WO2023241610A1 - 一种腺病毒包装与生产细胞系的构建方法及应用 - Google Patents

一种腺病毒包装与生产细胞系的构建方法及应用 Download PDF

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WO2023241610A1
WO2023241610A1 PCT/CN2023/100118 CN2023100118W WO2023241610A1 WO 2023241610 A1 WO2023241610 A1 WO 2023241610A1 CN 2023100118 W CN2023100118 W CN 2023100118W WO 2023241610 A1 WO2023241610 A1 WO 2023241610A1
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cell
adenovirus
hela
cells
region sequence
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方先龙
章康健
曹雪萍
顾锦法
张婷婷
王立申
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上海元宋生物技术有限公司
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Definitions

  • the present invention relates to a method for constructing an adenovirus packaging and production cell line and its application, and in particular to a method for constructing an adenovirus packaging and production cell line containing a modified E1 region and its application.
  • the cell line now widely used for large-scale production of adenovirus is the HEK293 cell line. Because its genome contains multiple copies of the E1 region gene fragment of wild-type adenovirus, it can spontaneously express high-level proteins encoded by the E1 region gene necessary for adenovirus replication.
  • the proteins encoded by the E1 region genes are E1A and E1B. Their main functions are: promoting and regulating the expression of subsequent genes in the adenovirus genome; regulating the cell cycle to create conditions for the packaging and replication of oncolytic adenovirus; inhibiting cell apoptosis to increase virus yield.
  • HEK293 is an adenovirus-susceptible cell, so it is widely used in the field of adenovirus packaging and production.
  • HEK293 cells have the advantages of high yield, easy culture, and fast growth rate, due to the presence of multiple copies of wild-type adenovirus E1 region gene fragments in the HEK293 genome, when adenovirus modified with the E1 region is packaged and produced in HEK293 cells, Wild-type E1 region adenovirus will inevitably be produced through homologous recombination, and as the amplification times increase, the proportion of wild-type E1 region adenovirus will increase significantly, seriously affecting the efficiency of E1 region-modified adenovirus. quality, and it is difficult to separate and remove it from the target product through existing technical means.
  • CRISPR/Cas9 gene editing technology can be used to integrate the modified adenovirus E1 region sequence into the cells of an adenovirus-susceptible cell line (for example, the cancer cell line HeLa S3).
  • an adenovirus-susceptible cell line for example, the cancer cell line HeLa S3
  • the cell line thus obtained can stably express the E1 region protein required for adenovirus replication and packaging, and the modified E1 region can avoid the possibility of producing adenovirus containing wild-type E1 region through homologous recombination.
  • the present invention completely solves the problem of producing adenovirus containing wild-type E1 region through homologous recombination during the packaging and production process of adenovirus without reducing the yield of adenovirus.
  • the present invention provides the following technical solutions.
  • a first aspect of the present invention provides an adenovirus packaging and production cell, wherein the genome of the cell contains a modified adenovirus E1 region sequence, and the modified adenovirus E1 region sequence is compared with a wild-type E1 region sequence.
  • the protein encoded by the modified adenovirus E1 region sequence has the same characteristics as the wild-type E1A and E1B proteins.
  • the same function, preferably, the protein encoded by the modified adenovirus E1 region sequence has the same amino acid sequence as the wild-type E1A and E1B proteins.
  • the modified adenovirus E1 region sequence also includes a promoter, which is located upstream of the E1 region coding sequence and is capable of initiating the expression of the E1 region gene.
  • a promoter Any promoter known in the art can be used in the present invention, as long as it can promote the expression of the E1 region gene in the host cell.
  • the promoter is selected from the group consisting of SV40 promoter and mPGK promoter.
  • the modified adenovirus E1 region sequence is selected from SEQ ID NO: 2 and SEQ ID NO: 4-7, preferably SEQ ID NO: 2.
  • the cells are human cells capable of adenovirus packaging and production, for example, derived from A549, A375, HeLa, SW620, 22RV1, MDA-MB-435S, ES-2 or HCC1806 cells, preferably HeLa S3 cells.
  • the genome of HeLa S3 cells does not contain part of the wild-type adenovirus genome, such as the E1 region, so wild-type adenovirus will not be produced through recombination.
  • HeLa S3 cells have a faster amplification speed than other virus-susceptible cells. , the advantage of high single cell yield.
  • the insertion site of the modified adenovirus E1 region sequence in the cell genome is selected from AAVS1, CCR5, ROSA26, LMO2, CCDN2, BMI1 ⁇ MECOM ⁇ PRDM1, H11, HBB03 , HBB04, FANCF02, RUNX1 and ZSCAN2, preferably the AAVS1 site.
  • Other sites known in the art may also be used in the present invention. These sites may
  • the cells are capable of packaging and producing replication-deficient adenovirus and replicating adenovirus, and no wild-type adenovirus is produced when the replicating adenovirus is serially passaged.
  • a second aspect of the present invention provides a method for constructing adenovirus packaging and production cells as described in the first aspect, including:
  • the modified adenovirus E1 region sequence is compared with the wild-type E1 region sequence: 1) the low-frequency codons of the wild-type E1 region sequence are replaced by the optimal synonymous codons in the cell; 2) the bases are repeated Reduce; 3) Avoid some restriction enzyme recognition sites in the wild-type E1 region sequence; 4) Increase the GC content; 5) Avoid the secondary structure of the mRNA obtained by transcription; 6) Avoid long repetitive sequences caused by codon duplication; so
  • the protein encoded by the modified adenovirus E1 region sequence has the same function as the wild-type E1A and E1B proteins.
  • the present invention also provides cells constructed according to the second aspect.
  • the third aspect of the present invention is the use of the adenovirus packaging and production cells described in the first aspect or the cells constructed in the second aspect in preparing replication-deficient adenovirus and replicating adenovirus.
  • a fourth aspect of the present invention provides a method for producing/packaging adenovirus, including:
  • Figure 1 shows the flow chart of pCRISPR/Cas9-T2 plasmid construction.
  • Figure 2 shows a schematic diagram of the alignment of the first 200 nucleotides of modified E1-M1 and wild-type E1.
  • Figure 3 shows the first round of monoclonal screening and identification after Donor-mPGK was transferred into HeLa S3 cells in Example 2.
  • Figure 4 shows the first round of monoclonal screening and identification after Donor-SV40 was transferred into HeLa S3 cells in Example 2.
  • Figure 5 shows the amplification of the complete inserted gene fragment identified in the modified cell line in Example 2.
  • Figure 6 shows the detection of E1A protein expression in each monoclonal cell line in Example 2.
  • Figure 7 shows the comparison of yields of each monoclonal cell line and HEK-293 cells in Example 3 (note: vp represents virus particles).
  • Figure 8 shows the second round of identification of inserted gene fragments in monoclonal cells of HeLa S3-mPGK in Example 3.
  • Figure 9 shows the relative expression level of E1A mRNA in monoclonal cells detected by PCR in Example 5.
  • Figure 10 shows the verification of puromycin resistance gene knockout in each selected monoclonal cell.
  • Figure 11 shows the verification of gene insertion into cells after Puro gene knockout.
  • Figure 12 shows the results of PCR detection of specific sequences in the E1 region of modified cells.
  • Figure 13 shows the PCR detection diagram between the homology arms of the inserted sequence in the modified cells.
  • Figure 14 shows the detection results of transformed cell E1B to the homology arm.
  • Figure 15 shows the quantitative detection of wild-type adenovirus content among 100,000 viruses.
  • 293-HeLa S3-SV40-3-1C-2G means that the virus inoculated into HeLa S3-SV40-3-1C-2G cells is a virus produced by continuous passage in HEK293.
  • Figure 16 shows the virus packaging process in HeLa S3-SV40-3-1C-2G cells.
  • CRISPR/Cas9 gene editing technology is the latest generation of gene editing technology, and its characteristics such as strong specificity and low cytotoxicity ensure its safety.
  • the realization of gene editing by this technology mainly relies on the Cas9 enzyme to specifically cut the DNA in the genome under the guidance of sgRNA (T2) to generate double-stranded breaks, and then relies on the HDR genome repair pathway of the cell to perform genome repair using the Donor plasmid as a template. Integrate the target gene fragment into the genome of the target cell to achieve gene editing.
  • T2 sgRNA
  • low-frequency codons refers to codons that appear with low frequency in Kazusa and genome databases, such as Ala (GCG), Leu (CUC), Arg (CGG and CGC), and Ser (UGG).
  • optimal synonymous codon also called optimal codon, refers to a specific one or several synonymous codons that organisms tend to use, that is, codon bias. ).
  • the AAVS1 site is a specific sequence located in the first intron of the human PPP1R12C gene.
