WO2022160512A1 - 一种基于诱导型昆虫细胞生产aav基因药物的方法 - Google Patents

一种基于诱导型昆虫细胞生产aav基因药物的方法 Download PDF

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WO2022160512A1
WO2022160512A1 PCT/CN2021/093906 CN2021093906W WO2022160512A1 WO 2022160512 A1 WO2022160512 A1 WO 2022160512A1 CN 2021093906 W CN2021093906 W CN 2021093906W WO 2022160512 A1 WO2022160512 A1 WO 2022160512A1
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plasmid
gene
cells
aav
rep
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董文吉
刘子瑾
赵忠亮
曹帆
程谟斌
张艳君
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中吉智药(南京)生物技术有限公司
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Definitions

  • the invention relates to the field of biotechnology, in particular to a method for producing AAV gene medicine based on inducible insect cells.
  • the basic principle of gene therapy is to use recombinant DNA technology to introduce normal genes and sequences required for their expression into diseased cells or somatic cells, to correct or repair DNA molecules, so as to achieve therapeutic purposes.
  • the advantage of gene therapy is to solve the problem at the root.
  • the gene therapy market size was US$3.61 billion in 2019 and is expected to reach US$35.67 billion by 2027, with a compound annual growth rate (CAGR) of 33.6% during the forecast period.
  • the viral vectors of gene medicine are mainly adeno-associated virus (AAV) and lentivirus. These two types of vectors are used for different gene therapy and indications according to their respective characteristics.
  • AAV adeno-associated virus
  • lentivirus lentivirus
  • Luxturna is a gene therapy developed by Spark (a subsidiary of Roche) and was approved by the US FDA in December 2017.
  • Luxturna uses AAV2 to carry the RPE65 gene and directly injects the virus into the eyeball by subretinal injection.
  • LCA2 Leber congenital amaurosis type 2
  • Figure 1 The packaging components Rep, Cap and the target gene sequence of adeno-associated virus were cloned into three baculovirus vectors, and three baculovirus vectors were used. A baculovirus infected insect sf9 cells and packaged AAV.
  • Baculovirus is a replicating virus. As a raw material for the production of AAV, a large number of replication and amplification are required for 3-4 generations, and the foreign gene will be lost. Repeated testing is required during production. Once lost, it needs to be re-amplified.
  • the present invention provides a method for producing AAV gene medicines based on inducible insect cells, which does not require the use of baculoviruses carrying foreign genes, and solves the problem of baculoviruses carrying foreign genes.
  • the problem of easy loss of exogenous genes in the process of large-scale replication and amplification is conducive to obtaining stable AAV products, further simplifying the production steps of AAV, improving production efficiency, realizing large-scale and efficient production of AAV and reducing production costs.
  • the main technical scheme adopted in the present invention includes:
  • the present invention provides a method for producing AAV gene medicine based on inducible insect cells, the method comprising:
  • Rep gene plasmid (2) respectively construct Rep gene plasmid, Cap gene plasmid and target gene plasmid for packaging adeno-associated virus; wherein Rep gene plasmid and Cap gene plasmid contain late promoter;
  • the Rep gene plasmid, the Cap gene plasmid, and the target gene plasmid respectively contain an early promoter and a screening tag.
  • the early promoter drives the expression of the screening tag to ensure that the stable transgenic cell line containing the Rep gene plasmid, the Cap gene plasmid and the target gene plasmid at the same time is obtained by final screening.
  • step (2) the three plasmids are respectively transfected into the Sf9 insect cells one by one, and after each plasmid is transfected, the plasmid corresponding to the screening tag in the plasmid is used. Antibiotics are screened to select surviving cells for transfection of the next plasmid, and finally a Sf9 stably transfected cell line that can produce AAV is obtained.
  • the Rep gene plasmid is pBSK-HR5-IE-G2P-IEter-HR5-p10-Rep intron-sv40, which includes the pBSK commercial vector sequence, HR5 enhancer, IE early promoter, G2P (GFP-2A-puro, GFP and puromycin double marker) selection tag, IEter transcription termination sequence, p10 late promoter, and Rep intron expression cassette gene sequence for expressing Rep protein.
  • the Cap gene plasmid is pBSK-HR5-IE-BSD-IEter-HR5-pH-Cap intron-sv40, which comprises pBSK commercialized vector sequence, HR5 enhancer, IE early promoter, BSD selection tag, IEter transcription termination sequence, pH late promoter, and Cap intron expression cassette gene sequence for expressing Cap protein.
  • the target gene plasmid is pBSK-HR5-IE-Neo-IEter-AAV-Target Gene, which includes pBSK commercial vector sequence, HR5 enhancer, IE early promoter , Neo screening tag, IEter transcription termination sequence, and adeno-associated virus genome sequence expressing Target Gene.
  • the target gene Target Gene is a codon-optimized SMN1 gene or a gene sequence encoding a soluble extracellular region ACE2 protein; wherein, the codon-optimized SMN1 gene sequence is as follows Shown in SEQ ID No: 12, the gene sequence encoding the soluble extracellular region ACE2 protein is shown in SEQ ID No: 13 or SEQ ID No: 14.
  • the early promoter IE in the plasmid can drive the expression of Puro screening tag, BSD screening tag, and Neo screening tag (these screening tags are actually antibiotic resistance genes), so that cells can acquire resistance, and can be used in the medium with corresponding antibiotics.
  • the stably transfected cell line was successfully transformed into the Rep gene plasmid, the Cap gene plasmid and the target gene plasmid in the genome of Sf9 insect cells.
  • the corresponding antibiotics are Puromycin, Blasticidin, G418.
  • step (3) in step (3), in step (3), the baculovirus that does not carry foreign genes is an empty baculovirus; the baculovirus is infected with an empty baculovirus; Stable transfection of the cell line enables the cells to enter a late virus-like effect, activates the late promoter p10 and polyhedrin, drives the expression of Rep gene and Cap gene, and packages and produces AAV in cells.
  • step (4) wherein the Sf9 insect cells induced to express the Rep gene and the Cap gene are subjected to repeated freezing and thawing, lysing the cells to release AAV virus particles, and finally subjected to affinity chromatography. Purification to obtain AAV virus product.
  • the present invention provides a method for producing a stable transfected cell line carrying a target gene adeno-associated virus, comprising the following steps:
  • Step S1 respectively constructing Rep gene plasmid, Cap gene plasmid and target gene plasmid for packaging adeno-associated virus;
  • Rep gene plasmid, Cap gene plasmid and target gene plasmid contain early promoter and screening tag respectively; Rep gene plasmid and Cap gene plasmid also contain late promoter;
  • Step S2 The Rep gene plasmid, the Cap gene plasmid and the target gene plasmid are respectively transfected into the Sf9 cells one by one. After each plasmid is transfected, the antibiotics corresponding to the screening tags in the plasmid are used for screening, and the selected cells are selected. The surviving cells were selected and used for the transfection of the next plasmid, and finally a surviving cell containing three plasmids of Rep gene plasmid, Cap gene plasmid and target gene plasmid was obtained, and the surviving cells were stable cells for the production of AAV. Transduced cell lines.
  • the Rep gene plasmid contains the IE early promoter and the Puro screening tag; the Cap gene plasmid contains the IE early promoter and the BSD screening tag; the target gene plasmid contains the IE early promoter and Neo respectively. Filter tags.
  • step S2 after transfecting Sf9 cells with the Rep gene plasmid, the antibiotic Puromycin is used for screening to select the surviving Sf9 cells; after transfecting the Sf9 cells with the Cap gene plasmid, the antibiotic Blasticidin is used for screening. Perform screening to select surviving Sf9 cells; after transfecting the Sf9 cells with the target gene plasmid, screening with antibiotic G418 to select surviving Sf9 cells, which are stable transfected cell lines for producing AAV.
