Adeno-associated virus susceptible cell lines and uses thereof

Abstract

Provided herein is an adeno-associated virus susceptible cell strain and its application. Specifically, provided herein is an adeno-associated virus susceptible cell strain, and the application of the cell strain. The cell strain is human kidney epithelial cell 293T-AH10 cell with preservation number CCTCC NO: C202331, which moderately and stably expresses three genes of adeno virus (Ad5): E4 gene (Orf6), E2A gene and VARNA gene, and is highly susceptible to adeno-associated virus (AAV) of different serotypes, and can be used for infection titer quantitative test of AAV.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a human renal epithelial cell line 293T-AH10 that stably expresses three gene segments of adenovirus (Ad5): the E4 gene (Orf6), the E2A gene, and the VARNA gene, based on HEK293T cells. This invention also relates to the application of this cell line in determining AAV infection titers. Background Technology

[0002] With the widespread application of bioengineering technology in scientific research, food, chemical engineering, and medicine, various DNA-based gene vectors are widely used, and the method of using viruses as DNA delivery vectors is widely applied. Among them, AAV (adeno-associated virus) is a type of single-stranded linear DNA-deficient virus that requires co-infection with a helper virus (adenovirus or herpesvirus) to replicate in vivo. Its advantages include high safety (there are currently no reports of AAV causing disease in humans), low immunogenicity, broad infectious spectrum, long expression time, strong diffusivity, and high stability. Therefore, rAAV (recombinant AAV) is suitable as a DNA vector for treating gene-related clinical diseases.

[0003] The determination of rAAV titer is a crucial vector quality assessment indicator in scientific research, clinical research, and biopharmaceutical engineering, particularly in cell gene therapy (CGT). Its accuracy is of paramount importance for these AAV-related applications. Currently, the main method used in production is to directly determine the rAAV viral genome titer (Vg) via qPCR. However, this method cannot reflect the viability of the AAV virus sample and its actual ability to infect cells; it only infers the number of viral particles from the DNA copy number in the sample. To more accurately reflect the viability of rAAV and its actual ability to infect cells, it is necessary to determine the rAAV infectious titer. AAV infection does not produce cytopathic effects, therefore its infectious titer cannot be determined by plaque assays. However, with the participation of adenovirus (Ad) accessory proteins, AAV can be induced to replicate within cells and express genes from the viral genome. Infected cells can then be detected by detecting the expression of the replicated viral genome or reporter gene, and the infectious titer of the original AAV virus can be extrapolated based on the gradient dilution ratio.

[0004] Several commonly used methods for determining infection titers include: measuring the median infectious dose (TCID) of AAV. 50One method involves co-infecting HeLa cell lines stably expressing AAV2 rep and cap genes with serially diluted AAV2 and a certain concentration of adenovirus (Ad). After AAV2 replicates within the cells for a certain period, the copy number of the newly replicated AAV2 genome sequence is detected using qPCR, and the original AAV2 viral infection titer is then inferred. Another method is ICA (infectious center assay). The detection method is similar to the previous one, using Ad5-assisted serially diluted AAV2 to co-infect HeLa cell lines stably expressing AAV2 rep and cap genes. The cells are then seeded onto nylon membranes coated with hybridization probes, and the viral infection titer is calculated by analyzing the number of positive cells on the membrane. Alternatively, the infection titer can be determined by detecting the intensity of the reporter gene expressed by the infected cells. This method typically involves serially diluting purified virus and infecting a certain number of susceptible cells, then calculating the viral infection titer by calculating the number of positive cells expressing the reporter gene and the dilution ratio. In order to improve susceptibility to AAV, this method often uses adenovirus co-infection to obtain the expression of helper genes when used to detect AAV infection titers.

