WO2025129387A1 - 一种杆状病毒-rAAV生产系统中转移载体pAAV-Donor的制备方法及应用 - Google Patents

一种杆状病毒-rAAV生产系统中转移载体pAAV-Donor的制备方法及应用 Download PDF

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WO2025129387A1
WO2025129387A1 PCT/CN2023/139457 CN2023139457W WO2025129387A1 WO 2025129387 A1 WO2025129387 A1 WO 2025129387A1 CN 2023139457 W CN2023139457 W CN 2023139457W WO 2025129387 A1 WO2025129387 A1 WO 2025129387A1
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aav
gene expression
cap
preparation
rep
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文鹏杰
王培培
徐富强
韩丹
朱续涛
齐玉祥
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Shenzhen Institute of Advanced Technology of CAS
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a preparation method and application of a transfer vector pAAV-Donor in a baculovirus-rAAV production system.
  • Recombinant adeno-associated virus is a star vector for gene therapy delivery and an important tool for analyzing brain structure and functional networks.
  • the most commonly used methods for packaging AAV include adherent or suspended cell/three-plasmid transfection system and insect cell/baculovirus production system.
  • research-grade rAAV can be produced by transient transfection of adherent human embryonic kidney (HEK) 293 cells with three plasmids, this process cannot be expanded.
  • HEK293 cell/three-plasmid transient transfection method is fast, suitable for all serotypes, and can be fermented on a large scale, it has limited yield, obvious batch differences in empty-full capsid ratio, and high production costs.
  • the baculovirus-based AAV production system has attracted much attention due to its scalability, low cost, and predictable biosafety.
  • 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.
  • the corresponding transfer vector In order to obtain rAAV of different serotypes or containing different core expression elements, the corresponding transfer vector must be prepared again.
  • Golden Gate cloning technology is a powerful tool for efficiently assembling complex vectors through a one-step reaction, which makes it possible to solve the above-mentioned rate-limiting step.
  • the GoldenBac system has been developed using this technology to simply and efficiently construct multi-gene expression vectors, unlike the expression of recombinant proteins, the encoding of each AAV element is much more complicated: 1) In addition to translating the three capsid subunits VP1, VP2 and VP3 from different start codons, the Cap gene also encodes assembly activation proteins MAAP, AAP, etc., 2) In the baculovirus AAV preparation system, the Rep gene encodes two proteins Rep78 and Rep52 required for viral replication, and compared with Rep52, the lower expression abundance of Rep78 is conducive to higher vector yields, 3)
  • the two ends of the core expression element are two T-shaped inverted terminal repeat sequences (ITRs) used as the origin of viral replication and packaging signals. ITRs are easily deleted and affect viral packaging and infection, not to mention
  • the present invention provides a method for preparing and using a transfer vector pAAV-Donor in a baculovirus-rAAV production system, the purpose of which is to place 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, so as to freely assemble the above three plasmids in one step according to demand by Golden gate, thereby improving the convenience, flexibility and efficiency of the transfer vector construction in the baculovirus-rAAV production system, and further shortening the cycle of rAAV production using baculovirus.
  • the present invention provides a method for preparing a transfer vector pAAV-Donor in a baculovirus-rAAV production system, comprising the following steps:
  • the modification method is selected from one or more of 1)-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 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) inserting an intron splicing acceptor sequence to regulate the relative expression of VP1, VP2, and VP3;
  • 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, and replacing the start codon of the Rep78 gene with a non-ATG sequence; 2) inserting an intron splicing acceptor sequence to weaken the expression of the Rep78 protein;
  • the AAV-Core backbone plasmid belongs to the Golden gate assembly donor plasmid, and contains two IIS type restriction endonuclease sites of the same type and two AAV-ITR sequences;
  • the AAV-Cap backbone plasmid belongs to the Golden gate assembly donor plasmid, and contains two IIS type restriction endonuclease sites, a baculovirus promoter, and transcriptional and post-transcriptional regulatory sequences that are the same as those of the AAV-Core backbone plasmid;
  • the AAV-Rep backbone plasmid belongs to the Golden gate assembly receptor plasmid, and contains two IIS type 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 the AAV-Cap backbone plasmid;
  • step (3) 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;
  • step (4) The three plasmids obtained in step (4) are assembled in one step using the Golden gate cloning technique, transformed into Stbl3 competent cells, and positive clones are screened to obtain the transfer vector pAAV-Donor.
  • the AAV serotype is selected from one or more of AAV1-13 and derived serotypes thereof;
  • the IIS type restriction enzyme recognition site in the AAV Cap gene expression frame that is 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);
  • 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);
  • the mutation to remove the IIS type restriction enzyme recognition site is to perform a codon synonymous substitution or an amino acid homology mutation on the IIS type restriction enzyme recognition site;
  • 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.
  • the method of modifying the AAV Cap gene expression frame in step (2) is to replace the ATG start codon of VP1 with the suboptimal translation start codon CTG and insert a GCCGCC sequence at the 5' end of CTG, and mutate the ATG codon between the VP1 start codon and the VP3 start codon;
  • the method of modifying the AAV Rep gene expression frame in step (2) is to remove all ATG sequences between the Rep78 start codon and the Rep52 start codon, and the ATG translation start codon of Rep78 is replaced with CTG and the GCCGCC sequence is inserted at the 5' end of CTG.
  • the IIS type 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);
  • 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);
  • the baculovirus promoter in step (3) is derived from AcMNPV or BmNPV, and includes Pp10, Pph, Pp6.9, Pgp64, Pie-1, and constitutive promoters formed by their combination or by adding enhancers before the promoter;
  • the core element 5' ⁇ 3' in the AAV-Core backbone plasmid in step (3) is GGTCTCATACT-ITR-MCS-ITR-CCATTGAGACC;
  • the core element 5' ⁇ 3' in the AAV-Cap backbone plasmid in step (3) is GGTCTCAAGTA-Pp10-MCS-HSV TKpoly(A)-CTCCTGAGACC;
  • the p10 promoter in the AAV-Cap backbone plasmid in step (3) is selected from AcMNPV;
  • 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;
  • the ph promoter in the AAV-Rep backbone plasmid in step (3) is preferably selected from AcMNPV;
  • the negative selection marker genes in the AAV-Rep backbone plasmid in step (3) include but are not limited to ccdB and sacB.
