CN116376843B - Adeno-associated virus susceptibility cell strain and application thereof - Google Patents

Adeno-associated virus susceptibility cell strain and application thereof Download PDF

Info

Publication number
CN116376843B
CN116376843B CN202310586411.2A CN202310586411A CN116376843B CN 116376843 B CN116376843 B CN 116376843B CN 202310586411 A CN202310586411 A CN 202310586411A CN 116376843 B CN116376843 B CN 116376843B
Authority
CN
China
Prior art keywords
adeno
cells
gene
cell
associated virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202310586411.2A
Other languages
Chinese (zh)
Other versions
CN116376843A (en
Inventor
孙秀莲
赵晟
郭典
朴顺福
陈瑶
王志民
仲明
张华�
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yiming Jinan Biotechnology Co ltd
Original Assignee
Jinan Yiming Medical Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jinan Yiming Medical Technology Co ltd filed Critical Jinan Yiming Medical Technology Co ltd
Priority to CN202310586411.2A priority Critical patent/CN116376843B/en
Publication of CN116376843A publication Critical patent/CN116376843A/en
Application granted granted Critical
Publication of CN116376843B publication Critical patent/CN116376843B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present disclosure provides adeno-associated virus-susceptible cell strains and uses thereof, and in particular, provides adeno-associated virus-susceptible cell strains and uses thereof. The cell strain is a cell strain with a preservation number of CCTCC NO: human kidney epithelial cell 293-AH cell of C202338 which stably expresses three genes of adenovirus (Ad 5): the E4 gene (Orf 6), the E2A gene and the VARNA gene have high susceptibility to adeno-associated viruses (AAV) of different serotypes, and can be used for quantitative test of infection titer of AAV.