[0005] In summary, conventional methods for detecting rAAV infection titers typically require the presence of helper viruses such as adenoviruses. Introducing and preparing these other viruses not only involves the waste of materials due to the addition of another virus and the long testing time (generally more than a week), but adenovirus infection also causes cytopathic effects, exhibiting high toxicity and leading to significant cell death, thus affecting data analysis. Furthermore, co-infection with multiple viruses can introduce data instability between different test batches. Moreover, for test protocols requiring cell lines stably expressing AAV2 rep and cap, the toxicity of Rep and Cap proteins to cells can also affect titer determination, necessitating the design of appropriately expressed cell lines to obtain sustainable and stable cell lines. Therefore, there is an urgent need in the field to design novel helper virus-free cell lines for detecting infection titers of various AAV serotypes. Summary of the Invention

[0006] On the one hand, this article provides human renal epithelial cells 293T-AH10 with accession number CCTCC NO: C202331.

[0007] The human kidney epithelial cell line 293T-AH10 used in this article is deposited at the China Center for Type Culture Collection, Wuhan University, China, on February 7, 2023, with accession number CCTCCNO: C202331.

[0008] On the other hand, this article provides the application of the aforementioned human renal epithelial cells 293T-AH10 in detecting the viral titer of adeno-associated virus.

[0009] In some implementations, the adeno-associated virus may be selected from various serotypes of AAV, such as common AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 or other special serotypes of AAV.

[0010] In some implementations, the adeno-associated virus is a recombinant adeno-associated virus.

[0011] On the other hand, this article provides a kit for detecting the viral titer of adeno-associated virus, which includes the aforementioned human renal epithelial cells 293T-AH10.

[0012] On the other hand, this article provides that the adeno-associated virus can be selected from various serotypes of AAV, such as the common AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 or other special serotypes of AAV.

[0013] On the other hand, this article provides that the adeno-associated virus is a recombinant adeno-associated virus.

[0014] The human renal epithelial cell line 293T-AH10 described in this article can stably express three gene segments of adenovirus (Ad5): E4 gene (Orf6), E2A gene, and VARNA gene. Compared with HEK293 or HEK293T cells, it can improve the expression of the target gene after rAAV infection, and can increase the sensitivity and accuracy of AAV infection titer determination in the absence of helper virus. Attached Figure Description

[0015] Figure 1 : A pHelper plasmid map containing the target gene plasmid.

[0016] Figure 2 Fluorescence microscopy results of human renal epithelial cells 293T-AH10 and HEK293T infected with rAAV-GL. When human renal epithelial cells 293T-AH10 and HEK293T were infected with the same number of MOI (1E+05) cells of different serotypes of rAAV-GL (e.g., serotypes 8 and 9), the fluorescence intensity of 293T-AH10 cells was significantly higher than that of HEK293T cells.

[0017] Figure 3The detection sensitivity of human renal epithelial cells 293T-AH10 and HEK293 cells for rAAV-GL (e.g., serotype 8) infection was compared. The detection sensitivity of human renal epithelial cells 293T-AH10 (significantly different from the virus-free control) was more than 16 times that of HEK293 cells.

[0018] Figure 4 The detection sensitivity of human renal epithelial cells 293T-AH10 and HEK293T cells for rAAV-GL (e.g., serotype 8) infection was compared. The detection sensitivity of human renal epithelial cells 293T-AH10 (significantly different from the virus-free control) was more than 4 times that of HEK293T cells. Detailed Implementation

[0019] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0020] Experimental methods not specifically described in this invention are performed according to the relevant product instructions. Unless otherwise specified, all biological reagents used in this invention are commercially available. Those skilled in the art can make various changes, modifications, and substitutions without departing from the spirit of this invention.

[0021] The main materials involved in this invention include plasmids synthesized by Anhui General Biotechnology Co., Ltd.; HEK293T cells (ATCC Center, USA); HEK293 cells (ATCC Center, USA); polyethyleneimine (PEI, Polyplus, USA); recombinant AAV carrying the NeonGreen gene and luciferase reporter gene (teLuc) (serotype 8: (rAAV8-GL, serotype 9: (rAAV9-GL) were purchased from Yiming Cell; the plasmid pHelper involved in cell preparation contained the mini-CMV promoter, and the genes including the E4 gene (Orf6), E2A gene, VARNA gene, and puromycin resistance gene were chemically synthesized (General Biotechnology).