  • the introduction of exogenous nucleotide sequences within this region has been shown to not affect the expression of the PPP1R12C gene or other endogenous genes and has little toxicity to cultured cells. Therefore, in theory, various foreign genes and regulatory sequences can be introduced into this section to achieve the purpose of controllable expression of target genes and treatment of corresponding diseases.
  • the E1 region involved in the present application may include a promoter, which is located upstream of the E1 region coding sequence and is capable of initiating the expression of the E1 region gene.
  • a promoter which is located upstream of the E1 region coding sequence and is capable of initiating the expression of the E1 region gene.
  • the E1 region sequence with mPGK as the promoter is used to promote the expression of E1A; or SV40 is used as the promoter.
  • Other promoters known in the art that can initiate gene transcription and expression can also be used in the present invention.
  • E1A not only activates other early genes, but also promotes cell apoptosis, which will significantly affect virus production.
  • E1B-55K has the effect of anti-apoptosis and promoting late virus RNA export from the nucleus.
  • E1B-19K is homologous to the expression product of the cell Bcl-2 gene and can prevent cell apoptosis or necrosis by inactivating and clearing Bax family members. .
  • part of the nucleic acid sequence of the E1 region in the above two designs has been optimized for homologous codons and humanization to avoid the production of wild-type adenovirus due to homologous recombination during the packaging and production process of the modified oncolytic adenovirus.
  • the sequence After optimization After the sequence is integrated into the cell genome, it can stably express the E1 region proteins required for adenovirus production and packaging: E1A, E1B-19K and E1B-55K.
  • the present invention uses CRISPR/Cas9 gene editing technology to modify the nucleotide sequence of the E1 region of the adenovirus through homologous codon substitution, humanization optimization, promoter replacement, etc., and then inserts it into the cancer cell line Hela- which is susceptible to adenovirus.
  • the safety site in the genome of S3 allows it to stably express the E1 region protein required for adenovirus replication and packaging. Without reducing the adenovirus yield, it completely solves the problem of homologous recombination during the packaging and production process of adenovirus. There are problems with the generation of adenoviruses containing the wild-type E1 region, and with the production of cassettes from replication-deficient adenoviruses in background cells.
  • the pCRISPR/Cas9-T2 plasmid is a vector plasmid based on CRISPR/Cas9 technology. Its function is to express wild-type spCas9 protein and sgRNA (T2), thereby specifically targeting the 5'-ggggccactagggacagGATTGG- at the AAVS1 site on chromosome 19 of the human genome. 3' (between lowercase and uppercase bases) produces a double-stranded break on chromosome 19 (Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells. Stem Cell Res. 2014 May; 12(3) :630-7).
  • T2 a double-stranded DNA fragment with sticky ends
  • 1.2 BbsI-HF digests the pCRISPR/Cas9-GFP vector and inserts the DNA fragment formed in 1.1. After ligation, transform into DH5a competent cells, select single clones, identify and amplify pCRISPR/Cas9-T2 carrying the inserted fragment. plasmid.
  • Wild-type E1 region sequence (including E1A coding region, IRES, E1B coding region):
  • E1-Modified-1 (referred to as E1-M1), that is, the E1 region contained in the recombinant plasmids Donor-mPGK and Donor-SV40 in the examples: the E1A and E1B coding region sequences were modified, and the homology was compared with the wild-type sequence. is 96%.
  • E1-Modified-2 (referred to as E1-M2), that is, the IRES-Donor E1 region in the examples: the entire E1 region has been modified, and the homology with the wild-type sequence is 17.22%.
  • the design of the RES-Donor E1 region is to completely transform the E1 region into something different from the wild-type E1 region.
  • the sequence of genes in the E1 region has also been adjusted to make the wild-type E1 region.
  • the gene sequence E1A ⁇ E1B19K ⁇ E1B55K in the E1 region is changed to E1B19K ⁇ E1B55K ⁇ E1A.
  • E1-Modified-3 (referred to as E1-M3): The E1A coding region and part of the E1B55K coding region are modified, and the homology with the wild-type sequence is 93.4%.
  • E1-Modified-4 (referred to as E1-M4): The entire E1 coding region is modified, and the homology with the wild-type sequence is 85.3%.
  • E1-Modified-5 (referred to as E1-M5): The homology with the wild-type sequence is 96.3%.
  • E1-Modified-6 (referred to as E1-M6): The homology with the wild-type sequence is 93.6%.
  • sequence of the SV40 promoter in this article is:
  • the function of the Donor plasmid is to co-transfect target cells with the pCRISPR/Cas9-T2 plasmid and serve as a template for homologous repair after DNA double-strand breaks.
  • the modified adenovirus E1 region sequence is inserted into the target site through homologous repair.
  • the AAVS1-T2 site in the cell genome allows the target cells to stably express the inserted E1 region gene, creating favorable conditions for adenovirus replication while avoiding homologous recombination to produce adenovirus with wild-type E1 region.
  • the upstream sequence of AAVS1-T2 site in the genome is:
  • sequence downstream of the AAVS1-T2 site in the genome is:
  • the plasmid obtained in Example 1 was transfected into target cells, the modified E1 gene was inserted into the target cell genome, and stable Monoclonal cell lines that express E1A protein and E1B protein.
  • all oncolytic adenovirus-susceptible cell lines can be used as target cell lines for gene editing, such as A549, A375, HeLa, SW620, 22RV1, MDA-MB-435S, ES-2, HCC1806 cells and other human cells.
  • This example uses the HeLa S3 (ATCC CCL-2.2) cell line.
  • the specific method is as follows: digest the resistant clones screened in step 1, dilute the cell suspension to 10 cells/ml, add puromycin with a final concentration of 0.25 ⁇ g/ml to the diluted cell suspension to maintain the selection pressure, and divide the cell suspension into Inoculate into six 96-well plates at 100 ⁇ l/well. Observe cell status every 48 hours and mark wells with only a single cell population. Change the medium every 96 hours. Passage the cells when the marked wells grow to obvious contact inhibition. Expand to a 48-well plate and observe the cell density every 48 hours. Change the medium every 96 hours. After full growth, expand to 24-well plates, 12-well plates, 6-well plates, and up to 6cm petri dishes.
  • HeLa S3-mPGK-1 represents the No. 1 cell clone in the first round of monoclonal selection after Donor-mPGK was stably transferred into HeLa S3 cells;
  • HeLa S3-SV40-1 represents the No. 1 cell clone after Donor-SV40 was stably transferred into HeLa S3 cells.
  • HeLa S3-SV40-1, HeLa S3-SV40-22, HeLa S3-SV40-3, HeLa S3-mPGK-4 and HeLa S3 cells and spread them into a 6-well plate at 5 *10 cells per well.
  • RIPA lysis buffer was used to lyse the cells, and the protein was collected.
  • the total protein concentration of each sample was measured by BCA method, and each sample was treated with 5 ⁇ SDS buffer and boiled at 100°C for 10 min.
  • HeLa S3-SV40-1, HeLa S3-SV40-2, HeLa S3-SV40-3 and HeLa S3-mPGK-4 can all express E1A protein, while HeLa S3 does not express it at all. This shows that the inserted target genes can be expressed normally.
  • HeLa S3, HeLa S3-SV40-1, HeLa S3-SV40-2, HeLa S3-mPGK-4 and HEK-293A cells and inoculate them with 1.6*10 7 cells per T175 square flask.
  • test virus YSCH-01 was inoculated at an infection rate of 500vp/cell.
  • the above virus samples were harvested after 60 hours, frozen and thawed three times, centrifuged at 2000 rpm for 5 min, the supernatant was taken, and filtered using a 0.22 ⁇ m syringe filter.
  • HPLC (Shimadzu, LC-2030C) was used to detect the virus particle concentration of each sample.
  • the viral titer of each sample was detected using an adenovirus titer detection kit (Cell Biolabs, VPK-109). The results are shown in Figure 7.
  • the HeLa S3-mPGK-4 cell line was subjected to the second round of monoclonal screening.
  • the limiting dilution method was used to select monoclonal cell lines according to Example 2. There were 5 cells in total. After the cells grew to 6 cm dish, the cells were named HeLa S3-mPGK-. 1A, HeLa S3-mPGK-1B, HeLa S3-mPGK-1C, HeLa S3-mPGK-1D, HeLa S3-mPGK-1E.
  • HeLa S3-mPGK-1A represents the number 1 selected in the second round of monoclonal screening of HeLa S3-mPGK-4 cells
  • HeLa S3-mPGK-1B represents the number 2 selected in the second round of monoclonal screening of HeLa S3-mPGK-4 cells. No., and so on.
  • the genome of the second round of monoclonal cell lines was extracted for PCR identification, and the results are shown in Figure 8.
  • HeLa S3 and HEK 293 Plate cells at a density of 80%, with HeLa S3 and HEK 293 plating at 3*10 6 cells/dish; HeLa S3-SV40-1, HeLa S3-SV40-2, and HeLa S3-SV40-3 at 4*10 6 cells/dish. ; HeLa S3-mPGK-1A, HeLa S3-mPGK-1B, and HeLa S3-mPGK-1E were plated at 3.8*10 6 cells/dish.
  • YSCH-01 virus was transfected at 600vp/cell.
  • the virus was harvested about 48 hours later. After repeated freezing and thawing three times, the supernatant was collected by centrifugation at 2000 rpm for 5 minutes and filtered using a 0.22 ⁇ m syringe filter.
