Method for preparing transfer vector paav-donor in baculovirus-raav production system and use

Abstract

Disclosed in the present invention are a method for preparing a transfer vector pAAV-Donor in a baculovirus-rAAV production system and the use. The preparation method comprises: removing type IIS restriction enzyme recognition sites from an AAV Cap gene expression cassette by means of mutation; modifying the obtained AAV Cap gene expression cassette; preparing AAV-Core, AAV-Cap and AAV-Rep backbone plasmids; sequentially inserting the modified Cap gene expression cassette, a heterologous functional gene expression element and the modified AAVRep gene expression cassette into the multiple cloning sites of the AAV-Cap, AAV-Core and AAV-Rep backbone plasmids to obtain pAAV-Rep, pAAV-Cap and pAAV-Core; and assembling the three plasmids in one step by means of the Golden Gate cloning technology, transforming same into Stbl3 competent cells, and screening positive clones to obtain pAAV-Donor.

Description

Preparation method and application of transfer vector pAAV-Donor in baculovirus-rAAV production system Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of a transfer vector pAAV-Donor in a baculovirus-rAAV production system. Background Art

[0002] Recombinant adeno-associated virus (rAAV) is a star vector for gene therapy delivery and an important tool for analyzing brain structure and functional networks. Currently, the most commonly used methods for packaging AAV include adherent or suspension cell / three-plasmid transfection systems and insect cell / baculovirus production systems. Although research-grade rAAV can be produced by transient transfection of adherent human embryonic kidney (HEK) 293 cells with three plasmids, this process cannot be scaled up. While the suspension HEK293 cell / three-plasmid transient transfection method is rapid, applicable to all serotypes, and can be fermented on a large scale, it has limited yields, significant batch variability in the empty-to-full capsid ratio, and high production costs. In contrast, the baculovirus-based AAV production system has attracted much attention due to its scalability, low cost, and predictable biosafety.

[0003] At present, there are three main methods for large-scale production of rAAV using baculovirus: 1) two baculovirus systems; 2) one baculovirus system that relies on a packaging cell line; 3) one baculovirus system based on a shuttle plasmid that does not rely on a packaging cell line (OneBac system). However, the cycle of these preparation processes is relatively long, and it takes several weeks to execute the processes from transfer vector construction, recombinant bacmid preparation, transfection, virus amplification to downstream separation and purification. For the third method that can flexibly and efficiently produce different types of rAAV, the construction of the transfer vector is the rate-limiting step and the difficulty, because the Cap gene, Rep gene and ITR core expression elements of AAV need to be constructed into a transfer vector. To obtain rAAV of different serotypes or containing different core expression elements, the corresponding transfer vector must be prepared again.

[0004] Golden Gate cloning technology is a powerful tool for efficiently assembling complex vectors in a single step, offering the potential to address the aforementioned rate-limiting step. Although this technology has been used to develop the GoldenBac system for simple and efficient construction of multigene expression vectors, unlike recombinant protein expression, the encoding of AAV components is much more complex: 1) The Cap gene, in addition to translating the three capsid subunits VP1, VP2, and VP3 from distinct start codons, also encodes assembly activation proteins such as MAAP and AAP. 2) In the baculovirus AAV production system, the Rep gene encodes two proteins required for viral replication, Rep78 and Rep52. Compared to Rep52, Rep78 is expressed at a lower abundance, facilitating higher vector yields. 3) The core expression element is flanked by two T-shaped inverted terminal repeats (ITRs), which serve as the viral replication origin and packaging signal. ITRs are easily lost and can affect viral packaging and infection. Furthermore, AAV components contain type IIS restriction enzyme recognition sites, making this method unsuitable for constructing transfer vectors in the One Bac system.

[0005] Summary of the Invention

[0006] In response to the above-mentioned defects of the prior art, the present invention provides a method for preparing and applying a transfer vector pAAV-Donor in a baculovirus-rAAV production system. The purpose of the present invention is to improve the convenience, flexibility and efficiency of the construction of the transfer vector in the baculovirus-rAAV production system, thereby improving the convenience, flexibility and efficiency of the construction of the transfer vector in the baculovirus-rAAV production system, and further shortening the cycle of rAAV production using baculovirus, by placing heterologous functional gene expression elements in the AAV-Core backbone plasmid, the serotype Cap gene in the AAV-Cap backbone plasmid, and the Rep gene in the AAV-Rep backbone plasmid.

