WO2019042307A1 - 降低可复制性腺病毒产生的细胞株及构建方法和应用 - Google Patents

降低可复制性腺病毒产生的细胞株及构建方法和应用 Download PDF

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WO2019042307A1
WO2019042307A1 PCT/CN2018/102882 CN2018102882W WO2019042307A1 WO 2019042307 A1 WO2019042307 A1 WO 2019042307A1 CN 2018102882 W CN2018102882 W CN 2018102882W WO 2019042307 A1 WO2019042307 A1 WO 2019042307A1
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cell
promoter
hek293
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adenovirus
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PCT/CN2018/102882
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French (fr)
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朱涛
崔海燕
陈伟伟
段磊
李军强
马金
莘春林
邵忠琦
宇学峰
毛慧华
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康希诺生物股份公司
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Priority to EP18851666.0A priority Critical patent/EP3670650B1/en
Publication of WO2019042307A1 publication Critical patent/WO2019042307A1/zh
Priority to US16/806,013 priority patent/US11795475B2/en

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    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2710/10351—Methods of production or purification of viral material
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Definitions

  • the invention relates to the field of cell line biotechnology, in particular to a cell line for reducing reproducible adenovirus production, a construction method and application thereof.
  • the type 5 non-proliferating adenovirus vector currently used removes the region necessary for replication and amplification of adenoviruses such as the E1/E3 gene, and loses the ability to multiply in non-permissive cells, and thus needs to be produced in producing cells (such as HEK293 cells).
  • the E1 gene is inserted to ensure normal packaging and expansion of the non-proliferating adenoviral vector of the recombinant foreign gene.
  • the technical problem to be solved by the present invention is to provide a cell line which reduces the production of reproducible adenovirus, and solves the risk of producing replicable adenovirus RCA in the process of producing Ad5 recombinant adenovirus by the existing cell line.
  • Another technical problem to be solved by the present invention is to provide a method for constructing the above-described cell strain which reduces the production of replicable adenovirus.
  • Another technical problem to be solved by the present invention is to provide the above-described use of a cell strain which reduces the production of reproducible adenovirus.
  • the technical solution of the present invention is:
  • a cell strain which reduces the production of reproducible adenovirus which is a cell line HEK293.CS, which is a heterologous control element in which the non-coding region of the HEK293E1 gene is replaced by a heterologous control element.
  • the cell line for reducing the replication-producible adenovirus is the cell line HEK293.CS, and the cell line HEK293.CS is the ITR and E1A Promoter sequence of the cell line HEK293E1 gene is replaced with a heterologous control element.
  • the cell line for reducing reproducible adenovirus is the cell line HEK293.CS
  • the sequence of the cell line HEK293.CS is the ITR of the cell line HEK293 E1 and the sequence of the E1A Promoter is replaced with a heterologous control element.
  • the heterologous control element is PGK Promoter
  • the base sequence of the PGK promoter is the sequence shown in SEQ ID NO: 1 of the Sequence Listing.
  • the above-mentioned cell strain for reducing reproducible adenovirus is produced, and the ITR and E1A promoter are regions in the genome of the HEK293 cell line which are homologous to the gene sequence on the Ad5 adenovirus vector, and which are likely to undergo homologous recombination and lose the target gene. .
  • the four plasmids obtained in the step (3) are co-transfected into the HEK293 cells with the step (4) homologous repair plasmid, and subjected to antibiotic screening to obtain a preliminary modified cell strain by monoclonal purification, and if not completely replaced by the identification, Then, transfection and screening were continued until the ITR of the E1 gene and the E1A Promoter were completely replaced with PGK Promoter on the HEK293 cell line, that is, the transformed cell line was obtained.
  • the method for constructing a cell strain for reducing reproducible adenovirus production wherein the method for synthesizing sgRNA1, sgRNA2, sgRNA3, sgRNA4 in the step (1) is to screen a sgRNA targeting site, and design and synthesize sgRNA1T, sgRNA1B, sgRNA2T, sgRNA2B, sgRNA3T, sgRNA3B, sgRNA4T, sgRNA4B.
  • sgRNA1T and sgRNA1B were respectively annealed to form double-stranded sgRNA1, sgRNA2, sgRNA3, sgRNA4.
  • the above method for constructing a cell strain for reducing reproducible adenovirus production wherein the screening marker used in the step (1) is Puromycin.
  • the method for constructing a cell line for reducing reproducible adenovirus production is as follows.
  • the cell line after the step (5) is subjected to gene sequencing to identify whether the cell line is completely replaced, and the transformed cell line is determined to be non-detectable by RCA detection. RCA.
  • the above-mentioned cell strain which reduces the production of replicable adenovirus is used to reduce the production of RCA in the process of vaccine and antibody production.
  • the above-mentioned cell strain for reducing reproducible adenovirus production is used for large-scale culture of recombinant human type 5 adenovirus.
  • Another aspect of the invention provides a modified cell or a passage cell thereof for expression of a foreign gene, the ITR and E1A Promoter sequence of the cell or its passage cell containing the E1 gene being replaced with a heterologous control element, the heterologous source
  • the sequence of the control element is less than 35% similar to the ITR+E1A Promoter sequence.
  • the E1 protein expressed by the E1 gene is a related protein of Ad5 adenovirus replication early, which plays a very important role in viral replication.
  • the deletion of the E1 gene causes the Ad5 adenovirus to be unable to replicate and form a replication defect.
  • E1 is divided into two proteins, E1A and E1B, wherein the E1A promoter determines the expression of the E1 gene as E1 protein.
  • ITR Inverted terminal repeat
  • ITR Inverted terminal repeat
  • the present application abbreviates the ITR and E1A Promoter sequences as ITR+E1A Promoter.
