one or more nucleic acids or polynucleotides into a target cell or tissue. Typically, a vector is used to introduce foreign DNA into another cell or tissue. A vector may contain a bacterial resistance gene for growth in bacteria and a promoter for expression of a target protein in an organism. The DNA can be produced in vitro by PCR or one or more suitable techniques known to those skilled in the art.

[0041] The first aspect of the present invention provides 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-acting element.

[0042] In one embodiment, the nucleic acid construct further includes a polynucleotide encoding a baculovirus recombinant homologous region.

[0043] In some embodiments, the nucleotide sequence of the nucleic acid construct includes SEQ ID NO.1, or includes 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 shown in SEQ ID NO.1 below.

[0044] SEQ ID NO:1:

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053] In some embodiments, the AAV element is 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. Different serotypes of AAV recognize different receptors on the cell surface, thus their infection efficiency in different tissues and cells can vary greatly, exhibiting a certain degree of organ-targeting specificity. Appropriate serotypes of AAV can be selected according to specific experimental objectives, thereby achieving highly efficient transduction of specific serotypes of AAV to specific types of cells and tissues.

[0054] In some embodiments, 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. In some embodiments, 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.

[0055] The Cap protein is a structural protein, typically comprising VP1, VP2, and VP3 structural proteins. It is believed that in some embodiments, the three structural proteins can assemble into the capsid protein of the AAV.

[0056] The Rep protein is a non-structural protein, typically including Rep78 and 52 proteins. It is believed that in some embodiments, the Rep protein regulates DNA replication and packaging.

[0057] In one embodiment, the AAV cis-acting element is selected from inverted terminal repeat sequences (ITR sequences). It is believed that in some embodiments, the ITR sequence can fold into a hairpin structure and is the only known cis-acting element required for AAV DNA replication initiation and packaging of recombinant AAV viral particles.

[0058] In some embodiments, the nucleic acid construct further includes a foreign target gene. In some embodiments, the foreign target gene is carried by an AAV element, i.e., the foreign target gene is embedded in an AAV element.

[0059] Specifically, in some embodiments, the exogenous target gene is embedded between the two ITR sequences.

[0060] Preferably, in some embodiments, the size of the exogenous target gene is ≤3.5kb. It is believed that in some embodiments, the size of the exogenous target gene is generally not too large due to the size limitation between the two ITR sequences, otherwise it may lead to poor packaging effect.

[0061] The types of exogenous genes vary depending on the experimental purpose. These exogenous genes can be genes related to the treatment of various diseases, such as AADC, FVIII, and FIX, or commonly used laboratory tool genes such as EGFP and mCherry genes.

[0062] It is believed that in some embodiments, the nucleic acid construct further includes a 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 includes Gp64. In some embodiments, the promoter of the IE protein gene includes pH. In some embodiments, the promoter of the IE protein gene includes p6.9. In some embodiments, the promoter of the IE protein gene includes p10.

[0063] Preferably, in some embodiments, the promoter of the IE protein gene is selected from a pH-strong promoter. It is believed that in some embodiments, adding an extra copy of ie to the nucleic acid construct and expressing it under pH control of a strong promoter, thereby increasing its level above the viral endogenous level, increases the survival of late-stage cells after baculovirus infection.

[0064] In some embodiments, the IE protein is selected from proteins encoded by one or more (e.g., two or three) genes of 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 shown in SEQ ID NO. 10, and the amino acid sequence of the IE0 protein is shown 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 shown in SEQ ID NO. 12, and the amino acid sequence of the IE1 protein is shown 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 shown in SEQ ID NO. 14, and the amino acid sequence of the IE2 protein is shown in SEQ ID NO. 15.

[0065] The sequences of SEQ ID NO.10-15 are shown below:

[0066] SEQ ID NO.10:

[0067]

[0068] SEQ ID NO.11:

[0069]

[0070] SEQ ID NO.12:

[0071]

[0072]

[0073] SEQ ID NO.13:

[0074]

[0075] SEQ ID NO.14:

[0076]

[0077] SEQ ID NO.15:

[0078]

[0079] It is believed that in some embodiments, the IE1 protein is a product of the baculovirus immediate early gene 1 (ie1), and the IE protein can also be referred to as a baculovirus transcriptional regulatory factor protein. It is a multifunctional protein capable of activating early and late viral genes and participating in viral genome replication, thus participating in the regulation of the viral cycle.

