WO2017049759A1 - 一种重组杆状病毒及其应用 - Google Patents

一种重组杆状病毒及其应用 Download PDF

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WO2017049759A1
WO2017049759A1 PCT/CN2015/095751 CN2015095751W WO2017049759A1 WO 2017049759 A1 WO2017049759 A1 WO 2017049759A1 CN 2015095751 W CN2015095751 W CN 2015095751W WO 2017049759 A1 WO2017049759 A1 WO 2017049759A1
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recombinant baculovirus
gene
seq
raav
sequence
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吴阳
徐富强
何晓斌
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中国科学院武汉物理与数学研究所
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Definitions

  • the invention belongs to the field of gene therapy, and more particularly to a recombinant baculovirus and its use.
  • rAAV Recombinant adeno-associated virus
  • VG VG, virus genomes
  • the main process of the two baculovirus system is to integrate the Rep gene and the Cap gene of AAV into one baculovirus genome, integrate the ITR core expression element into another baculovirus genome, and then combine the two recombination rods.
  • the virus co-infects the host cells to produce rAAV.
  • the main process of a baculovirus system that relies on packaging cell lines is to first establish a packaging cell line that induces expression of the Rep gene and the Cap gene.
  • This packaging cell line integrates the Rep gene and the Cap gene expression element, and the Rep gene and the Cap gene are respectively The hr2 enhancer sequence and the Rep protein binding sequence of AAV were added upstream of the PpH promoter under the control of PpH, a strong promoter of baculovirus late gene expression. Integration of ITR core expression elements in infection After the recombinant baculovirus, the Rep gene and the Cap gene in the packaging cell line were induced to express, thereby producing rAAV.
  • the former is inefficient in co-infecting cells with two baculoviruses, unable to fully utilize the productivity of each cell, and the infection is a random process, which is easy to produce empty-shell defective rAAV particles without nucleic acid, and the preparation process conditions are optimized.
  • Complex, rAAV quality prepared in different batches is unstable.
  • the latter relies on the packaging cell line that induces the expression of Rep gene and Cap gene, and the construction is complicated.
  • the screening of excellent packaging cell lines is difficult.
  • To prepare rAAV with different serotypes it is necessary to establish a packaging cell line expressing the corresponding serotype Cap gene, which is flexible. Sex and versatility are poor and difficult to promote.
  • the present invention provides a recombinant baculovirus and its use, which aims to integrate a Rep gene, a Cap gene and an ITR core expression element of an adeno-associated virus (AAV) into a rod.
  • AAV adeno-associated virus
  • rAAV recombinant adeno-associated virus
  • a recombinant baculovirus characterized in that the recombinant baculovirus genome integrates a Rep gene of AAV, a Cap gene, and an ITR core expression element.
  • the recombinant baculovirus has a sequence of a Rep gene which is codon-optimized according to a ribosome leak scanning principle, preferably as SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3. The sequence shown.
  • the recombinant baculovirus has a sequence of a Cap gene which is codon-optimized according to a ribosome leak scanning principle, preferably SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6. The sequence shown.
  • the recombinant baculovirus has an ITR core expression element linked to the core via a 5' end
  • the acid fragment and the 3'-end joining nucleic acid fragment are linked to the expression cassette of the Rep gene and the Cap gene, and include an end inverted repeat (ITR) in the AAV genome at both ends and a target gene sequence located in the middle;
  • ITR sequence is preferably a sequence as shown in SEQ ID No. 7.
  • the recombinant baculovirus wherein the 5' or 3' end joining nucleic acid fragment is a ligated nucleic acid sequence of between 80 bp and 140 bp in length, preferably the sequence is SEQ ID No. 8 or SEQ ID No. 9 is shown.
  • the recombinant baculovirus wherein the adeno-associated virus is a dienotype-associated virus.
  • the application comprises the following steps:
  • said application, step (1) utilizes a pFast.Bac.Dual shuttle vector, based on the Bac to Bac system.
  • the recombinant baculovirus disclosed by the present invention provides a replication packaging element required for preparing rAAV by using a baculovirus by genetic design and optimization, namely, AAV Rep gene, Cap gene and ITR core expression element, and maintains the organism. Activity, breaking the bottleneck of ITR compatibility with the other two genes.
  • rAAV can be produced by simple cell infection, in which the proportion of intact rAAV particles is high and the virus quality is high. Moreover, the rAAV production capacity of individual cells is also greatly improved.
  • the Bac-A system of the invention has high flexibility, high versatility, high virus quality and high yield, and is suitable for large-scale scale production, and can effectively solve the problem of large-scale preparation of rAAV.
  • FIG. 1 is a schematic diagram showing the structure of recombinant baculovirus, i.e., a schematic diagram of AcMNPV Bacmid incorporating the Rep gene of AAV, the Cap gene and the ITR core expression element.
  • the baculovirus AcMNPV genome is a double-stranded circular DNA with a full length of 133,894 bp, a sequence and map reference (Virology, 1994.202(2): p.586-605.), and a structural reference for AcMNPV Bacmid (bMON14272). J Virol, 1993. 67(8): p. 4566-79.), the Rep gene, Cap gene and ITR core expression element of the integrated AAV were inserted into the Tn7 site of bacmid (bMON14272).
  • FIG. 2 is a schematic diagram showing the principle of the recombinant baculovirus system and the preparation of rAAV in Examples 1 and 2.