  • the heterologous functional gene expression element in step (4) does not contain at least the same IIS type restriction endonuclease site as the AAV-Core or AAV-Cap or AAV-Rep backbone plasmid in step (3);
  • the heterologous functional gene expression element in step (4) at least comprises a promoter, a gene expression cassette, and transcriptional and post-transcriptional regulatory sequences;
  • 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.
  • 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;
  • the heterologous functional gene expression element, the modified Cap gene expression frame and the modified AAV Rep gene expression frame in step (4) do not contain the IIS type restriction endonuclease recognition site BsaI or Eco31I (GGTCTC).
  • 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 enzyme ligation method.
  • the dosage of the transformed Stbl3 competent cells in step (5) is 5 ⁇ L.
  • the present invention also provides a transfer vector pAAV-Donor in the baculovirus-rAAV production system obtained by the above preparation method.
  • 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.
  • the present invention further provides a method for producing rAAV, comprising the following steps:
  • the transfer vector pAAV-Donor was transposed into the baculovirus genome through the Bac-to-Bac system to obtain the recombinant bacmid;
  • the obtained P1 generation recombinant baculovirus BEV is infected with a host or a host cell line after continuous passage, and after a certain period of infection, the cells are harvested and purified to obtain rAAV;
  • the baculovirus genome is AcMNPV, BmNPV or ApNPV;
  • the host cell is derived from the ovary, testis, embryo, imaginal disc, midgut, fat body or blood cell of an insect;
  • Bac-to-Bac system is an Autographa californica baculovirus (AcMNPV) expression system;
  • the host cell is Expi-sf9.
  • the pAAV-Donor preparation method provided by the present invention loads the heterologous functional gene expression elements, serotype Cap gene, and Rep gene required for AAV packaging on three plasmids respectively, so that the three plasmids can be freely assembled in one step by 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.
  • the present invention can significantly shorten the cycle of rAAV production using baculovirus
  • the present invention is compatible with the existing baculovirus-rAAV production system.
  • Figure 1 is a flow chart of the preparation of the transfer vector pAAV-Donor of the present invention.
  • a Schematic diagram of the assembly strategy of pAAV-Cap plasmid, pAAV-Core plasmid, pAAV-Rep plasmid and pAAV-Donor;
  • b Assembly reaction system;
  • c Assembly conditions of the three plasmids Rep, Cap and Core;
  • Figure 2 is a diagram showing the effect of preparing rAAV after mutating and removing the BsaI recognition site in the Cap gene expression frame of pAAV-RC in Example 1.
  • a Schematic diagram of mutating and removing the BsaI recognition site in the Cap gene expression frame of AAV types 1, 5, and 8;
  • b HEK293T cell line 3 Schematic diagram of rAAV packaging by plasmid co-transfection and evaluation indicators;
  • ce Silver staining detection 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 detection of activity of infected HEK293T cells;
  • Figure 3 is the one-step assembly effect of the transfer vector pAAV-Donor in the baculovirus-rAAV production system in Example 2.
  • a-c 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 plasmid;
  • d The growth of Stbl3 competent cells coated with LB plates containing gentamicin after the one-step assembly of the three plasmids Cap, Core and Rep; e. PCR identification of pAAV-Donor positive clone colonies;
  • AcMNPV Autographa acalifornica-baculovirus
  • the Cap gene expression frame of AAV1, AAV5, and AAV8 contains the IIS type restriction enzyme recognition site BsaI or Eco31I (GGTCTC 1/5), which hinders the modularization of the three gene elements of AAV Cap, Rep, and Core into the transfer vector pAAV-Donor required in 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 takes 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 the method of mutating and removing the IIS type restriction enzyme recognition site in the AAV-Cap gene expression frame.
  • HEK293T cells/three-plasmid transient transfection system was used to package AAV1, AAV5, and AAV8 with mutated Cap gene expression frame BsaI recognition site ( Figure 2b), where the pTrans plasmid (i.e., core plasmid) carries the eGFP expression element to facilitate the 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 best 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 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 AAV1 mutant was slightly lower than that of wild-type AAV1, while the titers of AAV5 and AAV8 mutants were higher than those of the corresponding wild-type AAV.
  • the method for 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 infection activity and lacking the IIS type restriction enzyme recognition site in the Cap expression frame.
  • Example 2 One-step assembly of the transfer vector pAAV-Donor in the baculovirus-rAAV production system
  • the Cap backbone plasmid 9A00 (pBACKBONE_Ac-AAV-Cap-BsaI-SmR, the nucleic acid sequence is as shown in SEQ ID NO.1) was constructed based on Addgene-47984; 2) the Core backbone plasmid 2001 (pBACKBONE_AAV-Core-cw3sl-BsaI-SmR, the nucleic acid sequence is as shown in SEQ ID NO.2) was constructed based on Addgene-47985, in which ITR -CW3SL sequence was synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.; 3) Rep backbone plasmid T79Ac00-0-0 (pBACKBONE_Ac-AAV-Rep-BsaI-ccdB-Gen, nucleic acid sequence as SEQ ID NO.3) was constructed, of which the
  • DB3.1 competent cells purchased from Shanghai Weidi Biotechnology Co., Ltd., DL1040M
  • This example takes the preparation of pAAV8-CMV-EGFP-hGH and pAAV9-CMV-EGFP-hGH transfer vectors as an example to demonstrate the one-step assembly method of pAAV-Donor in the baculovirus-rAAV production system.
  • the BsaI recognition site in the terminator hGH in the CMV-EGFP-hGH expression frame was mutated by fusion PCR (the nucleic acid sequence of the modified CMV-EGFP-hGH is shown in SEQ ID NO.6) and the homology arm was introduced at the same time.
  • the Core backbone plasmid 2001 was double-digested with restriction endonucleases BspDI/RsrII, and the CMV-EGFP-hGH expression frame with the BsaI recognition site removed by mutation was inserted between the ITRs, as shown in FIG3b, thereby obtaining AAV-Core plasmid 2006;
  • 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 shown in SEQ ID NO.8).
  • T79A09-2006-4 i.e., the transfer vector pAAV9-CMV-EGFP-hGH (nucleic acid sequence as shown in SEQ ID NO.9 ), as shown in Figures 3a-c, 5 ⁇ L 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 can be seen to be distributed in LB culture plates in granular form (as shown in Figure 3d).