Description

Adeno-associated virus susceptibility cell strain and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to three genes for stably expressing adenovirus (Ad 5) based on HEK293 cells: cell lines of E4 gene (Orf 6), E2A gene and VARNA gene. The invention also relates to the application of the cell strain in determining AAV infection titer.
Background
Along with the popularization of bioengineering technology in various fields of scientific research, food, chemical industry, medical treatment and the like, various DNA-based gene vectors are widely used, and a method using viruses as DNA delivery vectors is widely used. Among them, AAV (adeno-associated virus), a single-stranded linear DNA-deficient virus, requires a helper virus (adenovirus or herpes virus) to replicate in vivo when infected at the same time. Its advantages are high safety (no report of AAV to human), low immunogenicity, broad spectrum, long expression time, high dispersivity and stability. Thus rAAV (recombinant AAV) is suitable as a DNA vector for the treatment of gene-related clinical diseases.
The determination of rAAV titer is one of the very important vector quality assessment indicators in the fields of scientific research, clinical research and bioengineering pharmacy, in particular in the field of Cellular Gene Therapy (CGT), and the accuracy is remarkable in the practical significance of AAV-related application. The main measurement mode in production at present is to directly measure the genome titer (Vg) of rAAV by qPCR, and the method has the defects that the activity of AAV virus samples and the actual capability of infected cells cannot be reflected, and the number of virus particles can only be estimated by the DNA copy number in the samples; to more accurately reflect the viability of the rAAV and its actual ability to infect cells, it is necessary to determine the infection titer (informatios titer) of the rAAV. AAV infection does not produce cytopathic effects and therefore cannot be assayed for infectious titer by plaque assay, but in the case of co-participation of adenovirus (Ad) helper proteins, AAV can be induced to replicate in cells and express genes in the viral genome, whereby infected cells can be detected by detecting the expression of the replicated viral genome or reporter gene, and then the infectious titer of the original AAV virus can be estimated from the gradient dilution ratio.
There are several more common methods of measuring infection titres including: determination of the half-number infection of AAV (TCID) 50 ) The method comprises the steps of carrying out a first treatment on the surface of the The method uses AAV2 diluted in gradient and adenovirus (Ad) with certain concentration to co-infect existing AAV2repAndcap hela cell line with stable gene expression, and after AAV2 is replicated in cell for a certain period of time, the copy number of the newly replicated AAV2 genome sequence is detected by qPCR methodFrom this, the original AAV2 virus infection titer was estimated. Another method is ICA (infectious center assay); the detection method is similar to the previous method, AAV2 is still diluted in a gradient mode with the aid of Ad5, and the existing AAV2 is co-infectedrepAndcap the Hela cell line with stable gene expression is inoculated onto nylon membrane coated with hybridized probe, so that the infection titer of virus is calculated by analyzing the positive cell number on the membrane. In addition, the titer of infection can be determined by detecting the intensity of a reporter gene carried by the virus expressed by the infected cells. The method is to dilute the purified virus gradient, infect a certain amount of susceptible cells, and calculate the positive cell number expressing the reporter gene and the dilution ratio to calculate the infection titer of the virus. When used to detect infectious titres of AAV, adenovirus co-infection is often used to obtain expression of accessory genes in order to increase susceptibility to AAV.
In summary, when these conventional methods detect rAAV infection titers, the presence of helper viruses such as adenovirus is often required, the introduction and preparation of other virus assays involve not only waste of additional production material of another virus, long testing time (typically more than one week is required), but also adenovirus infection can lead to cytopathic effects, greater toxicity, and massive death of the test cells, affecting data statistics. Meanwhile, co-infection of multiple viruses can also cause data instability between different test batches. Furthermore, for test protocols requiring the use of cell lines stably expressing AAV2 Rep and Cap, the titer test is also affected by the toxicity of the Rep and Cap proteins to the cells. Thus, there is a need in the art to design new helper-free cells for detecting infectious titers of individual AAV serotypes.