  • FIG. 1 is a schematic diagram of a production AAV in the prior art.
  • FIG. 2 is a schematic diagram of another production AAV in the prior art.
  • Figure 3 is a schematic diagram of the present invention for producing AAV.
  • Figure 4 shows the expression of Rep and Cap proteins in the stably transfected cell line detected by Western Blot before and after inducing the Sf9 stably transfected cell line with empty Baculovirus infection.
  • FIG. 3 it is a schematic diagram of the method for producing the AAV (AAV gene drug) carrying the target gene of the present invention, namely constructing the Rep gene plasmid, the Cap gene plasmid and the target gene plasmid respectively, and then the three plasmids are respectively passed through the transfection Enter Sf9 cells by way of antibiotics, and screen out the stable transfected cells that were successfully transfected into the genome of insect sf9 cells by combining the three plasmids with antibiotics.
  • AAV AAV gene drug
  • the baculovirus that does not carry the foreign gene is de-infected, so that the sf9 cells enter a virus-like state and the late promoter is activated, and the Rep, Cap and target genes are expressed, and AAV can be produced in large quantities.
  • This method can avoid the problem of easy loss of exogenous genes in the process of large-scale replication and amplification using baculoviruses carrying exogenous genes.
  • the AAV packaging plasmids to be constructed include Rep gene plasmid, Cap gene plasmid and GFP gene plasmid, which are recorded as:
  • Rep gene plasmid pBSK-HR5-IE-G2P-IEter-HR5-p10-Rep intron-sv40;
  • Cap gene plasmid pBSK-HR5-IE-BSD-IEter-HR5-pH-Cap intron-sv40;
  • GFP gene plasmid pBSK-HR5-IE-Neo-IEter-AAV-GFP.
  • Rep gene plasmid contains pBSK commercial vector sequence, HR5 enhancer, early promoter IE, screening tag G2P (GFP-2A-puro, namely GFP and puromycin double tag), IE ter is transcription termination sequence, p10 late promoter, and Rep intron expression cassette gene sequence for expressing Rep protein.
  • the Cap gene plasmid contains pBSK commercial vector sequence, HR5 enhancer, early promoter IE, screening tag BSD, IE ter is transcription termination sequence, pH late promoter, and Cap intron expression cassette gene sequence for expressing Cap protein.
  • the GFP gene plasmid contains the pBSK commercial vector sequence, HR5 enhancer, early promoter IE, screening tag Neo, IE ter is the transcription termination sequence, and the genome sequence of adeno-associated virus expressing GFP.
  • pBSK is a commercial vector (Stratagene 2 12205)
  • HR5 sequence is SEQ ID No: 1
  • IE sequence is SEQ ID No: 2
  • G2P sequence is SEQ ID No: 3
  • IEter sequence is SEQ ID No: 4
  • p10 The sequence is SEQ ID No:5, the Rep intron sequence is SEQ ID No:6, the BSD sequence is SEQ ID No:7, the pH sequence is SEQ ID No:8, the Cap intron sequence is SEQ ID No:9, and the Neo sequence is SEQ ID No:9 ID No: 10,
  • AAV-GFP sequence is SEQ ID No: 11.
  • HR5 is an enhancer
  • IE is an early promoter, which is used to drive the expression of screening tags
  • pH and p10 are late promoters, which are used to drive the expression of Rep and Cap proteins.
  • the Rep gene plasmid is recorded as pBSK-HR5-IE-G2P-IEter-HR5-p10-Rep intron-sv40, and its construction steps are as follows:
  • the pBSK plasmid was double digested with restriction enzymes KpnI and XhoI at 37°C for 1 h, and the pBSK vector fragment was recovered by gel cutting after agarose electrophoresis;
  • the HR5-IE-G2P-IE ter gene fragment was subjected to PCR amplification, and the 5' and 3' ends were respectively added with protective bases and KpnI/XhoI restriction sites, and the amplified PCR fragments were amplified by KpnI and XhoI. Double-enzyme digestion was performed at 37°C for 1 h, and the HR5-IE-G2P-IE ter gene fragment was recovered by cutting the gel after agarose electrophoresis.
  • the pBSK-HR5-IE-G2P-IE ter plasmid was double-digested with restriction endonuclease XbaI at 37°C for 1 h, and the pBSK-HR5-IE-G2P-IE ter vector fragment was recovered by cutting the gel after agarose electrophoresis. , the recovered pBSK-HR5-IE-G2P-IE ter vector fragment was reacted with alkaline phosphatase (CIAP) at 37 °C for 30 min, and then heat inactivated at 65 °C for 15 min to obtain pBSK-HR5-IE-G2P- IE ter CIAP vector fragment.
  • CIAP alkaline phosphatase
  • PCR amplification was performed on the HR5-p10-Rep intron-sv40 gene fragment, the 5' end and the 3' end were respectively added with protective bases and NheI restriction sites, and the amplified PCR fragment was purified by NheI at 37 Double-enzyme digestion was performed at °C for 1 h, and the HR5-p10-Rep intron-sv40 gene fragment was recovered by cutting the gel after agarose electrophoresis.
  • Transform the ligation product into E. coli Take the ligation product to transform competent DH5a, mix gently, ice bath for 30 minutes; heat shock at 42°C for 80s, immediately ice bath for 4 minutes, add antibiotic-free LB medium and shake at 37°C for 60 minutes , using a sterile glass spreader to evenly spread the bacterial solution onto an LB agar plate containing ampicillin, and invert at 37°C for 14h.
  • Cap gene plasmid is recorded as pBSK-HR5-IE-BSD-IEter-HR5-pH-Cap intron-sv40, and its construction steps are as follows:
  • the pBSK plasmid was double digested with restriction enzymes KpnI and XhoI at 37°C for 1 h, and the pBSK vector fragment was recovered by gel cutting after agarose electrophoresis;
  • the HR5-IE-BSD-IE ter gene fragment was subjected to PCR amplification, and the 5' end and the 3' end were respectively added with protective bases and KpnI/XhoI restriction sites, and the amplified PCR fragments were obtained using KpnI and XhoI. Double-enzyme digestion was performed at 37°C for 1 h, and the HR5-IE-BSD-IE ter gene fragment was recovered by cutting the gel after agarose electrophoresis.
  • the pBSK-HR5-IE-BSD-IE ter plasmid was double-digested with restriction enzymes XbaI and NotI at 37°C for 1 h, and the pBSK-HR5-IE-BSD-IE ter was recovered by cutting the gel after agarose electrophoresis. vector fragment.
  • Transform the ligation product into E. coli Take the ligation product to transform competent DH5a, mix gently, ice bath for 30 minutes; heat shock at 42°C for 80s, immediately ice bath for 4 minutes, add antibiotic-free LB medium and shake at 37°C for 60 minutes , using a sterile glass spreader to evenly spread the bacterial solution onto an LB agar plate containing ampicillin, and invert at 37°C for 14h.
  • the GFP gene plasmid is recorded as pBSK-HR5-IE-Neo-IEter-AAV-GFP, and its construction steps are as follows:
  • the pBSK plasmid was double digested with restriction enzymes KpnI and XhoI at 37°C for 1 h, and the pBSK vector fragment was recovered by gel cutting after agarose electrophoresis;
  • the HR5-IE-Neo-IE ter gene fragment was subjected to PCR amplification, and the 5' and 3' ends were respectively added with protective bases and KpnI/XhoI restriction sites, and the amplified PCR fragments were amplified by KpnI and XhoI. Double-enzyme digestion was performed at 37°C for 1 h, and the HR5-IE-Neo-IE ter gene fragment was recovered by cutting the gel after agarose electrophoresis.