  • Example 3 Flexible and efficient method for preparing pAAV-Donor using Autographa californica-baculovirus (AcMNPV) expression system for production of rAAV2, rAAV8, and rAAV9
  • AcMNPV Autographa californica-baculovirus
  • the prepared pAAV-Donor was transformed into DH10Bac competent cells (purchased from Thermo Fisher Scientific, 10361012) to obtain the AcMNPV recombinant bacmid, and BEV was further rescued by transfecting Expi-sf9 cells (purchased from Thermo Fisher Scientific, A35243). BEV was used to infect Expi-sf9 cells to verify whether rAAV was produced.
  • Example 2 pAAV-Donor T79A08-2006-4 and T79A09-2006-4 of rAAV8 and rAAV9 were obtained, and the same method was used to prepare pAAV-Donor T79A02-2006-4 of rAAV2 (nucleic acid sequence as SEQ ID NO.10).
  • the rAAV in Expi-sf9 cells was purified by iodixanol density gradient centrifugation purification method (method reference Aslanidi et al., 2009, Proc. Natl Acad. Sci. USA, 206: 5059-5064), and virus characteristic detection including protein silver staining, real-time fluorescence quantitative qPCR, and infection of 293T cells were performed. Protein silver staining and qPCR showed that rAAV2, rAAV8 and rAAV9 were successfully produced, and the titer of rAAV9 reached 4.099E+12VG/mL ( Figure 4d-e).
  • 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.

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Abstract

本发明公开一种杆状病毒-rAAV生产系统中转移载体pAAV-Donor的制备方法及应用，制备方法包括：突变去除AAVCap基因表达框中的IIS型限制酶识别位点；修饰获得的AAV Cap基因表达框；制备AAV-Core、AAV-Cap和AAV-Rep骨架质粒；将经修饰的Cap基因表达框、异源功能性基因表达元件、经修饰的AAVRep基因表达框依次插入至AAV-Cap、AAV-Core、AAV-Rep骨架质粒的多克隆位点中，得到pAAV-Rep、pAAV-Cap和pAAV-Core；将三个质粒通过Golden gate克隆技术一步组装，转化Stbl3感受态细胞，筛选阳性克隆，即得pAAV-Donor。

Description

一种杆状病毒-rAAV生产系统中转移载体pAAV-Donor的制备方法及应用 技术领域
本发明属于生物技术领域，具体涉及一种杆状病毒-rAAV生产系统中转移载体pAAV-Donor的制备方法及应用。
背景技术
重组腺相关病毒(recombinant Adeno-Associated Virus,rAAV)是基因治疗递送的明星载体，也是解析大脑结构与功能网络的重要工具。当前，包装AAV最常用的方法包括贴壁或悬浮细胞/三质粒转染系统和昆虫细胞/杆状病毒生产系统。尽管通过三质粒瞬时转染贴壁的人胚肾(HEK)293细胞可生产研究级的rAAV，不过这一工艺无法扩大。而悬浮HEK293细胞/三质粒瞬时转染的方法虽然快捷、适用于所有的血清型、也可规模化发酵，但产量有限、空-全衣壳比率批次差异明显而且生产成本高昂。相比之下，基于杆状病毒的AAV生产系统因其具有可扩展性、低成本和可预测的生物安全性而备受关注。
目前，利用杆状病毒大规模生产rAAV主要有三种方法：1)两杆状病毒系统；2)依赖包装细胞系的一杆状病毒系统；3)不依赖包装细胞系的基于穿梭质粒的一杆状病毒系统(OneBac system)。然而该些制备工艺的周期均较长，需要耗费数周时间执行从转移载体构建、重组杆粒制备、转染、病毒扩增到下游分离纯化等流程。对于可灵活、高效生产不同种类rAAV的第3种方法而言，转移载体的构建是限速步骤也是难点，因为AAV的Cap基因、Rep基因和ITR核心表达元件需要被构建到一个转移载体上，欲获得不同血清型或含有不同核心表达元件的rAAV就必须重新制备对应的转移载体。
Golden Gate克隆技术是一种通过一步反应高效组装复杂载体的强大工具，这为解决上述限速步骤提供了可能。尽管利用该技术已经开发可简单、高效构建多基因表达载体的GoldenBac系统，但与表达重组蛋白不同的是，AAV各元件的编码要复杂的多：1)Cap基因除了通过来自不同的起始密码子翻译三个衣壳亚基VP1、VP2和VP3外还编码组装活化蛋白MAAP、AAP等，2)在杆状病毒AAV制备系统中，Rep基因编码病毒复制所需的两种蛋白Rep78和Rep52，且与Rep52相比，Rep78表达丰度较低有利于较高的载体产量，3)核心表达元件两端是用作病毒复制起点和包装信号的两个T形反向末端重复序列(ITR)，ITR容易缺失并影响病毒包装和感染，更不用说AAV元件内部包含的IIS型限制酶识别位点，因此该 方法并不适用于One Bac system中转移载体的构建。