Disclosure of Invention
In one aspect, provided herein is a CCTCC NO: human kidney epithelial cell 293-AH cells of C202338.
The cell strain 293-AH is deposited in China center for type culture Collection, address: university of chinese martial arts, date of preservation: 2023, 2 and 14 days, deposit number: cctccc NO: C202338.
in another aspect, provided herein is the use of the human kidney epithelial cell 293-AH cell described above for detecting viral titers of adeno-associated viruses.
In some embodiments, the human kidney epithelial cells 293-AH cells stably moderately express the three genes of adenovirus (Ad 5) by using a low promoter (minimal CMV): the E4 gene (Orf 6), the E2A gene and the VARNA gene reduce the toxicity of the expression products of the three genes to the 293-AH cells of the human kidney epithelial cells, thereby screening and obtaining stable cell strains.
In some embodiments, the adeno-associated virus may be selected from various serotypes of AAV, such as common AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, or other particular serotypes of AAV.
In some embodiments, the adeno-associated virus is a recombinant adeno-associated virus.
In another aspect, provided herein is a kit for detecting viral titer of adeno-associated virus comprising the human kidney epithelial cell 293-AH cell described above.
In another aspect, provided herein are adeno-associated viruses that can be selected from a wide variety of serotypes of AAV, such as common AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, or other particular serotypes of AAV.
In another aspect, provided herein is that the adeno-associated virus is a recombinant adeno-associated virus.
The human kidney epithelial cell 293-AH cell line provided herein stably expresses three genes of adenovirus (Ad 5): e4 gene (Orf 6), E2A gene and VARNA gene. Compared with HEK293 cells, the expression of target genes after rAAV infection cells can be improved, and the sensitivity and accuracy of measuring AAV infection titer can be improved under the condition of no helper virus.
Drawings
Fig. 1: pHelper plasmid map containing the gene plasmid of interest.
Fig. 2: the results were photographed under a fluorescence microscope after infection of rAAV-GL with 293-AH and HEK 293. 293-AH and HEK293 are infected with rAAV-GL of the same MOI (1E+05) and different serotypes (1/2/5/6), and the fluorescence intensity of the expression of the 293-AH is obviously higher than that of HEK293 cells. Graphs a-D: fluorescence microscopy pictures of rAAV-GL infected with HEK293 cells of different serotypes (1/2/5/6), respectively; graphs E-H: fluorescence microscopy photographs of rAAV-GL of different serotypes (1/2/5/6) were infected with 293-AH cells, respectively.
Fig. 3: the results were photographed under a fluorescence microscope after infection of rAAV-GL with 293-AH and HEK 293. 293-AH and HEK293 are infected with rAAV-GL of the same MOI (1E+05) and different serotypes (7/8/9), and the fluorescence intensity of the expression of the 293-AH is obviously higher than that of HEK293 cells. Graphs I-K: fluorescence microscopy pictures of rAAV-GL infected with HEK293 cells of different serotypes (7/8/9), respectively; graph L-N: fluorescence microscopy photographs of rAAV-GL of 293-AH cells infected with different serotypes (7/8/9), respectively.
Fig. 4: infection detection sensitivity of rAAV8-GL was detected by using 293-AH and HEK293 cells. The sensitivity of detection of 293-AH (significantly different from the virus-free blank) was more than 2-fold that of HEK293 cells.
Detailed Description
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
The experimental methods not specifically described in the invention are all carried out according to the specifications of related products. The biological agents used in the present invention, without specific description, are commercially available. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the spirit of the invention.
The main materials involved in the invention comprise plasmids synthesized by Anhui general biological company; HEK293 cells (american ATCC center); polyethylenimine (PEI, polyplus company, usa); recombinant AAV serotype 1 (rAAV 1-GL), serotype 2 (rAAV 2-GL), serotype 5 (rAAV 5-GL), serotype 6 (rAAV 6-GL), serotype 7 (rAAV 7-GL), serotype 8 (rAAV 8-GL) and serotype 9 (rAAV 9-GL) carrying the green fluorescent protein (NeonGreen) gene and the luciferase reporter gene (teLuc) were all purchased from Yiming cells, the plasmid pHelper involved in cell preparation, the genes comprising the E4 gene (Orf 6), the E2A gene and the VARNA gene and the puromycin resistance gene were chemically synthesized (general organisms).