  • the pBSK-HR5-IE-Neo-IE ter plasmid was double-digested with restriction enzymes XhoI and XbaI at 37°C for 1 h. After agarose electrophoresis, the pBSK-HR5-IE-Neo-IE ter was recovered by cutting the gel. vector fragment.
  • Transform the ligation product into E. coli Take the ligation product to transform competent DH5a, mix gently, ice bath for 30 minutes; heat shock at 42°C for 80s, immediately ice bath for 5 minutes, add antibiotic-free LB medium and shake at 37°C for 60 minutes , using a sterile glass spreader to evenly spread the bacterial solution onto an LB agar plate containing ampicillin, and invert at 37°C for 16h.
  • the three plasmids constructed above were transfected into Sf9 cells, and the cell lines that successfully integrated the above three plasmids into the genome of Sf9 insect cells were screened by antibiotics and screening tags.
  • the specific steps are as follows:
  • Insect cells Sf9 were spread on a 35mm plate at 2 ⁇ 10 ⁇ 6/well, and cultured at 27°C for 1 h to make the cells adherent.
  • Sf9 cells were changed to medium containing 8 ⁇ g/ml Puromycin and continued to culture at 27°C, and fresh medium of 8 ⁇ g/ml Puromycin was changed every three days until the cells no longer died and survived The cells were all resistant to Puromycin, so far the Sf9 cell line successfully transferred into the Rep gene plasmid was obtained.
  • step (4) spread the Sf9 cell line successfully transferred into the Rep gene plasmid obtained in step (4) on a 35mm plate at 2 ⁇ 10 ⁇ 6/well, and culture at 27°C for 1 h to make the cells adhere to the wall.
  • the Sf9 cells were replaced with a medium containing 25 ⁇ g/ml Blasticidin, and continued to be cultured at 27°C, and a fresh 25 ⁇ g/ml Blasticidin medium was changed every three days until the cells no longer died and survived. All the cells are Blasticidin-resistant cells, so far the Sf9 cell line that has been successfully transformed into the Rep gene plasmid+Cap gene plasmid is obtained.
  • step (8) spread the Sf9 cell line obtained in step (8) that has been successfully transferred into the Rep gene plasmid+Cap gene plasmid on a 35mm plate at 2 ⁇ 10 ⁇ 6/well, and cultured at 27°C for 1h to make the cells adhere to the wall.
  • the Sf9 cells were replaced with a medium containing 100 ⁇ g/ml G418 and continued to culture at 27°C, and a fresh 100 ⁇ g/ml G418 medium was changed every three days until the cells no longer died and survived.
  • the cells were all anti-G418 cells, and thus the Sf9 cell line that induced AAV production was obtained, that is, the stable transfection cell line.
  • the integrated sf9 cell line was performed with an empty Baculovirus. Induction, the cells before and after induction were lysed by repeated freezing and thawing with PBS to verify the AAV packaging system.
  • the cells were lysed by repeated freezing and thawing at -80°C and 37°C three times to release the AAV virus.
  • the amount of virus produced is an average of 1*10 ⁇ 5vg/cell.
  • the target gene plasmid can be represented by the general formula pBSK-HR5-IE-Neo-IEter-AAV-CMV-Target Gene, wherein Target Gene can be various target genes, such as Target Gene When it is the SMN1 gene (as shown in SEQ ID No: 12), AAV gene drugs for the treatment of spinal muscular atrophy can be produced, or the Target Gene is the gene sequence encoding the soluble extracellular domain ACE2 protein (SEQ ID No: 13 or SEQ ID No: 13 or SEQ ID No: 13).