  • the cell or its passage cell contains 1 copy or more copies of the ITR+E1A Promoter.
  • HEK293 cells contain 6 copies of ITR+E1A Promoter; 911 cells contain 1 copy of ITR+E1A Promoter and the like.
  • a partial or full copy of the ITR+E1A Promoter in the cell or its passage cell is replaced with a heterologous control element.
  • a heterologous control element For example, among the 6 copies of the ITR+E1A Promoter of HEK293 cells, 5 copies, 4 copies, 3 copies, 2 copies, 1 copy of the ITR+E1A Promoter are replaced with heterologous control elements; or 6 The copies of the ITR+E1A Promoter are all replaced with heterogeneous control elements. Preferably, all copies of the ITR+E1A Promoter are replaced with heterogeneous control elements.
  • the heterologous control element is a promoter capable of initiating expression of E1A in the cell or its passage cell.
  • the heterologous control element is a promoter with high affinity for RNA polymerase that directs expression of a large amount of E1A protein.
  • the sequence of the heterologous control element is less than 35% similar to the ITR+E1A Promoter sequence, and the lower the similarity to the ITR+E1A Promoter sequence, the more advantageous.
  • the sequence of the heterologous control element is less than or equal to 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25 in the ITR+E1A Promoter sequence. %, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.5%, etc.
  • the ITR+E1A Promoter sequence is the sequence set forth in SEQ ID NO. 2 or a homologous sequence thereof.
  • the homologous sequence has about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% of homology with the original sequence. Or above, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% Or above, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% Or above, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more 99.7% or more, 99.8% or more, or 99.9% or more.
  • the control element is a group consisting of a chicken beta-actin promoter, a CMV promoter, an HSV TK promoter and a PGK promoter.
  • the sequence of the PGK promoter is 33% similar to the sequence of the ITR+E1A Promoter; the similarity of the sequence of the chicken ⁇ -actin promoter to the ITR+E1A Promoter is 19.44%; the CMV promoter The similarity of the promoter to the sequence of the ITR+E1A Promoter was 19.31%; the similarity of the sequence of the HSV TK promoter to the ITR+E1A Promoter was 12.64%.
  • the heterologous control element is a PGK promoter.
  • sequence of the heterologous control element is the sequence set forth in SEQ ID NO.
  • the modified cell for exogenous gene expression or a passage cell thereof is a group consisting of HEK293 cells, 911 cells, pTG6559 cells, and N52.E6 cells, preferably, the cells are HEK293 cells.
  • the ITR+E1A Promoter in HEK293 cells is completely replaced by PGK promoter, which has stable genetic characteristics and can be widely used as an engineering strain for preparing adenovirus.
  • the modified cell for the expression of the foreign gene or the passage cell thereof is HEK293.CS cells, and the HEK293.CS cells are completely treated by the ITR+E1A Promoter in HEK293 cells. Replaced with PGK promoter.
  • the cell or its passage cell is used for the preparation of an adenovirus carrying or not carrying the gene of interest.
  • the cell or its passage cell is used for the preparation of an Ad5 adenovirus carrying or not carrying the gene of interest.
  • a further aspect of the invention also provides a method of producing an adenovirus comprising infecting said cell or a passage cell thereof with an adenovirus.
  • Ad5 adenovirus when Ad5 adenovirus is prepared, it is obtained by infecting HEK293 cells in which the ITR+E1A Promoter is completely replaced with a heterologous control element (for example, PGK promoter) by Ad5 adenovirus infection.
  • the HEK293.CS cell strain is infected with the Ad5 adenovirus, and the HEK293.CS cell line is completely replaced by the ITR+E1A Promoter in the HEK293 cell.
  • Source control element eg PGK promoter
  • the above-mentioned cell line HEK293.CS which reduces the production of replicable adenovirus is a gene fragment homologous to the Ad5 adenovirus E1 gene in HEK293, and a template plasmid is provided to replace it with a non-homologous sequence capable of stabilizing the expression of the E1 gene.
  • a safe adenovirus producing cell line was constructed, and the growth ability and virus-producing ability of HEK293.CS were not decreased compared with the unmodified HEK293 cell line, but no detectable RCA was produced. The specific effects are as follows:
  • sgRNA, Cas9n and homologous template repair plasmid were simultaneously transfected into cells, used to knock out the E1 gene ITR and E1A Promoter sequences of HEK293 cells, and replaced with PGK Promoter;
  • SCR7 was added when sgRNA, Cas9n and homologous template repair plasmid were simultaneously transfected into cells, which was used to inhibit non-homologous repair, improve homologous repair efficiency, and promote the replacement of E1 gene ITR and E1A Promoter into PGK Promoter in HEK293 cells;
  • the sgRNA, Cas9n and the homologous template repair plasmid were simultaneously transfected into the cells, and the E1 gene ITR and E1A Promoter sequences were screened and identified, and transfected again to ensure that the final HEK293.CS cells did not contain the E1A Promoter sequence;
  • modified HEK293.CS and the pre-engineered HEK293 were simultaneously subjected to RCA detection to confirm that the modified HEK293.CS cells did not produce detectable RCA.
  • Figure 1 is a specific target site map of ETR gene ETR and E1A Promoter in HEK293 cells using Crispr/Cas9n technology;
  • Figure 2 is a plasmid map of a homologous repair template
  • Figure 3 is a verification method for verifying the modified cells and verifying whether the ITR and E1A Promoter are replaced;
  • Figure 4 is a graph showing the results of E1 gene sequencing of HEK293.CS cells after transformation.
  • ITR+E1A Promoter sequence in HEK293 cells was replaced by PGK promoter as an example.