  • HPLC was used to test the vp of each virus sample, and the two cell lines with the best yield results were selected, namely HeLa S3-SV40-3 and HeLa S3-mPGK-1E cells (see Table 6 for detailed yield results).
  • the virus amplification ability of the modified cell line is of the same order of magnitude as that of the starting cell line HeLa S3.
  • two cell lines, HeLa S3-mPGK-1E and HeLa S3-SV40-3 had better virus amplification ability. These two cell lines were subsequently selected to knock out the Puromycin resistance gene.
  • the IRES-Donor E1 region (SEQ ID NO: 3) was used to transform the cell line.
  • Cre/LoxP plasmid Transfection of Cre/LoxP plasmid to knock out the Puromycin resistance gene in each monoclonal cell line, Cre/LoxP plasmid
  • HeLa S3-SV40-3 and HeLa S3-mPGK-1E cells were plated in 1 well each, with 5.5 ⁇ 10 5 cells.
  • Use Effectene transfection reagent QIAGEN: 301425) to transfect 0.4 ⁇ g Cre/LoxP plasmid into the above wells for knocking out the Puromycin resistance gene.
  • monoclonal cells were selected by limiting dilution method. Specifically: digest the cells, dilute the cell suspension to 4 cells/ml, and then plate 6 96-well plates at 100 ⁇ l/well. Observe cell status every 48 hours and mark wells with only a single cell colony. Change the medium every 96 hours, and expand to a 48-well plate when the marked wells grow to obvious contact inhibition. Keep observing the cell density every 48 hours, change the medium every 96 hours, and expand to a 24-well plate when full.
  • the first round of Puro resistance screening Divide the cells in the 24-well plate into two and divide them equally into 2 wells. Add 2.5 ⁇ g/ml Puro resistance to one well and not to the other well. Observe whether the cells in the wells where Puro resistance is added die. If they die, it means that the Puro resistance gene has been successfully knocked out. When the cells in the corresponding wells without Puro resistance are full, pass them to a 12-well plate, and then pass them to In a 6-well plate, expand to 6cm dish.
  • HeLa SV40-3-1A The cells obtained after Puro killing screening are named HeLa SV40-3-1A, etc., where HeLa SV40-3-1 represents the first round of monocloning in HeLa SV40-3 cells after knocking out the Puromycin resistance gene, and "A" represents the cell
  • HeLa SV40-3-1A the first one expanded to 6cm dish
  • HeLa SV40-3-1B the second one expanded to 6cm dish
  • the monoclonal cells that successfully knocked out the Puromycin resistance gene are: HeLa S3-SV40-3-1B; HeLa S3-SV40-3-1C; HeLa S3-SV40-3-1E; HeLa S3- SV40-3-1G; HeLa S3-SV40-3-1H; HeLa S3-SV40-3-1I; HeLa S3-SV40-3-1J; HeLa S3-SV40-3-1K; HeLa S3-SV40-3-1M ; HeLa S3-SV40-3-1O and HeLa S3-mPGK-1E-1A; HeLa S3-mPGK-1E-1B.
  • the virus amplification ability of the modified cell line is of the same order of magnitude as that of the starting cell line HeLa S3.
  • HeLa S3-SV40-3-1C and HeLa S3-mPGK-1E-1B have the highest yields, so HeLa S3-SV40-3-1C and HeLa S3-mPGK-1E-1B cells were selected for puromycin knockout. Round of monoclonal selection.
  • the second round of monoclonal screening after puromycin resistance gene knockout digest the cells, dilute the cell suspension to 4 cells/ml, and plate 100 ⁇ l/well of the cell suspension into three 96-well plates. Observe cell status every 48 hours and mark wells with only a single cell population. Change the medium every 96 hours. When the marked wells grow to obvious contact inhibition, expand to a 48-well plate and observe the cell density every 48 hours. Change the medium every 96 hours. After it is full grown, expand it to a 24-well plate, a 12-well plate, then a 6-well plate, and so on to a 6cm dish.
  • HeLa S3-SV40-3-1C-2A After the cells were expanded to 6cm dish, they were named HeLa S3-SV40-3-1C-2A, etc., indicating that The cells are the first cells of the second round of monoclonal screening from the HeLa S3-SV40-3-1C monoclonal cells after the first round of Purok resistance gene knockout to reach 6cm dish.
  • the virus amplification ability of the modified cell line is of the same order of magnitude as that of the starting cell line HeLa S3.
  • HeLa S3-SV40-3-1C-2G and HeLa S3-mPGK-1E-1B-2D cells had the best toxin production effects.
  • tissue/cell genome extraction kit uses the tissue/cell genome extraction kit to extract the genome from the cells, and perform PCR to amplify the inserted gene fragments in the sample genome. The results are shown in Figure 12-14.
  • HeLa S3-SV40-3-1C-2G and HeLa S3-SV40-3-1C-2J cells not only successfully knocked out the Puromycin resistance gene, but the site and length of the inserted sequence were consistent with expectations, making them homozygous cells.
  • Puromycin was successfully knocked out in HeLa S3-mPGK-1E-1B-2A, HeLa S3-mPGK-1E-1B-2B, HeLa S3-mPGK-1E-1B-2C, and HeLa S3-mPGK-1E-1B-2D cells, The resistance gene and the site and length of the inserted sequence were consistent with expectations, but these two cells were heterozygous cells.
  • LIP3000 transfection reagent (Thermo Fisher: L3000-015) to transfect 0.5 ⁇ g of the pBXYA07-U2-EGFP plasmid that was successfully digested with PacI (NEB: R0547L) into 100,000 HeLa S3-SV40-3-1B cells. (Note: Since it takes a long time to select monoclones and encapsulate viruses, cells from the first round of monoclonals are used for virus encapsulation verification.)
  • OncoMul-V2-EGFP virus is successfully packaged.
  • 3*10 6 cells are spread on every 10cm dish of HEK 293 cells (ATCC: CRL-1573) and HeLa S3 cells (ATCC CCL-2.2), and 4*10 6 cells are spread on every 10cm dish of HeLa S3- SV40-3-1C-2G cells, each cell was plated on one plate. Add 400 ⁇ l of diseased cells to each. The poison is collected after the cells have become damaged. Test each sample in 15.5 on HPLC machine.
  • Example 9 The cell line obtained in Example 9 was used for virus amplification. After amplification for different generations, the wild-type virus content in the harvested viruses was detected.
  • Each T75 square flask is plated with 3*10 6 HeLa S3-Mock and HEK 293 cells, and each T75 square bottle is plated with 6*10 6 HeLa S3-SV40-3-1C-2G cells, of which 2 are plated for each cell.
  • the virus amplified using HEK 293 cells in Example 9 was added to HEK 293 cells, and the virus amplified using HeLa S3-SV40-3-1C-2G cells in Example 9 was added to other cells, and the virus was inoculated at 400vp/cell. Perform second-pass viral amplification. Then continue to amplify the virus for 10 generations in this way.
  • HeLa S3-SV40-3-1C-2G is spread with 3 T75
  • one is used for cell counting when receiving viruses one is added with the virus amplified for the third time by HeLa S3-SV40-3-1C-2G cells, and the last one is added with the virus amplified by HEK 293 cells for the third time. All poisons are received according to 400vp/cell. VP detection was performed after harvesting the virus.
  • the amplification results of the 5th and 10th passage viruses are shown in Table 14. The experimental results are shown in Figure 15.
  • Passage through HEK 293 means that the virus added to HeLa S3-SV40-3-1C-2G cells during the fourth generation of virus amplification is the virus produced by HEK 293 cells in the third generation.
  • Table 16 Wild-type adenovirus quantitative detection qPCR sample loading table
  • Table 18 Wild-type adenovirus quantitative detection standard dilution table
  • Example 11 Packaging and amplification of replication-deficient adenovirus
  • Ad-EGFP Ad-EGFP
  • the packaging and amplification of replication-deficient adenovirus by the cell line constructed in the present invention was tested.
  • HEK 293, HeLa S3, and HeLa S3-SV40-3-1C-2G were plated on a 6-well plate.
  • HEK 293 and HeLa S3 were plated with 2* 10 cells per well, and HeLa S3-SV40-3-1C-2G was plated in each well. 4*10 5 .
  • the cells in each well were transfected with 1 ⁇ g of PacI-digested non-replicating adenovirus packaging plasmid pAd-EGFP using PEI transfection reagent.
  • HeLa S3-SV40-3-1C-2G cells showed lesions and high fluorescence intensity. However, HeLa S3 fell off and died due to excessive cell growth and had no fluorescence, indicating that replication-deficient viruses cannot be packaged in HeLa S3.
  • the changes in cell morphology during virus packaging in HeLa S3-SV40-3-1C-2G cells are shown in Figure 16.
  • 3*10 6 cells are spread on each 10cm dish with HeLa S3 and HEK 293, 6*10 6 cells are spread on each 10cm dish with HeLa S3-SV40-3-1C-2G, where each cell is spread with 2 10cm dishes, 1 One was used to receive the poison, and one was used as a control.
  • the above cells were cultured for 18 hours and then inoculated with 500 ⁇ l of Ad-EGFP virus packaged by HeLa S3-SV40-3-1C-2G cells.
  • HEK 293 cell lesions were observed under the microscope and the virus was collected.
  • HeLa S3-SV40-3-1C-2G cell lesions were observed under the microscope and the virus was collected.
  • no HeLa S3 lesions were observed, and no obvious fluorescence was observed under a fluorescence microscope, indicating that HeLa S3 cells are not suitable for amplification of newly packaged (low virus concentration) replication-defective adenovirus.
  • the modified cell HeLa S3-SV40-3-1C-2G can not only package replication-deficient adenovirus, but also rapidly amplify newly packaged (low virus concentration) replication-deficient adenovirus in it, which is significantly stronger than HeLa S3 cells. .
  • the modified HeLa S3-SV40-3-1C-2G cells are not only able to amplify the replicating adenovirus present in the E1 region without introducing wild-type adenovirus, but the amplification ability is not weaker than that of HEK293 cells. , can also package and amplify adenovirus with defective E1 region function.