[0007] The specific technical solutions of the present invention are as follows:

[0008] The present invention provides a method for preparing a transfer vector pAAV-Donor in a baculovirus-rAAV production system, comprising the following steps:

[0009] (1) Mutation to remove the type IIS restriction enzyme recognition site in the AAV Cap gene expression frame;

[0010] (2) Modifying the AAV Cap gene expression frame obtained in step (1): the modification method is selected from one or more of 1) to 4): 1) inserting an additional out-of-frame start codon at the 5' end of the VP1 ATG start codon; 2) replacing the ATG start codon of VP1 with a suboptimal translation start codon, an ATG codon within the VP1 expression frame outside the mutated VP3 subunit expression frame; 3) inserting a coding sequence for one or more amino acid residues between the non-ATG translation start codon and the codon encoding the amino acid residue of the AAV capsid coding sequence; 4) inserting an intron splicing acceptor sequence to regulate the relative expression of VP1, VP2, and VP3;

[0011] Modification of the AAV Rep gene expression cassette: The modification method is selected from one of 1)-2): 1) mutating all ATG sequences between the Rep78 start codon and the Rep52 start codon, replacing the start codon of the Rep78 gene with a non-ATG sequence; 2) inserting an intron splice acceptor sequence to weaken the expression of the Rep78 protein;

[0012] (3) Prepare AAV-Core, AAV-Cap, and AAV-Rep backbone plasmids respectively;

[0013] The AAV-Core backbone plasmid belongs to the Golden Gate assembly donor plasmid and contains two identical type IIS restriction endonuclease sites and two AAV-ITR sequences;

[0014] The AAV-Cap backbone plasmid is a Golden gate assembly donor plasmid, and contains two type IIS restriction endonuclease sites, a baculovirus promoter, and transcriptional and post-transcriptional regulatory sequences identical to those of the AAV-Core backbone plasmid.

[0015] The AAV-Rep backbone plasmid belongs to the Golden gate assembly receptor plasmid, and contains two type IIS restriction endonuclease sites, a baculovirus promoter, transcriptional and post-transcriptional regulatory sequences, a negative selection marker gene, and a Tn7 transposable element that are the same as those of the AAV-Cap backbone plasmid;

[0016] (4) inserting the modified Cap gene expression cassette obtained in step (2) into the multiple cloning site of the AAV-Cap backbone plasmid in step (3), inserting the heterologous functional gene expression element into the multiple cloning site of the AAV-Core backbone plasmid in step (3), and inserting the modified AAV Rep gene expression cassette obtained in step (2) into the multiple cloning site of the AAV-Rep backbone plasmid in step (3) to obtain pAAV-Rep, pAAV-Cap, and pAAV-Core;

[0017] (5) The three plasmids obtained in step (4) were assembled in one step using the Golden Gate cloning technique, transformed into Stbl3 competent cells, and positive clones were screened to obtain the transfer vector pAAV-Donor.

[0018] Furthermore, the AAV serotype is selected from one or more of AAV1-13 and their derivative serotypes;

[0019] The IIS type restriction enzyme recognition site in the AAV Cap gene expression frame mutated and removed in step (1) includes one of BsaI or Eco31I (GGTCTC 1 / 5), BsmBI (CGTCTC 1 / 5), BbsI (GAAGAC 2 / 6), and SapI (GCTCTTC 1 / 4);

[0020] Preferably, the IIS type restriction enzyme recognition site in the AAV Cap gene expression frame that is mutated and removed in step (1) is BsaI or Eco31I (GGTCTC);

[0021] Preferably, the mutation to remove the IIS type restriction enzyme recognition site is to perform a codon synonymous substitution or an amino acid homologous mutation on the IIS type restriction enzyme recognition site;

[0022] Preferably, the mutation to remove the IIS type restriction enzyme recognition site includes synonymous substitution of codons in the VP3 subunit expression frame, amino acid homology mutations in the overlapping expression frames of VP1 / VP2 and MAAP / AAP subunits, and synonymous substitution of codons in the remaining Cap expression frames except the VP3 / MAAP / AAP subunit coding region.

[0023] Furthermore, the AAV Cap gene expression cassette is modified in step (2) by replacing the ATG start codon of VP1 with the suboptimal translation start codon CTG and inserting a GCCGCC sequence at the 5' end of CTG, while mutating the ATG codon between the VP1 start codon and the VP3 start codon;

[0024] Preferably, the AAV Rep gene expression cassette is modified in step (2) by removing all ATG sequences between the Rep78 start codon and the Rep52 start codon, replacing the ATG translation start codon of Rep78 with CTG and inserting a GCCGCC sequence at the 5' end of CTG.

[0025] Furthermore, the type IIS restriction endonuclease in step (3) comprises one of BsaI or Eco31I (GGTCTC 1 / 5), BsmBI (CGTCTC 1 / 5), BbsI (GAAGAC 2 / 6), and SapI (GCTCTTC 1 / 4);

[0026] Preferably, the two IIS type restriction endonucleases in the AAV-Core, AAV-Cap and AAV-Rep backbone plasmids in step (3) are BsaI or Eco31I (GGTCTC 1 / 5);

[0027] Preferably, the baculovirus promoter in step (3) is derived from AcMNPV or BmNPV, and comprises Pp10, Pph, Pp6.9, Pgp64, Pie-1, and their respective combinations or constitutive promoters formed by adding enhancers before the promoters;