  • Other heterologous control elements such as chicken ⁇ -actin promoter, CMV promoter, HSV TK promoter have Similar results for PGK promoter.
  • a method for transforming HEK293 cells by using Crispr/Cas9n technology comprising a specific target site sequence for E1 gene ITR and E1A Promoter of HEK293 cells, the base sequence of which is the sequence shown in SEQ ID NO: 2 of the sequence listing;
  • the sgRNA oligonucleotide sequence targeting ITR and E1A Promoter (ITR+E1A Promoter sequence as shown in SEQ ID NO: 2 of the Sequence Listing) has the base sequence shown in Table 1;
  • the homologous repair template PGK Promoter is shown, and its base sequence is the sequence shown in SEQ ID NO: 1 of the Sequence Listing.
  • the above method for transforming HEK293 cells using Crispr/Cas9n technology includes the following steps:
  • the sgRNA and the homologous repair template plasmid constructed in S2 and S3 were mixed and transfected into HEK293 cells, and the monoclonal cell strain in which the E1 gene was successfully replaced was screened, and the RCA forming ability after virus inoculation was detected. .
  • the final concentration of PX462.V2.0-sgRNA and homologous repair template plasmid in step S4 is: PX462.V2.0-sgRNA1, PX462.V2.0-sgRNA2, PX462.V2.0-sgRNA3, PX462.V2.0
  • the -sgRNA4 was 20 ng/ ⁇ L and the PGK Promoter repair template plasmid was 25 ng/ ⁇ L.
  • a method for transforming the E1 gene of HEK293 cells using the Crispr/Cas9n system is as follows:
  • the E1 gene ITR E1 Promoter was selected as the target site to design sgRNA.
  • the target site sequence is shown in Figure 1, and the sgRNA sequence is shown in Table 1 above.
  • PX462.V2.0 was digested by BsaI site and ligated with sgRNA and designated as PX462.V2.0-sgRNA.
  • a homologous repair template plasmid was designed based on the target site sequence of the sgRNA.
  • HEK293 cells were seeded at a density of 4 ⁇ 10 5 /well in a six-well plate and transfected when grown to 70%-90% (about 18-20 h) fusion rate.
  • Premixed PX462.V2.0 -sgRNA, PGK Promoter template repair plasmid (final concentration of PX462.V2.0-sgRNA1, PX462.V2.0-sgRNA2, PX462.V2.0-sgRNA3, PX462.V2.0-sgRNA4 are 20ng/ ⁇ L
  • PGK Promoter repair template plasmid was 25ng/ ⁇ L), 20 ⁇ L
  • Lipo2000 transfection reagent 5 ⁇ L transfected cells 12h later added SCR7 non-homologous recombination inhibitor (final concentration 0.01mM), 36h later added Puromycin (final concentration 3 ⁇ g / mL) for screening.
  • the validation primers are shown in Table 2.
  • the sequencing results can be seen in Figure 4.
  • the ITR in HEK293.CS cells and E1 Promoter have been completely replaced with PGK Promoter.
  • the infected cells were harvested on the third day, at which time most of the cells had lysed and floated, indicating that the virus had replicated. After harvesting the cells and the supernatant, the virus was cleaved from the cells by repeated freezing/thawing for three freeze-thaw cycles, and the cell debris was removed by centrifugation, followed by purification by column chromatography.