[0080] In some embodiments, the baculovirus recombinant homologous region (hr) is selected from one or more (e.g., two, three, four or five) of hr1 (nucleotide sequence as shown in SEQ ID NO. 16), hr2 (nucleotide sequence as shown in SEQ ID NO. 17), hr3 (nucleotide sequence as shown in SEQ ID NO. 18), hr4 (nucleotide sequence of hr4left as shown in SEQ ID NO. 19, nucleotide sequence of hr4right as shown in SEQ ID NO. 20), and hr5 (nucleotide sequence as shown in SEQ ID NO. 21).

[0081] The sequences of SEQ ID NO.16-21 are shown below:

[0082] SEQ ID NO.16:

[0083]

[0084]

[0085] SEQ ID NO.17:

[0086]

[0087] SEQ ID NO.18:

[0088]

[0089] SEQ ID NO.19:

[0090]

[0091] SEQ ID NO.20:

[0092]

[0093] SEQ ID NO.21:

[0094]

[0095]

[0096] Preferably, in some embodiments, the baculovirus recombinant homologous region is selected from hr1. It is believed that in some embodiments, hr1 is a repetitive sequence dispersed in the baculovirus genome, and hr1 serves as both the origin of baculovirus replication and an enhancer. Although the enhancing effect is not obvious in the early stages of infection, it becomes more significant in the later stages. In some embodiments, the baculovirus recombinant homologous region (hr) includes hr2. In some embodiments, the baculovirus recombinant homologous region (hr) includes hr3. In some embodiments, the baculovirus recombinant homologous region (hr) includes hr4. In some embodiments, the baculovirus recombinant homologous region (hr) includes hr5.

[0097] In some embodiments, the nucleic acid construct further includes a baculovirus promoter. In some embodiments, the baculovirus promoter is selected from one or more (e.g., two, three, or four) of a polyhedral promoter (pH), Gp64, p6.9, or p10. It is believed that in some embodiments, the hr1 cis-linked baculovirus promoter can promote IE-mediated transactivation, while the IE binds to hr1 in a dimer form, increasing the expression of downstream proteins.

[0098] In some embodiments, the structure of the nucleic acid construct includes: IE gene expression box - Cap gene expression box - hr1 - ITR - exogenous target gene expression box - ITR - Rep gene expression box. In some embodiments, the structure of the nucleic acid construct is: IE gene expression box - Cap gene expression box - ITR - exogenous target gene expression box - ITR - Rep gene expression box.

[0099] The gene expression cassette includes the gene and its promoter.

[0100] In a preferred embodiment, the nucleic acid construct comprises: pA-IE1-pH-pA-Cap-p6.9p10-hr1-ITR-exogenous target gene and its promoter-pA-ITR-pH-Rep-pA. In another preferred embodiment, 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.

[0101] 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.

[0102] In some embodiments, the adeno-associated virus vector or recombinant baculovirus vector further includes a vector backbone.

[0103] In some embodiments, the carrier skeleton may be selected from suitable carrier skeletons available on the market, such as pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB, or pFastBacHTC.

[0104] In one embodiment, the carrier skeleton is pFastBacdual.

[0105] In some embodiments, the rAAV titer obtained by packaging with the nucleic acid construct is at least 1 x 10⁻⁶. 12 VG / mL (e.g., at least 1.5 x 10⁻⁶) 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 VG / mL), meaning that the number of AAV genome copies per ml of viral culture medium is at least 1 x 10^6. 12 (For example, 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 129x10 12 9.5x10 12 1x10 13 5x10 13 or 1x10 14 ).

[0106] In some embodiments, the rAAV titer obtained by packaging with the nucleic acid construct is at least 2.60E+12VG / mL, that is, the number of AAV genome copies contained in each ml of viral culture medium is at least 2.60E+12.

[0107] The yield of rAAV was detected by qPCR, and the detection steps are as follows:

[0108] The standard used for quantitative PCR was the linearized pAAV-MCS plasmid digested with Pvu I-HF (NEB).

[0109] The primer sequences used for quantitative PCR are:

[0110] ITR Forward primer 5'-GGAACCCCTAGTGATGGAGTT-3'(SEQ ID NO:2)

[0111] ITR Reverse primer 5'-CGGCCTCAGTGAGCGA-3'(SEQ ID NO:3)

[0112] The quantitative PCR program was: 95℃ for 60s, (95℃ for 15s, 60℃ for 30s, 40 cycles). After plotting the standard curve based on the CT values ​​obtained from the quantitative PCR and the standard concentrations, the sample titer was calculated.