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Abstract

提供一种腺病毒包装与生产的细胞系、其构建方法及应用。与传统HEK293细胞相比，该腺病毒包装与生产细胞系在不降低腺病毒产量的前提下，解决了E1区修饰的腺病毒在包装与生产过程中通过同源重组产生野生型E1区腺病毒的问题。

Description

一种腺病毒包装与生产细胞系的构建方法及应用
本申请要求于2022年06月15日提交中国专利局、申请号为202210674162.8、发明名称为“一种腺病毒包装与生产细胞系的构建方法及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及一种腺病毒包装与生产细胞系的构建方法及其应用，尤其涉及一种包含修饰的E1区的腺病毒包装与生产细胞系的构建及其应用。
背景技术
现在被广泛用于腺病毒大规模生产的细胞系为HEK293细胞系。由于其基因组中含有多拷贝数的野生型腺病毒的E1区基因片段，能自发的高水平表达腺病毒复制所必须的E1区基因编码蛋白。E1区基因编码蛋白为E1A和E1B，主要功能有：促进和调节腺病毒基因组后续有关基因的表达；调节细胞周期为溶瘤腺病毒的包装和复制创造条件；抑制细胞凋亡以提高病毒单产。并且HEK293为腺病毒易感型细胞，因此被广泛运用于腺病毒的包装和生产领域。
HEK293细胞虽然有高产、易培养、生长速度快等优点，但由于HEK293基因组中存在多拷贝数的野生型腺病毒E1区基因片段，经E1区修饰的腺病毒在HEK293细胞中包装和生产时，会不可避免地通过同源重组而产生野生型E1区的腺病毒，且随着扩增代次的增加，野生型E1区的腺病毒比例会显著上升，严重影响经E1区修饰的腺病毒的质量，且很难通过现有技术手段将其从目的产物中分离去除。
发明内容
本发明的发明人在研究中意外发现，利用CRISPR/Cas9基因编辑技术，将序列修饰后的腺病毒E1区序列定点整合至对腺病毒易感的细胞系(例如，癌细胞系HeLa S3)的基因组中，由此获得的细胞系能稳定表达腺病毒复制包装所需的E1区蛋白，同时所述修饰后的E1区可避免通过同源重组产生包含野生型E1区腺病毒的可能性。与经典的腺病毒生产细胞 HEK293细胞相比，本发明在不降低腺病毒产量的前提下，完全解决了腺病毒在包装与生产过程中通过同源重组产生包含野生型E1区的腺病毒的问题。
为了达到上述目的，本发明提供以下技术方案。
本发明的第一个方面，提供一种腺病毒包装与生产细胞，其中所述细胞的基因组包含改造的腺病毒E1区序列，所述改造的腺病毒E1区序列与野生型E1区序列相比：1)野生型E1区序列的低频密码子被所述细胞中最优的同义密码子替换；2)碱基重复减少；3)避免野生型E1区序列中的一些限制酶识别位点；4)GC含量提高；5)避免转录获得的mRNA具有二级结构；6)避免密码子重复导致的长重复序列；所述改造的腺病毒E1区序列编码的蛋白具有与野生型E1A和E1B蛋白相同的功能，优选地，所述改造的腺病毒E1区序列编码的蛋白具有与野生型E1A和E1B蛋白相同的氨基酸序列。
在本发明的一些实施方案中，所述改造的腺病毒E1区序列还包含启动子，所述启动子位于E1区编码序列上游，能够启动E1区基因的表达。现有技术已知的启动子均可用于本发明，只要能启动E1区基因在宿主细胞的表达即可。优选地，所述启动子选自SV40启动子和mPGK启动子。
在本发明的另一些实施方案中，所述改造的腺病毒E1区序列选自SEQ ID NO:2和SEQ ID NO:4-7，优选为SEQ ID NO:2。
在本发明的另一些实施方案中，所述细胞为能进行腺病毒包装与生产细胞的人源细胞，例如来源于A549、A375、HeLa、SW620、22RV1、MDA-MB-435S、ES-2或HCC1806细胞，优选为HeLa S3细胞。HeLa S3细胞的基因组中不含有野生型腺病毒的部分基因组，例如E1区，因此不会通过重组产生野生型腺病毒，此外，HeLa S3细胞跟其他病毒易感细胞相比，具有扩增速度快、单细胞产量高的优势。
在本发明的另一些实施方案中，所述改造的腺病毒E1区序列在所述细胞基因组中的插入位点选自AAVS1、CCR5、ROSA26、LMO2、CCDN2、BMI1\MECOM\PRDM1、H11、HBB03、HBB04、FANCF02、RUNX1和ZSCAN2，优选为AAVS1位点。现有技术已知的其它位点也可用于本发明，这些位点可
在本发明的另一些实施方案中，所述细胞能够包装与生产复制缺陷型腺病毒和复制型腺病毒，在复制型腺病毒连续传代时不产生野生型腺病毒。
本发明的第二个方面，提供一种构建如第一个方面所述的腺病毒包装与生产细胞的方法，包括：
对野生型E1区序列进行改造，获得改造的腺病毒E1区序列；
将所述改造的腺病毒E1区序列插入宿主细胞基因组；
其中，所述改造的腺病毒E1区序列与野生型E1区序列相比：1)野生型E1区序列的低频密码子被所述细胞中最优的同义密码子替换；2)碱基重复减少；3)避免野生型E1区序列中的一些限制酶识别位点；4)GC含量提高；5)避免转录获得的mRNA具有二级结构；6)避免密码子重复导致的长重复序列；所述改造的腺病毒E1区序列编码的蛋白具有与野生型E1A和E1B蛋白相同的功能。
本发明还提供第二个方面构建获得的细胞。
本发明的第三个方面，第一个方面所述的腺病毒包装与生产细胞或第二个方面所构建的细胞在制备复制缺陷型腺病毒和复制型腺病毒中的用途。
本发明的第四个方面，提供一种生产/包装腺病毒的方法，包括：
1)将腺病毒导入权利要求1-10任一所述的腺病毒包装与生产细胞；
2)培养步骤1)获得的细胞；和
3)收集病毒。
附图说明
图1示pCRISPR/Cas9-T2质粒构建流程图。
图2示改造后E1-M1与野生型E1的前200个核苷酸的比对示意图。
图3示实施例2中Donor-mPGK转入HeLa S3细胞后第一轮单克隆筛选鉴定。
图4示实施例2中Donor-SV40转入HeLa S3细胞后第一轮单克隆筛选鉴定。
图5示实施例2中鉴定改造细胞系中扩增完整插入基因片段。
图6示实施例2中各单克隆细胞株中E1A蛋白表达检测。
图7示实施例3中各单克隆细胞株与HEK-293细胞的单产比较(注：vp代表病毒颗粒)。
图8示实施例3中HeLa S3-mPGK第二轮单克隆细胞中插入基因片段鉴定。
图9示实施例5中PCR检测单克隆细胞的E1A的mRNA相对表达水平。
图10示各挑选的单克隆细胞敲除Puromycin抗性基因验证。
图11示Puro基因敲除后细胞插入基因验证。
图12示改造细胞E1区特定序列PCR检测结果。
图13示改造细胞插入序列同源臂间的PCR检测图。
图14示改造细胞E1B至同源臂的检测结果图。
图15示10万个病毒中野生型腺病毒含量定量检测。注：293-HeLa S3-SV40-3-1C-2G表示接种至HeLa S3-SV40-3-1C-2G细胞的病毒为在HEK293中连续传代所生产的病毒。
图16示HeLa S3-SV40-3-1C-2G细胞包装病毒过程。
具体实施方式
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。
尽管本发明的广义范围所示的数字范围和参数近似值，但是具体实施例中所示的数值尽可能准确的进行记载。然而，任何数值本来就必然含有一定的误差，其是由它们各自的测量中存在的标准偏差所致。另外，本文公开的所有范围应理解为涵盖其中包含的任何和所有子范围。例如记载的“1至10”的范围应认为包含最小值1和最大值10之间(包含端点)的任何和所有子范围；也就是说，所有以最小值1或更大起始的子范围，例如1至6.1，以及以最大值10或更小终止的子范围，例如5.5至10。另外，任何称为“并入本文”的参考文献应理解为以其整体并入。
另外应注意，如本说明书中所使用的，单数形式包括其所指对象的复数形式，除非清楚且明确的限于一个所指对象。术语“或”可与术语“和/或”互换使用，除非上下文另有清楚指明。
现有技术已知的能够将目的片段整合入基因组的方法均可应用于本发明，只要能将本发明所述改造的腺病毒E1区序列插入基因组即可。例如Cre-lox系统、Zinc Finger Nucleases(ZFN)、CRISPR或TALEN，优选为CRISPR。CRISPR/Cas9基因编辑技术是最新一代的基因编辑技术，其特异性强细胞毒性小等特点保证了其安全性。此技术实现基因编辑主要依赖于Cas9酶在sgRNA(T2)的引导下定点剪切基因组中的DNA产生双链断裂，而后依赖于细胞的HDR基因组修复通路进行以Donor质粒为模板的基因组修复，从而将目的基因片段定点整合到靶细胞基因组中实现基因编辑。
术语“低频密码子”是指在Kazusa和基因组数据库中，以低频率出现的密码子，例如Ala(GCG)、Leu(CUC)、Arg(CGG和CGC)和Ser(UGG)。术语“最优的同义密码子”，也称最优密码子(Optimal codon)，是指生物体中倾向于使用的特定一种或几种同义密码子，即密码子偏好性(codon bias)。
AAVS1位点是位于人类PPP1R12C基因第一个内含子中的一段特定序列。在该区域内引入外源核苷酸序列已被证明不会影响PPP1R12C基因或其他内源基因的表达，并且对培养细胞的毒性很小。因此,理论上可以通过在此区段引入各种外源基因及调控序列，以实现可控表达目的基因、治疗相应疾病的目的。现有技术已知的基因组其它安全插入位点也可以被被发明采用，例如AAVS1、CCR5、ROSA26、LMO2、CCDN2、BMI1\MECOM\PRDM1、H11、HBB03、HBB04、FANCF02、RUNX1、ZSCAN2(Safe harbours for the integration of new DNA in the human genome,Nature Reviews Cancer volume 12,pages51–58(2012)；Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11locus in pigs,Scientific Reports volume 5,Article number:14253(2015)；Directed evolution of CRISPR-Cas9 to increase its specificity,Nature Communications volume 9,Article number:3048(2018))。
本申请中涉及的E1区可包含启动子，所述启动子位于E1区编码序列上游，能够启动E1区基因的表达。例如以mPGK为启动子的E1区序列，用于启动E1A的表达；或以SV40为启动子。现有技术已知的能够启动基因转录和表达的其它启动子也可以运用到本发明中。
mPGK、SV40是真核系统中常用的组成型启动子，但同一启动子在不同的细胞中，往往启动下游基因表达的能力不一样，所以本专利采用了两种不同的启动子进行筛选。E1A不仅具有激活其他早期基因的作用，同时也有促进细胞凋亡的作用，会显著影响病毒产量，而 E1B-55K则具有抗凋亡和促进病毒晚期RNA出核的作用，E1B-19K与细胞Bcl-2基因的表达产物同源，可以通过灭活和清除Bax家族成员来防止细胞发生凋亡或坏死。
此外，上述2种设计中E1区的部分核酸序列进行同源密码子及人源化优化，避免改造的溶瘤腺病毒在包装和生产过程中因同源重组产生野生型腺病毒，同时优化后的序列整入细胞基因组后能够稳定表达腺病毒生产和包装所需的E1区蛋白：E1A、E1B-19K和E1B-55K。
本发明采用CRISPR/Cas9基因编辑技术，将腺病毒E1区核苷酸序列通过同源密码子替代、人源化优化以及启动子替换等改造后，插入对腺病毒易感的癌细胞系Hela-S3的基因组中的安全位点，使其能稳定表达腺病毒复制包装所需的E1区蛋白，在不降低腺病毒产量的前提下，完全解决了腺病毒在包装与生产过程中通过同源重组产生包含野生型E1区的腺病毒的问题，以及复制缺陷型腺病毒在本底细胞中的包装盒生产问题。
实施例
下面结合具体实施例，进一步阐述本发明的一些优选的实施方式和方面，这些实施例不应被解释为限制本发明的范围。
实施例1质粒构建
构建用于基因编辑的pCRISPR/Cas9-T2质粒、携带目的基因的Donor-mPGK和Donor-SV40质粒
1.构建pCRISPR/Cas9-T2质粒
构建流程参见图1。pCRISPR/Cas9-T2质粒是基于CRISPR/Cas9技术的载体质粒，其功能是表达野生型spCas9蛋白和sgRNA(T2)，从而特异性的在人类基因组19号染色体的AAVS1位点处5’-ggggccactagggacagGATTGG-3’(小写和大写碱基之间)产生19号染色体双链断裂(Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells.Stem Cell Res.2014 May；12(3):630-7)。
1.1使用High-Efficiency gRNA-Cas9-GFP Plasmid Assembly Kit(南京金斯瑞生物科技有限公司：L00692-10)依照其说明书合成如表1所示的引物。
表1
变性后梯度退火，形成带粘性末端的双链DNA片段，命名为T2。
1.2 BbsI-HF(NEB：R3539S)酶切pCRISPR/Cas9-GFP载体并插入1.1形成的DNA片段，连接后转化DH5a感受态细胞，挑取单克隆鉴定并扩增携带插入片段的pCRISPR/Cas9-T2质粒。
1.3制备pCRISPR/Cas9-T2质粒用于后续实验。
2.构建目的基因敲入所需的供体模板质粒：Donor-mPGK、Donor-SV40
野生型E1区序列(包括E1A编码区、IRES、E1B编码区)：