[0028] Preferably, the core element 5'→3' in the AAV-Core backbone plasmid in step (3) is GGTCTCATACT-ITR-MCS-ITR-CCATTGAGACC;

[0029] Preferably, the core element 5'→3' in the AAV-Cap backbone plasmid in step (3) is GGTCTCAAGTA-Pp10-MCS-HSV TKpoly(A)-CTCCTGAGACC;

[0030] Preferably, the p10 promoter in the AAV-Cap backbone plasmid in step (3) is selected from AcMNPV;

[0031] Preferably, the core element 5'→3' in the AAV-Rep backbone plasmid in step (3) is Tn7R-Gen-GGAGTGAGACC-ccdB-GGTCTCACCAT-Pph-MCS-SV40poly(A)-Tn7L;

[0032] Preferably, the ph promoter in the AAV-Rep backbone plasmid in step (3) is preferably selected from AcMNPV;

[0033] Preferably, the negative selection marker genes in the AAV-Rep backbone plasmid in step (3) include but are not limited to ccdB and sacB.

[0034] Furthermore, the heterologous functional gene expression element in step (4) does not contain at least the same type IIS restriction endonuclease site as the AAV-Core, AAV-Cap or AAV-Rep backbone plasmid in step (3);

[0035] Preferably, the heterologous functional gene expression element in step (4) comprises at least a promoter, a gene expression cassette, and transcriptional and post-transcriptional regulatory sequences;

[0036] Preferably, the transcriptional and post-transcriptional regulatory sequences in the heterologous functional gene expression element in step (4) include but are not limited to cw3sl, WPRE, hGHpolyA, and bGHpolyA.

[0037] Furthermore, the heterologous functional gene expression element, the modified Cap gene expression cassette, and the modified AAV Rep gene expression cassette in step (4) do not contain a type IIS restriction endonuclease recognition site;

[0038] Preferably, the heterologous functional gene expression element, the modified Cap gene expression cassette and the modified AAV Rep gene expression cassette in step (4) do not contain the IIS type restriction endonuclease recognition site BsaI or Eco31I (GGTCTC).

[0039] Furthermore, in step (4), the modified Cap gene expression cassette is inserted into the multiple cloning site of the AAV-Cap backbone plasmid, the heterologous functional gene expression element is inserted into the multiple cloning site of the AAV-Core backbone plasmid, and the modified AAV Rep gene expression cassette is inserted into the multiple cloning site of the AAV-Rep backbone plasmid using homologous recombination cloning technology or enzyme ligation method.

[0040] Furthermore, the Golden Gate assembly conditions of the three plasmids in step (5) are as follows: 1) plasmid assembly requirement (ng) = 0.0345 × plasmid size of pAAV-Rep or pAAV-Cap or pAAV-Core, T4 DNA Ligase Buffer (10×) 2 μL, Golden Gate Enzyme Mix 1 μL, supplemented with ddH2O to 20 μL; 2) 37°C (1 h) → 60°C (5 min) → 4°C (∞);

[0041] Preferably, the dosage of the transformed Stbl3 competent cells in step (5) is 5 μL.

[0042] The present invention also provides a transfer vector pAAV-Donor in the baculovirus-rAAV production system obtained by the above preparation method.

[0043] The present invention also provides the preparation method or the use of the transfer vector pAAV-Donor in the baculovirus-rAAV production system in producing rAAV.

[0044] The present invention further provides a method for producing rAAV, comprising the following steps:

[0045] The transfer vector pAAV-Donor was transposed into the baculovirus genome using the Bac-to-Bac system to obtain a recombinant bacmid.

[0046] Extract the corresponding recombinant bacmid and transfect host cells to rescue the recombinant baculovirus BEV (P1 generation);

[0047] The obtained P1 generation recombinant baculovirus BEV is serially passaged and then infected into a host or host cell line. After a certain period of infection, the cells are harvested and purified to obtain rAAV;

[0048] Preferably, the baculovirus genome is AcMNPV, BmNPV or ApNPV;

[0049] Preferably, the host cell is derived from the ovary, testis, embryo, imaginal disc, midgut, fat body or blood cell of an insect;

[0050] Preferably, the Bac-to-Bac system is the Autographa californica baculovirus (AcMNPV) expression system;

[0051] Preferably, the host cell is Expi-sf9.