  • the sequence shown in ID No: 14 the former encodes the soluble extracellular domain ACE2 1-620 fragment, the latter encodes the soluble extracellular domain ACE2 1-740 fragment), can make cells express soluble ACE2 protein outside the cell (ACE2 protein can be It specifically binds to the S protein of the coronavirus), thereby preparing an AAV gene drug that antagonizes SARS-CoV infection.

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Abstract

一种基于诱导型昆虫细胞生产AAV基因药物的方法，所述方法包括：(1)分别构建用于包装腺相关病毒的Rep基因质粒、Cap基因质粒和目的基因质粒；其中Rep基因质粒和Cap基因质粒含有晚期启动子；(2)将构建的三个质粒整合到Sf9昆虫细胞基因组上，得到可生产AAV的稳转细胞株；(3)大量扩增、培养所述稳转细胞株；在需要生产AAV时，用不携带外源基因的杆状病毒，感染所述稳转细胞株，以诱导稳转细胞株中的晚期启动子激活并驱动Rep基因和Cap基因表达，包装生产AAV。该方法不需要使用携带外源基因的杆状病毒，因此不会带来因杆状病毒大量复制扩增所带来的外源基因丢失的问题，有利于获得稳定的AAV产物，进一步简化AAV生产步骤，提高生产效率，实现AAV的大规模化高效生产并降低成本。

Description

一种基于诱导型昆虫细胞生产AAV基因药物的方法 技术领域
本发明涉及生物技术领域，尤其是一种基于诱导型昆虫细胞生产AAV基因药物的方法。
背景技术
基因治疗的基本原理是运用重组DNA的技术，将具有正常基因及其表达所需的序列导入到病变细胞或体细胞，对DNA分子进行纠正或修复，从而达到治疗目的。相比于其他药物，基因治疗的优势在于从根本上解决问题。根据市场调研机构Fortune Business Insights发布的报告，2019年基因治疗市场规模为36.1亿美元，预计到2027年将达到356.7亿美元，预测期内的年均复合增长率(CAGR)为33.6％。目前，基因药物的病毒类载体主要是腺相关病毒(AAV)和慢病毒两大类，这两类载体根据各自的特点，分别用于不同的基因疗法和适应症。
全球已有多款获批的遗传性疾病AAV载体基因疗法。Uniqure，诺华，Spark Therapeutics等重要机构在市场获得了先机。除此之外，也有一些重要制药企业的候选疗法处于临床阶段。2012年，欧盟委员会(EC)批准了西方世界首个基因治疗药物Glybera，该药来自荷兰生物技术公司UniQure，用于治疗一种极其罕见的遗传性疾病--脂蛋白脂肪酶缺乏症(LPLD)，其高达100万美元的定价创造了昂贵现代医药的新纪录。Glybera也是目前已经上市的唯一利用杆状病毒昆虫细胞生产的AAV载体的基因治疗药物。此外，Luxturna是由Spark(罗氏子公司)研发的一款基因疗法，于2017年12月获得美国FDA批准上市，Luxturna使用AAV2携带RPE65基因，采用在视网膜下腔直接注射的方法将病毒导入眼球中，用于治疗Leber先天性黑蒙2型(LCA2)的患者。
未来几年将有十多款基因治疗的新药上市，基因治疗创新药物的发展离不开病毒载体生产技术的进步。目前基因药物高昂的价格与病毒载体的生产成本较高有密切关系，尤其是腺相关病毒载体。国际上除UniQure、Biomarin等少数企业利用杆状病毒昆虫系统解决临床级AAV载体生产问题外，大多数药企和CDMO平台仍然用传统的293T细胞质粒转染的方式生产，无法实现规模化生产以有效降低AAV的生产成本。利用杆状病毒昆虫细胞生产AAV是解决临床级病毒大规模生产的重要手段。
目前，利用杆状病毒昆虫系统生产AAV问题的基本原理，如图1所示：将腺相关病毒的包装组分Rep,Cap和目的基因序列，分别克隆到三个杆状病毒载体上，利用三个杆状病毒感染昆虫sf9细胞，包装得到AAV。为了进一步简化流程，提高操作的稳定性，一种改进方法原理如图2所示：将腺相关病毒的包装组分Rep,Cap整合到昆虫sf9细胞的基因组上，构建成诱导型细胞系，然后将目的基因的序列克隆到杆状病毒载体上，利用杆状病毒载体感染昆虫sf9细胞并诱导Rep,Cap表达，包装生产AAV。这两种方法均在如下技术问题：
(1)该两种方法，都利用携带外源基因的杆状病毒感染昆虫sf9细胞，然而携带了外源基因的杆状病其传代不稳定，外源基因会丢失，造成生产过程不稳定。杆状病毒是复制型病毒，作为生产AAV的原料，要大量复制扩增，扩增3-4代，外源基因就会丢失。生产时需要反复检测。一旦丢失，要重新扩增。
(2)Rep,Cap蛋白有细胞毒性，如果持续表达，将不利于昆虫sf9细胞的大量扩增和培养，难以实现AAV的规模化生产和降低成本。此外，现有技术方法仍存在生产步骤不够简化及生产效率低等问题。
发明内容
(一)要解决的技术问题
鉴于现有技术的上述缺点、不足，本发明提供一种基于诱导型昆虫 细胞生产AAV基因药物的方法，该方法不需要使用携带外源基因的杆状病毒，解决携带外源基因的杆状病毒在大量复制扩增过程中容易丢失外源基因的问题，有利于获得稳定的AAV产物，并进一步简化AAV生产步骤，提高生产效率，实现AAV的大规模化高效生产并降低生产成本。
(二)技术方案
为了达到上述目的，本发明采用的主要技术方案包括：
第一方面，本发明提供一种基于诱导型昆虫细胞生产AAV基因药物的方法，所述方法包括：
(1)分别构建用于包装腺相关病毒的Rep基因质粒、Cap基因质粒和目的基因质粒；其中Rep基因质粒和Cap基因质粒含有晚期启动子；
(2)将构建的三个质粒整合到Sf9昆虫细胞基因组上，得到可生产AAV的稳转细胞株；
(3)大量扩增、培养所述稳转细胞株；在需要生产AAV时，用不携带外源基因的杆状病毒，感染所述稳转细胞株，以诱导稳转细胞株中的晚期启动子激活并驱动Rep基因和Cap基因表达，包装生产AAV。
根据本发明的较佳实施例，步骤(1)中，Rep基因质粒、Cap基因质粒、目的基因质粒中分别含有早期启动子和筛选标签。
所述早期启动子驱动筛选标签表达，以确保最终筛选得到同时包含Rep基因质粒、Cap基因质粒、目的基因质粒三个质粒的稳转细胞株。
根据本发明的较佳实施例，步骤(2)中，分别将所述三个质粒逐一通过转染进入Sf9昆虫细胞中，每转染完一种质粒后，利用与该质粒中筛选标签相应的抗生素进行筛选，选出存活的细胞，用于下一种质粒的转染，最终得到可生产AAV的Sf9稳转细胞株。
根据本发明的较佳实施例，步骤(1)中，Rep基因质粒为pBSK-HR5-IE-G2P-IEter-HR5-p10-Rep intron-sv40，其包含pBSK商业化载体序列、HR5增强子、IE早期启动子、G2P(GFP-2A-puro，GFP和puromycin双标记)筛选标签、IEter转录终止序列、p10晚期启动子、及表 达Rep蛋白的Rep intron表达盒基因序列。
根据本发明的较佳实施例，步骤(1)中，Cap基因质粒为pBSK-HR5-IE-BSD-IEter-HR5-pH-Cap intron-sv40，其包含pBSK商业化载体序列、HR5增强子、IE早期启动子、BSD筛选标签、IEter转录终止序列、pH晚期启动子、及表达Cap蛋白的Cap intron表达盒基因序列。
根据本发明的较佳实施例，步骤(1)中，目的基因质粒为pBSK-HR5-IE-Neo-IEter-AAV-Target Gene，其包含pBSK商业化载体序列、HR5增强子、IE早期启动子、Neo筛选标签、IEter转录终止序列、及表达Target Gene的腺相关病毒基因组序列。
根据本发明的较佳实施例，步骤(1)中，所述目的基因Target Gene为密码子优化的SMN1基因或编码可溶性胞外区ACE2蛋白的基因序列；其中，密码子优化的SMN1基因序列如SEQ ID No:12所示，所述编码可溶性胞外区ACE2蛋白的基因序列如SEQ ID No:13或SEQ ID No:14所示。
筛选时，质粒中早期启动子IE可驱动Puro筛选标签、BSD筛选标签、Neo筛选标签表达(这些筛选标签实为抗生素抗性基因)，使细胞获得抗性，而能够在具有相应抗生素的培养基中存活下来，从而筛选出成功在Sf9昆虫细胞的基因组中同时转入了Rep基因质粒、Cap基因质粒和目的基因质粒三种质粒的稳转细胞株。
可理解的，所述相应抗生素为Puromycin、Blasticidin、G418。