发明人经过多次尝试，对野生型E1区序列进行同义密码子改造：
1)野生型E1区序列的低频密码子被所述细胞中最优的同义密码子替换；2)碱基重复减少；3)避免野生型E1区序列中的一些限制酶识别位点；4)GC含量提高；5)避免转录获得的mRNA具有二级结构；6)避免密码子重复导致的长重复序列。此外，考虑翻译动力学、翻译后折叠和代谢水平等因素。
获得的一些改造后E1区序列示例如下：
E1-Modified-1(简称E1-M1)，即实施例中重组质粒Donor-mPGK和Donor-SV40包含的E1区：对E1A和E1B编码区序列进行了改造，与野生型序列比对同源性为96％。


作为示例，改造后E1-M1与野生型E1的前200个核苷酸的比对如图3所示。
E1-Modified-2(简称E1-M2)，即实施例中的IRES-Donor E1区：全E1区均经过改造，与野生型序列比对同源性为17.22％。RES-Donor E1区的设计是为了彻底地将E1区改造成与野生型E1区不同，在完成人源同义密码子优化的基础上，还调整了E1区内基因的排列顺序，将野生型E1区中的基因顺序E1A\E1B19K\E1B55K改变为E1B19K\E1B55K\E1A。

E1-Modified-3(简称E1-M3)：对E1A编码区和部分E1B55K编码区进行改造，与野生型序列比对同源性为93.4％。

E1-Modified-4(简称E1-M4)：在整个E1编码区范围进行改造，与野生型序列比对同源性为85.3％。

E1-Modified-5(简称E1-M5)：与野生型序列比对同源性为96.3％。

E1-Modified-6(简称E1-M6)：与野生型序列比对同源性为93.6％。


本文中mPGK启动子的序列为：
本文中SV40启动子的序列为：
以上片段均可通过基因合成获得。
Donor质粒的作用是与pCRISPR/Cas9-T2质粒共转染靶细胞，作为DNA双链断裂后同源修复的模板，将序列修饰后的腺病毒E1区序列通过同源修复的方式定点插入到靶细胞基因组中的AAVS1-T2位点，使靶细胞稳定表达插入的E1区基因，为腺病毒的复制创造有利条件，同时避免同源重组产生带有野生型E1区的腺病毒。
2.1从NCBI上查询人类基因组AAVS1-T2位点上游和下游各800bp的基因组序列(参见下文)作为Donor载体上的同源臂，分别置于待敲入基因的上下游，为靶细胞内Cas9切断DNA后的同源修复提供同源片段；根据野生型腺病毒E1区序列设计插入目的序列；再在目的序列下游添加Puromycin抗性筛选标记基因；在Puromycin抗性筛选标记基因的两端添加Cre/LoxP重组酶系统识别位点loxP，用于后期去除重组细胞株基因组中的Puromycin抗性基因。
基因组中AAVS1-T2位点上游序列为：