[0052] Beneficial effects of the present invention:

[0053] (1) The pAAV-Donor preparation method provided by the present invention is to load the heterologous functional gene expression elements required for AAV packaging, the serotype Cap gene, and the Rep gene on three plasmids respectively, so that the three plasmids can be freely assembled in one step of Golden Gate according to the production requirements of rAAV, thereby realizing the convenient, flexible, and efficient construction of the transfer vector in the baculovirus-rAAV production system. That is, for the preparation of different serotype rAAVs carrying different heterologous functional gene fragments, it is only necessary to select the appropriate plasmid from the AAV-Cap plasmid library and the AAV-Core plasmid library and assemble it with the AAV-Rep plasmid Golden Gate, without repeatedly relying on the action of restriction endonucleases, DNA ligases or homologous recombinases to connect the Cap gene, Rep gene and ITR core expression elements to the pFBD (pFastBac Dual) vector;

[0054] (2) Based on the convenience, flexibility, and efficiency of rAAV-Donor preparation, the present invention can significantly shorten the production cycle of rAAV using baculovirus;

[0055] (3) The present invention is compatible with the existing baculovirus-rAAV production system. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 is a flow chart for the preparation of the transfer vector pAAV-Donor of the present invention. a. Schematic diagram of the assembly strategy for the pAAV-Cap plasmid, pAAV-Core plasmid, pAAV-Rep plasmid, and pAAV-Donor; b. Assembly reaction system; c. Assembly conditions for the Rep, Cap, and Core plasmids;

[0057] Figure 2 shows the results of rAAV preparation after mutation removal of the BsaI recognition site in the Cap gene expression frame of pAAV-RC in Example 1. a. Schematic diagram of mutation removal of the BsaI recognition site in the Cap gene expression frame of AAV types 1, 5, and 8; b. Schematic diagram of rAAV packaging by co-transfection of three plasmids into HEK293T cells and evaluation indicators; ce. Silver staining of purified rAAV types 1, 5, and 8 and mutant proteins; fh. qPCR titer determination of purified rAAV types 1, 5, and 8 and mutants and activity detection of infected HEK293T cells;

[0058] Figure 3 shows the one-step assembly of the transfer vector pAAV-Donor in the baculovirus-rAAV production system described in Example 2. a. Schematic diagram of the construction of 9A08, 9A09, 2006, and T79Ac00-0-4, required for preparing the transfer vectors pAAV8-CMV-EGFP-hGH and pAAV9-CMV-EGFP-hGH based on the backbone plasmids; d. Growth of Stbl3 competent cells transformed with the Cap, Core, and Rep plasmids and coated on LB plates containing gentamicin; e. PCR identification of pAAV-Donor positive clones;

[0059] Figure 4 is a diagram of the production of rAAV2, rAAV8 and rAAV9 using the Autographa californica-baculovirus (AcMNPV) expression system based on the flexible and efficient pAAV-Donor preparation method in Example 3; a. Schematic diagram of the transposition of the Cap, Core and Rep elements in pAAV-Donor into the baculovirus genome; b. MOI = 1 infection of suspended Expi-sf9 cells (2E+06 cells / mL, 50 ml), and the expression of fluorescent protein in the cells after 3 days; c. Protein immunoblotting to detect the expression levels of Rep and Cap in Expi-sf9 cells 3 days after infection with BEV; d. Protein silver staining to detect the purity and capsid protein ratio of the prepared rAAV; e. qPCR to quantify the titer of rAAV; f. rAAV produced based on the flexible and efficient pAAV-Donor system was infected with 293T cells to detect viral activity. DETAILED DESCRIPTION

[0060] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0061] Example 1 Mutation to remove the BsaI recognition site in the Cap gene expression frame

[0062] Among the commonly used AAV serotypes 1, 2, 5, 8, and 9, the Cap gene expression frame of AAV1, AAV5, and AAV8 contains the IIS type restriction enzyme recognition site BsaI or Eco31I (GGTCTC 1 / 5). This hinders the modularization of the Cap, Rep, and Core gene elements of AAV into the transfer vector pAAV-Donor required for the baculovirus-rAAV production system in one step. Therefore, it is necessary to mutate and remove the GGTCTC sequence in the Cap gene expression frame. This example uses AAV1, AAV5, and AAV8 (Addgene, 112862, 104964, and 112864) as examples, combined with the HEK293T cell / three-plasmid transient transfection AAV packaging system, to demonstrate how to mutate and remove the IIS type restriction enzyme recognition site in the AAV-Cap gene expression frame.

[0063] (1) Single-base mutation of the BsaI recognition site within the Cap gene expression frame in the pAAV-RC plasmid, as shown in FIG2a ;

[0064] 1.1 The codon encoding VP1-467 serine (467S) in AAV-Cap1 was replaced synonymously, and "tct" was mutated to "tca";

[0065] 1.2 Mutate the leucine (58L) at position 58 of the VP1-encoded protein in AAV-Cap5 to the same amino acids: valine (58V) and isoleucine (58I), and mutate "ctc" to "gtc" and "atc" respectively;

[0066] 1.3 The arginine at position 117 (117R) encoding MAAP in AAV-Cap8 was mutated to the same amino acid - lysine (117K), and "aga" was mutated to "aaa".