  • 2A is a schematic diagram of the packaging of rAAV;
  • FIG. 2B is a schematic diagram of a shuttle plasmid pFast.Bac.Dual (pFBD) of the baculovirus expression system;
  • FIG. 2C is a schematic diagram showing the structure of a recombinant shuttle plasmid integrating the components required for preparation of rAAV; It is a schematic diagram of the process of preparing rAAV by recombinant baculovirus infection of Sf9 cells;
  • Figure 3 is a graph showing the activity verification of recombinant baculovirus-infected Sf9 cells prepared by the three combination schemes in Example 1.
  • 3A is a fluorescence microscopic image of the Sf9 cell control group
  • FIG. 3B is a fluorescence microscopic image of the experimental group 3 days after the recombinant baculovirus prepared by the protocol 1 to 3 is infected with Sf9 cells;
  • Figure 4 is a graph showing the activity verification of rAAV-infected cells prepared by the three combination schemes in Examples 1 and 2.
  • 4A is the activity verification result of rAAV prepared by the protocol 1 at the levels of HEK293 cells and Sf9 cells
  • FIG. 4B is the activity verification result of the rAAV-infected HEK293 cells prepared by the protocol 2
  • FIG. 4C is the activity of the rAAV-infected HEK293 cells prepared by the protocol 3;
  • Validation results is the activity verification results;
  • FIG. 5 is a transmission electron micrograph of the purified rAAV particles of Example 4.
  • 5A is a transmission electron micrograph after negative dyeing of rAAV particles
  • FIG. 5B is a partial enlarged view (2.5 times) of FIG. 5A;
  • FIG. 6 is a graph showing the activity verification of purified rAAV in Example 5 at the level of HEK293 cells and mouse brain.
  • 6A is a fluorescence microscopic image of purified rAAV-infected HEK293 cells
  • FIG. 6B is a fluorescence microscopic image of hippocampal cortical neurons in purified rAAV-infected mice
  • FIG. 6C is a partial enlarged view of FIG. 6B (FIG. 6C). 2 times).
  • the recombinant baculovirus provided by the present invention has a genome diagram as shown in Fig. 1, and is preferably based on AcMNPV clone C6 (genomic sequence such as: Genbank accession No. NC_001623.1), integrating AAV Rep gene, Cap gene and ITR core.
  • the expression element, the Rep gene and the Cap gene were codon optimized according to the ribosome leak scanning principle.
  • the ITR core expression element includes an end inverted repeat (ITR) in the AAV genome at both ends, and a carrying target gene (GOI) fragment in the middle, and the nucleic acid fragment and the 3'-end nucleic acid fragment are ligated through the 5' end.
  • the Rep gene is linked to the expression cassette of the Cap gene. Among them, both the Rep gene and the Cap gene can be upstream or downstream of the ITR core expression element.
  • the Rep gene was codon-optimized according to the principle of ribosome leakage scanning, and one mRNA was transcribed by the PpH promoter to realize functional expression of Rep72 and Rep52 replication proteins.
  • the sequence thereof is preferably as shown in one of SEQ ID No. 1 to 3.
  • Cap gene codon optimization also utilizes the principle of ribosome leakage scanning to transcribe an mRNA through the P10 promoter to achieve functional expression of three capsid proteins of VP1, VP2 and VP3 in a near natural ratio (1:1:10). .
  • the sequence thereof is preferably as shown in one of SEQ ID Nos. 4 to 6.
  • the ITR core expression element is ligated to the expression cassette of the Rep gene and the Cap gene through the ligated nucleic acid fragment at both ends.
  • the ITR is a terminal inverted repeat in the AAV genome, preferably the ITR sequence of type 2 AAV, as set forth in SEQ ID No. 7.
  • the 5' or 3' end joining nucleic acid sequence is preferably a ligated nucleic acid sequence of between 80 bp and 140 bp in length, as set forth in SEQ ID No. 8 or SEQ ID No. 9.
  • the ITR core expression element is selected in the example containing a CMV promoter, a GFP gene,
  • the GFP gene expression cassette of the SV40 ploy A component facilitates the validation of the technical protocol.
  • the recombinant baculovirus provided by the present invention can be prepared as follows:
  • the codon-optimized Rep gene and Cap gene were obtained by gene artificial synthesis.
  • the ITR core expression elements were obtained by gene synthesis, PCR amplification and restriction enzyme ligation.
  • step A The Rep gene, Cap gene and ITR core expression element obtained in step A were constructed on the pFast.Bac.Dual (pFBD) shuttle vector by molecular cloning method, and the recombinant baculovirus was obtained according to the Bac to Bac system operation method. .
  • pFBD pFast.Bac.Dual
  • the rAAV vector DNA generally replaces the AAV encoding gene with an exogenous gene expression element, retaining only the ITR sequences required for viral replication and packaging.
  • Preparation of rAAV requires trans compensation of the Rep gene, Cap gene and helper viral function factors, as shown in Figure 2A. It is very difficult to integrate AAV's Rep gene, Cap gene and ITR core expression elements into a baculovirus.
  • the combination design and operation of the three clones are more complicated and difficult. Each of them has a key role in the successful preparation of rAAV, and the combination of the three cannot affect their respective functions.
  • the present invention optimizes the Rep gene and the Cap gene sequence on the one hand through a large number of experiments, and explores the ligation sequence at both ends of the ITR core expression element on the other hand, and integrates the Rep gene, the Cap gene and the ITR on the pFBD shuttle vector with extremely limited loading capacity.
  • the core expression component more rarely, maintains the biological functions of each of the three, so that it is convenient to mass produce rAAV with stable quality.
  • the recombinant baculovirus provided by the invention can be applied to prepare recombinant adeno-associated virus for gene therapy, and the specific steps are as follows:
  • the recombinant baculovirus provided by the present invention can optimize three functional element combinations by using a pFast.Bac. Dual (pFBD) shuttle vector as a skeleton carrier (as shown in FIG. 2B) (as shown in FIG. 2C).