基因组中AAVS1-T2位点下游序列为：
2.2直接合成所需DNA片段(上海捷瑞生物工程有限公司)，然后插入到pMD19-T载体上(Donor-SV40插入位点是SmaI，Donor-mPGK插入位点是MluI/XhoI)，得到重组质粒Donor-mPGK、Donor-SV40。
2.3将上述重组质粒转化DH5a，挑取单克隆，鉴定质粒，制备无内毒素质粒。构建成功的供体模板序列Donor-mPGK(SEQ ID NO:27)和Donor-SV40参见序列表(SEQ ID NO:28)。
实施例2稳定细胞系的第一轮筛选和鉴定
将实施例1获得的质粒转染靶细胞，在靶细胞基因组中插入改造后的E1基因，筛选稳 定表达E1A蛋白和E1B蛋白的单克隆细胞株。
现有技术中溶瘤腺病毒易感的细胞系均可作为靶细胞系进行基因编辑，例如A549、A375、HeLa、SW620、22RV1、MDA-MB-435S、ES-2、HCC1806细胞等人源细胞。本实施例使用HeLa S3(ATCC CCL-2.2)细胞系。
1.转染和抗性筛选
使用Effectene转染试剂(QIAGEN：301425)共转1μg pCRISPR/Cas9-T2质粒和4μg Donor质粒至4*105HeLa S3细胞。转染后48h，使用含2.5μg/ml puromycin及10％FBS(Sunrise)的高糖DMEM(Gibco)完全培养基筛选具有puromycin抗性的HeLa S3克隆，持续筛选10天左右后挑选单克隆细胞。
2.筛选单克隆细胞株
使用有限稀释法挑选和培养步骤1抗性筛选后的单克隆细胞株。具体方式如下：消化步骤1筛选后的抗性克隆，将细胞悬液稀释至10cells/ml，向稀释后的细胞悬液中加入终浓度0.25μg/ml的Puromycin以保持选择压力，将细胞悬液以100μl/well接种至6块96孔板中。每48h观察细胞状态，并标记只有单一细胞群落的孔。每96h换液，至标记孔生长至有明显接触抑制时传代，扩大至48孔板并保持每48h观察细胞密度，96h换液。长满后扩大至24孔板、12孔板、6孔板，直至6cm培养皿。
3.鉴定阳性细胞株
3.1 PCR鉴定插入片段
提取上述单克隆细胞株基因组，PCR扩增AAVS1-T2位点DNA片段，检测插入基因是否正确存在于细胞基因组中指定插入位点。具体方式如下：
胰酶消化上步6cm培养皿中细胞，计数，取出约106个细胞进行离心，重悬于200μl培养基中，剩余细胞转移至10cm培养皿中扩大培养。
使用磁珠法基因组提取试剂盒提取上述细胞样品的基因组。
使用PCR鉴定样品基因组中是否插入目的基因片段，体系如下：
表2 PCR体系

表3引物
PCR反应程序如表4：
PCR实验结果如图3和图4所示。
注：HeLa S3-mPGK-1代表Donor-mPGK稳转入HeLa S3细胞后第一轮单克隆挑选中的1号细胞克隆；HeLa S3-SV40-1代表Donor-SV40稳转入HeLa S3细胞后第一轮单克隆挑选中的1号细胞克隆，依此类推。
结论：由图3和图4可知，在挑选的所有单克隆中，均成功插入了目的基因片段。
使用PCR鉴定样品基因组中是否插入完整目的基因片段
使用提取的基因组作为模板，用50μl Taq酶体系扩增其中插入基因片段，进行琼脂糖凝胶成像，切胶回收目的片段并送测序。引物信息如下：
表5引物
电泳结果如图5所示。
结论：从图5可知，HeLa S3-mPGK-4和HeLa S3-SV40-1、HeLa S3-SV40-2、HeLa S3-SV40-3均能扩增出目的片段。
3.2WB检测E1A蛋白的表达
取HeLa S3-SV40-1、HeLa S3-SV40-22、HeLa S3-SV40-3、HeLa S3-mPGK-4和HeLa S3细胞，以每孔5*105个细胞铺至6孔板。
24h后使用RIPA裂解液裂解细胞，收取蛋白，BCA法测定各样品总蛋白浓度，使用5×SDS缓冲液处理各样品，100℃煮沸10min。
各样品取15μg进行SDS-PAGE电泳，转膜，WB检测E1A的表达，结果如图6所示。
结论：HeLa S3-SV40-1、HeLa S3-SV40-2、HeLa S3-SV40-3和HeLa S3-mPGK-4均能表达E1A蛋白，HeLa S3本底不表达。说明插入的目的基因均能正常表达。
实施例3细胞株的单细胞病毒产量测试和第二轮筛选
检测实施例2筛选获得的各单克隆细胞株的单细胞病毒产量(单产)
1.单细胞病毒产量(单产)测试
取HeLa S3、HeLa S3-SV40-1、HeLa S3-SV40-2、HeLa S3-mPGK-4和HEK-293A细胞，以每个T175方瓶1.6*107个细胞进行接种。
24h后以500vp/cell的感染比例接种测试病毒YSCH-01。
60h后收获上述病毒样品，反复冻融3次后以2000rpm离心5min，取上清，使用0.22μm针头滤器进行过滤。
HPLC(岛津，LC-2030C)上机检测各样品的病毒颗粒浓度。
使用腺病毒滴度检测试剂盒(Cell Biolabs，VPK-109)检测各样品的病毒滴度。结果如图7所示。
结论：由图7可知，HeLa S3-SV40-1、HeLa S3-SV40-2和HeLa S3-mPGK-4的病毒单产都显著高于HeLa S3对照组，且高于常规腺病毒生产使用的HEK-293A细胞，说明各单克隆细胞株中插入的目的基因均能正常发挥功能，促进腺病毒的复制。
2.HeLa S3-mPGK细胞株第二轮筛选及鉴定
对HeLa S3-mPGK-4细胞株进行第二轮单克隆筛选，参照实施例2进行有限稀释法挑选单克隆细胞株，共5株，待细胞长至6cm dish后对细胞命名HeLa S3-mPGK-1A、HeLa S3-mPGK-1B、HeLa S3-mPGK-1C、HeLa S3-mPGK-1D、HeLa S3-mPGK-1E。HeLa S3-mPGK-1A表示HeLa S3-mPGK-4细胞第二轮单克隆筛选时选择的1号，HeLa S3-mPGK-1B表示HeLa S3-mPGK-4细胞第二轮单克隆筛选时选择的2号，以此类推。提取第二轮单克隆细胞株的基因组进行PCR鉴定，结果如图8所示。
结论：由图8可知，HeLa S3-mPGK第二轮单克隆挑选的5株均扩增出与目的片段大小相符合的片段。
实施例4 HeLa S3-SV40和HeLa S3-mPGK细胞株的单产比较
按照80％密度铺细胞，其中HeLa S3和HEK 293铺3*106细胞/dish；HeLa S3-SV40-1、HeLa S3-SV40-2、HeLa S3-SV40-3铺4*106细胞/dish；HeLa S3-mPGK-1A、HeLa S3-mPGK-1B、HeLa S3-mPGK-1E铺3.8*106细胞/dish。
第二天以600vp/cell转染YSCH-01病毒。
约48h后收获病毒，反复冻融3次后2000rpm离心5min收上清液，使用0.22μm针头滤器进行过滤。
HPLC上机测各病毒样品vp，选择单产结果最好的2株细胞，分别是HeLa S3-SV40-3和HeLa S3-mPGK-1E细胞(详细单产结果见表6)。
表6各细胞株的病毒产量结果
结论：经病毒扩增能力验证，改造后细胞株的病毒扩增能力与出发细胞株HeLa S3在同一数量级。其中，HeLa S3-mPGK-1E和HeLa S3-SV40-3这2株细胞病毒扩增能力较佳，后续选用这2株细胞敲除Puromycin抗性基因。
实施例5 IRES区改造后细胞株的挑选及单产毒比较
按照上述实施例方法，采用IRES-Donor E1区(SEQ ID NO:3)改造细胞株。
筛选获得单克隆后，PCR检测单克隆细胞的E1A的mRNA相对表达水平，结果如图9所示。可以看出，HeLa S3与其他HeLa S3-IRES的单克隆细胞间存在着显著差异。
IRES改造细胞单产毒比较，按照前述实施例进行操作。
数据分析结果如表7所示。
表7 IRES改造细胞单产毒比较结果