  • the virus particle/cell ratio is given as a measure of the cell productivity in the growth of the virus by dividing the total virus particles by the number of cells at the time of infection, thereby determining the virus-producing ability of the cells before and after the transformation.
  • the yields of the adenoviral vectors produced by HEK293 or HEK293.CS cells are shown by Table 3.
  • the Ad5-GP virus that is propagated in HEK293 cells can detect 1 RCA, and when the sample size is increased to 3 ⁇ 10 11 VP.
  • the Ad5-GP virus that was propagated in HEK293 cells was able to detect 13 RCAs, but the Ad5-GP virus propagated with HEK293.CS cells did not detect RCA in both sample sizes.

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Abstract

提供了一种降低可复制性腺病毒产生的细胞株HEK293.CS及构建方法和应用，HEK293.CS是通过敲除HEK293中与Ad5腺病毒E1基因同源的基因片段，同时提供模板质粒将其替换为能稳定E1基因表达的非同源序列，构建的一种安全腺病毒生产细胞系，与未经改造的HEK293细胞株相比，HEK293.CS的生长能力和产病毒能力没有下降，但不产生可检测的RCA。HEK293.CS可用于重组人5型腺病毒进行规模化培养，降低疫苗、抗体等药物制造过程中RCA的产生概率。

Description

降低可复制性腺病毒产生的细胞株及构建方法和应用 技术领域
本发明涉及细胞株生物技术领域，尤其是降低可复制性腺病毒产生的细胞株及构建方法和应用。
背景技术
Crispr/Cas9被证明可以通过RNA介导切割任意给定的DNA，Crispr/Cas9对目标基因的切割引入DNA双链断裂(double-strand breaks，DSBs)，DSBs通过非同源末端结合(non-homologous end joining,NHEJ)方式或者同源修复(homologous-directed repair HDR)方式进行修复。NHEJ修复在Cas9切割处常造成目的基因的移码突变，引入功能缺失突变。HDR修复方式在外源序列指导下可敲入基因，引入功能获得突变。另外利用DSBs，同时提供供体，可以在基因组中定点插入或者突变目标序列。但Crispr/Cas9在哺乳动物细胞中会引起严重的脱靶效应，为了解决脱靶效应，利用Cas9n切口酶(Cas9-D10，突变的活性中心)加上两个sgRNA可以大大降低脱靶效应，但是这两条要结合在不同的链上，并且要足够近，同时两个sgRNA的PAM要相背着，这样两个近距离的单链断裂会组成双链断裂。而在潜在的脱靶序列处，Cas9n切口酶只会存在一定几率造成单链断裂，而单链断裂都会通过碱基切除修复途径修复，在这个过程中，很少会引起突变，从而大大降低脱靶效率。
目前所用的5型非增殖型腺病毒载体去除了E1/E3基因等腺病毒复制扩增所必需的区域，丧失了在非允许细胞内繁殖的能力,因此需要在生产细胞(如HEK293细胞)中插入E1基因从而保证重组外源基因的非增殖型腺病毒载体的正常包装和扩增。但在Ad5腺病毒载体与HEK293细胞间仍有E1基因的E1A Promoter重叠区域，同源序列间发生重组，理论上有丢失目的基因、产生可复制性腺病毒(RCA)的可能性。
发明内容
本发明所要解决的技术问题在于提供一种降低可复制性腺病毒产生的细胞株，解决了现有细胞株生产Ad5重组腺病毒过程中产生可复制腺病毒RCA的风险。
本发明所要解决的另一技术问题在于提供上述降低可复制性腺病毒产生的细胞株的构建方法。
本发明所要解决的另一技术问题在于提供上述降低可复制性腺病毒产生的细胞株的应用。
为解决上述技术问题，本发明的技术方案是：
一种降低可复制性腺病毒产生的细胞株，为细胞株HEK293.CS，所述细胞株HEK293.CS为细胞株HEK293E1基因的非编码区被替换为异源控制元件。
优选的，上述降低可复制性腺病毒产生的细胞株，为细胞株HEK293.CS，所述细胞株HEK293.CS为细胞株HEK293E1基因的ITR及E1A Promoter序列被替换为异源控制元件。
优选的，上述降低可复制性腺病毒产生的细胞株，为细胞株HEK293.CS，所述细胞株HEK293.CS为细胞株HEK293 E1基因的ITR及E1A Promoter的序列被替换为异源控制元件，所述异源控制元件为PGK Promoter，所述PGK promoter的碱基序列如序列表SEQ ID NO：1所示序列。
优选的，上述降低可复制性腺病毒产生的细胞株，所述ITR和E1A promoter为HEK293细胞株基因组中与Ad5腺病毒载体上的基因序列同源、很容易发生同源重组而丢失目的基因的区域。
上述降低可复制性腺病毒产生的细胞株的构建方法，具体步骤如下：
(1)设计合成并退火得到sgRNA1,sgRNA2,sgRNA3,sgRNA4；
(2)准备含Cas9n酶的PX462.V2.0质粒,并用Bsal酶切质粒；
(3)将步骤(1)得到的四条双链sgRNA分别与步骤(2)酶切胶回收后的质粒连接；
(4)准备同源模版修复质粒，在PGK Promoter两端加上左同源臂PSG4序列和右同源臂Ad5序列，将合成的序列连接至PUC57载体质粒上，质粒图谱如图1所示；
(5)将步骤(3)得到的四个质粒与步骤(4)同源修复质粒共转染至HEK293细胞中，经过抗生素筛选，单克隆纯化得到初步改造的细胞株，经鉴定如无完全替换，则继续转染、筛选，直至HEK293细胞株上E1基因的ITR以及E1A Promoter被完全替换为PGK Promoter，即得到改造后的细胞系。