  • pFBD Dual shuttle vector
  • the Bac to Bac system is used to facilitate the advantages of recombinant baculovirus.
  • rAAV is mainly present in cell pellets. By performing the separation and purification operation of the prepared rAAV, the rAAV required for further application can be obtained.
  • the Bac-A system disclosed in the present invention provides a replication, packaging element and ITR core expression element required for preparing rAAV by using a recombinant baculovirus, and produces a complete ratio of rAAV particles with high virus quality. Moreover, the rAAV production capacity of single cells has also been greatly improved, reaching the international advanced level.
  • the Bac-A system has high flexibility, versatility, high virus quality and high output, and is suitable for large-scale scale production, which can effectively solve the problem of large-scale preparation of rAAV.
  • AAV2 dienotype-associated virus
  • Example 1 Preparation and amplification of recombinant baculovirus (BEV)
  • the Cap gene, the Rep gene, and the core expression element containing the ITR were placed in a recombinant baculovirus.
  • the Rep gene based on type 2 AAV was codon optimized according to the principle of ribosome leakage scanning, and the Rep gene was placed in PpH. Functional expression of Rep72, Rep52 replication proteins is achieved under the control of the promoter.
  • the Rep gene sequence is a sequence as shown in SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3 (3 types corresponding to RepA, RepB, RepC).
  • the Cap gene based on type 2 AAV was codon optimized according to the principle of ribosome leakage scanning.
  • the Cap gene was placed under the control of P10 promoter to realize three capsid proteins of VP1, VP2 and VP3 to close to the natural proportion (1). :1:10) Functional expression.
  • the Cap gene sequence is a sequence as shown in SEQ ID No. 4, SEQ ID No. 5 or SEQ ID No. 6 (3 corresponding to CapA, CapB, CapC).
  • ITR core expression element in which the ITR selects the ITR nucleic acid sequence of type 2 AAV, ie the sequence shown in SEQ ID No. 7, the core expression element of ITR adopts the green fluorescent protein (GFP) expression cassette, which is controlled by the CMV promoter.
  • GFP green fluorescent protein
  • the expression of GFP facilitates the detection of rAAV activity.
  • the ITR core expression element is ligated to the expression cassette of the Rep gene and the Cap gene via a 5'-end ligation nucleic acid fragment and a 3'-end ligation nucleic acid fragment.
  • the 5'-end or 3'-end ligated nucleic acid fragment is the sequence shown in SEQ ID No. 8 (link A) or SEQ ID No. 9 (link B).
  • the main components of the recombinant baculovirus are selected from three representative combinations;
  • the IFB core expression element (ITR-GFP) was constructed by placing the ITR core expression element (ITR-GFP) between the P10 and PpH promoters of the pFBD vector by ligation of the nucleic acid fragment to construct the pFBD/Cap-(ITR-GFP)-Rep recombinant shuttle plasmid. 2C is shown.
  • the recombinant shuttle plasmid was transformed into DH10Bac bacteria containing the AcMNPV baculovirus genome, following the Bac to Bac system operating instructions.
  • the recombinant baculovirus genome (Bacmid) was obtained by Tn7 transposon mediated recombination.
  • Positive bacteria containing recombinant Bacmid were obtained by blue-white spot screening and PCR identification.
  • Recombinant Bacmid was purified by extraction and transfected into adherently cultured Sf9 cells. Sf9 cells were gradually infected with the recombinant baculovirus produced by transfection and showed significant cytopathic effect (CPE) of BV-infected Sf9 cells. The cell culture medium was centrifuged at 3000 rpm for 5 min, and the supernatant contained the produced weight. Group of baculoviruses.
  • the supernatant was infected with adherently cultured Sf9 cells and cultured for 3 days. It can be seen that the uninfected Sf9 cells in the control group were in a normal state and there was no expression of GFP, as shown in Fig. 3A.
  • Sf9 cells infected with BEV prepared by protocol 1, protocol 2, and protocol 3 all showed obvious CPE phenomenon, and all had obvious GFP expression, as shown in Fig. 3B. This indicates that the recombinant baculovirus was successfully prepared in all three experimental protocols.
  • the BEV prepared by transfecting Sf9 cells was infected with adherent or suspension cultured Sf9 cells.
  • the titer of the virus was determined by a method of real-time quantitative PCR, which is a reference (Proc Natl Acad Sci U S A, 2009. 106 (13): p. 5059-64).
  • Example 2 Infection of Sf9 insect cells with recombinant baculovirus (BEV) to prepare rAAV and verify its activity
  • CPE cytopathic effect
  • active rAAV was prepared.
  • the experimental method of virus infection of HEK293 cells and Sf9 cells confirmed that the Bac-A system prepared rAAV, and the experimental results are shown in Fig. 4A.
  • the specific implementation process and results were analyzed as follows: After the cell pellet was repeatedly freeze-thawed and lysed 3 times, the cell lysis supernatant was collected by centrifugation at 5000 rpm for 5 min.
  • rAAV has no envelope, the activity is not affected after treatment at 60 ° C for 30 minutes; while the recombinant baculovirus (BEV) has an envelope and is inactivated after treatment at 60 ° C for 30 minutes.
  • BEV baculovirus
  • the virus-containing supernatant was infected with HEK293 cells for 2 days,
  • the rAAV control group prepared by the traditional three-plasmid transfection of HEK293 cells showed GFP expression before and after treatment, indicating that rAAV has infectious activity before and after treatment.