根据本发明的较佳实施例，步骤(3)中，步骤(3)中，所述不携带外源基因的杆状病毒为空的Baculovirus杆状病毒；用空的Baculovirus杆状病毒感染所述稳转细胞株，使细胞进入晚期的病毒样效应，激活晚期启动子p10和polyhedrin，驱动Rep基因和Cap基因的表达，在细胞中包装产生AAV。
根据本发明的较佳实施例，还包含步骤(4)，将经诱导表达Rep基因和Cap基因的Sf9昆虫细胞，进行反复冻融、裂解细胞，释放出AAV 病毒颗粒，最后经亲和层析纯化，得到AAV病毒产品。
第二方面，本发明提供一种用于生产携带目的基因腺相关病毒的稳转细胞系的制备方法，其包含如下步骤：
步骤S1：分别构建用于包装腺相关病毒的Rep基因质粒、Cap基因质粒和目的基因质粒；
其中，Rep基因质粒、Cap基因质粒和目的基因质粒分别含有早期启动子和筛选标签；Rep基因质粒和Cap基因质粒中还含有晚期启动子；
步骤S2：分别将Rep基因质粒、Cap基因质粒和目的基因质粒逐一通过转染进入Sf9细胞中，每次转染完一种质粒后，采用与该质粒中筛选标签相应的抗生素进行筛选，选出存活的细胞，选出存活的细胞，用于下一种质粒的转染，最终得到同时包含Rep基因质粒、Cap基因质粒、目的基因质粒三个质粒的存活细胞，该存活细胞为生产AAV的稳转细胞株。
根据本发明较佳实施例，步骤S1中，Rep基因质粒中含有IE早期启动子和Puro筛选标签；Cap基因质粒含有IE早期启动子和BSD筛选标签；目的基因质粒分别含有IE早期启动子和Neo筛选标签。
根据本发明较佳实施例，步骤S2中，用Rep基因质粒转染Sf9细胞后，采用抗生素Puromycin进行筛选，选出存活的Sf9细胞；用Cap基因质粒转染所述Sf9细胞后，用抗生素Blasticidin进行筛选，选出存活的Sf9细胞；用目的基因质粒转染所述Sf9细胞后，用抗生素G418进行筛选，选出存活的Sf9细胞，为生产AAV的稳转细胞株。
(三)有益效果
本发明技术效果包括以下几个方面：
(1)通过转染方式获得包含Rep基因质粒、Cap基因质粒和目的基因质粒的稳转细胞株，可以进一步简化AAV生产步骤，提高生产效率，实现AAV的规模化生产和降低成本。
(2)本发明中，只需用不携带外源的杆状病毒对Sf9稳转细胞株进 行感染和诱导，全程不需要使用携带外源基因的杆状病毒，因此不会带来因杆状病毒大量复制扩增所带来的外源基因丢失的问题，有利于获得稳定的AAV产物。
(3)本发明的基于诱导型昆虫细胞生产AAV基因药物的方法，在对Sf9稳转细胞株进行大量扩增培养时，有细胞毒性的Rep、Cap蛋白不会表达，只有在需要生产AAV时，使用不携带外源的杆状病毒感染稳转细胞使晚期启动子p10和polyhedrin才会被激活，驱动Rep、Cap基因表达，包装AAV颗粒。因此，Rep、Cap蛋白不会对Sf9稳转细胞株的大量扩增和培养产生抑制。
附图说明
图1为现有技术中一种生产AAV的原理图。
图2为现有技术中另一种生产AAV的原理图。
图3为本发明生产AAV的原理图。
图4为使用空的Baculovirus感染诱导Sf9稳转细胞株前后，Western Blot检测稳转细胞株Rep、Cap蛋白的表达情况。
具体实施方式
为了更好的解释本发明，以便于理解，下面结合附图，通过具体实施方式，对本发明作详细描述。
如图3所示，为本发明生产携带目的基因的AAV(AAV基因药物)的方法原理图，即分别构建Rep基因质粒、Cap基因质粒和目的基因质粒，然后将三种质粒分别通过转染的方式进入Sf9细胞中，通过抗生素结合筛选标签，筛选出三种质粒均成功转染到昆虫sf9细胞基因组上的稳转株细胞，在需要生产AAV时，只需将这个细胞系扩增起来，用不携带外源基因的杆状病毒去感染，使sf9细胞进入病毒样状态晚期启动子激活，Rep,Cap和目的基因表达，即可大量生产AAV。该方法可以避免使用携带外源基因的杆状病毒在大量复制扩增过程中，外源基因易丢失的问题。
现以目的基因为GFP为例，对本发明方案、可实现性和技术效果进行说明。实施方法如下：
一、需构建的质粒
要构建的AAV包装质粒包括Rep基因质粒、Cap基因质粒和GFP基因质粒，分别记录为：
Rep基因质粒：pBSK-HR5-IE-G2P-IEter-HR5-p10-Rep intron-sv40；
Cap基因质粒：pBSK-HR5-IE-BSD-IEter-HR5-pH-Cap intron-sv40；
GFP基因质粒：pBSK-HR5-IE-Neo-IEter-AAV-GFP。
Rep基因质粒包含pBSK商业化载体序列，HR5增强子，早期启动子IE，筛选标签G2P(GFP-2A-puro,即GFP和puromycin双标记)，IE ter为转录终止序列，p10晚期启动子，及表达Rep蛋白的Rep intron表达盒基因序列。
Cap基因质粒包含pBSK商业化载体序列，HR5增强子，早期启动子IE，筛选标签BSD，IE ter为转录终止序列，pH晚期启动子，及表达Cap蛋白的Cap intron表达盒基因序列。
GFP基因质粒包含pBSK商业化载体序列，HR5增强子，早期启动子IE，筛选标签Neo，IE ter为转录终止序列，及表达GFP的腺相关病毒基因组序列。
其中，pBSK为商业化载体(Stratagene 2 12205)，HR5序列为SEQ ID No:1，IE序列为SEQ ID No:2，G2P序列为SEQ ID No:3，IEter序列为SEQ ID No:4，p10序列为SEQ ID No:5，Rep intron序列为SEQ ID No:6，BSD序列为SEQ ID No:7，pH序列为SEQ ID No:8，Cap intron序列为SEQ ID No:9，Neo序列为SEQ ID No:10，AAV-GFP序列为SEQ ID No:11。
HR5为增强子，IE为早期启动子，用于驱动筛选标签表达，pH和p10均为晚期启动子，用于驱动Rep、Cap蛋白的表达。
二、质粒的构建过程
(一)Rep基因质粒记录为pBSK-HR5-IE-G2P-IEter-HR5-p10-Rep  intron-sv40，其构建步骤如下：
(1)将pBSK质粒用限制性内切酶KpnI和XhoI于37℃进行双酶切1h，琼脂糖电泳后切胶回收pBSK载体片段；
将HR5-IE-G2P-IE ter基因片段进行PCR扩增，5’端和3’端分别加上保护碱基和KpnI/XhoI酶切位点，将扩增得到的PCR片段利用KpnI和XhoI于37℃进行双酶切1h，琼脂糖电泳后切胶回收HR5-IE-G2P-IE ter基因片段。
(2)将胶回收试剂盒分别回收的pBSK载体片段和HR5-IE-G2P-IE ter基因片段，采用T4 DNA连接酶连接，室温反应15min。
(3)将连接产物转化至大肠杆菌：取连接产物转化感受态DH5a，轻轻混匀，冰浴30min；42℃热休克100s，立刻冰浴4min，加入无抗生素的LB培养液37℃振荡60min，用无菌玻璃涂布器将菌液均匀涂布至含有氨苄青霉素的LB琼脂平板上，37℃倒置培养14h。
(4)挑取单克隆菌落接种于含有氨苄青霉素LB液体培养液中，37℃振荡16h；用质粒提取试剂盒提取质粒pBSK-HR5-IE-G2P-IE ter质粒，进行KpnI和XhoI双酶切鉴定后进行测序鉴定，获得pBSK-HR5-IE-G2P-IE ter质粒。
(5)将pBSK-HR5-IE-G2P-IE ter质粒用限制性内切酶XbaI于37℃进行双酶切1h，琼脂糖电泳后切胶回收pBSK-HR5-IE-G2P-IE ter载体片段，将回收后的pBSK-HR5-IE-G2P-IE ter载体片段用碱性磷酸酶(CIAP)于37℃进行反应30min，再于65℃热失活15min，获得pBSK-HR5-IE-G2P-IE ter CIAP载体片段。
(6)将HR5-p10-Rep intron-sv40基因片段进行PCR扩增，5’端和3’端分别加上保护碱基和NheI酶切位点，将扩增得到的PCR片段利用NheI于37℃进行双酶切1h，琼脂糖电泳后切胶回收HR5-p10-Rep intron-sv40基因片段。
(7)将胶回收试剂盒分别回收的pBSK-HR5-IE-G2P-IE ter CIAP载 体片段和HR5-p10-Rep intron-sv40基因片段，采用T4 DNA连接酶连接，室温反应20min。
(8)将连接产物转化至大肠杆菌：取连接产物转化感受态DH5a，轻轻混匀，冰浴30min；42℃热休克80s，立刻冰浴4min，加入无抗生素的LB培养液37℃振荡60min，用无菌玻璃涂布器将菌液均匀涂布至含有氨苄青霉素的LB琼脂平板上，37℃倒置培养14h。