发明内容
针对现有技术的以上缺陷，本发明提供一种杆状病毒-rAAV生产系统中转移载体pAAV-Donor的制备方法及应用，其目的在于，通过将异源功能性基因表达元件置于AAV-Core骨架质粒、血清型Cap基因置于AAV-Cap骨架质粒、Rep基因置于AAV-Rep骨架质粒，从而根据需求一步Golden gate自由组装上述三质粒，由此提高杆状病毒-rAAV生产系统中转移载体构建的便捷性、灵活性和高效性，进一步缩短利用杆状病毒生产rAAV的周期。
本发明的具体技术方案如下：
本发明提供一种杆状病毒-rAAV生产系统中转移载体pAAV-Donor的制备方法，包括以下步骤：
(1)突变去除AAV Cap基因表达框中的IIS型限制酶识别位点；
(2)修饰步骤(1)中获得的AAV Cap基因表达框：所述修饰的方式选自1)-4)中的一种或几种：1)在VP1ATG起始密码子5’端插入额外框外起始密码子；2)将VP1的ATG起始密码子更换为次优翻译起始密码子、突变VP3亚基表达框外VP1表达框内的ATG密码子；3)在AAV衣壳编码序列的非ATG翻译起始密码子与编码氨基酸残基的密码子之间插入一个或多个氨基酸残基的编码序列；4)插入内含子剪接受体序列调控VP1、VP2、VP3的相对表达；
修饰AAV Rep基因表达框：所述修饰的方式选自1)-2)中的一种：1)突变Rep78起始密码子与Rep52起始密码子之间所有的ATG序列、更换Rep78基因的起始密码子为非ATG序列；2)插入内含子剪接受体序列弱化Rep78蛋白的表达；
(3)分别制备AAV-Core、AAV-Cap和AAV-Rep骨架质粒；
所述AAV-Core骨架质粒属于Golden gate组装供体质粒，包含两个相同类型的IIS型限制性内切酶位点、两个AAV-ITR序列；
所述AAV-Cap骨架质粒属于Golden gate组装供体质粒，包含两个与所述AAV-Core骨架质粒相同的IIS型限制性内切酶位点、杆状病毒启动子、转录和转录后调控序列；
所述AAV-Rep骨架质粒属于Golden gate组装受体质粒，包含两个与所述AAV-Cap骨架质粒相同的IIS型限制性内切酶位点、杆状病毒启动子、转录和转录后调控序列、负筛选标记基因、Tn7转座元件；
(4)将步骤(2)中获得的经修饰的Cap基因表达框插入至步骤(3)AAV-Cap骨架质粒的多克隆位点中，将异源功能性基因表达元件插入至步骤(3)AAV-Core骨架质粒的多克隆位点中，将步骤(2)中获得的经修饰的AAV Rep基因表达框插入步骤(3)AAV-Rep骨架质粒的多克隆位点中，得到pAAV-Rep、pAAV-Cap和pAAV-Core；
(5)将步骤(4)中获得的三个质粒通过Golden gate克隆技术一步组装，转化Stbl3感受态细胞，筛选阳性克隆，即可获得转移载体pAAV-Donor。
进一步地，所述AAV血清型选自AAV1-13及其衍生血清型中一种或几种；
步骤(1)中突变去除的AAV Cap基因表达框中IIS型限制酶识别位点包含BsaI或Eco31I(GGTCTC 1/5)、BsmBI(CGTCTC 1/5)、BbsI(GAAGAC 2/6)、SapI(GCTCTTC 1/4)的其中一个；
优选地，步骤(1)突变去除的AAV Cap基因表达框中IIS型限制酶识别位点为BsaI或Eco31I(GGTCTC)；
优选地，所述突变去除IIS型限制酶识别位点为对IIS型限制酶识别位点进行密码子同义替换或氨基酸同属性突变；
优选地，所述突变去除IIS型限制酶识别位点包括VP3亚基表达框中密码子同义替换、VP1/VP2与MAAP/AAP亚基重叠的表达框中氨基酸同属性突变、除了VP3/MAAP/AAP亚基编码区外其余Cap表达框中密码子同义替换。
进一步地，步骤(2)中所述修饰AAV Cap基因表达框的方式为将VP1的ATG起始密码子更换为次优翻译起始密码子CTG且在CTG 5’端插入GCCGCC序列，同时突变VP1起始密码子到VP3起始密码子之间的ATG密码子；
优选地，步骤(2)中所述修饰AAV Rep基因表达框的方式为去除Rep78起始密码子与Rep52起始密码子之间所有的ATG序列，并且Rep78的ATG翻译起始密码子更换为CTG同时在CTG 5’端插入GCCGCC序列。
进一步地，步骤(3)所述IIS型限制性内切酶包含BsaI或Eco31I(GGTCTC 1/5)、BsmBI(CGTCTC 1/5)、BbsI(GAAGAC 2/6)、SapI(GCTCTTC 1/4)的其中一个；
优选地，步骤(3)所述AAV-Core、AAV-Cap和AAV-Rep骨架质粒中的两个IIS型限制性内切酶为BsaI或Eco31I(GGTCTC 1/5)；
优选地，步骤(3)所述杆状病毒启动子来源于AcMNPV或BmNPV，包含Pp10、Pph、Pp6.9、Pgp64、Pie-1及其相互联合或启动子前添加增强子形成的组成型启动子；
优选地，步骤(3)所述AAV-Core骨架质粒中核心元件5’→3’为GGTCTCATACT-ITR-MCS-ITR-CCATTGAGACC；
优选地，步骤(3)所述AAV-Cap骨架质粒中核心元件5’→3’为GGTCTCAAGTA-Pp10-MCS-HSV TKpoly(A)-CTCCTGAGACC；
优选地，步骤(3)所述AAV-Cap骨架质粒中p10启动子选自AcMNPV；
优选地，步骤(3)所述AAV-Rep骨架质粒中核心元件5’→3’为Tn7R-Gen-GGAGTGAGACC-ccdB-GGTCTCACCAT-Pph-MCS-SV40poly(A)-Tn7L；
优选地，步骤(3)所述AAV-Rep骨架质粒中ph启动子优选自AcMNPV；
优选地，步骤(3)所述AAV-Rep骨架质粒中负筛选标记基因包括但不限于ccdB、sacB。
进一步地，步骤(4)所述异源功能性基因表达元件中至少不含有与步骤(3)所述AAV-Core或AAV-Cap或AAV-Rep骨架质粒相同的IIS型限制性内切酶位点；
优选地，步骤(4)所述异源功能性基因表达元件至少包含启动子、基因表达框、转录和转录后调控序列；
优选地，步骤(4)所述异源功能性基因表达元件中转录和转录后调控序列包括但不限于cw3sl、WPRE、hGHpolyA、bGH polyA。
进一步地，步骤(4)所述异源功能性基因表达元件、经修饰的Cap基因表达框与经修饰的AAV Rep基因表达框均不含有IIS型限制性内切酶识别位点；