EXAMPLE 1 screening of stably surviving monoclonal cells
This example describes the preparation of transgenic cell lines and the screening process, briefly as follows.
1) The plasmid pHelper was synthesized and the genes contained in this plasmid included the E4 gene (Orf 6), E2A gene, VARNA gene, and puromycin resistance gene (Puromycin R) (see FIG. 1);
2) Culturing HEK293 cells to logarithmic growth phase, inoculating 6-well cell culture plate, 2E6 cells/well;
3) The plasmid described in 1) was transfected by the PEI method in a daily dose of 1. Mu.g, PEI to plasmid ratio of 3:1, a step of;
4) After 72 hours of transfection, the adherent cells are digested, and after uniform mixing, monoclonal is selected in a 48-well plate, and 2 mug/mL puromycin is added into each well;
5) Screening out cells (HEK 293-1# -HEK293-16 #) which can be stably amplified.
Example 2 selection of cell lines
1) Culturing HEK293-1# -HEK293-16# selected in example 1; and HEK293 cells (HEK 293-0#) to log phase, 24 well cell plates, 5E5 cells/well;
5) Cells with stronger fluorescence in all the monoclonal cells were selected 48h after infection with rAAV8-GL (MOI 1E+05).
The relative fluorescence intensities (converted to comparison with the fluorescence intensity of cell line HEK293-0 #) are shown in Table 1.
。
We named the cell line HEK293-2# which can be stably passaged 293-AH, deposited with the chinese collection of typical cultures at: the preservation date of the university of Chinese Wuhan is: 2023, 2 and 14 days with deposit number: cctccc NO: C202338.
EXAMPLE 3 expression of the target protein after infection of rAAV by 293-AH cells
This example looks at infection of HEK293 cells with AAV of different serotypes.
The experimental procedure was as follows:
1) HEK293 cells and 293-AH cells (passaged 10 times) were cultured to log phase and inoculated with 24-well cell culture plates, 5E5 cells/well;
2) The two cells in the next day are respectively infected with rAAV1-GL, rAAV2-GL, rAAV5-GL, rAAV6-GL, rAAV7-GL, rAAV8-GL and rAAV9-GL, and MOI is 1E+05;
3) 48h and images were observed and acquired using a fluorescence microscope (488/520 green filter set).
As shown in FIGS. 2A-2H and FIGS. 3I-3N, the fluorescence intensity of 293-AH expression was significantly higher for different serotypes of AAV than for HEK293 cells.
Example 4 comparison of sensitivity of 293-AH cells to HEK293 cells after infection with rAAV
This example quantitatively examines the difference in sensitivity of 293-AH cells and HEK293 cells to rAAV8 infection.
The experimental procedure was as follows:
1) Inoculating cells: the 293-AH cells and HEK293 cells (HEK 293 cells transfected with pHelper plasmid and antibiotic selection as in example 1) were inoculated into bottom-permeabilized white 96-well plates, 2-thousand/well, 100. Mu.L per well volume, 37℃and 5% CO at 2-3 passages after resuscitation 2 Culturing for 16h;
2) Viral infection: 10 mu L of rAAV8-GL diluted in a 2-fold gradient is added into each hole, and infection is carried out for 48-72 hours;
3) Cell lysis: taking out the infected culture plate, allowing each hole to act for 5 min at room temperature by using Triton-X100 with a final concentration of 1%, and then placing under a fluorescence enzyme-labeling instrument for luciferase activity measurement;
4) Luciferase activity assay: luciferase substrate was added per well: diphenyl terazine (DTZ, final concentration 30. Mu.M), shaking and mixing well, and detecting the luminous intensity.
As shown in FIG. 4, 293-AH cells infected rAAV8 at least 2-fold more sensitive than HEK293 transfected with pHelper plasmid.
Conclusion(s)
The cell strain 293-AH stably expresses three genes of adenovirus (Ad 5): e4 gene (Orf 6), E2A gene and VARNA gene. Compared with HEK293 cells, 293-AH cells can improve the expression of target genes after rAAV infection cells, and can increase the sensitivity and accuracy of measuring AAV infection titer under the condition of no helper virus. The cell strain 293-AH provided by the invention is based on HEK293 cells commonly used in AAV production, and is prepared by using a low-promoter-activity promoter (minimum CMV) to stably and moderately express three genes of adenovirus (Ad 5): the E4 gene (Orf 6), the E2A gene and the VARNA gene reduce the toxicity of the exogenous adenovirus proteins to cells, unexpectedly obtain stable cell strains, and can be directly used for detecting AAV infection titer. The invention makes the AAV infection titer test faster (only 2-3 days), convenient, sensitive, accurate, stable and low cost, as no additional helper virus is required.