[0113] A second aspect of the present invention provides a recombinant baculovirus, wherein the recombinant baculovirus is obtained by constructing any of the nucleic acid constructs using a baculovirus system, or by constructing nucleic acid constructs comprising any element of any of the nucleic acid constructs using a baculovirus system.

[0114] The nucleic acid construct comprising any element from any of the aforementioned nucleic acid constructs primarily refers to: nucleic acid constructs comprising AAV elements, nucleic acid constructs comprising polynucleotides encoding IE proteins, and polynucleotides encoding baculovirus homologous regions. The nucleic acid construct comprising AAV elements may also be selected from nucleic acid constructs comprising polynucleotides encoding Cap proteins, nucleic acid constructs comprising polynucleotides encoding Rep proteins, and nucleic acid constructs comprising AAV cis-acting elements. The recombinant baculovirus can be constructed from the above nucleic acid constructs using a baculovirus system.

[0115] The baculovirus system is selected from the Bac-to-Bac system (from ThermoFisher / Invitrogen), flashBac / BacMagic system (from Mirus / EMD / OET / Nextgen), BaculoDirect system (from ThermoFisher / Invitrogen), or BacPAK6 / Baculogold system (from BD Biosciences / Clonetech).

[0116] Specifically, the recombinant baculovirus is obtained by transforming competent cells with the nucleic acid construct, extracting Bacmid, and then transfecting SF9 insect cells.

[0117] The competent cells can be selected from any competent cells applicable in the art, as long as they do not limit the purpose of the invention. For example, the competent cells can be DH10Bac.

[0118] A third aspect of this invention provides an adeno-associated virus (AAV) obtained by packaging any of the recombinant baculoviruses described above. The AAV can be used to treat various diseases, such as hemophilia, spinal muscular atrophy, Duchenne muscular dystrophy, Parkinson's disease, and age-related macular degeneration.

[0119] A fourth aspect of the present invention provides a cell line, said cell line being a cell line infected with any of the recombinant baculoviruses described above.

[0120] In some embodiments, the cell line is an insect cell line, such as SF9 cells, SF21 cells, or High5 cells.

[0121] A fifth aspect of the present invention provides an adeno-associated virus vector system, the adeno-associated virus vector system comprising a baculovirus system and the nucleic acid construct.

[0122] The sixth aspect of the present invention provides a method for constructing the nucleic acid construct, the method comprising integrating an AAV element carrying an exogenous target gene and a polynucleotide encoding an IE protein into a baculovirus vector backbone.

[0123] The construction method also includes integrating polynucleotides encoding the recombinant homologous region of baculovirus into the baculovirus vector backbone.

[0124] In some embodiments, the AAV element includes a polynucleotide encoding the Cap protein, a polynucleotide encoding the Rep protein, and an AAV cis-acting element.

[0125] In some embodiments, the AAV cis-acting element is selected from inverted terminal repeat sequences (ITR sequences).

[0126] Specifically, in some embodiments, the polynucleotide encoding the IE protein is linked to a strong promoter pH.

[0127] In some embodiments, the polynucleotide encoding the baculovirus recombinant homologous region protein is cis-linked to the baculovirus promoter. In some embodiments, the baculovirus promoter is p6.9 or p10.

[0128] In some embodiments, the baculovirus vector backbone is selected from pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB, or pFastBacHTC, etc.

[0129] In some embodiments, the baculovirus recombinant homologous region is selected from one or more of hr1, hr2, hr3, hr4 or hr5 (e.g., two, three, four or five).

[0130] The seventh aspect of the present invention provides a method for producing adeno-associated virus, the method comprising the following steps: infecting an insect cell line with the recombinant baculovirus.

[0131] The method for producing adeno-associated virus can increase the yield of adeno-associated virus.

[0132] In some embodiments, the cell line is selected from insect cell lines. In one embodiment, the insect cell line is selected from SF9 cells.

[0133] The eighth aspect of the present invention provides a method for treating a disease, the method comprising administering to a patient an effective amount of any of the aforementioned adeno-associated viruses.

[0134] In some embodiments, the diseases include, for example, hemophilia, spinal muscular atrophy, Duchenne muscular dystrophy, Parkinson's disease, age-related macular degeneration, etc.

[0135] This invention also includes any of the following numbered paragraphs:

[0136] 1. A nucleic acid construct, characterized in that the nucleic acid construct comprises: an AAV element, 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-acting element.

[0137] 2. The nucleic acid construct according to paragraph 1, characterized in that the nucleic acid construct further includes a polynucleotide encoding a baculovirus recombinant homologous region.