优选地，步骤(4)所述异源功能性基因表达元件、经修饰的Cap基因表达框与经修饰的AAV Rep基因表达框均不含有IIS型限制性内切酶识别位点BsaI或Eco31I(GGTCTC)。
进一步地，步骤(4)中经修饰的Cap基因表达框插入至AAV-Cap骨架质粒的多克隆位点，异源功能性基因表达元件插入至AAV-Core骨架质粒的多克隆位点，经修饰的AAV Rep基因表达框插入AAV-Rep骨架质粒的多克隆位点采取同源重组克隆技术或酶切连接法。
进一步地，步骤(5)所述三个质粒Golden gate组装条件为：1)质粒的组装需求量(ng)＝0.0345×pAAV-Rep或pAAV-Cap或pAAV-Core的质粒大小，T4DNA Ligase Buffer(10×)2μL，Golden Gate Enzyme Mix 1μL，补充ddH2O至20μL；2)37℃(1h)→60℃(5min)→4℃(∞)；
优选地，步骤(5)所述转化Stbl3感受态细胞的剂量为5μL。
本发明还提供上述制备方法得到的杆状病毒-rAAV生产系统中转移载体pAAV-Donor。
本发明还提供所述制备方法或所述杆状病毒-rAAV生产系统中转移载体pAAV-Donor在生产rAAV中的应用。
本发明进一步提供一种生产rAAV的方法，包括以下步骤：
通过Bac-to-Bac系统，将转移载体pAAV-Donor转座至杆状病毒基因组中，获得重组杆粒；
提取相应的重组杆粒，并转染宿主细胞拯救出重组杆状病毒BEV(P1代)；
将获得的P1代重组杆状病毒BEV经连续传代后感染宿主或宿主细胞系，感染一定时间后，收获细胞纯化即得到rAAV；
优选地，所述杆状病毒基因组为AcMNPV、BmNPV或ApNPV；
优选地，所述宿主细胞来源于昆虫的卵巢、精巢、胚胎、成虫盘、中肠、脂肪体或血细胞；
优选地，所述Bac-to-Bac系统为苜蓿银纹夜蛾杆状病毒(AcMNPV)表达系统；
优选地，所述宿主细胞为Expi-sf9。
本发明的有益效果：
(1)本发明提供的pAAV-Donor制备方法，通过将AAV包装所需的异源功能性基因表达元件、血清型Cap基因、Rep基因分别负载于3个质粒上，从而根据rAAV的生产需求可以一步Golden gate自由组装上述三质粒，实现杆状病毒-rAAV生产系统中转移载体的便捷、灵活、高效构建，即对于制备携带不同异源功能性基因片段的不同血清型rAAV，只需要在AAV-Cap质粒库、AAV-Core质粒库中选择恰当的质粒与AAV-Rep质粒Golden gate组装即可，无需多次重复依赖限制性核酸内切酶、DNA连接酶或同源重组酶作用将Cap基因、Rep基因和ITR核心表达元件连接在pFBD(pFastBac Dual)载体上；
(2)本发明基于rAAV-Donor制备的便捷性、灵活性、高效性，可大幅缩短利用杆状病毒生产rAAV的周期；
(3)本发明可兼容现有的杆状病毒-rAAV生产系统。
附图说明
图1是本发明转移载体pAAV-Donor的制备流程图。a.pAAV-Cap质粒、pAAV-Core质粒、pAAV-Rep质粒及pAAV-Donor组装策略示意图；b.组装反应体系；c.Rep、Cap及Core三质粒组装条件；
图2是实施例1中突变去除pAAV-RC中Cap基因表达框内BsaI识别位点后rAAV的制备效果。a.突变去除1、5、8型AAV Cap基因表达框内BsaI识别位点示意图；b.HEK293T细胞三 质粒共转染包装rAAV示意图及评估指标；c-e.纯化后的1、5、8型rAAV及突变体蛋白银染检测；f-h.纯化后的1、5、8型rAAV及突变体qPCR滴度测定及感染HEK293T细胞活性检测；
图3是实施例2中杆状病毒-rAAV生产系统中转移载体pAAV-Donor的一步组装效果。a-c.基于骨架质粒制备转移载体pAAV8-CMV-EGFP-hGH和pAAV9-CMV-EGFP-hGH所需的9A08、9A09、2006、T79Ac00-0-4构建示意图；d.Cap、Core及Rep三质粒一步组装后转化Stbl3感受态细胞涂布含有庆大霉素的LB平板生长情况；e.pAAV-Donor阳性克隆菌落PCR鉴定；
图4是实施例3中基于灵活、高效的pAAV-Donor制备方法利用苜蓿银纹夜蛾-杆状病毒(AcMNPV)表达系统生产rAAV2、rAAV8和rAAV9；a.pAAV-Donor中的Cap、Core和Rep元件转座至杆状病毒基因组示意图；b.MOI＝1感染悬浮Expi-sf9细胞(2E+06个/mL，50ml)，3天后细胞中荧光蛋白的表达情况；c.蛋白免疫印迹检测被BEV感染3天后Expi-sf9细胞中Rep和Cap的表达量；d.对制备的rAAV纯度及衣壳蛋白比例进行蛋白银染检测；e.qPCR定量rAAV的滴度；f.基于灵活、高效制备pAAV-Donor系统生产的rAAV感染293T细胞检测病毒活性。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
实施例1突变去除Cap基因表达框内BsaI识别位点
在常用AAV血清型1、2、5、8、9中，AAV1、AAV5、AAV8的Cap基因表达框中含有IIS型限制酶识别位点BsaI或Eco31I(GGTCTC 1/5)，这阻碍了以模块化方式将AAV的Cap、Rep、Core三个基因元件一步快速组装成杆状病毒-rAAV生产系统中所需的转移载体pAAV-Donor，因此需要突变去除Cap基因表达框中的GGTCTC序列。本实施例以AAV1、AAV5、AAV8(Addgene，112862、104964、112864)为例，结合HEK293T细胞/三质粒瞬时转染AAV包装系统，以展示突变去除AAV-Cap基因表达框中IIS型限制酶识别位点的方式。
(1)对pAAV-RC质粒中Cap基因表达框内的BsaI识别位点进行单碱基突变，如附图2a所示；
1.1将AAV-Cap1中编码VP1-467位丝氨酸(467S)进行密码子同义替换，“tct”突变为“tca”；
1.2将AAV-Cap5中编码VP1-58位亮氨酸(58L)突变为同属性氨基酸-缬氨酸(58V)、异亮氨酸(58I)，“ctc”分别突变为“gtc”、“atc”；
1.3将AAV-Cap8中编码MAAP-117位精氨酸(117R)突变为同属性氨基酸-赖氨酸(117K),“aga”分别突变为“aaa”。