  • BEV infected Sf9 cells in the supernatant group untreated with strong GFP expression, only a small amount of GFP expression after treatment, indicating that BEV can infect 293 cells, BEV is not infected after inactivation, but a small amount in the supernatant rAAV is still able to infect.
  • untreated GFP was strongly expressed. After treatment, the GFP intensity was decreased but there was still strong fluorescence, indicating that BEV was not infected after inactivation, but a large amount of rAAV in the supernatant was still able to be infected. .
  • the results showed that the rAAV control group prepared by the conventional three-plasmid transfection of HEK293 cells showed no GFP fluorescence, indicating that rAAV was not infectious to Sf9 cells.
  • the supernatant of the medium after BEV infection of Sf9 cells showed strong GFP expression before inactivation, and no GFP expression after inactivation.
  • untreated GFP was expressed, and no GFP expression was observed after treatment.
  • the supernatant was extracted with chloroform, and the extracted supernatant was further purified by a two-phase precipitation method using a solution containing 13.2% of (NH 4 ) 2 SO 4 and 10% of PEG 8000. Reference method (J Virol Methods, 2007. 139(1): p. 61-70. and J Virol Methods, 2012. 179(1): p.276-80.).
  • the supernatant after the two-phase precipitation was subjected to dialysis desalting treatment with a PBS solution, and concentrated by centrifugation to a final volume of 1 ml using an Amicon ultra-4 (100 KD cutoff) dialysis column, and frozen at -80 ° C after aseptic dispensing.
  • the titer of rAAV was determined by fluorescence quantitative PCR, and the titer unit was expressed by VG/ml (VG, virus genomes).
  • the rAAV yield of the purification process is shown in Table 1.
  • the experimental results show that the yield of rAAV in a single Sf9 cell can reach 1.78 ⁇ 10 5 VG. After purification by this method, the recovery rate is about 32.9%.
  • Example 4 Electron microscopy and integrity determination of rAAV particles
  • a drop of purified rAAV of about 10 ⁇ l was adsorbed with a 200 mesh carbon coated copper mesh for 5 min, washed 4 times with ultrapure water, then a drop of 1% uranyl acetate was added, and the sample was dried in air for 5 min. Finally, the virus particles were observed by a transmission electron microscope.
  • the intact solid rAAV particles are hexagonal uniform particles, and the hollow nucleic acid-free defective rAAV particles are dyed dark in the middle, as shown in FIG.
  • the results of electron micrographs show that the ratio of intact rAAV particles is more than 75%, and the results between different preparation batches can be repeated.