结论：在A组和B组中HeLa S3-IRES的几株单克隆细胞产毒能力没有另外2种改造细胞好，在之后的验证中不对此种改造的细胞株进行验证
实施例6敲除改造后细胞株中的Puromycin抗性基因
转染Cre/LoxP质粒敲除各单克隆细胞株中的Puromycin抗性基因，Cre/LoxP质粒
在六孔板中，HeLa S3-SV40-3和HeLa S3-mPGK-1E细胞各铺1孔，5.5×105个细胞。使用Effectene转染试剂(QIAGEN：301425)转染0.4μg Cre/LoxP质粒至上述孔中，用于敲除Puromycin抗性基因。
转染48h后，进行有限稀释法挑选单克隆细胞。具体为：消化细胞，将细胞悬液稀释至4cells/ml，然后以100μl/well铺板6块96孔板。每48h观察细胞状态，并标记只有单一细胞群落的孔。每96h换液，至标记孔生长至有明显接触抑制时传代扩大至48孔板，保持每48h观察细胞密度，96h换液，长满后扩大至24孔板。
第一轮Puro抗性筛选：将24孔板中的细胞一分为二，均分至2个孔中，一孔中加入2.5μg/ml的Puro抗性，另一孔不加。观察加入Puro抗性的孔中细胞是否死亡，若死亡则说明Puro抗性基因被成功敲除，待对应不加Puro抗性的孔中的细胞长满，传代至12孔板中，然后传代至6孔板中，再扩大至6cm dish。(经Puro杀伤筛选后得到的细胞命名为HeLa SV40-3-1A等，其中HeLa SV40-3-1表示HeLa SV40-3细胞敲除Puromycin抗性基因后第1轮单克隆，“A”表示细胞扩大到6孔板时的排序，即第1个扩大至6cm dish的为HeLa SV40-3-1A，第2个扩大至6cm dish的为HeLa SV40-3-1B)。
消化上述6cm dish中的细胞，计数，取出约106个细胞进行离心，重悬于200μl培养基中，剩余细胞接种至10cm dish中扩大培养。
使用磁珠法基因组提取试剂盒提取细胞样品基因组。
使用PCR鉴定样品基因组中是否敲除Puromycin抗性基因。
表8 PCR引物
实验结果如图10A、10B所示。
结论：由图10可知，成功敲除Puromycin抗性基因的单克隆细胞有：HeLa S3-SV40-3-1B；HeLa S3-SV40-3-1C；HeLa S3-SV40-3-1E；HeLa S3-SV40-3-1G；HeLa S3-SV40-3-1H；HeLa S3-SV40-3-1I；HeLa S3-SV40-3-1J；HeLa S3-SV40-3-1K；HeLa S3-SV40-3-1M；HeLa S3-SV40-3-1O和HeLa S3-mPGK-1E-1A；HeLa S3-mPGK-1E-1B。
实施例7检测各细胞株的病毒产量
使用敲除Puromycin抗性基因后的单克隆细胞进行病毒扩增测试，选取扩增结果较好的细胞株进行后期实验。
1.检测细胞株对复制型病毒的生产能力
3*106个细胞每10cm dish铺HeLa S3和HEK 293细胞；4*106个细胞每10cm dish铺HeLa S3-SV40-3-1B、HeLa S3-SV40-3-1C、HeLa S3-SV40-3-1E、HeLa S3-SV40-3-1G、HeLa S3-SV40-3-1H、HeLa S3-SV40-3-1I、HeLa S3-SV40-3-1J、HeLa S3-SV40-3-1K、HeLa S3-SV40-3-1M、HeLa S3-SV40-3-1O细胞；3.8*106个细胞每10cm dish铺HeLa S3-mPGK-1E-1A、HeLa S3-mPGK-1E-1B。其中每个细胞铺2盘。
第二天取其中1盘10cm dish细胞消化计数，向另一盘细胞中以600vp/cell转染YSCH-01病毒。收集病毒进行vp检测，结果见表9。
表9细胞株包装病毒的产量

结论：经病毒扩增能力验证，改造后细胞株的病毒扩增能力与出发细胞株HeLa S3在同一数量级。其中，HeLa S3-SV40-3-1C和HeLa S3-mPGK-1E-1B的产量最高，因此选用HeLa S3-SV40-3-1C和HeLa S3-mPGK-1E-1B细胞进行puromycin敲除后第二轮单克隆挑选。
2.检测基因组中的插入片段
进行PCR扩增，检测各样品基因组中插入基因片段，结果如图11A-D所示。
表10 PCR引物
结论：HeLa S3-SV40-3-1C细胞成功敲除Puromycin抗性基因，且敲除后插入目的序列的位置和长度均符合预期。HeLa S3-mPGK-1E-1A和HeLa S3-mPGK-1E-1B扩增出目的条带，还存在一些杂带，需进行第二次单克隆挑选。
实施例8对HeLa S3-SV40-3-1C和HeLa S3-mPGK-1E-1B细胞进行第二轮单克隆挑选
第二轮Puromycin抗性基因敲除后单克隆筛选：消化细胞，将细胞悬液稀释至4cells/ml，将细胞悬液100μl/well铺板3块96孔板。每48小时观察细胞状态，并标记只有单一细胞群落的孔。每96小时换液，至标记孔生长至有明显接触抑制时传代扩大至48孔板并保持每48小时观察细胞密度，96小时换液。长满后扩大至24孔板，12孔板中，然后6孔板以此类推扩大至6cm dish。(待细胞扩大至6cm dish后命名为HeLa S3-SV40-3-1C-2A等，表明 细胞是从第一轮Purok抗性基因敲除后的HeLa S3-SV40-3-1C单克隆细胞中进行第二轮单克隆筛选的第1个长至6cm dish的细胞)
消化上述步骤中6cm dish中细胞，细胞计数，取出约106个细胞离心，重悬于200μl体积PBS/培养基中，剩余细胞扩大培养至10cm dish中扩大培养。
按照底面积的80％铺细胞，每个细胞铺2个10cm dish，第二天待细胞稳定后取其中一盘进行计数。将计数后的细胞接种病毒，其中HEK 293和HeLa S3细胞按照600vp/cell转染YSCH-01病毒，其余细胞按照300vp/cell转染YSCH-01病毒。
表11第二轮单克隆细胞株单产结果
结论：经病毒扩增能力验证，改造后细胞株的病毒扩增能力与出发细胞株HeLa S3在同一数量级。其中，HeLa S3-SV40-3-1C-2G和HeLa S3-mPGK-1E-1B-2D细胞的产毒效果最佳。
使用组织/细胞基因组提取试剂盒提取细胞中的基因组，进行PCR扩增样品基因组中插入基因片段，结果如图12-14所示。
表12 PCR扩增引物
结论：HeLa S3-SV40-3-1C-2G、HeLa S3-SV40-3-1C-2J细胞不仅成功敲除Puromycin抗性基因，且插入序列的位点和长度与预期相符，为纯合子细胞。但是HeLa S3-mPGK-1E-1B-2A、HeLa S3-mPGK-1E-1B-2B、HeLa S3-mPGK-1E-1B-2C、HeLa S3-mPGK-1E-1B-2D细胞虽然成功敲除Puromycin抗性基因且插入序列的位点和长度与预期相符，但是这2个细胞是杂合子细胞。
实施例9检测单克隆细胞系的病毒包装能力
使用LIP3000转染试剂(赛默飞：L3000-015)转染0.5μg经PacI(NEB：R0547L)酶切鉴定成功的pBXYA07-U2-EGFP质粒至10万HeLa S3-SV40-3-1B细胞。(注：由于挑单克隆和包毒时间都比较久，因此使用第一轮单克隆的细胞进行包毒验证。)
经显微镜观察，直到所有细胞发荧光或者细胞出现病变后进行收毒，此时成功包装出OncoMul-V2-EGFP病毒。
验证病毒包装是否成功，3*106个细胞每10cm dish铺HEK 293细胞(ATCC：CRL-1573)、HeLa S3细胞(ATCC CCL-2.2)，4*106个细胞每10cm dish铺HeLa S3-SV40-3-1C-2G细胞，其中每个细胞铺1盘。均加入400μl病变的细胞。待细胞病变后进行收毒。HPLC上机测15.5中的各个样品。
表13改造细胞包装、扩增病毒结果
结论：由表13可知，经Puromycin抗性基因敲除后的改造细胞能够包装出病毒，再次表明构建成功。
实施例10细胞连续扩增病毒后野生型病毒的鉴定
将实施例9获得的细胞系用于病毒的扩增，扩增不同代数后，检测收获的病毒中野生型病毒的含量。
每个T75方瓶铺3*106个HeLa S3-Mock、HEK 293细胞，每个T75方瓶铺6*106个HeLa S3-SV40-3-1C-2G细胞，其中每个细胞铺2个方瓶，1个用于计数，另1个用于接种病毒。
HEK 293细胞中加入实施例9中使用HEK 293细胞扩增的病毒，其他细胞加入实施例9中使用HeLa S3-SV40-3-1C-2G细胞扩增的病毒，均按照400vp/cell接毒，进行第2代病毒扩增。然后照此连续扩增10代病毒，在第4代产毒时，HeLa S3-SV40-3-1C-2G铺3个T75 方瓶，接毒时1个用于细胞计数，1个加HeLa S3-SV40-3-1C-2G细胞第3次扩增的病毒，最后1个加HEK 293细胞第3次扩增的病毒，均按照400vp/cell接毒。收获病毒后进行vp检测。第5代和第10代病毒的扩增结果见表14。实验结果如图15所示。
表14第5代和第10代病毒的扩增结果
注：经HEK 293传代表示在第4代病毒扩增时HeLa S3-SV40-3-1C-2G细胞加入的毒种为HEK 293细胞第3代产生的病毒。
提取第10代病毒的基因组，进行qPCR定量包含野生型E1区序列的病毒，引物见表15，加样体系见表16，扩增程序见表17，标品稀释见表18。
表15包含野生型E1区序列的病毒定量检测引物
表16野生型腺病毒定量检测qPCR加样表
表17 qPCR扩增程序
表18野生型腺病毒定量检测标准品稀释表