(2)利用HEK293T细胞/三质粒瞬时转染系统包装突变Cap基因表达框内BsaI识别位点的AAV1、AAV5、AAV8(附图2b)，其中pTrans质粒(即核心质粒)携带eGFP表达元件便于检测包装的rAAV特性。通过蛋白银染和实时荧光定量qPCR测定滴度和纯度、感染293T细胞比较感染活性，获取Cap基因表达框中不含BsaI位点的最佳突变体，蛋白银染显示突变去除Cap基因表达框中BsaI识别位点后的AAV1-tct467tca、AAV5-L58V、AAV8-MAAP-R117K均能产生rAAV，而AAV5-L58I无法被包装(附图2c-e)；实时荧光定量qPCR与感染293T细胞显示AAV1突变体的滴度略低于野生型AAV1，而AAV5和AAV8突变体滴度均高于对应的野生型AAV，AAV1、AAV5和AAV8突变体均能感染293T细胞且感染效果接近野生型AAV(附图2f-h)。综上，通过本发明描述的突变去除AAV-Cap基因表达框内IIS型限制酶识别位点的方法可以筛选出具有感染活性且Cap表达框内缺失IIS型限制酶识别位点的AAV突变体。
实施例2一步组装杆状病毒-rAAV生产系统中转移载体pAAV-Donor
为了实现携带AAV三元件(Rep/Cap/Core)转移载体pAAV-Donor的简便、快速构建，1)基于Addgene-47984构建了Cap骨架质粒9A00(pBACKBONE_Ac-AAV-Cap-BsaI-SmR，核酸序列如SEQ ID NO.1)；2)基于Addgene-47985构建了Core骨架质粒2001(pBACKBONE_AAV-Core-cw3sl-BsaI-SmR，核酸序列如SEQ ID NO.2)，其中ITR-CW3SL序列由北京六合华大基因科技有限公司合成；3)构建了Rep骨架质粒T79Ac00-0-0(pBACKBONE_Ac-AAV-Rep-BsaI-ccdB-Gen，核酸序列如SEQ ID NO.3)，其中ccdB序列由北京擎科生物科技有限公司合成，其余序列从pFastBac Dual表达载体(购自赛默飞世尔科技公司，由本实验室保存)中扩增获得，载体构建过程中使用DB3.1感受态细胞(购自上海唯地生物技术有限公司，DL1040M)。本实施例以制备pAAV8-CMV-EGFP-hGH和pAAV9-CMV-EGFP-hGH转移载体为例，展示杆状病毒-rAAV生产系统中pAAV-Donor的一步组装法。
(1)分别构建AAV Cap、Core和Rep质粒；
1.1通过PCR分别在Cap8(来源于实施例1，已突变去除BsaI识别位点)和Cap9(来源Addgene，由本实验室保存)基因表达框两端引入同源臂，并将起始密码子“ATG”均突变为 “CTG”后在CTG 5’端增加“gccgcc”序列(修饰后的AAV8和AAV9Cap基因表达框的核酸序列分别如SEQ ID NO.4和SEQ ID NO.5)，接着利用同源重组克隆技术将修饰后的Cap8和Cap9基因表达框分别插入Cap骨架质粒9A00的多克隆位点，如附图3a所示，从而分别获得AAV-Cap质粒9A08和9A09；
1.2利用融合PCR突变CMV-EGFP-hGH表达框内终止子hGH中的BsaI识别位点(修饰后CMV-EGFP-hGH的核酸序列如SEQ ID NO.6)的同时引入同源臂，随后限制性内切酶BspDI/RsrII双酶切Core骨架质粒2001，将突变去除BsaI识别位点的CMV-EGFP-hGH表达框插入ITR之间，如附图3b所示，从而获得AAV-Core质粒2006；
1.3通过PCR在修饰后的Rep2基因表达框两端引入同源臂(修饰后的Rep2基因表达框的核酸序列如SEQ ID NO.7)，接着利用同源重组克隆技术将该片段插入Rep骨架质粒T79Ac00-0-0的多克隆位点，如附图3a所示，从而获得AAV-Rep质粒T79Ac00-0-4；
(2)利用Goldengate组装试剂盒(NEB公司，E1601L)对AAV Cap、Core和Rep质粒进行一步组装，组装条件如附图1b-c所示，9A08+2006+T79Ac00-0-4组装形成T79A08-2006-4即转移载体pAAV8-CMV-EGFP-hGH(核酸序列如SEQ ID NO.8)，9A09+2006+T79Ac00-0-4组装形成T79A09-2006-4即转移载体pAAV9-CMV-EGFP-hGH(核酸序列如SEQ ID NO.9)，如附图3a-c所示，取Goldengate组装后的产物5μL转化stbl3化学感受态细胞，涂布在含有庆大霉素的LB固体培养基上，经过37℃过夜培养可以看到菌落呈颗粒状分布于LB培养板(如附图3d所示)，分别在T79Ac08-2006-4和T79Ac09-2006-4平板中挑选10个单克隆菌落进行菌落PCR并筛选阳性克隆(如附图3e所示)，选取T79Ac08-2006-4和T79Ac09-2006-4各2个阳性克隆过夜培养，提取质粒进行测序，经序列比对正确。这表明本发明提供的转移载体pAAV-Donor制备方法可根据rAAV的生产需求通过一步Golden gate自由组装实现杆状病毒-rAAV生产系统中转移载体的便捷、灵活、高效构建。
实施例3基于灵活、高效制备pAAV-Donor的方法利用苜蓿银纹夜蛾-杆状病毒 (AcMNPV)表达系统生产rAAV2、rAAV8和rAAV9
为了验证上述一步组装法快速获得的pAAV-Donor的有效性，将所制备的pAAV-Donor转化至DH10Bac感受态细胞(购自赛默飞世尔科技公司，10361012)中以获得AcMNPV重组杆粒，进一步通过转染Expi-sf9细胞(购自赛默飞世尔科技公司，A35243)拯救出BEV，利用BEV感染Expi-sf9细胞验证是否有rAAV产生。
(1)分别制备rAAV2、rAAV8和rAAV9的AcMNPV重组杆粒；
在实施例2中已获得rAAV8和rAAV9的pAAV-Donor T79A08-2006-4和T79A09-2006-4，使用相同的方法制备rAAV2的pAAV-Donor T79A02-2006-4(核酸序列如SEQ ID NO.10)。按照Bac-to-Bac表达系统使用说明书，如附图4a所示，通过将T79A02-2006-4、T79A08-2006-4和T79A09-2006-4分别转座至AcMPNV基因组中，分别制备重组杆粒AcMNPV-T79A02-2006-4(rAAV2)、AcMNPV-T79A08-2006-4(rAAV8)、AcMNPV-T79A09-2006-4(rAAV9)；
(2)利用杆状病毒(AcMNPV)表达系统生产rAAV；