  • Example 5 Detection of rAAV activity at the in vitro cell level and in vivo mouse brain levels
  • HEK293 cells were seeded at 1 x 10 4 cells/well into 96-well plates, and after 6 h, the corresponding concentration gradients of purified rAAV were infected. After 48 hours of infection, the expression of GFP was observed by fluorescence microscopy, as shown in Fig. 6A.
  • the purified rAAV was microinjected into the hippocampus of C57 mice. After three weeks of injection, the mouse brain sections were taken and the brain neurons of rAAV-transduced mice were observed under a fluorescence microscope. Green fluorescence is shown as GFP expressed after rAAV infection, and blue fluorescence shows neuronal nuclei not labeled with DAPI dye, as shown in Figure 6B.

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Abstract

提供一种重组杆状病毒及其应用。所述重组杆状病毒整合了腺相关病毒的Rep基因，Cap基因以及ITR核心表达元件，可应用于制备基因治疗用重组腺相关病毒载体。

Description

一种重组杆状病毒及其应用 【技术领域】
本发明属于基因治疗领域，更具体地，涉及一种重组杆状病毒及其应用。
【背景技术】
重组腺相关病毒(rAAV)具有安全性高、免疫原性低、宿主范围广、能介导外源基因在动物体内长期稳定表达等特点，是基因治疗领域最具应用前景的载体之一。rAAV在神经科学研究以及疾病的基因治疗等领域具有重要作用和巨大需求。有研究数据表明，一次大型灵长类动物试验或临床基因治疗试验就需要约1015VG(VG，virus genomes)的rAAV，是一般体外细胞试验或小鼠试验用量的成百上千倍。利用传统的三质粒共同转染HEK293细胞的方法，生产1014VG的rAAV病毒颗粒，就需要转染多达5000多个175cm2培养瓶的细胞，这种传统方法显然无法满足大规模试验的需求。
目前利用杆状病毒表达系统大规模制备rAAV的方法主要有以下两种：两杆状病毒系统(Two Bac system)和依赖包装细胞系的一杆状病毒系统(One Bac system)。两杆状病毒系统的主要流程是，将AAV的Rep基因和Cap基因整合在一个杆状病毒基因组中，将ITR核心表达元件整合到另外一个杆状病毒基因组中，然后将上述两种重组杆状病毒共同感染宿主细胞，产生出rAAV。依赖包装细胞系的一杆状病毒系统的主要流程是，先建立诱导表达Rep基因和Cap基因的包装细胞系，这种包装细胞系整合了Rep基因和Cap基因表达元件，Rep基因和Cap基因分别置于杆状病毒晚期基因表达强启动子PpH调控下，在PpH启动子的上游加入了hr2增强子序列和AAV的Rep蛋白结合序列。在感染整合了ITR核心表达元 件的重组杆状病毒后，包装细胞系中的Rep基因和Cap基因被诱导表达，进而产生rAAV。
然而，前者由于两种杆状病毒共同感染细胞效率较低，无法充分利用每个细胞的产能，而且感染是个随机的过程，容易产生不含核酸的空壳缺陷rAAV颗粒，制备工艺条件优化起来比较复杂，不同批次制备的rAAV质量不稳定。后者由于依赖诱导表达Rep基因和Cap基因的包装细胞系，构建复杂，优良包装细胞系的筛选难度大，制备不同血清型的rAAV，就需要建立表达相应血清型Cap基因的包装细胞系，灵活性和通用性较差，难以推广。
【发明内容】
针对现有技术的以上缺陷或改进需求，本发明提供了一种重组杆状病毒及其应用，其目的在于通过将腺相关病毒(AAV)的Rep基因、Cap基因和ITR核心表达元件整合在杆状病毒基因组中作为重组腺相关病毒(rAAV)的表达载体，由此解决现有的大规模制备rAAV方法存在的灵活性差、通用性低、构建复杂或者病毒产量低、质量不稳定、生产成本高的技术问题。
为实现上述目的，按照本发明的一个方面，提供了一种重组型杆状病毒，其特征在于，所述重组杆状病毒基因组整合了AAV的Rep基因、Cap基因以及ITR核心表达元件。
优选地，所述重组型杆状病毒，其Rep基因其序列为依据核糖体泄露扫描原理进行密码子优化的序列，优选为如SEQ ID No.1、SEQ ID No.2或SEQ ID No.3所示的序列。
优选地，所述重组型杆状病毒，其Cap基因其序列为依据核糖体泄露扫描原理进行密码子优化的序列，优选为如SEQ ID No.4、SEQ ID No.5或SEQ ID No.6所示的序列。
优选地，所述重组型杆状病毒，其ITR核心表达元件通过5’端连接核 酸片段和3’端连接核酸片段与所述Rep基因和Cap基因的表达框相连，其包括位于两端的AAV基因组中的一段末端反向重复序列(ITR)以及位于中间的目标基因序列；所述ITR序列优选为如SEQ ID No.7所示的序列。
优选地，所述重组型杆状病毒，其所述5’端或3’端连接核酸片段为长度在80bp-140bp之间的连接核酸序列，优选序列如SEQ ID No.8或SEQ ID No.9所示。
优选地，所述重组型杆状病毒，其所述腺相关病毒为二型腺相关病毒。
按照本发明的另一方面，提供了一种所述重组型杆状病毒的应用，其应用于制备基因治疗用重组腺相关病毒载体。
优选地，所述应用，其包括以下步骤：
(1)将目标基因作为功能基因构建在如权利要求1至6所述的重组型杆状病毒的基因组上，制备重组型杆状病毒；