优选的，上述降低可复制性腺病毒产生的细胞株的构建方法，所述步骤(1)中设计合成sgRNA1,sgRNA2,sgRNA3,sgRNA4的方法为筛选sgRNA靶向位点，设计并合成sgRNA1T,sgRNA1B,sgRNA2T,sgRNA2B,sgRNA3T,sgRNA3B,sgRNA4T,sgRNA4B。将sgRNA1T与sgRNA1B,sgRNA2T与sgRNA2B,sgRNA3T与sgRNA3B,sgRNA4T与sgRNA4B分别退火、形成双链sgRNA1,sgRNA2,sgRNA3,sgRNA4。
优选的，上述降低可复制性腺病毒产生的细胞株的构建方法，所述步骤(1)中使用的筛选标记为Puromycin(嘌呤霉素)。
优选的，上述降低可复制性腺病毒产生的细胞株的构建方法，所述步骤(5)中改造后的细胞系经基因测序鉴定是否替换完全，经RCA检测确定改造后的细胞系不产生可检测的RCA。
上述降低可复制性腺病毒产生的细胞株用于在疫苗、抗体制造过程中降低RCA的产 生。
上述降低可复制性腺病毒产生的细胞株用于重组人5型腺病毒的规模化培养。
本发明另一方面提供用于外源基因的表达的修饰的细胞或其传代细胞，所述细胞或其传代细胞含有E1基因的ITR及E1A Promoter序列被替换为异源控制元件，所述异源控制元件的序列与ITR+E1A Promoter序列的相似性低于35％。
E1基因所表达的E1蛋白是Ad5腺病毒复制早起的相关蛋白，其对病毒复制起着非常重要的作用，E1基因的缺失会导致Ad5腺病毒无法复制，形成复制缺陷。E1分为E1A和E1B两个蛋白，其中，E1A启动子决定着E1基因表达为E1蛋白。ITR(Inverted terminal repeat)为反向末端重复序列，其能够激活E1A的转录。为了方便描述，本申请将ITR及E1A Promoter序列简写为ITR+E1A Promoter。
在本发明的一个具体实施方式中，所述细胞或其传代细胞含有1个拷贝或1个以上拷贝的所述ITR+E1A Promoter。例如HEK293细胞中含有6个拷贝的ITR+E1A Promoter；911细胞中含有1个拷贝的ITR+E1A Promoter等。
在本发明的一个具体实施方式中，所述细胞或其传代细胞中的所述ITR+E1A Promoter的部分拷贝或全部拷贝被替换为异源控制元件。例如，HEK293细胞的6个拷贝的ITR+E1A Promoter中，其中5个拷贝、4个拷贝、3个拷贝、2个拷贝、1个拷贝的ITR+E1A Promoter被替换为异源控制元件；或者6个拷贝的ITR+E1A Promoter全部被替换为异源控制元件。优选地，所述ITR+E1A Promoter的全部拷贝被替换为异源控制元件。
在本发明的一个具体实施方式中，所述异源控制元件为启动子，其在所述细胞或其传代细胞中能够启动E1A的表达。优选地，所述异源控制元件为对RNA聚合酶有很高亲和力的启动子，能指导表达大量的E1A蛋白。
在本发明的一个具体实施方式中，所述异源控制元件的序列与ITR+E1A Promoter序列的相似性低于35％，且其与ITR+E1A Promoter序列的相似性越低越有利。例如所述异源控制元件的序列与ITR+E1A Promoter序列的相似性低于或者等于34％、33％、32％、31％、30％、29％、28％、27％、26％、25％、24％、23％、22％、21％、20％、19％、18％、17％、16％、15％、14％、13％、12％、11％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％或0.5％等。
上述所述的相似性是利用DNAman软件，替换的异源控制元件序列与ITR+E1A Promoter序列进行比对而得。
在本发明的一个具体实施方式中，所述ITR+E1A Promoter序列如SEQ ID NO.2所示的序列或其同源序列。
示例性地，所述同源序列与原序列的同源性约60％或以上、约70％或以上、71％或以 上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1或以上、99.2或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上。
在本发明的一个具体实施方式中，所述控制元件为chicken β-actin启动子、CMV启动子、HSV TK启动子和PGK promoter组成的组。所述PGK promoter的序列与所述ITR+E1A Promoter的序列的相似性为33％；所述chicken β-actin启动子与所述ITR+E1A Promoter的序列的相似性为19.44％；所述CMV promoter启动子与所述ITR+E1A Promoter的序列的相似性为19.31％；所述HSV TK启动子与所述ITR+E1A Promoter的序列的相似性为12.64％。优选地，所述异源控制元件为PGK promoter。
在本发明的一个具体实施方式中，所述异源控制元件的序列如SEQ ID NO.1所示的序列。
在本发明一个具体实施方式中，所述用于外源基因表达的修饰的细胞或其传代细胞为HEK293细胞、911细胞、pTG6559细胞和N52.E6细胞组成的组，优选地，所述细胞为HEK293细胞。HEK293细胞中的ITR+E1A Promoter完全被替换为PGK promoter，其遗传特性稳定，可以作为制备腺病毒的工程菌株而被广泛的应用。
在本发明的一个具体实施方式中，所述用于外源基因表达的修饰的细胞或其传代细胞为HEK293.CS细胞，所述HEK293.CS细胞通过将HEK293细胞中的ITR+E1A Promoter完全被替换为PGK promoter而得。
在本发明的一个具体实施方式中，所述细胞或其传代细胞用于腺病毒的制备，所述腺病毒上携带或者不携带目的基因。优选地，所述细胞或其传代细胞用于Ad5腺病毒的制备，所述Ad5腺病毒上携带或者不携带目的基因。
本发明又一方面还提供一种制备腺病毒的方法，其包括使用腺病毒感染上述细胞或其传代细胞。例如，制备Ad5腺病毒时，利用Ad5腺病毒感染ITR+E1A Promoter被完全替换为异源控制元件(例如PGK promoter)的HEK293细胞而获得。
在本发明的一个具体实施方式中，制备Ad5腺病毒时，利用Ad5腺病毒感染HEK293.CS细胞株而获得，所述HEK293.CS细胞株为HEK293细胞中的ITR+E1A Promoter被完全替换为异源控制元件(例如PGK promoter)所得。
本发明的有益效果：
上述降低可复制性腺病毒产生的细胞株HEK293.CS是通过敲除HEK293中与Ad5腺 病毒E1基因同源的基因片段,同时提供模板质粒将其替换为能稳定E1基因表达的非同源序列，构建的一种安全腺病毒生产细胞系，与未经改造的HEK293细胞株相比，HEK293.CS的生长能力和产病毒能力没有下降，但不产生可检测的RCA。具体效果如下：