[0022] Example 1: Screening of Stable Surviving Monoclonal Cells

[0023] This embodiment describes the preparation and screening process of transgenic cell lines, and the brief steps are as follows.

[0024] 1) Synthesize plasmid pHelper, which contains genes including the mini-CMV promoter, E4 gene (Orf6), E2A gene, VARNA gene, and puromycin resistance gene (PuromycinR) (see...). Figure 1 );

[0025] 2) Culture HEK293T cells to the logarithmic growth phase and seed them into 6-well cell culture plates at 2E6 cells / well;

[0026] 3) The next day, the plasmid described in 1) was transfected using the PEI method. The amount of plasmid transfected was 1 μg, and the ratio of PEI to plasmid was 3:1.

[0027] 4) 72 hours after transfection, the adherent cells were digested, mixed, and screened for single clones in a 48-well plate. 2 μg / mL of puromycin was added to each well.

[0028] 5) Screen out cells that can be stably expanded (HEK293T-1#-HEK293T-10#).

[0029] Example 2: Screening of cell lines

[0030] 1) Culture HEK293T-1#-HEK293T-10# and HEK293T cells (HEK293-0#) to the logarithmic growth phase, and seed them into 24-well cell culture plates at 5E5 cells / well;

[0031] 2) Infect cells with AAV(rAAV8-GL) (MOI 1E+05), and select cells with strong fluorescence from all single clones after 48 hours.

[0032] The relative fluorescence intensities among them (converted to compare with the fluorescence intensity of cell line HEK293T-0#) are shown in Table 1.

[0033] Table 1: Relative fluorescence intensity of different cell lines infected with AAV (rAAV8-GL)

[0034] cell lines Relative fluorescence intensity HEK293T-0# 1.00 HEK293T-1# 6.00 HEK293T-2# 3.90 HEK293T-3# 9.49 HEK293T-4# 4.87 HEK293T-5# 4.11 HEK293T-6# 9.67 HEK293T-7# 7.55 HEK293T-8# 6.63 HEK293T-9# 1.05 HEK293T-10# 18.09

[0035] We named the stable cell line HEK293T-10# human kidney epithelial cells 293T-AH10 and deposited it at the China Center for Type Culture Collection, Wuhan University, China, on February 7, 2023, with accession number CCTCC NO: C202331.

[0036] Example 3: Expression of the target protein in human renal epithelial cells 293T-AH10 infected with rAAV

[0037] This embodiment examines the infection of HEK293T cells with different serotypes of AAV.

[0038] The experimental procedure is as follows:

[0039] 1) Culture HEK293T cells and human kidney epithelial cells 293T-AH10 cells (passage 10 times) to the logarithmic growth phase, and seed them into 24-well cell culture plates at 5E5 cells / well;

[0040] 2) The next day, the two cells were infected with AAV (rAAV8-GL and rAAV9-GL) respectively, with an MOI of 1E+05;

[0041] 3) After 48 hours, observe and acquire images using a fluorescence microscope (488 / 520 green filter group).

[0042] The results are as follows Figure 2 As shown, for different serotypes of AAV, the fluorescence intensity of human renal epithelial cells 293T-AH10 was significantly higher than that of HEK293T cells.

[0043] Example 4: Comparison of sensitivity of human renal epithelial cells 293T-AH10 and HEK293 cells after infection with rAAV

[0044] This embodiment quantitatively investigates the difference in sensitivity of human renal epithelial cells 293T-AH10 and HEK293 cells to AAV(rAAV8) infection.