[0138] 3. The nucleic acid construct according to paragraph 2 is characterized in that the IE protein is selected from one or more proteins encoded by Acie0, Acie01 or Acie02, and / or the baculovirus recombinant homologous region is selected from one or more of hr1, hr2, hr3, hr4 or hr5.

[0139] 4. The nucleic acid construct according to any one of paragraphs 1-3, characterized in that the nucleic acid construct further includes a promoter of the IE protein gene, wherein the promoter of the IE protein gene is selected from one or more of Gp64, pH, p6.9 or p10.

[0140] 5. The nucleic acid construct according to any one of paragraphs 1-4, characterized in that the nucleic acid construct further includes a baculovirus promoter, the baculovirus promoter being linked to a baculovirus recombination homologous region, and the baculovirus promoter preferably being one or more of pH, Gp64, p6.9 or p10.

[0141] 6. The nucleic acid construct according to any one of paragraphs 1-5, characterized in that the AAV cis-acting element is selected from an ITR sequence.

[0142] 7. The nucleic acid construct according to any one of paragraphs 1-6, characterized in that the nucleic acid construct further includes an exogenous target gene, wherein the exogenous target gene is embedded in an AAV element.

[0143] 8. The nucleic acid construct according to any one of paragraphs 1-7, characterized in that the structure of the nucleic acid construct is: IE gene expression box - Cap gene expression box - ITR - exogenous target gene expression box - ITR - Rep gene expression box.

[0144] 9. The nucleic acid construct according to any one of paragraphs 1-8, characterized in that the nucleotide sequence of the nucleic acid construct is as shown in SEQ ID NO.1.

[0145] 10. The nucleic acid construct according to any one of paragraphs 1-9, characterized in that the nucleic acid construct is an adeno-associated virus vector or a recombinant baculovirus vector, wherein the recombinant baculovirus vector is preferably a recombinant baculovirus shuttle vector.

[0146] 11. A recombinant baculovirus, characterized in that the recombinant baculovirus is obtained by constructing a baculovirus system from any of the nucleic acid constructs described in paragraphs 1-10, or by constructing a baculovirus system from nucleic acid constructs including any element of any of the nucleic acid constructs described in paragraphs 1-10.

[0147] 12. An adeno-associated virus, characterized in that the adeno-associated virus is obtained by packaging cells after infection with the recombinant baculovirus described in paragraph 11.

[0148] 13. A cell line, characterized in that the cell line is a cell line infected with the recombinant baculovirus described in paragraph 11.

[0149] 14. An adeno-associated virus vector system, characterized in that the adeno-associated virus vector system comprises a baculovirus system and any one of the nucleic acid constructs described in paragraphs 1-10.

[0150] 15. A method for constructing any one of the nucleic acid constructs described in paragraphs 1-10, characterized in that the construction method includes integrating an AAV element carrying an exogenous target gene and a polynucleotide encoding an IE protein into a baculovirus vector backbone.

[0151] 16. The construction method according to paragraph 15, characterized in that the construction method includes one or more of the following features:

[0152] 1) The AAV element comprises a polynucleotide encoding the Cap protein, a polynucleotide encoding the Rep protein, and an AAV cis-acting element, wherein the AAV cis-acting element is preferably an ITR sequence;

[0153] 2) The IE protein is encoded by one or more of the Acie0, Acie01, or Acie02 genes;

[0154] 3) The baculovirus vector backbone is selected from one of pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB or pFastBacHTC;

[0155] 4) The baculovirus recombinant homologous region is selected from one or more of hr1, hr2, hr3, hr4 or hr5;

[0156] 5) Integrate the polynucleotide encoding the recombinant homologous region of the baculovirus into the baculovirus vector backbone.

[0157] 17. A method for producing adeno-associated virus, characterized in that the method comprises the following steps: infecting an insect cell line with the recombinant baculovirus described in paragraph 11.

[0158] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0159] 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 specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0160] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0161] The following examples use EGFP as the exogenous target gene.

[0162] Example 1: Construction of Recombinant Baculovirus Shuttle Vector and Obtaining Recombinant Baculovirus

[0163] Relevant genes were identified: the genes for Cap and Rep proteins were derived from the AAV2 genome (GenBank: AF043303.1). Point mutations were performed on the bases in the gene according to the ribosome leakage scanning mechanism reference (Smith RH, Levy JR, Kotin RM: Asimplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells. MolTher 2009, 17(11): 1888-1896.). Acie01 (GenBank: NC_001623.1) and the baculovirus recombinant homology region hr1 (GenBank: M14313.1).