悬浮培养的Expi-sf9细胞以2E+06个/孔接种于6孔细胞培养板中，贴壁培养1h后利用TransIT-Insect Transfection Reagent(购自Mirusbio公司，MIR6105)分别转染2μg步骤(1)中制备的不同AAV重组杆粒，24h后对细胞进行换液，再经过3天的培养使用qPCR对上清中的第一代BEV(P1)进行定量检测。进一步，取适量的悬浮Expi-sf9细胞接种在摇瓶中(2E+06个/mL，50mL)，按照MOI＝1分别接种BEV(P1)，经过3天的感染收集细胞，荧光显微镜观察到绿色荧光蛋白表达(附图4b)，这表明重组BEV感染了细胞并介导eGFP的表达；蛋白免疫印迹检测细胞中的Rep和Cap表达，Rep52的表达丰度高于Rep78且VP1:VP2:VP3的表达量接近1：1：10(附图4c)，同时上清中的大量BEV(P2)可作为种子再次感染Expi-sf9细胞生产rAAV。接着，采用碘克沙醇密度梯度离心分离纯化法(方法参考Aslanidi等,2009,Proc.NatlAcad.Sci.USA,206:5059-5064)纯化Expi-sf9细胞中的rAAV，并进行病毒特性检测包括蛋白银染、实时荧光定量qPCR、感染293T细胞，蛋白银染和qPCR显示rAAV2、rAAV8和rAAV9被成功生产，其中的rAAV9滴度最高达到4.099E+12VG/mL(附图4d-e)，取3μL制备的rAAV2、rAAV8和rAAV9分别感染贴壁的293T细胞，仅感染36h就观察到荧光蛋白表达(附图4f)，这表明生产的rAAV具有感染活性。综上，本发明提供的灵活、高效制备转移载体pAAV-Donor的方法能够用于杆状病毒表达系统生产rAAV。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (28)

  1. 一种杆状病毒-rAAV生产系统中转移载体pAAV-Donor的制备方法，其特征在于，包括以下步骤：
    (1)突变去除AAV Cap基因表达框中的IIS型限制酶识别位点；
    (2)修饰步骤(1)中获得的AAV Cap基因表达框：所述修饰的方式选自1)-4)中的一种或几种：1)在VP1 ATG起始密码子5’端插入额外框外起始密码子；2)将VP1的ATG起始密码子更换为次优翻译起始密码子、突变VP3亚基表达框外VP1表达框内的ATG密码子；3)在AAV衣壳编码序列的非ATG翻译起始密码子与编码氨基酸残基的密码子之间插入一个或多个氨基酸残基的编码序列；4)插入内含子剪接受体序列调控VP1、VP2、VP3的相对表达；
    修饰AAV Rep基因表达框：所述修饰的方式选自1)-2)中的一种：1)突变Rep78起始密码子与Rep52起始密码子之间所有的ATG序列、更换Rep78基因的起始密码子为非ATG序列；2)插入内含子剪接受体序列弱化Rep78蛋白的表达；
    (3)分别制备AAV-Core、AAV-Cap和AAV-Rep骨架质粒；
    所述AAV-Core骨架质粒属于Golden gate组装供体质粒，包含两个相同类型的IIS型限制性内切酶位点、两个AAV-ITR序列；
    所述AAV-Cap骨架质粒属于Golden gate组装供体质粒，包含两个与所述AAV-Core骨架质粒相同的IIS型限制性内切酶位点、杆状病毒启动子、转录和转录后调控序列；
    所述AAV-Rep骨架质粒属于Golden gate组装受体质粒，包含两个与所述AAV-Cap骨架质粒相同的IIS型限制性内切酶位点、杆状病毒启动子、转录和转录后调控序列、负筛选标记基因、Tn7转座元件；
    (4)将步骤(2)中获得的经修饰的Cap基因表达框插入至步骤(3)AAV-Cap骨架质粒的多克隆位点中，将异源功能性基因表达元件插入至步骤(3)AAV-Core骨架质粒的多克隆位点中，将步骤(2)中获得的经修饰的AAV Rep基因表达框插入步骤(3)AAV-Rep骨架质粒的多克隆位点中，得到pAAV-Rep、pAAV-Cap和pAAV-Core；
    (5)将步骤(4)中获得的三个质粒通过Golden gate克隆技术一步组装，转化Stbl3感受态细胞，筛选阳性克隆，即可获得转移载体pAAV-Donor。
  2. 如权利要求1所述的制备方法，其特征在于，所述AAV血清型选自AAV1-13及其衍生血清型中一种或几种；
    步骤(1)中突变去除的AAV Cap基因表达框中IIS型限制酶识别位点包含BsaI或Eco31I(GGTCTC 1/5)、BsmBI(CGTCTC 1/5)、BbsI(GAAGAC 2/6)、SapI(GCTCTTC 1/4)的其中一个。
  3. 如权利要求1所述的制备方法，其特征在于，步骤(1)突变去除的AAV Cap基因表达框中IIS型限制酶识别位点为BsaI或Eco31I(GGTCTC)。
  4. 如权利要求1所述的制备方法，其特征在于，步骤(1)突变去除IIS型限制酶识别位点为对IIS型限制酶识别位点进行密码子同义替换或氨基酸同属性突变。
  5. 如权利要求1所述的制备方法，其特征在于，步骤(1)突变去除IIS型限制酶识别位点包括VP3亚基表达框中密码子同义替换、VP1/VP2与MAAP/AAP亚基重叠的表达框中氨基酸同属性突变、除了VP3/MAAP/AAP亚基编码区外其余Cap表达框中密码子同义替换。
  6. 如权利要求1所述的制备方法，其特征在于，步骤(2)中所述修饰AAV Cap基因表达框的方式为将VP1的ATG起始密码子更换为次优翻译起始密码子CTG且在CTG 5’端插入GCCGCC序列，同时突变VP1起始密码子到VP3起始密码子之间的ATG密码子。
  7. 如权利要求1所述的制备方法，其特征在于，步骤(2)中所述修饰AAV Rep基因表达框的方式为去除Rep78起始密码子与Rep52起始密码子之间所有的ATG序列，并且Rep78的ATG翻译起始密码子更换为CTG同时在CTG 5’端插入GCCGCC序列。
  8. 如权利要求1所述的制备方法，其特征在于，步骤(3)所述IIS型限制性内切酶包含BsaI或Eco31I(GGTCTC 1/5)、BsmBI(CGTCTC 1/5)、BbsI(GAAGAC 2/6)、SapI(GCTCTTC 1/4)的其中一个。
  9. 如权利要求1所述的制备方法，其特征在于，步骤(3)所述AAV-Core、AAV-Cap和AAV-Rep骨架质粒中的两个IIS型限制性内切酶为BsaI或Eco31I(GGTCTC 1/5)。
  10. 如权利要求1所述的制备方法，其特征在于，步骤(3)所述杆状病毒启动子来源于AcMNPV或BmNPV，包含Pp10、Pph、Pp6.9、Pgp64、Pie-1及其相互联合或启动子前添加增强子形成的组成型启动子。