[0067] (2) HEK293T cells / three-plasmid transient transfection system was used to package AAV1, AAV5, and AAV8 with a mutant Cap gene expression frame containing the BsaI recognition site (Figure 2b), where the pTrans plasmid (i.e., core plasmid) carries the eGFP expression element to facilitate detection of the packaged rAAV characteristics. The titer and purity were determined by protein silver staining and real-time fluorescence quantitative qPCR, and the infection activity was compared by infecting 293T cells to obtain the optimal mutant without the BsaI site in the Cap gene expression frame. Protein silver staining showed that AAV1-tct467tca, AAV5-L58V, and AAV8-MAAP-R117K after mutation to remove the BsaI recognition site in the Cap gene expression frame could all produce rAAV, while AAV5-L58I could not be packaged (Figures 2c-e); real-time fluorescence quantitative qPCR and infection of 293T cells showed that the titer of the AAV1 mutant was slightly lower than that of the wild-type AAV1, while the titers of the AAV5 and AAV8 mutants were higher than those of the corresponding wild-type AAV. The AAV1, AAV5, and AAV8 mutants could all infect 293T cells and the infection effect was close to that of the wild-type AAV (Figures 2f-h). In summary, the method of removing the IIS type restriction enzyme recognition site in the AAV-Cap gene expression frame by mutation described in the present invention can screen out AAV mutants with infectious activity and lacking the IIS type restriction enzyme recognition site in the Cap expression frame.

[0068] Example 2 One-step assembly of the transfer vector pAAV-Donor in the baculovirus-rAAV production system

[0069] To facilitate the rapid and simple construction of the AAV triple-element (Rep / Cap / Core) transfer vector pAAV-Donor, 1) the Cap backbone plasmid 9A00 (pBACKBONE_Ac-AAV-Cap-BsaI-SmR, with a nucleic acid sequence such as SEQ ID NO. 1) was constructed based on Addgene-47984. 2) the Core backbone plasmid 2001 (pBACKBONE_AAV-Core-cw3sl-BsaI-SmR, with a nucleic acid sequence such as SEQ ID NO. 2) was constructed based on Addgene-47985, in which the ITR-CW3SL sequence was synthesized by Beijing Liuhe BGI Genomics Co., Ltd. 3) the Rep backbone plasmid T79Ac00-0-0 (pBACKBONE_Ac-AAV-Rep-BsaI-ccdB-Gen, with a nucleic acid sequence such as SEQ ID NO. 3) was constructed, in which the ccdB sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. and the remaining sequences were obtained from pFastBac Dual expression vector (purchased from Thermo Fisher Scientific and maintained in this laboratory) was amplified using DB3.1 competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd., DL1040M). This example demonstrates the one-step assembly of pAAV-Donor in the baculovirus-rAAV production system, using the preparation of pAAV8-CMV-EGFP-hGH and pAAV9-CMV-EGFP-hGH transfer vectors as examples.

[0070] (1) Construct AAV Cap, Core, and Rep plasmids respectively;

[0071] 1.1 By PCR, homology arms were introduced at both ends of the Cap8 (derived from Example 1, mutated to remove the BsaI recognition site) and Cap9 (sourced from Addgene, stored by this laboratory) gene expression cassettes, and the start codon "ATG" was mutated to "CTG" and a "gccgcc" sequence was added to the 5' end of CTG (the nucleic acid sequences of the modified AAV8 and AAV9Cap gene expression cassettes are shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively). Then, the modified Cap8 and Cap9 gene expression cassettes were inserted into the multiple cloning site of the Cap backbone plasmid 9A00, respectively, using homologous recombination cloning technology, as shown in Figure 3a, to obtain AAV-Cap plasmids 9A08 and 9A09, respectively;

[0072] 1.2 Using fusion PCR, the BsaI recognition site in the terminator hGH within the CMV-EGFP-hGH expression frame was mutated (the nucleic acid sequence of the modified CMV-EGFP-hGH is shown in SEQ ID NO. 6) and homology arms were introduced. Subsequently, the Core backbone plasmid 2001 was double-digested with restriction endonucleases BspDI / RsrII, and the CMV-EGFP-hGH expression cassette with the BsaI recognition site removed was inserted between the ITRs, as shown in FIG3b , thereby obtaining AAV-Core plasmid 2006;

[0073] 1.3 Homology arms were introduced at both ends of the modified Rep2 gene expression cassette by PCR (the nucleic acid sequence of the modified Rep2 gene expression cassette is shown in SEQ ID NO. 7). This fragment was then inserted into the multiple cloning site of the Rep backbone plasmid T79Ac00-0-0 using homologous recombination cloning technology, as shown in Figure 3a, to obtain AAV-Rep plasmid T79Ac00-0-4;