The minimal CMV promoter and gene sequences mentioned herein are as follows:
minimal CMV promoter: (SEQ ID NO: 1)
taggcgtgtacggtgggaggcctatataagcagagctggtttagtgaaccgtcagatcgcctggagtcgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggccc
Ad5 E4Orf6: (SEQ ID NO: 2)
ctacatgggggtagagtcataatcgtgcatcaggatagggcggtggtgctgcagcagcgcgcgaataaactgctgccgccgccgctccgtcctgcaggaatacaacatggcagtggtctcctcagcgatgattcgcaccgcccgcagcataaggcgccttgtcctccgggcacagcagcgcaccctgatctcacttaaatcagcacagtaactgcagcacagcaccacaatattgttcaaaatcccacagtgcaaggcgctgtatccaaagctcatggcggggaccacagaacccacgtggccatcataccacaagcgcaggtagattaagtggcgacccctcataaacacgctggacataaacattacctcttttggcatgttgtaattcaccacctcccggtaccatataaacctctgattaaacatggcgccatccaccaccatcctaaaccagctggccaaaacctgcccgccggctatacactgcagggaaccgggactggaacaatgacagtggagagcccaggactcgtaaccatggatcatcatgctcgtcatgatatcaatgttggcacaacacaggcacacgtgcatacacttcctcaggattacaagctcctcccgcgttagaaccatatcccagggaacaacccattcctgaatcagcgtaaatcccacactgcagggaagacctcgcacgtaactcacgttgtgcattgtcaaagtgttacattcgggcagcagcggatgatcctccagtatggtagcgcgggtttctgtctcaaaaggaggtagacgatccctactgtacggagtgcgccgagacaaccgagatcgtgttggtcgtagtgtcatgccaaatggaacgccggacgtagtcat
Ad5 E2A: (SEQ ID NO: 3)
atggccagtcgggaagaggagcagcgcgaaaccacccccgagcgcggacgcggtgcggcgcgacgtcccccaaccatggaggacgtgtcgtccccgtccccgtcgccgccgcctccccgggcgcccccaaaaaagcggatgaggcggcgtatcgagtccgaggacgaggaagactcatcacaagacgcgctggtgccgcgcacacccagcccgcggccatcgacctcggcggcggatttggccattgcgcccaagaagaaaaagaagcgcccttctcccaagcccgagcgcccgccatcaccagaggtaatcgtggacagcgaggaagaaagagaagatgtggcgctacaaatggtgggtttcagcaacccaccggtgctaatcaagcatggcaaaggaggtaagcgcacagtgcggcggctgaatgaagacgacccagtggcgcgtggtatgcggacgcaagaggaagaggaagagcccagcgaagcggaaagtgaaattacggtgatgaacccgctgagtgtgccgatcgtgtctgcgtgggagaagggcatggaggctgcgcgcgcgctgatggacaagtaccacgtggataacgatctaaaggcgaacttcaaactactgcctgaccaagtggaagctctggcggccgtatgcaagacctggctgaacgaggagcaccgcgggttgcagctgaccttcaccagcaacaagacctttgtgacgatgatggggcgattcctgcaggcgtacctgcagtcgtttgcagaggtgacctacaagcatcacgagcccacgggctgcgcgttgtggctgcaccgctgcgctgagatcgaaggcgagcttaagtgtctacacggaagcattatgataaataaggagcacgtgattgaaatggatgtgacgagcgaaaacgggcagcgcgcgctgaaggagcagtctagcaaggccaagatcgtgaagaaccggtggggccgaaatgtggtgcagatctccaacaccgacgcaaggtgctgcgtgcacgacgcggcctgtccggccaatcagttttccggcaagtcttgcggcatgttcttctctgaaggcgcaaaggctcaggtggcttttaagcagatcaaggcttttatgcaggcgctgtatcctaacgcccagaccgggcacggtcaccttttgatgccactacggtgcgagtgcaactcaaagcctgggcacgcgccctttttgggaaggcagctaccaaagttgactccgttcgccctgagcaacgcggaggacctggacgcggatctgatctccgacaagagcgtgctggccagcgtgcaccacccggcgctgatagtgttccagtgctgcaaccctgtgtatcgcaactcgcgcgcgcagggcggaggccccaactgcgacttcaagatatcggcgcccgacctgctaaacgcgttggtgatggtgcgcagcctgtggagtgaaaacttcaccgagctgccgcggatggttgtgcctgagtttaagtggagcactaaacaccagtatcgcaacgtgtccctgccagtggcgcatagcgatgcgcggcagaacccctttgatttttaaacggcgcagacggcaagggtgggggtaaataatcacccgagagtgtacaaataaaagcatttgcctttattgaaagtgtctctagtacattatttttacatgtttttcaagtgacaaaaagaagtggcgctcctaatctgcgcactgtggctgcggaagtagggcgagtggcgctccaggaagctgtagagctgttcctggttgcgacgcagggtgggctgtacctggggactgttgagcatggagttgggtaccccggtaataaggttcatggtggggttgtgatccatgggagtttggggccagttggcaaaggcgtggagaaacatgcagcagaatagtccacaggcggccgagttgggcccctgtacgctttgggtggacttttccagcgttatacagcggtcgggggaagaagcaatggcgctacggcgcaggagtgactcgtactcaaactggtaaacctgcttgagtcgctggtcagaaaagccaaagggctcaaagaggtagcatgtttttgagtgcgggttccaggcaaaggccatccagtgtacgcccccagtctcg
Ad5 VA RNA: (SEQ ID NO: 4)
Cagaagcaccatgtccttgggtccggcctgctgaatgcgcaggcggtcggccatgccccaggcttcgttttgacatcggcgcaggtctttgtagtagtcttgcatgagcctttctaccggcacttcttcttctccttcctcttgtcctgcatctcttgcatctatcgctgcggcggcggcggagtttggccgtaggtggcgccctcttcctcccatgcgtgtgaccccgaagcccctcatcggctgaagcagggctaggtcggcgacaacgcgctcggctaatatggcctgctgcacctgcgtgagggtagactggaagtcatccatgtccacaaagcggtggtatgcgcccgtgttgatggtgtaagtgcagttggccataacggaccagttaacggtctggtgacccggctgcgagagctcggtgtacctgagacgcgagtaagccctcgagtcaaatacgtagtcgttgcaagtccgcaccaggtactggtatcccaccaaaaagtgcggcggcggctggcggtagaggggccagcgtagggtggccggggctccgggggcgagatcttccaacataaggcgatgatatccgtagatgtacctggacatccaggtgatgccggcggcggtggtggaggcgcgcggaaagtcgcggacgcggttccagatgttgcgcagcggcaaaaagtgctccatggtcgggacgctctggccggtcaggcgcgcgcaatcgttgacgctctagaccgtgcaaaaggagagcctgtaagcgggcactcttccgtggtctggtggataaattcgcaagggtatcatggcggacgaccggggttcgagccccgtatccggccgtccgccgtgatccatgcggttaccgcccgcgtgtcgaacccaggtgtgcgacgtcagacaacgggggagtgctccttttggcttccttccaggcgcggcggctgctgcgctagcttttttggccactggccgcgcgcagcgtaagcggttaggctggaaagcgaaagcattaagtggctcgctccctgtagccggagggttattttccaagggttgagtcgcgggacccccggttcgagtctcggaccggccggactgcggcgaacgggggtttgcctccccgtcatgcaagaccccgcttgcaaattcctccggaaacagggacgagcccctttttt