(9)挑取单克隆菌落接种于含有氨苄青霉素LB液体培养液中，37℃振荡16h；用质粒提取试剂盒提取质粒pBSK-HR5-IE-G2P-IEter-HR5-p10-Rep intron-sv40质粒，进行NheI和BglII双酶切鉴定后进行测序鉴定，获得pBSK-HR5-IE-G2P-IEter-HR5-p10-Rep intron-sv40质粒。
(二)Cap基因质粒记录为pBSK-HR5-IE-BSD-IEter-HR5-pH-Cap intron-sv40，其构建步骤如下：
(1)将pBSK质粒用限制性内切酶KpnI和XhoI于37℃进行双酶切1h，琼脂糖电泳后切胶回收pBSK载体片段；
将HR5-IE-BSD-IE ter基因片段进行PCR扩增，5’端和3’端分别加上保护碱基和KpnI/XhoI酶切位点，将扩增得到的PCR片段利用KpnI和XhoI于37℃进行双酶切1h，琼脂糖电泳后切胶回收HR5-IE-BSD-IE ter基因片段。
(2)将胶回收试剂盒分别回收的pBSK载体片段和HR5-IE-BSD-IE ter基因片段，采用T4 DNA连接酶连接，室温反应20min。
(3)将连接产物转化至大肠杆菌：取连接产物转化感受态DH5a，轻轻混匀，冰浴30min；42℃热休克80s，立刻冰浴4min，加入无抗生素的LB培养液37℃振荡70min，用无菌玻璃涂布器将菌液均匀涂布至含有氨苄青霉素的LB琼脂平板上，37℃倒置培养16h。
(4)挑取单克隆菌落接种于含有氨苄青霉素LB液体培养液中，37℃振荡16h；用质粒提取试剂盒提取质粒pBSK-HR5-IE-BSD-IE ter质粒， 进行KpnI和XhoI双酶切鉴定后进行测序鉴定，获得pBSK-HR5-IE-BSD-IEter质粒。
(5)将pBSK-HR5-IE-BSD-IE ter质粒用限制性内切酶XbaI和NotI于37℃进行双酶切1h，琼脂糖电泳后切胶回收pBSK-HR5-IE-BSD-IE ter载体片段。
(6)将HR5-pH-Cap9intron-sv40基因片段进行PCR扩增，5’端和3’端分别加上保护碱基XbaI和NotI酶切位点，将扩增得到的PCR片段利用XbaI和NotI于37℃进行双酶切1h，琼脂糖电泳后切胶回收HR5-pH-Cap9intron-sv40基因片段。
(7)将胶回收试剂盒分别回收的pBSK-HR5-IE-BSD-IE ter载体片段和HR5-pH-Cap9intron-sv40基因片段，采用T4 DNA连接酶连接，室温反应20min。
(8)将连接产物转化至大肠杆菌：取连接产物转化感受态DH5a，轻轻混匀，冰浴30min；42℃热休克80s，立刻冰浴4min，加入无抗生素的LB培养液37℃振荡60min，用无菌玻璃涂布器将菌液均匀涂布至含有氨苄青霉素的LB琼脂平板上，37℃倒置培养14h。
(9)挑取单克隆菌落接种于含有氨苄青霉素LB液体培养液中，37℃振荡16h；用质粒提取试剂盒提取质粒pBSK-HR5-IE-BSD-IEter-HR5-pH-Cap9intron-sv40质粒，进行NheI和NotI双酶切鉴定后进行测序鉴定，获得pBSK-HR5-IE-BSD-IEter-HR5-pH-Cap9intron-sv40质粒。
(三)GFP基因质粒记录为pBSK-HR5-IE-Neo-IEter-AAV-GFP，其构建步骤如下：
(1)将pBSK质粒用限制性内切酶KpnI和XhoI于37℃进行双酶切1h，琼脂糖电泳后切胶回收pBSK载体片段；
将HR5-IE-Neo-IE ter基因片段进行PCR扩增，5’端和3’端分别加上保护碱基和KpnI/XhoI酶切位点，将扩增得到的PCR片段利用KpnI和 XhoI于37℃进行双酶切1h，琼脂糖电泳后切胶回收HR5-IE-Neo-IE ter基因片段。
(2)将胶回收试剂盒分别回收的pBSK载体片段和HR5-IE-Neo-IE ter基因片段，采用T4 DNA连接酶连接，室温反应20min。
(3)将连接产物转化至大肠杆菌：取连接产物转化感受态DH5a，轻轻混匀，冰浴30min；42℃热休克80s，立刻冰浴4min，加入无抗生素的LB培养液37℃振荡70min，用无菌玻璃涂布器将菌液均匀涂布至含有氨苄青霉素的LB琼脂平板上，37℃倒置培养16h。
(4)挑取单克隆菌落接种于含有氨苄青霉素LB液体培养液中，37℃振荡16h；用质粒提取试剂盒提取质粒pBSK-HR5-IE-Neo-IE ter质粒，进行KpnI和XhoI双酶切鉴定后进行测序鉴定，获得pBSK-HR5-IE-Neo-IE ter质粒。
(5)将pBSK-HR5-IE-Neo-IE ter质粒用限制性内切酶XhoI和XbaI于37℃进行双酶切1h，琼脂糖电泳后切胶回收pBSK-HR5-IE-Neo-IE ter载体片段。
(6)将AAV-CMV-GFP基因片段进行PCR扩增，5’端和3’端分别加上保护碱基是SalI和XbaI酶切位点，将扩增得到的PCR片段利用SalI和XbaI于37℃进行双酶切1h，琼脂糖电泳后切胶回收AAV-CMV-GFP基因片段。
(7)将胶回收试剂盒分别回收的pBSK-HR5-IE-Neo-IE ter载体片段和AAV-CMV-GFP基因片段，采用T4 DNA连接酶连接，室温反应20min。
(8)将连接产物转化至大肠杆菌：取连接产物转化感受态DH5a，轻轻混匀，冰浴30min；42℃热休克80s，立刻冰浴5min，加入无抗生素的LB培养液37℃振荡60min，用无菌玻璃涂布器将菌液均匀涂布至含有氨苄青霉素的LB琼脂平板上，37℃倒置培养16h。
(9)挑取单克隆菌落接种于含有氨苄青霉素LB液体培养液中，37℃振荡16h；用质粒提取试剂盒提取质粒pBSK-HR5-IE-Neo-IEter-AAV-GFP 质粒，进行NheI和BglII双酶切鉴定后进行测序鉴定，获得pBSK-HR5-IE-Neo-IEter-AAV-GFP质粒。
三、转染及筛选稳转细胞株
将上述构建的三种质粒，通过转染进入Sf9细胞中，通过抗生素配合筛选标签筛选出将上述三种质粒成功整合到Sf9昆虫细胞基因组的细胞系，具体步骤如下：
(1)、将昆虫细胞Sf9以2x10^6/孔铺一块35mm平皿，27℃培养1h，使细胞贴壁。
(2)、取Rep基因质粒2μg用Grace培养基稀释至100μl，轻弹混匀，室温静置5min，取8μl Cellfectin II转染试剂用Grace培养基稀释至100μl，轻弹混匀，室温静置5min，稀释的转染试剂加入稀释的质粒中，轻弹混匀，室温静置15min。
(3)、将转染试剂质粒混合物加入贴壁的Sf9细胞中，于27℃培养6h后换成新鲜的培养基。
(4)、转染后24h，Sf9细胞换成含有8μg/ml Puromycin的培养基，继续于27℃培养，每三天换新鲜的8μg/ml Puromycin的培养基，直至细胞不再出现死亡，存活的细胞均为抗Puromycin的细胞，至此获得成功转入Rep基因质粒的Sf9细胞系。
(5)、将步骤(4)中获得的成功转入Rep基因质粒的Sf9细胞系以2x10^6/孔铺一块35mm平皿，27℃培养1h，使细胞贴壁。
(6)、取Cap基因质粒2μg用Grace培养基稀释至100μl，轻弹混匀，室温静置5min，取8μl CellfectinII转染试剂用Grace培养基稀释至100μl，轻弹混匀，室温静置5min，稀释的转染试剂加入稀释的质粒中，轻弹混匀，室温静置15min。
(7)、将转染试剂质粒混合物加入贴壁的已成功转入Rep基因质粒的Sf9细胞系中，于27℃培养6h后换成新鲜的培养基。
(8)、转染后24h，Sf9细胞换成含有25μg/ml Blasticidin的培养基， 继续于27℃培养，每三天换新鲜的25μg/ml Blasticidin的培养基，直至细胞不再出现死亡，存活的细胞均为抗Blasticidin的细胞，至此获得已成功转入Rep基因质粒+Cap基因质粒的Sf9细胞系。
(9)、将步骤(8)中获得的已成功转入Rep基因质粒+Cap基因质粒的Sf9细胞系以2x10^6/孔铺一块35mm平皿，27℃培养1h，使细胞贴壁。
(10)、取GFP基因质粒2μg用Grace培养基稀释至100μl，轻弹混匀，室温静置5min，取8μl CellfectinII转染试剂用Grace培养基稀释至100μl，轻弹混匀，室温静置5min，稀释的转染试剂加入稀释的质粒中，轻弹混匀，室温静置15min。
(11)、将将转染试剂质粒混合物加入贴壁的已成功转入Rep基因质粒+Cap基因质粒的Sf9细胞系中，于27℃培养6h后换成新鲜的培养基。