[0074] (2) AAV Cap, Core, and Rep plasmids were assembled in one step using the Goldengate assembly kit (NEB, E1601L). The assembly conditions are shown in Figures 1b-c. 9A08+2006+T79Ac00-0-4 was assembled to form T79A08-2006-4, i.e., the transfer vector pAAV8-CMV-EGFP-hGH (nucleic acid sequence as SEQ ID NO. 8). 9A09+2006+T79Ac00-0-4 was assembled to form T79A09-2006-4, i.e., the transfer vector pAAV9-CMV-EGFP-hGH (nucleic acid sequence as SEQ ID NO. 8). NO.9), as shown in Figures 3a-c, 5 μL of the product after Goldengate assembly was taken to transform stbl3 chemical competent cells and spread on LB solid culture medium containing gentamicin. After overnight culture at 37°C, colonies were seen to be distributed in granular form on the LB culture plate (as shown in Figure 3d). Ten monoclonal colonies were selected from the T79Ac08-2006-4 and T79Ac09-2006-4 plates, respectively, for colony PCR and screening of positive clones (as shown in Figure 3e). Two positive clones each from T79Ac08-2006-4 and T79Ac09-2006-4 were selected for overnight culture, and the plasmids were extracted for sequencing, which was correct after sequence alignment. This shows that the preparation method of the transfer vector pAAV-Donor provided by the present invention can realize the convenient, flexible and efficient construction of the transfer vector in the baculovirus-rAAV production system through one-step Golden gate free assembly according to the production requirements of rAAV.

[0075] Example 3 Production of rAAV2, rAAV8, and rAAV9 Using the Autographa californica-Baculovirus (AcMNPV) Expression System Based on a Flexible and Efficient Method for Preparing pAAV-Donor

[0076] To verify the effectiveness of the pAAV-Donor rapidly obtained by the one-step assembly method, the prepared pAAV-Donor was transformed into DH10Bac competent cells (purchased from Thermo Fisher Scientific, 10361012) to obtain the AcMNPV recombinant bacmid. BEV was further rescued by transfecting Expi-sf9 cells (purchased from Thermo Fisher Scientific, A35243), and BEV was used to infect Expi-sf9 cells to verify whether rAAV was produced.

[0077] (1) Preparation of rAAV2, rAAV8, and rAAV9 AcMNPV recombinant bacmids;

[0078] In Example 2, pAAV-Donor T79A08-2006-4 and T79A09-2006-4 for rAAV8 and rAAV9 were obtained. The same method was used to prepare pAAV-Donor T79A02-2006-4 for rAAV2 (nucleic acid sequence shown in SEQ ID NO. 10). According to the instructions for use of the Bac-to-Bac expression system, as shown in FIG4a , T79A02-2006-4, T79A08-2006-4, and T79A09-2006-4 were transposed into the AcMNPV genome to prepare recombinant bacmids AcMNPV-T79A02-2006-4 (rAAV2), AcMNPV-T79A08-2006-4 (rAAV8), and AcMNPV-T79A09-2006-4 (rAAV9), respectively.

[0079] (2) rAAV production using the baculovirus (AcMNPV) expression system;

[0080] Expi-sf9 cells in suspension culture were seeded at 2E+06 cells / well in a 6-well cell culture plate. After 1 hour of adherent culture, 2 μg of different AAV recombinant bacmids prepared in step (1) were transfected using TransIT-Insect Transfection Reagent (purchased from Mirusbio, MIR6105). After 24 hours, the cell medium was replaced. After another 3 days of culture, the first generation BEV (P1) in the supernatant was quantitatively detected using qPCR. Furthermore, an appropriate amount of suspended Expi-sf9 cells were inoculated in a shake flask (2E+06 cells / mL, 50 mL), and BEV (P1) was inoculated at an MOI of 1. After 3 days of infection, the cells were collected, and green fluorescent protein expression was observed under a fluorescence microscope (Figure 4b), indicating that the recombinant BEV infected the cells and mediated the expression of eGFP; protein immunoblotting was used to detect Rep and Cap expression in the cells. The expression abundance of Rep52 was higher than that of Rep78, and the expression ratio of VP1:VP2:VP3 was close to 1:1:10 (Figure 4c). At the same time, a large amount of BEV (P2) in the supernatant can be used as seeds to infect Expi-sf9 cells again to produce rAAV. Next, the rAAV in Expi-sf9 cells was purified by iodixanol density gradient centrifugation purification (method reference Aslanidi et al., 2009, Proc. Natl Acad. Sci. USA, 206: 5059-5064), and viral characteristics were detected including protein silver staining, real-time fluorescence quantitative qPCR, and infection of 293T cells. Protein silver staining and qPCR showed that rAAV2, rAAV8, and rAAV9 were successfully produced, among which the titer of rAAV9 reached 4.099E+12VG / mL (Figures 4d-e). 3 μL of the prepared rAAV2, rAAV8, and rAAV9 were taken to infect adherent 293T cells respectively. Fluorescent protein expression was observed after only 36 hours of infection (Figure 4f), indicating that the produced rAAV has infectious activity. In summary, the flexible and efficient method for preparing the transfer vector pAAV-Donor provided by the present invention can be used to produce rAAV in a baculovirus expression system.