[0164] The following sequences were synthesized by a commissioned gene company: the Cap gene sequence (nucleotide sequence SEQ ID NO:4), the Rep gene sequence (nucleotide sequence SEQ ID NO:5), the hr1 and p6.9, p10 promoter combination (hr1p6.9p10, nucleotide sequence SEQ ID NO:6), and the pH promoter and Acie01 combination (pH-Acie01, nucleotide sequence SEQ ID NO:7). Using homologous recombination methods well-known in the art, the above sequences and the ITR-CMV-EGFP sequence from the pAAV-EGFP vector (modified by Kanglin Biotechnology (Hangzhou) Co., Ltd.) were cloned into pFastBacdual (Invitrogen). After sequencing and identification, pFBd-Cap-ITR-Rep was obtained. Figure 1 and Figure 2 Nucleotide sequence SEQ ID NO:8) and pFBd-IE-hr1Cap-ITR-Rep ( Figure 3 and Figure 4 Two recombinant baculovirus shuttle vectors (nucleotide sequence SEQ ID NO:9).

[0165] The sequences of SEQ ID NO.4-9 are shown below:

[0166] SEQ ID NO:4:

[0167]

[0168]

[0169] SEQ ID NO:5:

[0170]

[0171]

[0172] SEQ ID NO:6:

[0173]

[0174]

[0175] SEQ ID NO:7:

[0176]

[0177]

[0178] SEQ ID NO:8:

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186] SEQ ID NO:9:

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195] The two recombinant vectors were transformed into DH10Bac competent cells (Invitrogen), and blue-white screening was performed. White positive clones were selected and cultured using PureLink. TM Baculovirus plasmids were extracted using the HiPure Plasmid DNA Purification Kits (Invitrogen). Two types of baculovirus plasmids were transfected into adherent SF9 cells (Gbico), and primary recombinant baculoviruses were harvested after 3 days and amplified to passage P2. The resulting P2 generation recombinant baculoviruses were named BV-Cap-ITR-Rep and BV-IE-hr1Cap-ITR-Rep, respectively, and viral titers were determined using the TCID50 method. For detailed instructions on this section, please refer to the Bac to Bac Expression System manual (Invitrogen).

[0196] Example 2: Packaging and titer determination of rAAV2

[0197] Two P2 generation recombinant baculoviruses were used to infect 25 mL of suspended SF9 cells (density 3.0E+06 cells / mL) with an MOI of 0.1. After 4 days, cells were harvested and lysed using 5 mL of TNT lysis buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 10 mmol / L MgCl2) at room temperature for 1 h. Nuclease was added to a final concentration of 50 U / mL and digested at 37°C for 2 h. Samples were then taken and the capsid was digested with proteinase K (Tiangen) at 56°C for 1 h. The titer was determined by qPCR, following the steps outlined below:

[0198] The standard used for quantitative PCR was the linearized pAAV-EGFP plasmid digested with Pvu I-HF (NEB) (the plasmid sequence is shown in SEQ ID NO:22 below).

[0199] SEQ ID NO.22:

[0200]

[0201]

[0202]

[0203] The primer sequences used for quantitative PCR are:

[0204] ITR Forward primer 5'-GGAACCCCTAGTGATGGAGTT-3'(SEQ ID NO:2)

[0205] ITR Reverse primer 5'-CGGCCTCAGTGAGCGA-3'(SEQ ID NO:3)

[0206] The quantitative PCR program was: 95℃ for 60s, (95℃ for 15s, 60℃ for 30s, 40 cycles).

[0207] After plotting the standard curve based on the Ct value obtained from quantitative PCR and the standard concentration, the sample titer was calculated. The rAAV yields of BV-Cap-ITR-Rep and BV-IE-hr1Cap-ITR-Rep packages were calculated to be 2.39E+04VG / cell (equivalent to 7.18E+10VG / mL) and 8.67E+05VG / cell (equivalent to 2.60E+12VG / mL), respectively.

[0208] Example 3: Comparison of EGFP expression during cell infection and Cap protein expression in cell lysate.

[0209] Cells infected with the two baculoviruses were observed under an inverted fluorescence microscope at 48h, 72h, and 96h after infection. The optimized recombinant baculovirus significantly reduced the expression of the target gene (EGFP in this example). Figure 5 The reduction in the expression of the target gene (EGFP) appears to have reduced the interference with intracellular rAAV packaging, making it more conducive to the expression of rAAV packaging-related structural and functional proteins.