(1)使用Crispr/Cas9n技术改造HEK293细胞，能最大程度降低脱靶效应；
(2)在HEK293细胞E1基因ITR及E1A Promoter的两端均设计靶向sgRNA序列可以提高Cas9n切割活性，保证原始序列的去除；
(3)将sgRNA、Cas9n与同源模版修复质粒同时转染至细胞，用于敲除HEK293细胞E1基因ITR及E1A Promoter序列，同时替换为PGK Promoter；
(4)将sgRNA、Cas9n与同源模版修复质粒同时转染细胞时添加了SCR7,用于抑制非同源修复、提高同源修复效率，促进HEK293细胞E1基因ITR及E1A Promoter替换为PGK Promoter；
(5)将sgRNA、Cas9n与同源模版修复质粒同时转染细胞，筛选鉴定后如还有E1基因ITR及E1A Promoter序列，再次转染，保证最终的HEK293.CS细胞中不含E1A Promoter序列；
(6)将改造后的细胞用于腺病毒培养，HEK293.CS与改造前HEK293所收获腺病毒的产率一致；
(7)将改造后的HEK293.CS与改造前的HEK293同时进行RCA检测，确定改造后的HEK293.CS细胞不产生可检测的RCA。
附图说明
图1为利用Crispr/Cas9n技术改造HEK293细胞E1基因ITR及E1A Promoter的特异性靶位点图；
图2为同源修复模版质粒图谱；
图3为对改造后细胞进行验证，验证ITR及E1A Promoter是否被替换的验证方法图；
图4为改造后HEK293.CS细胞E1基因测序结果图。
具体实施方式
下面结合具体实施例对本发明所述技术方案作进一步的说明。
以下实施例中主要以PGK promoter替换HEK293细胞中的ITR+E1A Promoter序列为例进行示例性地验证说明，其他异源控制元件如chicken β-actin启动子、CMV启动子、HSV TK启动子具有与PGK promoter相似的结果。
一种利用Crispr/Cas9n技术改造HEK293细胞的方法，包括针对HEK293细胞E1基因ITR以及E1A Promoter的特异性靶位点序列，其碱基序列如序列表SEQ ID NO：2所示序列；包括特异性靶向ITR以及E1A Promoter(ITR+E1A Promoter序列如序列表SEQ ID NO： 2所示序列)的sgRNA寡核苷酸序列，其碱基序列如表1所示；包括设计合成的如图2所示同源修复模版PGK Promoter，其碱基序列如序列表SEQ ID NO：1所示序列。
表1 sgRNA序列
Figure PCTCN2018102882-appb-000001
上述利用Crispr/Cas9n技术改造HEK293细胞的方法，包括以下步骤：
S1.设计合成特异性靶向HEK293细胞E1基因ITR以及E1A Promoter的sgRNA,并退火形成双链；
S2.将双链sgRNA构建入PX462.V2.0载体中；
S3.设计合成同源修复模版质粒；
S4.将S2和S3中构建好的sgRNA及同源修复模板质粒混合后，转染至HEK293细胞中，筛选出E1基因被成功替换的单克隆细胞株，并检测其接种病毒后的RCA形成能力。
在步骤S4中PX462.V2.0-sgRNA、同源修复模板质粒终浓度为：PX462.V2.0-sgRNA1、PX462.V2.0-sgRNA2、PX462.V2.0-sgRNA3、PX462.V2.0-sgRNA4均为20ng/μL，PGK Promoter修复模版质粒为25ng/μL。
利用Crispr/Cas9n系统改造HEK293细胞E1基因的方法，具体步骤为：
(1)选择HEK293细胞E1基因ITR以及E1 Promoter的靶位点，并利用软件设计特异序列的sgRNA；
(2)设计合成sgRNA和同源修复模板质粒,并将sgRNA克隆到经BbsI酶切的载体骨架中得到PX462.V2.0-sgRNA；
(3)将得到的PX462.V2.0-sgRNA与同源修复模板质粒混合，终浓度为PX462.V2.0-sgRNA1、PX462.V2.0-sgRNA2、PX462.V2.0-sgRNA3、PX462.V2.0-sgRNA4均为20ng/μL，PGK Promoter修复模版质粒为25ng/μL。
(4)将混合好的PX462.V2.0-sgRNA与同源修复模板质粒转染入HEK293细胞中，然后经过抗性加压，筛选出E1基因ITR以及E1 Promoter完全被替换的基因编辑细胞HEK293.CS。
实施例1
针对HEK293细胞E1基因ITR以及E1 Promoter的Crispr/Cas9n系统构建
首先，根据NCBI中HEK293基因组序列，选择E1基因ITR E1 Promoter作为靶位点设计sgRNA,其靶位点序列如图1所示，sgRNA序列如上表1所示。
其次，含有特定sgRNA序列PX462.V2.0-sgRNA的构建：
(1)设计并合成识别E1基因ITR以及E1 Promoter的sgRNA；
(2)合成后的sgRNA寡核苷酸进行体外退火；
(3)将PX462.V2.0通过BsaⅠ位点进行酶切并与sgRNA连接，命名为PX462.V2.0-sgRNA。
最后，根据sgRNA的靶位点序列设计同源修复模板质粒。
实施例2
细胞转染
在六孔板中以4×10 5/孔的密度接种HEK293细胞，待其生长至70％-90％(约18-20h)融合率时开始转染，将预混好的PX462.V2.0-sgRNA、PGK Promoter模版修复质粒(终浓度为PX462.V2.0-sgRNA1、PX462.V2.0-sgRNA2、PX462.V2.0-sgRNA3、PX462.V2.0-sgRNA4均为20ng/μL，PGK Promoter修复模版质粒为25ng/μL)，取20μL，Lipo2000转染试剂5μL转染细胞，12h后加入SCR7非同源重组抑制剂(终浓度为0.01mM),36h之后加入Puromycin(终浓度为3μg/mL)进行筛选。
实施例3
筛选验证
用图3所示方法验证HEK293.CS细胞中的E1基因是否被完全替换，方法为：提取改造细胞的基因组，如果ProF/AdR引物对能扩增出条带则证明有原始序列被PGK Promoter 替换，如YSF/R引物对能扩增出片段则证明替换的细胞中还存在原始序列，没有替换完全，只有当ProF/AdR引物对能扩增出条带而YSF/R引物对不能扩增出条带才能证明HEK293细胞中ITR以及E1A Promoter被完全替换为PGK Promoter。而YZF/R引物对扩增出的片段测序结果如果是纯的PGK Promoter序列则进一步证明原始序列被替换完全。