(12)、转染后24h，Sf9细胞换成含有100μg/ml G418的培养基，继续于27℃培养，每三天换新鲜的100μg/ml G418的培养基，直至细胞不再出现死亡，存活的细胞均为抗G418的细胞，至此获得诱导性产AAV的Sf9细胞系，即稳转细胞株。
四、病毒包装体系的验证
用三个诱导表达包装腺相关病毒(AAV)的质粒瞬时转染sf9昆虫细胞，通过筛选标签筛选出基因组中整合了三种质粒的sf9细胞系，整合后的sf9细胞系，用空的Baculovirus进行诱导，诱导前后的细胞用PBS反复冻融裂解，验证AAV包装体系。
采用Western Blot验证诱导前后的sf9稳转细胞中包装蛋白Rep,Cap的表达情况。以293T细胞中转染Rep,Cap的载体质粒作为阳性对照；以野生型293T细胞和Sf9细胞作为阴性对照。
结果如图4所示，整合了三个质粒的sf9细胞系在杆状病毒诱导前Rep、Cap蛋白都没有表达，而被空的Baculovirus诱导后，sf9稳转细胞 中Rep、Cap蛋白表达明显。此结果说明了，诱导型系统避免诱导前Rep、Cap蛋白大量表达带来的细胞毒性(避免其毒性抑制sf9稳转细胞的扩增培养、减少稳转株细胞裂解液中Rep、Cap蛋白的含量，有利于AAV颗粒的纯化过程)，诱导后能有效表达Rep、Cap包装蛋白，是利用昆虫系统生产AAV的理想系统。
五、利用sf9稳转细胞，完成1升细胞体积的AAV小试生产及纯化的情况，具体方法如下：
(1)、将整合的产AAV-GFP的sf9稳转细胞系进行扩增，以2x10^7/皿，铺20皿150mm细胞培养皿的细胞，在27℃昆虫细胞培养箱中静置2h，使细胞贴壁。
(2)、用已经测好基因组滴度的不带外源基因的杆状病毒，按照MOI1感染sf9稳转细胞系，感染后72h后收集细胞，每皿用2ml的PBS垂悬后冻于-80℃。
(3)、在-80℃和37℃反复冻融3次裂解细胞，释放出AAV病毒。
(4)、用50U/ml的核酸酶于37℃消化1h。
(5)、将消化好的料液置于4℃离心机中10000rpm离心20min。
(6)、用5mlAAVX对料液进行层析纯化，流速1.5ml/min，用1xPBS平衡至电导、pH稳定。
(7)、将离心澄清的料液上样，流速1.5ml/min。
(8)、上完样品后再用1xPBS清洗AAVX柱子，清洗至电导、pH稳定后，用1xPBS+1MNacl以1.5ml/min清洗柱子至电导、pH稳定后，再次用1XPBS清洗柱子。
(9)、用50mM的柠檬酸和150M的NaCl，以1.5ml/min进行洗脱，洗脱后的样品由于pH较低，用1/10体积的1.5MTris pH8.8进行稀释。
(10)、用100mM柠檬酸和300mM的Nacl进行CIP，CIP后用1xPBS将电导和PH冲洗至稳定。
(11)、产出的病毒量是平均1*10^5vg/细胞。
上述实施例是以目的基因为GFP为例所进行的说明。在实际生产应用制备AAV基因药物时，根据治疗需求携带不同的目的基因，以达到基因修复或产生特定蛋白质等的治疗目的。因而，在上述实施例的基础上，目的基因质粒可以用通式pBSK-HR5-IE-Neo-IEter-AAV-CMV-Target Gene来表示，其中，Target Gene可以是各种目的基因，例如Target Gene是SMN1基因(如SEQ ID No:12所示)时，可以生产出治疗脊肌萎缩症的AAV基因药物，或者Target Gene是编码可溶性胞外区ACE2蛋白的基因序列(SEQ ID No:13或SEQ ID No:14所示序列，前者编码可溶性胞外区ACE2 1-620片段，后者编码可溶性胞外区ACE2 1-740的片段)，均可使细胞表达可溶性ACE2蛋白到胞外(ACE2蛋白可与冠状病毒S蛋白特异结合)，由此制得拮抗SARS-CoV感染的AAV基因药物。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Figure PCTCN2021093906-appb-000001
Figure PCTCN2021093906-appb-000002
Figure PCTCN2021093906-appb-000003
Figure PCTCN2021093906-appb-000004
Figure PCTCN2021093906-appb-000005
Figure PCTCN2021093906-appb-000006
Figure PCTCN2021093906-appb-000007
Figure PCTCN2021093906-appb-000008
Figure PCTCN2021093906-appb-000009
Figure PCTCN2021093906-appb-000010
Figure PCTCN2021093906-appb-000011
Figure PCTCN2021093906-appb-000012
Figure PCTCN2021093906-appb-000013
Figure PCTCN2021093906-appb-000014
Figure PCTCN2021093906-appb-000015

Claims (10)

  1. 一种基于诱导型昆虫细胞生产AAV基因药物的方法，其特征在于，所述方法包括：
    (1)分别构建用于包装腺相关病毒的Rep基因质粒、Cap基因质粒和目的基因质粒；其中Rep基因质粒和Cap基因质粒含有晚期启动子；
    (2)将构建的三个质粒整合到Sf9昆虫细胞基因组上，得到可生产AAV的稳转细胞株；
    (3)大量扩增、培养所述稳转细胞株；在需要生产AAV时，用不携带外源基因的杆状病毒，感染所述稳转细胞株，以诱导稳转细胞株中的晚期启动子激活并驱动Rep基因和Cap基因表达，包装生产AAV。
  2. 根据权利要求1所述的方法，其特征在于，步骤(1)中，Rep基因质粒、Cap基因质粒、目的基因质粒中分别含有早期启动子和筛选标签。
  3. 根据权利要求1所述的方法，其特征在于，步骤(2)中，分别将所述三个质粒逐一通过转染进入Sf9昆虫细胞中，每转染完一种质粒后，利用与该质粒中筛选标签相应的抗生素进行筛选，选出存活的细胞，用于下一种质粒的转染，最终得到包含Rep基因质粒、Cap基因质粒及目的基因质粒三个质粒的稳转细胞株，用于生产AAV。
  4. 根据权利要求1或2所述的方法，其特征在于，步骤(1)中，Rep基因质粒为pBSK-HR5-IE-G2P-IEter-HR5-p10-Rep intron-sv40，其包含pBSK商业化载体序列、HR5增强子、IE早期启动子、G2P筛选标签、IEter转录终止序列、p10晚期启动子、及表达Rep蛋白的Rep intron表达盒基因序列。
  5. 根据权利要求1或2所述的方法，其特征在于，步骤(1)中，Cap基因质粒为pBSK-HR5-IE-BSD-IEter-HR5-pH-Cap intron-sv40，其包含pBSK商业化载体序列、HR5增强子、IE早期启动子、BSD筛选标签、IEter转录终止序列、pH晚期启动子、及表达Cap蛋白的Cap intron表达盒基因序列。
  6. 根据权利要求1或2所述的方法，其特征在于，步骤(1)中，目的基因质粒为pBSK-HR5-IE-Neo-IEter-AAV-Target Gene，其包含pBSK商业化载体序列、HR5增强子、IE早期启动子、Neo筛选标签、IEter转录终止序列、及表达Target Gene的腺相关病毒基因组序列。
  7. 根据权利要求6所述的方法，其特征在于，所述目的基因Target Gene为密码子优化的SMN1基因或编码可溶性胞外区ACE2蛋白的基因序列；其中，密码子优化的SMN1基因序列如SEQ ID No:12所示，所述编码可溶性胞外区ACE2蛋白的基因序列如SEQ ID No:13或SEQ ID No:14所示。
  8. 根据权利要求1或2所述的方法，其特征在于，步骤(3)中，所述不携带外源基因的杆状病毒为空的Baculovirus杆状病毒，用空的Baculovirus杆状病毒感染所述稳转细胞株，使细胞进入晚期的病毒样效应，激活晚期启动子，驱动Rep基因和Cap基因的表达，在细胞中包装产生AAV；
    所述方法还包含步骤(4)：将经诱导表达Rep基因和Cap基因的Sf9昆虫细胞，进行反复冻融、裂解细胞，释放出AAV病毒颗粒，最后经亲和层析纯化，得到AAV病毒产品。
  9. 一种用于生产携带目的基因腺相关病毒的稳转细胞系的制备方法，其包含如下步骤：
    步骤S1：分别构建用于包装腺相关病毒的Rep基因质粒、Cap基因质粒和目的基因质粒；