[0210] Western blot analysis was performed on equal volumes of cell lysate using Cap protein antibody (Progen). Figure 6 The results showed that the optimized recombinant baculovirus significantly increased the expression level of Cap protein in cells after infection.

[0211] Example 4: Electron microscopy analysis of purified rAAV2 and determination of infectivity.

[0212] Using POROS TM CaptureSelect TM Cell lysates were purified using affinity chromatography (Thermo) and titers were determined by quantitative real-time PCR (method as in Example 2). The purified rAAV2 was then negatively stained and observed under a transmission electron microscope. Figure 7 The rAAV2 cells containing the genome were solid particles, with the nucleus-deficient rAAV particles stained dark in the center. Overall, the morphology was intact, and the empty shell rate was significantly reduced (~3%) compared to the traditional method (Benskey et al. 2016). This indicates that the optimized baculovirus vector construct significantly reduced the proportion of empty shell viruses in the packaged product, thus significantly reducing the burden on downstream steps such as empty shell virus removal.

[0213] The purified rAAV2 was serially diluted and used to infect cultured 293T cells (48-well plates, 5.0E+04 cells / well) with MOIs of 10000, 2000, 400, and 80, respectively. Two days after infection, EGFP expression was observed using a fluorescence microscope. The results showed that the rAAV2 prepared by this system had high in vitro infectious activity. Figure 8 ).

[0214] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A nucleic acid construct, characterized in that, The nucleic acid construct includes the following elements: an AAV element, a polynucleotide encoding the IE protein, and a polynucleotide encoding the baculovirus recombinant homologous region. The AAV element includes a polynucleotide encoding the Cap protein, a polynucleotide encoding the Rep protein, and an AAV cis-acting element. The structure of the nucleic acid construct includes: an IE gene expression cassette, a Cap gene expression cassette, a foreign target gene expression cassette, and a Rep gene expression cassette. The IE protein is encoded by the Acie01 gene, the baculovirus recombinant homologous region is hr1, and the Cap protein is expressed by the p6.9p10 promoter.

2. The nucleic acid construct according to claim 1, characterized in that, The nucleic acid construct also includes a promoter for the IE protein gene, wherein the promoter for the IE protein gene is selected from one or more of Gp64, pH, p6.9 or p10.

3. The nucleic acid construct according to claim 1, characterized in that, The nucleic acid construct also includes a baculovirus promoter, which is linked to the baculovirus recombination homologous region.

4. The nucleic acid construct according to claim 3, characterized in that, The baculovirus promoter is one or more of pH, Gp64, p6.9 or p10.

5. The nucleic acid construct according to claim 1, characterized in that, The AAV cis-acting element is selected from the ITR sequence.

6. The nucleic acid construct according to claim 1, characterized in that, The nucleic acid construct also includes an exogenous target gene, which is embedded in an AAV element.

7. The nucleic acid construct according to claim 6, characterized in that, The structure of the nucleic acid construct is as follows: IE gene expression box - Cap gene expression box - ITR - exogenous target gene expression box - ITR - Rep gene expression box.

8. The nucleic acid construct according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid construct is shown 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. The nucleic acid construct according to claim 9, characterized in that, The recombinant baculovirus vector is a recombinant baculovirus shuttle vector.

11. A recombinant baculovirus, characterized in that, The recombinant baculovirus is obtained by constructing the nucleic acid construct according to any one of claims 1-10 using a baculovirus system.

12. An adeno-associated virus, characterized in that, The adeno-associated virus is obtained by packaging cells after infection with the recombinant baculovirus as described in claim 11.

13. A cell line, characterized in that, The cell line is a cell line infected with the recombinant baculovirus as described in claim 11.

14. An adeno-associated virus vector system, characterized in that, The adeno-associated virus vector system includes a baculovirus system and the nucleic acid construct according to any one of claims 1-10.

15. A method for constructing a nucleic acid construct according to any one of claims 1-10, characterized in that, The construction method includes integrating an AAV element carrying a foreign target gene, a polynucleotide encoding an IE protein, and a polynucleotide encoding a baculovirus recombinant homologous region into the baculovirus vector backbone.

16. The construction method according to claim 15, characterized in that, The construction method includes the following features: the baculovirus vector backbone is selected from one of pFastBacdual, pFastBac1, pFastBacHTA, pFastBacHTB or pFastBacHTC.

17. A method for producing adeno-associated virus, characterized in that, The production method includes the following steps: infecting an insect cell line with the recombinant baculovirus of claim 11.

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