Claims (7)

1. The preservation number is CCTCC NO: human kidney epithelial cell 293-AH cells of C202338.
2. Use of the human kidney epithelial cell 293-AH cell of claim 1 for detecting viral titers of adeno-associated viruses.
3. The use of claim 2, wherein the adeno-associated virus is selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9.
4. The use of claim 2, wherein the adeno-associated virus is a recombinant adeno-associated virus.
5. A kit for detecting the viral titer of an adeno-associated virus comprising the human kidney epithelial cell 293-AH cell of claim 1.
6. The kit for detecting the viral titer of an adeno-associated virus as claimed in claim 5, wherein the adeno-associated virus is selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 or AAV9.
7. The kit for detecting the viral titer of an adeno-associated virus as claimed in claim 5 or 6, wherein the adeno-associated virus is a recombinant adeno-associated virus.
CN202310586411.2A 2023-05-24 2023-05-24 Adeno-associated virus susceptibility cell strain and application thereof Active CN116376843B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310586411.2A CN116376843B (en) 2023-05-24 2023-05-24 Adeno-associated virus susceptibility cell strain and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310586411.2A CN116376843B (en) 2023-05-24 2023-05-24 Adeno-associated virus susceptibility cell strain and application thereof

Publications (2)

Publication Number Publication Date
CN116376843A CN116376843A (en) 2023-07-04
CN116376843B true CN116376843B (en) 2023-08-18

Family

ID=86964246

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310586411.2A Active CN116376843B (en) 2023-05-24 2023-05-24 Adeno-associated virus susceptibility cell strain and application thereof

Country Status (1)

Country Link
CN (1) CN116376843B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1657632A (en) * 2004-11-30 2005-08-24 华中科技大学同济医学院附属同济医院 Combination adeno-associated virus of expression human CYP2J2 antigene and its preparation method
CN1834255A (en) * 2005-12-23 2006-09-20 上海交通大学附属第一人民医院 Method of fast increasing glandular related virus mediating gene in expression of inside of retina cell
CN112280801A (en) * 2020-10-30 2021-01-29 武汉枢密脑科学技术有限公司 Method and kit for rAAV infection titer detection by plasmid assistance
CN112912506A (en) * 2018-10-17 2021-06-04 葛兰素史克知识产权开发有限公司 Adeno-associated virus vector producing cell lines