[0045] The experimental procedure is as follows:

[0046] 1) Cell seeding: Human kidney epithelial cells 293T-AH10 and HEK293 cells (HEK293 cells were transfected with pHelper plasmid and screened for antibiotics according to the method in Example 1) were seeded into transparent white 96-well plates, 20,000 cells / well, 100 μL per well, and cultured at 37°C, 5% CO2 for 16 h.

[0047] 2) Viral infection: Add 10 μL of rAAV8-GL diluted 2-fold to each well and infect for 48-72 h;

[0048] 3) Cell lysis: Remove the infected culture plate, add 1% Triton-X100 to each well, incubate at room temperature for 5 min, and then place it under a fluorescence microplate reader to measure luciferase activity.

[0049] 4) Luciferase activity assay: Add luciferase substrate: Diphenylterazine (DTZ, final concentration 30 μM) to each well, shake to mix, and then measure the luminescence intensity.

[0050] The results are as follows Figure 3 As shown, human renal epithelial cells 293T-AH10 were at least 16 times more sensitive to AAV (rAAV8) infection than HEK293 cells transfected with the pHelper plasmid.

[0051] Example 5: Comparison of sensitivity of human renal epithelial cells 293T-AH10 and HEK293T cells after infection with rAAV

[0052] This embodiment quantitatively investigates the difference in sensitivity of human renal epithelial cells 293T-AH10 and HEK293T cells to AAV(rAAV8) infection.

[0053] The experimental procedure was carried out in accordance with Example 4.

[0054] The results are as follows Figure 4 As shown, human renal epithelial cells 293T-AH10 are at least 4 times more sensitive to AAV (rAAV8) infection than HEK293T cells transfected with pHelper plasmid.

[0055] in conclusion

[0056] The human renal epithelial cell line 293T-AH10 provided by this invention can stably express three gene segments of adenovirus (Ad5): the E4 gene (Orf6), the E2A gene, and the VARNA gene. Compared with HEK293 or HEK293T cells, human renal epithelial cell line 293T-AH10 can improve the expression of the target gene after rAAV infection, and can increase the sensitivity and accuracy of AAV infection titer determination without helper virus. The human renal epithelial cell line 293T-AH10 provided by this invention is based on HEK293T cells commonly used in AAV production. By using a promoter with low initiation activity (minimal CMV), it stably and moderately expresses three gene segments of adenovirus (Ad5): the E4 gene (Orf6), the E2A gene, and the VARNA gene, reducing the toxicity of these exogenous adenovirus proteins to cells, and unexpectedly obtaining a stable cell line that can be directly used to detect AAV infection titer. Because no additional auxiliary viruses are required, this invention makes AAV infection titer testing faster (only 2-3 days), more convenient, sensitive, accurate, stable, and low-cost.

[0057] The promoters or gene sequences mentioned in this article are as follows:

[0058] Mini-CMV: (SEQ ID NO: 1)

[0059]

[0060] Ad5 E4Orf6:(SEQ ID NO:2)

[0061]

[0062] Ad5 E2A: (SEQ ID NO: 3)

[0063]

[0064]

[0065] Ad5 VA RNA:(SEQ ID NO:4)

[0066]

Claims

1. Human renal epithelial cells 293T-AH10 cells with accession number CCTCC NO: C202331.

2. The application of human renal epithelial cells 293T-AH10 as described in claim 1 in detecting the viral titer of adeno-associated virus.

3. The application according to claim 2, characterized in that, The adeno-associated virus is selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9.

4. The application according to claim 2 or 3, characterized in that, The adeno-associated virus mentioned is a recombinant adeno-associated virus.

5. A kit for detecting the viral titer of adeno-associated virus, characterized in that, Including the human renal epithelial cells 293T-AH10 cells as described in claim 1.

6. The kit for detecting the viral titer of adeno-associated virus according to claim 5, characterized in that, The adeno-associated virus is selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9.

7. The kit for detecting the viral titer of adeno-associated virus according to claim 5 or 6, characterized in that, The adeno-associated virus mentioned is a recombinant adeno-associated virus.

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