统计结果如图15所示。由图15可知，以改造细胞包装出的OncoMul-V2-EGFP病毒为毒种，在HEK293细胞中连续扩增10代后会出现明显的野生型腺病毒，显著影响了重组腺病毒的质量；在HEK293细胞中连续扩增3代后再在HeLa S3-SV40-3-1C-2G细胞连续扩增7代，同样会出现明显的野生型腺病毒，甚至高于在HEK293细胞中连续扩增10代的量；相反地，在HeLa S3、HeLa S3-SV40-3-1C-2G细胞中连续扩增10代均检测不到野生型腺病毒，说明在HeLa S3、HeLa S3-SV40-3-1C-2G细胞中不会重组产生野生型腺病毒，表现出更好的安全性。
实施例11复制缺陷型腺病毒的包装、扩增
以Ad-EGFP为例，检测本发明构建的细胞系对复制缺陷型腺病毒的包装及扩增。
HEK 293、HeLa S3、HeLa S3-SV40-3-1C-2G进行铺6孔板，其中HEK 293、HeLa S3每孔铺2*105细胞，HeLa S3-SV40-3-1C-2G每孔铺4*105。
各孔细胞均经PEI转染试剂转染1μg经PacI酶切的非复制型腺病毒包装质粒pAd-EGFP。
转染2天后在荧光显微镜下观察，各孔均有荧光。转染7天后HeLa S3细胞生长过量且没有观察到荧光变多，HeLa S3-SV40-3-1C-2G虽然细胞生长变多，但荧光也在增加。此时HEK 293细胞已经出现病变，收集病毒，其余2个细胞继续培养，直至病变收毒。
转染23天后HeLa S3-SV40-3-1C-2G细胞出现病变，且荧光强度高。而HeLa S3由于细胞生长过多而脱落死亡，没有荧光，说明复制缺陷型病毒不能在HeLa S3中包装。HeLa S3-SV40-3-1C-2G细胞包装病毒过程中细胞形态变化参见图16。
3*106个细胞每个10cm dish铺HeLa S3和HEK 293，6*106个细胞每个10cm dish铺HeLa S3-SV40-3-1C-2G，其中每个细胞铺2个10cm dish，1个用于接毒，1个作为对照。
将上述细胞经过18h培养后进行接毒，接毒量为500μl的由HeLa S3-SV40-3-1C-2G细胞包装的Ad-EGFP病毒。
接毒4天后在镜下观察HEK 293细胞病变，对其进行收毒。接毒6天后在镜下观察HeLa S3-SV40-3-1C-2G细胞病变，收集病毒。但未观察到HeLa S3病变，在荧光显微镜下也未观察到明显荧光，说明HeLa S3细胞不适合用于扩增新包装(低病毒浓度)的复制缺陷型腺病毒。
对上述病毒进行vp检测，结果见表19。
表19复制缺陷型腺病毒第2次扩增结果
结论：改造细胞HeLa S3-SV40-3-1C-2G不仅可以包装复制缺陷型腺病毒，还能在其中快速扩增新包装(低病毒浓度)的复制缺陷型腺病毒，显著强于HeLa S3细胞。
对HEK 293和HeLa S3-SV40-3-1C-2G细胞扩增的Ad-EGFP病毒进行滴度测定，结果见表20。
表20 HEK 293与HeLa S3-SV40-3-1C-2G产毒滴度比较
结论：由表20可知，HeLa S3-SV40-3-1C-2G与HEK 293细胞扩增的Ad-EGFP的比滴度无明显差异。
以上结果说明，改造后的HeLa S3-SV40-3-1C-2G细胞不仅能够在不引入野生型腺病毒的前提下扩增E1区存在的复制型腺病毒，且扩增能力不弱于HEK293细胞，也能包装和扩增E1区功能缺陷的腺病毒。
本申请中提及的所有公开物和专利通过引用方式并入本文。不脱离本发明的范围和精神，本发明的所描述的方法和组合物的多种修饰和变体对于本领域技术人员是显而易见的。虽然通过具体的优选实施方式描述了本发明，但是应该理解所要求保护的本发明不应该被不 适当地局限于这些具体实施方式。事实上，那些对于相关领域技术人员而言显而易见的用于实施本发明的所描述的模式的多种变体意在包括在随附的权利要求的范围内。

Claims (14)

  1. 一种腺病毒包装与生产细胞，其中所述细胞的基因组包含改造的腺病毒E1区序列，所述改造的腺病毒E1区序列与野生型E1区序列相比：1)野生型E1区序列中的低频密码子被所述细胞中最优的同义密码子替换；2)碱基重复减少；3)避免野生型E1区序列中的一些限制酶识别位点；4)GC含量提高；5)避免转录获得的mRNA具有二级结构；6)避免密码子重复导致的长重复序列；
    所述改造的腺病毒E1区序列编码的蛋白具有与野生型E1A和E1B蛋白相同的功能，优选地，所述改造的腺病毒E1区序列编码的蛋白具有与野生型E1A和E1B蛋白相同的氨基酸序列。
  2. 如权利要求1所述的细胞，其中所述改造的腺病毒E1区序列选自SEQ ID NO:2和SEQ ID NO:4-7。
  3. 如权利要求2所述的细胞，其中所述改造的腺病毒E1区序列为SEQ ID NO:2。
  4. 如权利要求1-3任一所述的细胞，其中所述改造的腺病毒E1区序列还包含启动子，优选地，所述启动子选自SV40启动子和mPGK启动子。
  5. 如权利要求1-4任一所述的细胞，其中所述细胞是人源细胞。
  6. 如权利要求5所述的细胞，其中所述细胞选自来源于A549、A375、HeLa、SW620、22RV1、MDA-MB-435S、ES-2和HCC1806细胞。
  7. 如权利要求6所述的细胞，其中所述细胞为HeLa S3细胞。
  8. 如权利要求1-7任一所述的细胞，其中所述改造的腺病毒E1区序列在所述细胞基因组中的插入位点选自AAVS1、CCR5、ROSA26、LMO2、CCDN2、BMI1\MECOM\PRDM1、H11、HBB03、HBB04、FANCF02、RUNX1和ZSCAN2。
  9. 如权利要求8所述的细胞，其中所述改造的腺病毒E1区序列在所述细胞基因组中的插入位点为AAVS1位点。
  10. 如权利要求1-9任一所述的细胞，其中所述细胞能够包装与生产复制缺陷型腺病毒和复制型腺病毒，在复制型腺病毒连续传代时不产生野生型腺病毒。
  11. 一种构建如权利要求1-10任一所述的腺病毒包装与生产细胞的方法，包括：
    对野生型E1区序列进行改造，获得改造的腺病毒E1区序列；
    将所述改造的腺病毒E1区序列插入宿主细胞基因组；
    其中，所述改造的腺病毒E1区序列与野生型E1区序列相比：1)野生型E1区序列的低频密码子被所述细胞中最优的同义密码子替换；2)碱基重复减少；3)避免野生型E1区序列中的一些限制酶识别位点；4)GC含量提高；5)避免转录获得的mRNA具有二级结构；6)避免密码子重复导致的长重复序列；
    所述改造的腺病毒E1区序列编码的蛋白具有与野生型E1A和E1B蛋白相同的功能。
  12. 如权利要求11所述的方法构建获得的腺病毒包装与生产细胞。
  13. 如权利要求1-10任一所述的腺病毒包装与生产细胞和如权利要求11的方法构建的细胞在制备复制缺陷型腺病毒和复制型腺病毒中的用途。
  14. 一种生产/包装腺病毒的方法，包括：
    1)将腺病毒或编码腺病毒蛋白的多核苷酸导入权利要求1-10任一所述的腺病毒包装与生产细胞；
    2)培养步骤1)获得的细胞；和
    3)收集病毒。
PCT/CN2023/100118 2022-06-15 2023-06-14 一种腺病毒包装与生产细胞系的构建方法及应用 WO2023241610A1 (zh)

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