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4693244B2 (en) * 1999-03-18 2011-06-01 ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア Compositions and methods for helperless production of recombinant adeno-associated virus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1657632A (en) * 2004-11-30 2005-08-24 华中科技大学同济医学院附属同济医院 Combination adeno-associated virus of expression human CYP2J2 antigene and its preparation method
CN1834255A (en) * 2005-12-23 2006-09-20 上海交通大学附属第一人民医院 Method of fast increasing glandular related virus mediating gene in expression of inside of retina cell
CN112912506A (en) * 2018-10-17 2021-06-04 葛兰素史克知识产权开发有限公司 Adeno-associated virus vector producing cell lines
CN112280801A (en) * 2020-10-30 2021-01-29 武汉枢密脑科学技术有限公司 Method and kit for rAAV infection titer detection by plasmid assistance

Also Published As

Publication number Publication date
CN116376843A (en) 2023-07-04

Similar Documents

Publication Publication Date Title
Jungmann et al. Protocol for efficient generation and characterization of adeno-associated viral vectors
Liu et al. Neutralizing antibodies against AAV2, AAV5 and AAV8 in healthy and HIV-1-infected subjects in China: implications for gene therapy using AAV vectors
François et al. Accurate titration of infectious AAV particles requires measurement of biologically active vector genomes and suitable controls
JP2003310252A (en) Direct rescue of recombinant virus from cell dna of tissue, and method for amplification
CN112280801A (en) Method and kit for rAAV infection titer detection by plasmid assistance
WO2020224395A1 (en) Probe, primer pair, fluorescent quantitative pcr kit and method for detecting replicative lentivirus
US11926841B2 (en) AAV-based conditional expression system
WO2020224394A1 (en) Fluorescent probe, primer pair, fluorescent quantitative pcr kit and detection method for detecting lentivirus
CN101551392A (en) Method for detecting human papilloma virus neutralizing antibody
Trivedi et al. Comparison of highly pure rAAV9 vector stocks produced in suspension by PEI transfection or HSV infection reveals striking quantitative and qualitative differences
Ramsey et al. Overview of analytics needed to support a robust gene therapy manufacturing process
Mason Jr et al. Complementation between BK human papovavirus and a simian virus 40 tsA mutant
CN116376843B (en) Adeno-associated virus susceptibility cell strain and application thereof
WO2020078274A1 (en) Striatin interacting protein inhibitor and use thereof in preparation of anti-tumor drug
CN116836910A (en) Adeno-associated virus susceptibility cell strain and application thereof
Nagy et al. Engineered CHO cells as a novel AAV production platform for gene therapy delivery
Bowman et al. Transient expression of herpes simplex virus type 1 ICP22 represses viral promoter activity and complements the replication of an ICP22 null virus
Bleckwenn et al. Exploring vaccinia virus as a tool for large‐scale recombinant protein expression
US20230038151A1 (en) Methods of determining viral titer
JP2022526858A (en) Methods and Compositions for Improving Adeno-Associated Virus (AAV) Assembly
JP2023513903A (en) Methods for Determining the Loading State of AAV Particles by Nuclear Magnetic Resonance Relaxation Measurements
Leland et al. Virus isolation
US20230374468A1 (en) Formulations for viral vectors
CN112852883B (en) Method for titrating recombinant baculovirus based on coexpression secretory luciferase
CN117907597A (en) Method for determining adenovirus serotype neutralizing antibody

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CP03 Change of name, title or address

Address after: 3rd Floor, Building 12, Life Science City, Yaogu Industrial Park, No. 1777 Dazheng Road, High tech Zone, Jinan City, Shandong Province, 250101

Patentee after: Yiming (Jinan) Biotechnology Co.,Ltd.

Address before: 3rd Floor, Building 12, Life Science City, Yaogu Industrial Park, No. 1777 Dazheng Road, High tech Zone, Jinan City, Shandong Province, 250101

Patentee before: Jinan Yiming Medical Technology Co.,Ltd.

CP03 Change of name, title or address