(2)将步骤(1)中制备得到的重组型杆状病毒，感染宿主细胞，产生大量重组腺相关病毒；
(3)将步骤(2)中制备的重组腺相关病毒进行纯化。
优选地，所述应用，其步骤(1)利用pFast.Bac.Dual穿梭载体，基于Bac to Bac系统。
总体而言，通过本发明所构思的以上技术方案与现有技术相比，能够取得下列有益效果：
本发明公开的重组杆状病毒，通过基因设计和优化，利用一种杆状病毒提供了制备rAAV所需的复制包装元件，即AAV的Rep基因、Cap基因以及ITR核心表达元件，并保持了生物活性，打破了ITR与其他两个基因兼容的瓶颈。通过简单的细胞感染即能产出rAAV，其中的完整的rAAV颗粒比例较高，病毒质量高。而且，单个细胞的rAAV生产能力也大大提高。本发明Bac-A系统灵活性高、通用性强、病毒质量高、产量高，适于大规模放大生产，可有效解决rAAV大规模制备的难题。
【附图说明】
图1是重组杆状病毒结构示意图，即整合了AAV的Rep基因、Cap基因和ITR核心表达元件的AcMNPV Bacmid的示意图。其中杆状病毒AcMNPV基因组为双链环状DNA，的全长133,894bp，序列和图谱参考文献(Virology,1994.202(2):p.586-605.)，AcMNPV Bacmid(bMON14272)的结构参考文献(J Virol,1993.67(8):p.4566-79.)，被整合的AAV的Rep基因、Cap基因和ITR核心表达元件插入到bacmid(bMON14272)的Tn7位点。
图2是实施例1、2中重组杆状病毒系统及其制备rAAV的原理示意图。其中图2A是rAAV的包装原理图；图2B是杆状病毒表达系统穿梭质粒pFast.Bac.Dual(pFBD)的示意图；图2C是整合了制备rAAV所需组件的重组穿梭质粒结构示意图；图2D是重组杆状病毒感染Sf9细胞制备rAAV的过程示意图；
图3是实施例1中3种组合方案制备的重组杆状病毒感染Sf9细胞的活性验证图。其中图3A是Sf9细胞对照组的荧光显微成像图，图3B是方案1至3制备的重组杆状病毒感染Sf9细胞3天后的实验组荧光显微成像图；
图4是实施例1、2中3种组合方案制备的rAAV感染细胞的活性验证图。其中图4A是方案1制备的rAAV在HEK293细胞和Sf9细胞水平的活性验证结果，图4B是方案2制备的rAAV感染HEK293细胞的活性验证结果；图4C是方案3制备的rAAV感染HEK293细胞的活性验证结果；
图5是实例4中纯化的rAAV颗粒透射电镜照片。其中图5A是rAAV颗粒负染后的透射电镜图，图5B是图5A的局部放大图(2.5倍)；
图6是实施例5中纯化的rAAV在HEK293细胞水平和小鼠大脑水平的活性验证图。其中图6A是纯化后的rAAV感染HEK293细胞的荧光显微成像图，图6B是纯化后的rAAV感染小鼠大脑海马皮层神经元的荧光显微成像图，图6C是图6B的局部放大图(2倍)。
【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仅用以解释本发明，并不用于限定本发明。此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
本发明提供的重组型杆状病毒，其基因组示意图如图1所示，优选基于AcMNPV clone C6(基因组序列如：Genbank accession No.NC_001623.1)，整合了AAV的Rep基因、Cap基因和ITR核心表达元件，所述Rep基因和Cap基因依据核糖体泄露扫描原理进行了密码子优化。所述ITR核心表达元件，两端包括AAV基因组中的一段末端反向重复序列(ITR)，中间为携带的目标基因(GOI)片段，通过5’端连接核酸片段和3’端连接核酸片段与所述Rep基因和Cap基因的表达框相连。其中，Rep基因和Cap基因，皆可处于ITR核心表达元件的上游或下游。
所述Rep基因依据核糖体泄露扫描原理进行了密码子优化，通过PpH启动子转录出一条mRNA，实现Rep72、Rep52复制蛋白的功能性表达。其序列优选为如SEQ ID No.1至3之一所示。
所述Cap基因密码子优化同样利用核糖体泄露扫描原理，通过P10启动子转录出一条mRNA，实现VP1、VP2、VP3三种衣壳蛋白以接近天然比例(1:1:10)的功能性表达。其序列优选为如SEQ ID No.4至6之一所示。
ITR核心表达元件，通过两端的连接核酸片段与Rep基因、Cap基因的表达框连接。ITR为AAV基因组中的一段末端反向重复序列，优选2型AAV的ITR序列，如SEQ ID No.7所示。所述5’端或3’端连接核酸序列优选长度在80bp-140bp之间的连接核酸序列，如SEQ ID No.8或SEQ ID No.9所示。ITR核心表达元件在实例中选用含CMV启动子、GFP基因、 SV40 ploy A组件的GFP基因表达框，便于技术方案的验证。
整合到杆状病毒基因组上的Rep基因、Cap基因和ITR核心表达元件的优选组合方案，如图2C所示。
本发明提供的重组杆状病毒，可按照如下方法制备：
A、采用基因人工合成的方法获得密码子优化的Rep基因、Cap基因；采用基因人工合成、PCR扩增和酶切连接的方法获得ITR核心表达元件。
B、通过分子克隆的方法将步骤A中获得的Rep基因、Cap基因以及ITR核心表达元件构建到pFast.Bac.Dual(pFBD)穿梭载体上，根据Bac to Bac系统操作方法，获得重组杆状病毒。
rAAV载体DNA一般是用外源目的基因表达元件替换AAV的编码基因，仅保留了病毒复制和包装所需的ITR序列。制备rAAV需要反式补偿Rep基因、Cap基因和辅助病毒功能因子，如图2A所示。要将AAV的Rep基因、Cap基因和ITR核心表达元件整合到一个杆状病毒上存在很大的困难：首先，三者组合在一起的克隆构建设计和操作都比较复杂，难度较大；再者，三者各自对rAAV的成功制备都具有关键作用，三者的组合不能影响各自的功能。因此，目前要么采取将三者分置于两种杆状病毒中进行共感染，要么将三者分别至于宿主细胞和杆状病毒中。本发明通过大量实验，一方面优化Rep基因和Cap基因序列，另一方面摸索出ITR核心表达元件两端的连接序列，在装载能力极其有限的pFBD穿梭载体上，整合了Rep基因、Cap基因以及ITR核心表达元件，更难得的是保持了三者各自的生物学功能，因此能方便的大批量的生产出质量稳定的rAAV。在此基础上只需要依据不同的使用需求，替换ITR核心表达元件所携带的外源目标基因(GOI)片段，或者替换所需不同血清型AAV的Cap基因或Rep基因，就可以灵活制备出所需的rAAV，实现相应的基因给药。