  11. 如权利要求1所述的制备方法，其特征在于，步骤(3)所述AAV-Core骨架质粒中核心元件5’→3’为GGTCTCATACT-ITR-MCS-ITR-CCATTGAGACC。
  12. 如权利要求1所述的制备方法，其特征在于，步骤(3)所述AAV-Cap骨架质粒中核心元件5’→3’为GGTCTCAAGTA-Pp10-MCS-HSV TK poly(A)-CTCCTGAGACC。
  13. 如权利要求12所述的制备方法，其特征在于，步骤(3)所述AAV-Cap骨架质粒中p10启动子选自AcMNPV。
  14. 如权利要求1所述的制备方法，其特征在于，步骤(3)所述AAV-Rep骨架质粒中核心元件5’→3’为Tn7R-Gen-GGAGTGAGACC-ccdB-GGTCTCACCAT-Pph-MCS-SV40poly(A)-Tn7L。
  15. 如权利要求14所述的制备方法，其特征在于，步骤(3)所述AAV-Rep骨架质粒中ph启动子优选自AcMNPV。
  16. 如权利要求1所述的制备方法，其特征在于，步骤(3)所述AAV-Rep骨架质粒中负筛选标记基因包括但不限于ccdB、sacB。
  17. 如权利要求1所述的制备方法，其特征在于，步骤(4)所述异源功能性基因表达元件中至少不含有与步骤(3)所述AAV-Core或AAV-Cap或AAV-Rep骨架质粒相同的IIS型限制性内切酶位点。
  18. 如权利要求1所述的制备方法，其特征在于，步骤(4)所述异源功能性基因表达元件至少包含启动子、基因表达框、转录和转录后调控序列。
  19. 如权利要求18所述的制备方法，其特征在于，步骤(4)所述异源功能性基因表达元件中转录和转录后调控序列包括但不限于cw3sl、WPRE、hGH polyA、bGH polyA。
  20. 如权利要求1所述的制备方法，其特征在于，步骤(4)所述异源功能性基因表达元件、经修饰的Cap基因表达框与经修饰的AAV Rep基因表达框均不含有IIS型限制性内切酶识别位点。
  21. 如权利要求1所述的制备方法，其特征在于，步骤(4)所述异源功能性基因表达元件、经修饰的Cap基因表达框与经修饰的AAV Rep基因表达框均不含有IIS型限制性内切酶识别位点BsaI或Eco31I(GGTCTC)。
  22. 如权利要求1所述的制备方法，其特征在于，步骤(4)中经修饰的Cap基因表达框插入至AAV-Cap骨架质粒的多克隆位点，异源功能性基因表达元件插入至AAV-Core骨架质粒的多克隆位点，经修饰的AAV Rep基因表达框插入AAV-Rep骨架质粒的多克隆位点采取同源重组克隆技术或酶切连接法。
  23. 如权利要求1所述的制备方法，其特征在于，步骤(5)所述三个质粒Golden gate组装条件为：1)质粒的组装需求量(ng)＝0.0345×pAAV-Rep或pAAV-Cap或pAAV-Core的质粒大小，T4 DNA Ligase Buffer(10×)2μL，Golden Gate Enzyme Mix 1μL，补充ddH2O至20μL；2)37℃(1h)→60℃(5min)→4℃(∞)。
  24. 如权利要求1所述的制备方法，其特征在于，步骤(5)所述转化Stbl3感受态细胞的剂量为5μL。
  25. 权利要求1-24任一项所述制备方法得到的杆状病毒-rAAV生产系统中转移载体pAAV-Donor。
  26. 权利要求25所述杆状病毒-rAAV生产系统中转移载体pAAV-Donor在生产rAAV中的应用。
  27. 一种生产rAAV的方法，其特征在于，包括以下步骤：
    通过Bac-to-Bac系统，将权利要求25所述的转移载体pAAV-Donor转座至杆状病毒基因组中，获得重组杆粒；
    提取相应的重组杆粒，并转染宿主细胞拯救出重组杆状病毒BEV(P1代)；
    将获得的P1代重组杆状病毒BEV经连续传代后感染宿主或宿主细胞系，感染一定时间后，收获细胞纯化即得到rAAV。
  28. 如权利要求27所述的方法，其特征在于，所述杆状病毒基因组为AcMNPV、BmNPV或ApNPV；
    所述宿主细胞来源于昆虫的卵巢、精巢、胚胎、成虫盘、中肠、脂肪体或血细胞；
    所述Bac-to-Bac系统为苜蓿银纹夜蛾杆状病毒(AcMNPV)表达系统；
    所述宿主细胞为Expi-sf9。
PCT/CN2023/139457 2023-12-18 2023-12-18 一种杆状病毒-rAAV生产系统中转移载体pAAV-Donor的制备方法及应用 Pending WO2025129387A1 (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022166027A1 (zh) * 2021-02-04 2022-08-11 中吉智药(南京)生物技术有限公司 一种杆状病毒感染昆虫细胞生产aav基因药物的系统及方法
CN116355936A (zh) * 2023-04-10 2023-06-30 苏州左旋星生物科技有限公司 一种高稳定性载体质粒及其构建方法和应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022166027A1 (zh) * 2021-02-04 2022-08-11 中吉智药(南京)生物技术有限公司 一种杆状病毒感染昆虫细胞生产aav基因药物的系统及方法
CN116355936A (zh) * 2023-04-10 2023-06-30 苏州左旋星生物科技有限公司 一种高稳定性载体质粒及其构建方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YONGLUN LUO, LIN LIN, LARS BOLUND, CHARLOTTE BRANDT SøRENSEN: "Efficient construction of rAAV-based gene targeting vectors by Golden Gate cloning", BIOTECHNIQUES, vol. 56, no. 5, US , XP055746425, ISSN: 0736-6205, DOI: 10.2144/000114169 *

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