验证引物如表2所示，测序结果如图4可以看到HEK293.CS细胞中的ITR以及E1 Promoter已被完全替换为PGK Promoter。
表2 检测引物序列
名称 序列
ProF TCTCGCACATTCTTCACGTC,序列表SEQIDNO：11所示序列
AdR CGTTAACCACACACGCAATC,序列表SEQIDNO：12所示序列
YSF CTGCTTCGCCGAGTCTAAC,序列表SEQIDNO：13所示序列
YSR CCACATCCGTCGCTTACA,序列表SEQIDNO：14所示序列
YZF CTGTTCCAGAAGCCCTAT,序列表SEQIDNO：15所示序列
YZR ACACCTCCGTGGCAGATA,序列表SEQIDNO：16所示序列
实施例4
产病毒能力检测
将HEK293和HEK293.CS细胞分别接种至10cm细胞培养皿中，每种细胞分别以MOI＝10感染复苏的Ad5-EBOV(GP)和Ad5-TB(Ag85A)两种病毒感染。将所感染的细胞在第三天收获，此时大多数细胞已经裂解漂浮，表明病毒已经复制。收获细胞及上清液后经过3次冷冻/解冻反复冻融将病毒从细胞中裂解释放，并通过离心去除细胞碎片，之后通过柱层析纯化。通过将总病毒颗粒除以感染时间的细胞数，给出病毒颗粒/细胞比率作为对补充病毒生长中细胞生产力的量度，从而确定改造前后的细胞的产病毒能力。由表3显示HEK293或HEK293.CS细胞生产腺病毒载体的产率相当。
表3 改造前HEK293细胞与改造后HEK293.CS细胞产病毒能力比较
Figure PCTCN2018102882-appb-000002
实施例5
RCA检测
使用在HEK293或HEK293.CS繁殖后纯化的Ad5-GP的3×10 10或3×10 11个病毒颗粒， 采用现有生物测试法(重组腺病毒临床级基因治疗制品的质量控制.[J].张晓志，林鸿，杨晓燕等.中华医学杂志,2004,84(10),849-852.)张晓志等，中国医学杂志，2004)检测RCA。检测结果见表4，由表4可知检测样本量为3×10 10VP时，在HEK293细胞中繁殖的Ad5-GP病毒能检测到1个RCA，当检测样本量增加到3×10 11VP时，在HEK293细胞中繁殖的Ad5-GP病毒能检测到13个RCA，但用HEK293.CS细胞繁殖的Ad5-GP病毒在两种检测样本量中均未检测到RCA。
表4 改造前HEK293细胞与改造后HEK293.CS细胞RCA形成能力比较
病毒生产细胞 3×10 10个病毒中的RCA数目 3×10 11个病毒中的RCA数目
HEK293 1 13
HEK293.CS 0 0
上述参照实施例对该降低可复制性腺病毒产生的细胞株及构建方法和应用进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。
Figure PCTCN2018102882-appb-000003
Figure PCTCN2018102882-appb-000004
Figure PCTCN2018102882-appb-000005
Figure PCTCN2018102882-appb-000006
Figure PCTCN2018102882-appb-000007
Figure PCTCN2018102882-appb-000008
Figure PCTCN2018102882-appb-000009

Claims (19)

  1. 一种降低可复制性腺病毒产生的细胞株，其特征在于：为细胞株HEK293.CS，所述细胞株HEK293.CS为细胞株HEK293 E1基因的非编码区被替换为异源控制元件。
  2. 根据权利要求1所述的降低可复制性腺病毒产生的细胞株，其特征在于：所述细胞株HEK293.CS为细胞株HEK293 E1基因的ITR及E1A Promoter序列被替换为异源控制元件。
  3. 根据权利要求2所述的降低可复制性腺病毒产生的细胞株，其特征在于：所述异源控制元件为PGK Promoter，所述PGK promoter的碱基序列如序列表SEQ ID NO：1所示序列。
  4. 权利要求1所述降低可复制性腺病毒产生的细胞株的构建方法，其特征在于：具体步骤如下：
    (1)设计合成并退火得到sgRNA1,sgRNA2,sgRNA3,sgRNA4；
    (2)准备含Cas9n酶的PX462.V2.0质粒,并用Bsal酶切质粒；
    (3)将步骤(1)得到的四条双链sgRNA分别与步骤(2)酶切胶回收后的质粒连接；
    (4)准备同源模版修复质粒，在PGK Promoter两端加上左同源臂PSG4序列和右同源臂Ad5序列，将合成的序列连接至PUC57载体质粒上；
    (5)将步骤(3)得到的四个质粒与步骤(4)同源修复质粒共转染至HEK293细胞中，经过抗生素筛选，单克隆纯化得到初步改造的细胞株，经鉴定如无完全替换，则继续转染、筛选，直至HEK293细胞株上E1基因的ITR以及E1APromoter被完全替换为PGK Promoter，即得到改造后的细胞系。
  5. 根据权利要求4所述的降低可复制性腺病毒产生的细胞株的构建方法，其特征在于：所述步骤(1)中设计合成sgRNA1,sgRNA2,sgRNA3,sgRNA4的方法为筛选sgRNA靶向位点，设计并合成sgRNA1T,sgRNA1B,sgRNA2T,sgRNA2B,sgRNA3T,sgRNA3B,sgRNA4T,sgRNA4B；将sgRNA1T与sgRNA1B,sgRNA2T与sgRNA2B,sgRNA3T与sgRNA3B,sgRNA4T与sgRNA4B分别退火、形成双链sgRNA1,sgRNA2,sgRNA3,sgRNA4。
  6. 根据权利要求4所述的降低可复制性腺病毒产生的细胞株的构建方法，其特征在于：所述步骤(1)中使用的筛选标记为嘌呤霉素。
  7. 根据权利要求4所述的降低可复制性腺病毒产生的细胞株的构建方法，其特征在于：所述步骤(5)中改造后的细胞系经基因测序鉴定替换完全，且经生物测试法检测确定改造 后的细胞系不产生可检测的RCA。
  8. 权利要求1所述降低可复制性腺病毒产生的细胞株的用途，其用于在疫苗、抗体制造过程中降低可复制性腺病毒的产生。
  9. 如权利要求8所述的用途，所述腺病毒为重组人5型腺病毒。
  10. 用于外源基因表达的修饰的细胞或其传代细胞，其特征在于，所述细胞或其传代细胞含有E1基因的ITR及E1A Promoter序列(简写为ITR+E1A Promoter)被替换为异源控制元件，所述异源控制元件的序列与ITR+E1A Promoter序列的相似性低于35％。
  11. 如权利要求10所述的细胞或其传代细胞，其特征在于，所述细胞或其传代细胞含有1个拷贝或1个以上拷贝的所述ITR+E1A Promoter，所述ITR+E1A Promoter的部分拷贝或全部拷贝被替换为异源控制元件；优选地，所述ITR+E1A Promoter的全部拷贝被替换为异源控制元件。