    其中，Rep基因质粒、Cap基因质粒和目的基因质粒分别含有早期启动子和筛选标签；Rep基因质粒和Cap基因质粒中还含有晚期启动子；
    步骤S2：分别将Rep基因质粒、Cap基因质粒和目的基因质粒逐一通过转染进入Sf9细胞中，每次转染完一种质粒后，采用与该质粒中筛选标签相应的抗生素进行筛选，选出存活的细胞，选出存活的细胞，用于下一种质粒的转染，最终得到同时包含Rep基因质粒、Cap基因质粒、 目的基因质粒三个质粒的存活细胞，该存活细胞为生产AAV的稳转细胞株。
  10. 根据权利要求9所述的制备方法，其特征在于，步骤S1中，Rep基因质粒中含有IE早期启动子和Puro筛选标签；Cap基因质粒含有IE早期启动子和BSD筛选标签；目的基因质粒分别含有IE早期启动子和Neo筛选标签；
    步骤S2中，用Rep基因质粒转染Sf9细胞后，采用抗生素Puromycin进行筛选，选出存活的Sf9细胞；用Cap基因质粒转染所述Sf9细胞后，用抗生素Blasticidin进行筛选，选出存活的Sf9细胞；用目的基因质粒转染所述Sf9细胞后，用抗生素G418进行筛选，选出存活的Sf9细胞，为生产AAV的稳转细胞株。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106085972A (zh) * 2016-07-01 2016-11-09 王安平 一种高效生产重组禽腺联病毒的方法
CN106916793A (zh) * 2015-12-24 2017-07-04 中国科学院武汉物理与数学研究所 一种重组腺相关病毒的制备方法及重组杆状病毒
CN106987603A (zh) * 2016-01-20 2017-07-28 中国科学院武汉物理与数学研究所 一种重组腺相关病毒的制备方法
WO2017184879A1 (en) * 2016-04-21 2017-10-26 Virovek, Inc Aav production in insect cells, methods and compositions therefor
CN112852880A (zh) * 2021-01-28 2021-05-28 中吉智药(南京)生物技术有限公司 一种基于诱导型昆虫细胞生产aav基因药物的方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1570121A (zh) * 2003-07-25 2005-01-26 北京三诺佳邑生物技术有限责任公司 高效生产重组腺病毒伴随病毒的病毒载体、细胞系、方法及其组成的生产系统
EP2933335A1 (en) * 2014-04-18 2015-10-21 Genethon A method of treating peripheral neuropathies and motor neuron diseases
CA3084185A1 (en) * 2017-12-06 2019-06-13 Generation Bio Co. Gene editing using a modified closed-ended dna (cedna)

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106916793A (zh) * 2015-12-24 2017-07-04 中国科学院武汉物理与数学研究所 一种重组腺相关病毒的制备方法及重组杆状病毒
CN106987603A (zh) * 2016-01-20 2017-07-28 中国科学院武汉物理与数学研究所 一种重组腺相关病毒的制备方法
WO2017184879A1 (en) * 2016-04-21 2017-10-26 Virovek, Inc Aav production in insect cells, methods and compositions therefor
CN106085972A (zh) * 2016-07-01 2016-11-09 王安平 一种高效生产重组禽腺联病毒的方法
CN112852880A (zh) * 2021-01-28 2021-05-28 中吉智药(南京)生物技术有限公司 一种基于诱导型昆虫细胞生产aav基因药物的方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
LEI, QIUGANG: "Establishment of Production System of Recombinant Adeno-associated Viruses Based on Baculoviruses", JOURNAL OF ZHEJIANG SCI-TECH UNIVERSITY, vol. 27, no. 5, 1 September 2010 (2010-09-01), pages 1§ - 6, XP055953564, ISSN: 1673-3851 *
MARIO MIETZSCH, HENRIK HERING, EVA-MARIA HAMMER, MAVIS AGBANDJE-MCKENNA, SERGEI ZOLOTUKHIN, REGINE HEILBRONN: "OneBac 2.0: Sf 9 Cell Lines for Production of AAV1, AAV2, and AAV8 Vectors with Minimal Encapsidation of Foreign DNA", HUMAN GENE THERAPY METHODS, vol. 28, no. 1, 1 February 2017 (2017-02-01), pages 15 - 22, XP055417034, ISSN: 1946-6536, DOI: 10.1089/hgtb.2016.164 *
MICHAEL F. NASO, BRIAN TOMKOWICZ, WILLIAM L. PERRY, WILLIAM R. STROHL: "Adeno-Associated Virus (AAV) as a Vector for Gene Therapy", BIODRUGS, ADIS INTERNATIONAL LTD., NZ, vol. 31, no. 4, 1 August 2017 (2017-08-01), NZ , pages 317 - 334, XP055547503, ISSN: 1173-8804, DOI: 10.1007/s40259-017-0234-5 *
WU YANG, MEI TING, JIANG LIANGYU, HAN ZENGPENG, DONG RUPING, YANG TIAN, XU FUQIANG: "Development of Versatile and Flexible Sf9 Packaging Cell Line-Dependent OneBac System for Large-Scale Recombinant Adeno-Associated Virus Production", HUMAN GENE THERAPY METHODS, vol. 30, no. 5, 1 October 2019 (2019-10-01), pages 172 - 183, XP055845975, ISSN: 1946-6536, DOI: 10.1089/hgtb.2019.123 *
YANG WU, LIANGYU JIANG, HAO GENG, TIAN YANG, ZENGPENG HAN, XIAOBING HE, KUNZHANG LIN, FUQIANG XU: "A Recombinant Baculovirus Efficiently Generates Recombinant Adeno-Associated Virus Vectors in Cultured Insect Cells and Larvae", MOLECULAR THERAPY- METHODS & CLINICAL DEVELOPMENT, NATURE PUBLISHING GROUP, GB, vol. 10, 1 September 2018 (2018-09-01), GB , pages 38 - 47, XP055681042, ISSN: 2329-0501, DOI: 10.1016/j.omtm.2018.05.005 *

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