本发明提供的重组型杆状病毒，可应用于制备基因治疗用重组腺相关病毒，具体步骤如下：
(1)将目标基因(GOI)作为功能基因构建在所述重组型杆状病毒的基因组上，制备重组型杆状病毒；
优选地，本发明提供的重组型杆状病毒可通过以pFast.Bac.Dual(pFBD)穿梭载体为骨架载体(如图2B所示)，优化了三个功能元件组合(如图2C所示)，利用了Bac to Bac系统方便获得重组杆状病毒的优势。
(2)将步骤(1)中制备得到的重组型杆状病毒，感染宿主细胞，产生大量rAAV，具体可按照如下步骤操作：利用上述重组杆状病毒(BEV)感染Sf9昆虫细胞(如图2D所示)。以感染悬浮培养的Sf9细胞为例，摇瓶培养Sf9细胞至细胞密度达到3×106cells/ml，以感染复数(MOI＝5)感染重组杆状病毒(BEV)，27℃、120rpm/min摇床培养。感染后3天，将细胞悬液3000rpm离心5min，收集培养液上清和细胞沉淀。
(3)将步骤(2)中制备的重组腺相关病毒进行纯化。
rAAV主要存在于细胞沉淀中。对制备出的rAAV进行分离纯化操作，就能够得到进一步应用所需的rAAV。
本发明公开的Bac-A系统，利用一种重组杆状病毒提供了制备rAAV所需的复制、包装元件以及ITR核心表达元件，产出完整的rAAV粒子比例较高，病毒质量高。而且，单个细胞的rAAV生产能力也大大提高，达到国际先进水平。Bac-A系统灵活性高、通用性强、病毒质量高、产量高，适于大规模放大生产，可有效解决rAAV大规模制备的难题。
本发明适用于各种类型的腺相关病毒，下面仅以二型腺相关病毒(AAV2)为例，具体说明如下：
实施例1：重组杆状病毒(BEV)的制备与扩增
为了将rAAV制备所需的三种主要组件：Cap基因、Rep基因以及含有ITR的核心表达元件置于一个重组杆状病毒中。我们利用了杆状病毒表达系统Bac to Bac中的pFast.Bac.Dual(pFBD)穿梭载体。实例中基于2型AAV的Rep基因依据核糖体泄露扫描原理进行了密码子优化，Rep基因置于PpH 启动子的调控之下，实现Rep72、Rep52复制蛋白的功能性表达。Rep基因序列为如SEQ ID No.1、SEQ ID No.2或SEQ ID No.3(3种对应为RepA，RepB，RepC)所示的序列。实例中基于2型AAV的Cap基因依据核糖体泄露扫描原理进行了密码子优化，Cap基因置于P10启动子的调控之下，实现VP1、VP2、VP3三种衣壳蛋白以接近天然比例(1:1:10)的功能性表达。Cap基因序列为如SEQ ID No.4、SEQ ID No.5或SEQ ID No.6(3种对应为CapA，CapB，CapC)所示的序列。ITR核心表达元件，实例中ITR选用2型AAV的ITR核酸序列，即如SEQ ID No.7所示的序列，ITR的核心表达元件采用了绿色荧光蛋白(GFP)表达框，由CMV启动子控制GFP的表达，便于检测rAAV的活性。ITR核心表达元件通过5’端连接核酸片段和3’端连接核酸片段与Rep基因和Cap基因的表达框相连。5’端或3’端连接核酸片段为如SEQ ID No.8(linkA)或SEQ ID No.9(linkB)所示的序列。
在本实例中，重组杆状病毒中主要组件选取3种代表性的组合方案；
1.CapA-LinkA-(ITR-GFP)-linkA-RepA
2.CapB-LinkB-(ITR-GFP)-linkB-RepB
3.CapC-LinkA-(ITR-GFP)-linkB-RepC
利用常规分子克隆技术将ITR核心表达元件(ITR-GFP)通过连接核酸片段置于pFBD载体的P10和PpH启动子之间，构建pFBD/Cap-(ITR-GFP)-Rep重组穿梭质粒，如图2C所示。参照Bac to Bac系统操作说明，将该重组穿梭质粒转化含有AcMNPV杆状病毒基因组的DH10Bac菌。通过Tn7转座子元件介导的重组得到重组杆状病毒基因组(Bacmid)。通过蓝白斑筛选和PCR鉴定获得含有重组Bacmid的阳性菌。抽提纯化重组Bacmid，将其转染贴壁培养的Sf9细胞。Sf9细胞逐渐被转染后产生的重组杆状病毒完全感染并出现明显的BEV感染Sf9细胞的细胞病变(CPE)现象。将细胞培养液3000rpm离心5min，上清中就含有产生的重 组杆状病毒。
将上清液感染贴壁培养的Sf9细胞后继续培养3天，可以看到对照组未感染病毒的Sf9细胞状态正常，没有GFP的表达，如图3A。而在实验组感染了采用方案1、方案2、方案3所制备的BEV的Sf9细胞都出现了明显的CPE现象，都有明显的GFP表达，如图3B。这表明3种实验方案都成功制备出了重组杆状病毒。将转染Sf9细胞后制备的BEV感染贴壁或悬浮培养的Sf9细胞，感染3天后细胞出现明显CPE，将细胞培养液3000rpm离心5min，收上清即可获得大量BEV。病毒的滴度用荧光定量PCR的方法进行测定，参考文献(Proc Natl Acad Sci U S A,2009.106(13):p.5059-64)中的方法。
实施例2：利用重组杆状病毒(BEV)感染Sf9昆虫细胞制备rAAV并验证其活性
将实施例1中制备得到的BEV以感染复数(MOI＝5)感染悬浮培养的Sf9细胞，感染72小时后，将细胞培养液3000rpm离心5min，分别收集培养上清和细胞沉淀。BEV产生后，主要通过分泌释放到培养基上清中，在Sf9细胞中也有部分未释放的BEV。rAAV产生后，主要存在于Sf9细胞核中，由于Sf9细胞感染后发生细胞病变(CPE)，部分细胞会裂解，rAAV也会有部分释放到上清中。而因此上清和细胞沉淀中会同时存在BEV和rAAV。