  12. 如权利要求10或11所述的细胞或其传代细胞，其特征在于，所述异源控制元件为在所述细胞或其传代细胞中可启动E1A表达的启动子，且与ITR+E1A Promoter序列的相似性低于35％、33％、32％、31％、30％、29％、28％、26％、25％、23％、22％或20％。
  13. 如权利要求10-12中任一项所述的细胞或其传代细胞，其特征在于，所述ITR+E1A Promoter序列如SEQ ID NO.2所示的序列或其同源序列。
  14. 如权利要求10-13中任一项所述的细胞或其传代细胞，其特征在于，所述异源控制元件为chickenβ-actin启动子、CMV启动子、HSV TK启动子和PGK promoter组成的组。
  15. 如权利要求10-14中任一项所述的细胞或其传代细胞，其特征在于，所述异源控制元件为PGK promoter。
  16. 如权利要求10-15中任一项所述的细胞或其传代细胞，其特征在于，所述异源控制元件的序列如SEQ ID NO.1所示的序列。
  17. 如权利要求10-16中任一项所述的细胞或其传代细胞，其特征在于，所述细胞为HEK293细胞、911细胞、pTG6559细胞和N52.E6细胞组成的组，优选地，所述细胞为HEK293细胞，更优选为HEK293.CS细胞株。
  18. 如权利要求10-17中任一项所述的细胞或其传代细胞，其特征在于，所述细胞或其传代细胞用于腺病毒的制备，优选地，用于Ad5腺病毒的制备。
  19. 制备腺病毒的方法，其特征在于，包括使用腺病毒感染权利要求10-17中任一项所述的细胞或其传代细胞。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110923362A (zh) * 2019-12-19 2020-03-27 武汉中帜生物科技股份有限公司 用于同时检测单纯疱疹病毒i型/ii型胶体金层析试剂盒及其应用
WO2023241610A1 (zh) * 2022-06-15 2023-12-21 上海元宋生物技术有限公司 一种腺病毒包装与生产细胞系的构建方法及应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107630004B (zh) 2017-09-01 2021-01-15 康希诺生物股份公司 降低可复制性腺病毒产生的细胞株及构建方法和应用
CN108456696A (zh) * 2018-02-09 2018-08-28 复百澳（苏州）生物科技有限公司 一种用于病毒包装的293t细胞株的构建方法
CN114350615B (zh) * 2021-12-20 2024-04-16 北京镁伽科技有限公司 Stat2基因缺失细胞株及其制备方法和应用
CN115232835A (zh) * 2022-04-15 2022-10-25 郑州大学 一种降低可复制性腺病毒的细胞株nhek-c28的建立与应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011086509A1 (en) * 2010-01-12 2011-07-21 Vascular Biogenics Ltd. Methods of producing adenovirus vectors and viral preparations generated thereby
CN107630004A (zh) * 2017-09-01 2018-01-26 康希诺生物股份公司 降低可复制性腺病毒产生的细胞株及构建方法和应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070286845A1 (en) 2000-11-17 2007-12-13 Vascular Biogenics Ltd. Promoters exhibiting endothelial cell specificity and methods of using same for regulation of angiogenesis

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011086509A1 (en) * 2010-01-12 2011-07-21 Vascular Biogenics Ltd. Methods of producing adenovirus vectors and viral preparations generated thereby
CN107630004A (zh) * 2017-09-01 2018-01-26 康希诺生物股份公司 降低可复制性腺病毒产生的细胞株及构建方法和应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
RAUSCHHUBER, C. ET AL.: "Transcriptional Activity of Inverted Terminal Repeats of Various Human Adenovirus Serotypes", JOURNAL OF GENERAL VIROLOGY, vol. 92, no. 3, 1 March 2011 (2011-03-01), pages 669 - 674, XP055679440, ISSN: 0022-1317, DOI: 10.1099/vir.0.026542-0 *
ZHANG XIAOZHI ET AL., CHINESE JOURNAL OF MEDICINE, 2004
ZHANG XIAOZHILIN HONGYANG XIAOYAN ET AL., CHINESE MEDICAL JOURNAL, vol. 84, no. 10, 2004, pages 849 - 852

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110923362A (zh) * 2019-12-19 2020-03-27 武汉中帜生物科技股份有限公司 用于同时检测单纯疱疹病毒i型/ii型胶体金层析试剂盒及其应用
CN110923362B (zh) * 2019-12-19 2023-06-06 武汉中帜生物科技股份有限公司 用于同时检测单纯疱疹病毒i型/ii型胶体金层析试剂盒及其应用
WO2023241610A1 (zh) * 2022-06-15 2023-12-21 上海元宋生物技术有限公司 一种腺病毒包装与生产细胞系的构建方法及应用

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