[0081] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A preparation method of transfer vector pAAV-Donor in a baculovirus-rAAV production system, characterized in that, Including the following steps: (1) Mutate and remove the type IIS restriction enzyme recognition sites in the AAV Cap gene expression cassette; (2) Modify the AAV Cap gene expression cassette obtained in step (1): The modification method is selected from one or more of 1)-4): 1) Insert an additional out-of-frame start codon at the 5' end of the VP1 ATG start codon; 2) Replace the ATG start codon of VP1 with a suboptimal translation start codon and mutate the ATG codons within the VP1 expression cassette outside the VP3 subunit expression cassette; 3) Insert the coding sequence of one or more amino acid residues between the non-ATG translation start codon of the AAV capsid coding sequence and the codon encoding the amino acid residue; 4) Insert an intron splice acceptor sequence to regulate the relative expression of VP1, VP2, and VP3; Modify the AAV Rep gene expression cassette: The modification method is selected from one of 1)-2): 1) Mutate all ATG sequences between the Rep78 start codon and the Rep52 start codon and replace the start codon of the Rep78 gene with a non-ATG sequence; 2) Insert an intron splice acceptor sequence to weaken the expression of the Rep78 protein; (3) Prepare AAV-Core, AAV-Cap, and AAV-Rep backbone plasmids respectively; The AAV-Core backbone plasmid belongs to a Golden gate assembly donor plasmid and contains two identical types of type IIS restriction enzyme sites and two AAV-ITR sequences; The AAV-Cap backbone plasmid belongs to a Golden gate assembly donor plasmid and contains two type IIS restriction enzyme sites identical to those of the AAV-Core backbone plasmid, a baculovirus promoter, and transcriptional and post-transcriptional regulatory sequences; The AAV-Rep backbone plasmid belongs to a Golden gate assembly acceptor plasmid and contains two type IIS restriction enzyme sites identical to those of the AAV-Cap backbone plasmid, a baculovirus promoter, transcriptional and post-transcriptional regulatory sequences, a negative selection marker gene, and a Tn7 transposon element; (4) Insert the modified Cap gene expression cassette obtained in step (2) into the multiple cloning site of the AAV-Cap backbone plasmid obtained in step (3), insert the heterologous functional gene expression element into the multiple cloning site of the AAV-Core backbone plasmid obtained in step (3), and insert the modified AAV Rep gene expression cassette obtained in step (2) into the multiple cloning site of the AAV-Rep backbone plasmid obtained in step (3) to obtain pAAV-Rep, pAAV-Cap, and pAAV-Core; (5) Assemble the three plasmids obtained in step (4) in one step by the Golden gate cloning technique, transform Stbl3 competent cells, and screen for positive clones to obtain the transfer vector pAAV-Donor.

2. The preparation method according to claim 1, wherein, The AAV serotype is selected from one or more of AAV1-13 and their derived serotypes; In step (1), the type IIS restriction enzyme recognition site in the AAV Cap gene expression cassette removed by mutation includes one of BsaI or Eco31I (GGTCTC 1 / 5), BsmBI (CGTCTC 1 / 5), BbsI (GAAGAC 2 / 6), and SapI (GCTCTTC 1 / 4).

3. The preparation method according to claim 1, characterized in that, In step (1), the type IIS restriction enzyme recognition site in the AAV Cap gene expression cassette removed by mutation is BsaI or Eco31I (GGTCTC).

4. The preparation method according to claim 1, characterized in that, In step (1), the removal of the type IIS restriction enzyme recognition site by mutation is to perform synonymous codon substitution or amino acid conservative mutation on the type IIS restriction enzyme recognition site.

5. The preparation method according to claim 1, characterized in that, In step (1), the removal of the type IIS restriction enzyme recognition site by mutation includes synonymous codon substitution in the VP3 subunit expression cassette, amino acid conservative mutation in the overlapping expression cassette of VP1 / VP2 and MAAP / AAP subunits, and synonymous codon substitution in the Cap expression cassette except for the VP3 / MAAP / AAP subunit coding region.

6. The preparation method according to claim 1, characterized in that, In step (2), the method for modifying the AAV Cap gene expression cassette is to replace the ATG start codon of VP1 with the sub-optimal translation start codon CTG and insert the GCCGCC sequence at the 5'-end of CTG, and at the same time mutate the ATG codons between the VP1 start codon and the VP3 start codon.

7. The preparation method according to claim 1, characterized in that, In step (2), the method for modifying the AAV Rep gene expression cassette is to remove all ATG sequences between the Rep78 start codon and the Rep52 start codon, and replace the ATG translation start codon of Rep78 with CTG and insert the GCCGCC sequence at the 5'-end of CTG.

8. The preparation method according to claim 1, characterized in that, In step (3), the type IIS restriction endonuclease includes one of BsaI or Eco31I (GGTCTC 1 / 5), BsmBI (CGTCTC 1 / 5), BbsI (GAAGAC 2 / 6), and SapI (GCTCTTC 1 / 4).

9. The preparation method according to claim 1, wherein, In step (3), the two type IIS restriction endonucleases in the AAV-Core, AAV-Cap, and AAV-Rep backbone plasmids are BsaI or Eco31I (GGTCTC 1 / 5).