为了验证用重组杆状病毒感染Sf9细胞制备出了具有活性的rAAV。在此以实施例1中3种组合方案中的方案1为例，通过病毒感染HEK293细胞和Sf9细胞的实验方法证实Bac-A系统制备出了rAAV，实验结果如图4A。具体实施过程和结果分析如下：将细胞沉淀反复冻融3次裂解后，5000rpm离心5min收集细胞裂解上清液。因为rAAV无包膜，经60℃、30分钟处理，活性不受影响；而重组杆状病毒(BEV)有包膜，经60℃、30分钟处理，失去活性。含有病毒的上清液感染HEK293细胞2天后，利 用传统的三质粒转染HEK293细胞方法制备的rAAV对照组，在处理前后都有GFP表达，表明rAAV在处理前后都有感染活性。BEV感染Sf9细胞后的培养基上清组，未处理的有较强GFP表达，处理后仅有少量GFP表达，表明BEV可以感染293细胞，灭活处理后BEV不感染，但上清中少量的rAAV仍能够感染。细胞沉淀裂解液上清实验组，未处理的有较强GFP表达，处理后GFP强度有降低但仍有较强荧光，表明灭活处理后BEV不感染，但上清中大量的rAAV仍能够感染。用与感染HEK293细胞组相同的样品感染Sf9细胞2天后，结果显示：利用传统的三质粒转染HEK293细胞方法制备的rAAV对照组，没有GFP荧光，表明rAAV对Sf9细胞没有感染性。BEV感染Sf9细胞后的培养基上清组，灭活后前有较强GFP表达，灭活后的没有GFP表达。细胞沉淀裂解液上清实验组，未处理的有较强GFP表达，处理后的没有GFP表达。
按照上述方法，我们同样检测了采用实施例1中3种组合方案中的方案2和方案3所制备的rAAV感染HEK293细胞的情况。实验结果表明，用以上来源的rAAV感染HEK293细胞后都有明显的GFP表达，如图4B、4C所示。以上结果证明，该Bac-A系统制备出了具有活性的rAAV。
实施例3：rAAV的纯化与滴度测定
因为采用实施例1中3种组合方案制备出的rAAV实际上没有差别，所以对Bac-A系统制备出的rAAV进行的后续纯化，活性检测等实例，将选取方案1所制备的rAAV为范例。
将重组BEV感染后收集得到约为1×108cells的Sf9细胞沉淀，加入10ml裂解缓冲液(50mM Tris-Cl，150mM NaCl，2mM MgCl2，pH 8.0)，后反复冻融3次，5000rpm离心5min后收集上清，在上清中加入核酸酶Benzonase至终浓度50U/ml，37℃水浴处理60min。处理后5000rpm离心10min收上清。上清用氯仿抽提，抽提后的上清再用含13.2％的(NH4)2SO4和10％的PEG8000的溶液进行两相沉淀的方法进一步纯化，方法参考文献(J Virol  Methods,2007.139(1):p.61-70.和J Virol Methods,2012.179(1):p.276-80.)。两相沉淀后的上清，用PBS溶液进行透析脱盐处理，用Amicon ultra-4(100KD cutoff)透析柱离心浓缩至终体积1ml，无菌分装后-80℃冻存。rAAV的滴度测定采用荧光定量PCR的方法，滴度单位用VG/ml(VG，virus genomes)表示。
纯化过程的rAAV得率见表1。实验结果表明，单个Sf9细胞rAAV的产量可达1.78×105VG，经过本方法的纯化后，回收率达到32.9％左右。
表1 rAAV纯化过程的得率分析
Figure PCTCN2015095751-appb-000001
实施例4：rAAV颗粒的电镜检测与完整性测定
将一滴纯化的rAAV约10μl用200mesh的碳包被铜网吸附5min，用超纯水洗4次，然后加一滴1％乙酸铀酰，样品在空气中干燥5min。最后，用透射电子显微镜观察病毒颗粒。
完整的实心rAAV颗粒，呈六角形均匀颗粒，空心无核酸的缺陷rAAV颗粒中间被染成深色，如图5所示。电镜照片统计结果表明，完整rAAV颗粒的比率达75％以上，且不同制备批次间的结果可重复。
实施例5：rAAV在体外细胞水平与体内小鼠大脑水平的活性检测
将HEK293细胞以1×104cells/well接种到96孔板中，6h后感染相应浓度梯度含量的纯化的rAAV。感染48h后，用荧光显微镜观察GFP的表达情况，如图6A。将纯化的rAAV，显微注射C57小鼠的脑部海马区，注射三周后，取鼠脑切片处理，在荧光显微镜下观察rAAV转导鼠脑神经元的情况。绿色荧光显示为rAAV感染后表达的GFP，蓝色荧光显示未为DAPI 染料标记的神经元细胞核，如图6B。
以上结果表明，本发明的Bac-A系统制备得到的rAAV具有较好的细胞水平和动物水平活性。
本领域的技术人员容易理解，以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种重组型杆状病毒，其特征在于，所述病毒基因组整合了腺相关病毒的Rep基因、Cap基因以及ITR核心表达元件。
  2. 如权利要求1所述的重组型杆状病毒，其特征在于，所述Rep基因其序列为依据核糖体泄露扫描原理进行密码子优化的序列，优选为如SEQ ID No.1、SEQ ID No.2或SEQ ID No.3所示的序列。
  3. 如权利要求1所述的重组型杆状病毒，其特征在于，所述Cap基因其序列为依据核糖体泄露扫描原理进行密码子优化的序列，优选为如SEQ ID No.4、SEQ ID No.5或SEQ ID No.6所示的序列。
  4. 如权利要求1所述的重组型杆状病毒，其特征在于，所述ITR核心表达元件通过5’端连接核酸片段和3’端连接核酸片段与所述Rep基因和Cap基因的表达框相连，其包括位于两端的腺相关病毒基因组中的一段末端反向重复序列(ITR)以及位于中间的目标基因序列；所述反向重复序列优选为如SEQ ID No.7所示的序列。
  5. 如权利要求4所述的重组型杆状病毒，其特征在于，所述5’端或3’端连接核酸片段为长度在80bp-140bp之间的连接核酸序列，优选序列如SEQ ID No.8或SEQ ID No.9所示。
  6. 如权利要求1所述的重组型杆状病毒，其特征在于，所述腺相关病毒为二型腺相关病毒。
  7. 如权利要求1至6任意一项所述的重组型杆状病毒的应用，其应用于制备基因治疗用重组腺相关病毒载体。
  8. 如权利要求7所述的应用，其特征在于，包括以下步骤：
    (1)将目标基因作为功能基因构建在如权利要求1至6所述的重组型杆状病毒的基因组上，制备重组型杆状病毒；
    (2)将步骤(1)中制备得到的重组型杆状病毒，感染宿主细胞，产 生大量重组腺相关病毒；
    (3)将步骤(2)中制备的重组型腺相关病毒进行纯化。
  9. 如权利要求8所述的应用，其特征在于，所述步骤(1)利用pFast.Bac.Dual穿梭载体，基于Bac to Bac系统。
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