10. The preparation method according to claim 1, characterized in that, In step (3), the baculovirus promoter is derived from AcMNPV or BmNPV and includes constitutive promoters formed by Pp10, Pph, Pp6.9, Pgp64, Pie-1 and their combinations or addition of enhancers in front of the promoter.

11. The preparation method according to claim 1, characterized in that, In step (3), the core elements in the AAV-Core backbone plasmid are GGTCTCATACT-ITR-MCS-ITR-CCATTGAGACC from 5' to 3'.

12. The preparation method according to claim 1, characterized in that, In step (3), the core elements in the AAV-Cap backbone plasmid are GGTCTCAAGTA-Pp10-MCS-HSV TK poly(A)-CTCCTGAGACC from 5' to 3'.

13. The preparation method according to claim 12, characterized in that, In step (3), the p10 promoter in the AAV-Cap backbone plasmid is selected from AcMNPV.

14. The preparation method according to claim 1, characterized in that, In the AAV-Rep backbone plasmid described in step (3), the core elements are in the 5'→3' direction as Tn7R-Gen-GGAGTGAGACC-ccdB-GGTCTCACCAT-Pph-MCS-SV40poly(A)-Tn7L.

15. The preparation method according to claim 14, characterized in that, In the AAV-Rep backbone plasmid described in step (3), the ph promoter is preferably selected from AcMNPV.

16. The preparation method according to claim 1, characterized in that, In the AAV-Rep backbone plasmid described in step (3), the negative selection marker genes include but are not limited to ccdB and sacB.

17. The preparation method according to claim 1, characterized in that, In the heterologous functional gene expression element described in step (4), it does not contain at least the same IIS-type restriction endonuclease site as the AAV-Core or AAV-Cap or AAV-Rep backbone plasmid described in step (3).

18. The preparation method according to claim 1, characterized in that, The heterologous functional gene expression element described in step (4) at least includes a promoter, a gene expression cassette, and transcriptional and post-transcriptional regulatory sequences.

19. The preparation method according to claim 18, characterized in that, In the heterologous functional gene expression element described in step (4), the transcriptional and post-transcriptional regulatory sequences include but are not limited to cw3sl, WPRE, hGH polyA, and bGH polyA.

20. The preparation method according to claim 1, characterized in that, The heterologous functional gene expression element, the modified Cap gene expression cassette, and the modified AAV Rep gene expression cassette described in step (4) do not contain IIS-type restriction endonuclease recognition sites.

21. The preparation method according to claim 1, characterized in that, The heterologous functional gene expression element, the modified Cap gene expression cassette, and the modified AAV Rep gene expression cassette described in step (4) do not contain the IIS-type restriction endonuclease recognition sites BsaI or Eco31I (GGTCTC).

22. The preparation method according to claim 1, characterized in that, In step (4), the modified Cap gene expression cassette is inserted into the multiple cloning site of the AAV-Cap backbone plasmid, the heterologous functional gene expression element is inserted into the multiple cloning site of the AAV-Core backbone plasmid, and the modified AAV Rep gene expression cassette is inserted into the multiple cloning site of the AAV-Rep backbone plasmid by using homologous recombination cloning technology or restriction enzyme digestion and ligation method.

23. The preparation method according to claim 1, characterized in that, The Golden gate assembly conditions for the three plasmids described in step (5) are as follows: 1) The required amount of plasmid for assembly (ng) = 0.0345 × the plasmid size of pAAV-Rep or pAAV-Cap or pAAV-Core, 2 μL of T4 DNA Ligase Buffer (10×), 1 μL of Golden Gate Enzyme Mix, and ddH2O is added to make up to 20 μL; 2) 37°C (1 h) → 60°C (5 min) → 4°C (∞).

24. The preparation method according to claim 1, characterized in that, The dosage for transforming Stbl3 competent cells described in step (5) is 5 μL.

25. The transfer vector pAAV-Donor in the baculovirus-rAAV production system obtained by the preparation method according to any one of claims 1-24.

26. The application of the transfer vector pAAV-Donor in the baculovirus-rAAV production system according to claim 25 in the production of rAAV.

27. A method for producing rAAV, characterized in that, Comprising the following steps: The transfer vector pAAV-Donor described in claim 25 was transposed into the baculovirus genome through the Bac-to-Bac system to obtain recombinant bacmid; The corresponding recombinant bacmid was extracted and transfected into host cells to rescue recombinant baculovirus BEV (P1 generation); The obtained P1 generation recombinant baculovirus BEV was continuously passaged and then used to infect the host or host cell line. After infection for a certain period of time, the cells were harvested and purified to obtain rAAV.

28. The method according to claim 27, wherein The baculovirus genome is AcMNPV, BmNPV or ApNPV; The host cells are derived from the ovary, testis, embryo, imaginal disc, midgut, fat body or blood cells of insects; The Bac-to-Bac system is the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expression system; The host cell is Expi-sf9.

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