Detection method of replicative adeno-associated virus and kit thereof

Abstract

The invention provides a detection method of replicating adeno-associated viruses and a kit thereof. The detection method comprises three rounds of amplification and detection processes, wherein the detection process comprises the following steps: extracting virus DNA of a supernatant of the first round of amplification, a supernatant of the second round of amplification and / or a supernatant of the third round of amplification, and carrying out quantitative PCR genome titer detection on the virus DNA and a replicated adeno-associated virus standard substance through a specific primer and a Taqman probe; the sequence of the specific primer is as shown in SEQ ID No. 3-4, and the sequence of the Taqman probe is as shown in SEQ ID No. 5. The invention provides a detection method for rapidly detecting the content of the replicating adeno-associated virus in a recombinant adeno-associated virus product in a short time, the amplification of the replicating adeno-associated virus can be successfully realized by culturing the detection system for 24 hours, the sample detection period is greatly shortened, and the quality control pressure is relieved.

Description

Technical Field

[0001] This invention relates to the field of virus detection, specifically to a method and kit for detecting replicating adeno-associated virus, as well as nucleic acid molecules, recombinant plasmids, and adeno-associated virus. Background Technology

[0002] Adeno-associated virus (AAV) is a defective virus that cannot complete its replication cycle independently. It can only replicate and cause infection when co-infected with helper viruses such as adenovirus and herpesvirus. Recombinant adeno-associated virus (rAAV) has advantages over other viral vectors such as adenovirus, lentivirus, and retrovirus, including higher safety, a wider host range, non-pathogenicity, long-term stable expression of exogenous genes, low immunogenicity, and high stability. It has become one of the most widely used viral vectors in gene therapy.

[0003] Although the rAAV vector design has eliminated the Rep (replication) and Cap (capsid) genes, homologous or non-homologous recombination of the rAAV vector genome, Rep / Cap genes, and ITR sequence during viral packaging may result in a replicating adeno-associated virus (rcAAV). rcAAV carries the complete Rep gene, and its expressed Rep78 / 68 protein possesses helicase and site-specific endonuclease activities, potentially leading to host genome instability, helper virus-dependent reactivation, and enhanced immunogenicity. Its capsid protein may also trigger cytotoxic T-lymphocyte responses. Therefore, for safety reasons in the clinical application of rAAV drug production, rcAAV testing is essential.

[0004] Currently, there is no unified standard for rcAAV detection both domestically and internationally. It is broadly categorized into two types: direct qPCR detection and cell culture-based qPCR detection. Traditional qPCR methods rely on the specific binding of primer fragments to the target DNA fragment, achieving absolute quantification of the target fragment by referring to a standard curve. However, this method detects short fragments, and its sensitivity, specificity, and overall assessment of wild-type AAV often fall short of requirements. It may also be susceptible to false positives due to extremely low contamination levels. Cell culture-based qPCR detection is one of the most commonly used methods. This method amplifies rcAAV through continuous passage and uses highly sensitive qPCR for detection. Currently, there is no unified execution procedure or gold standard for this method both domestically and internationally. Leading domestic companies engaged in AAV gene therapy have all established their own detection methods for testing AAV virus stock solutions and finished products. CN113308492B provides a method for detecting replicating AAV. This cytological detection method does not rely on helper virus rcAAV, is safe and friendly to the environment and testing personnel, and has extremely high detection sensitivity and accuracy. However, this detection method only detects rcAAV in the cell culture supernatant and does not cover intracellular rcAAV, which may lead to false negatives. In addition, it requires at least 3 rounds of infection, with each round of infection taking 5 days, resulting in a long experimental cycle. The detection limit is 10 / 1E8, and there is still room for improvement. CN116004921A discloses a method and kit for determining the rcAAV content in recombinant adeno-associated virus (rAAV) vector products. This method uses 4-5 mL cell culture flasks to culture cells and infects them three or more times without loss of rcAAV. Then, quantitative PCR is performed on the ultrafiltration concentrates harvested from the first and last rounds of infection using gene-specific primers and specific TaqMan probes to detect the rcAAV content in the recombinant adeno-associated virus vector sample. However, this method uses large-scale single-sample operations in culture dishes, does not detect the virus recovery rate in the ultrafiltration concentration step, does not control the rcAAV inoculation during each round of infection, and cannot prove that "there is no loss of inoculum during infection." Furthermore, the detection limit is 1 rcAAV9 / 1E6vg AAV9, leaving room for improvement. Therefore, finding a method to detect rcAAV in rAAV that overcomes the above limitations is a technical problem urgently needing to be solved in this field. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a method and kit for detecting replicating adeno-associated virus (rAAV), which also includes a nucleic acid molecule, a recombinant plasmid, and adeno-associated virus. This method can detect the rcAAV content in rAAV products, featuring a short experimental cycle and high sensitivity. It can be used as a quality control method for AAV virus products, for the detection and characterization of both viral stock solutions and finished products.

[0006] To achieve the above objectives, the present invention first provides a method for detecting replicating adeno-associated virus, which includes the following steps: First round of amplification: The test sample, positive control and negative control were diluted to obtain the first round of diluted sample. The first round of diluted sample was used to infect HeLaRC32 cells. After infection, the culture medium was changed after a period of time. After a period of further culture, the cell suspension was collected and subjected to freeze-thaw, HuAd5 inactivation and centrifugation. After centrifugation, the supernatant of the first round of amplification of the test sample, positive control and negative control sample was collected. Second round of amplification: The supernatant from the first round of amplification was diluted a second time to obtain the second round of diluted samples. The second round of diluted samples were used to infect HeLaRC32 cells. After infection, the culture medium was changed after a period of culture. After a period of culture, the cell suspension was collected and subjected to freeze-thaw, HuAd5 inactivation, and centrifugation. After centrifugation, the supernatant from the second round of amplification of the test sample, positive control sample, and negative control sample was collected. Third round of amplification: The supernatant of the second round of amplification was diluted in the third round to obtain the third round of diluted sample. The third round of diluted sample was used to infect HeLaRC32 cells. After infection, the culture medium was changed after a period of culture. After a period of culture, the cell suspension was collected and subjected to freeze-thaw, HuAd5 inactivation, and centrifugation. After centrifugation, the supernatant of the third round of amplification of the test sample, positive control sample, and negative control sample was collected. Detection: Viral DNA was extracted from the supernatant of the first round of amplification, the supernatant of the second round of amplification, and / or the supernatant of the third round of amplification. The viral DNA and replicating adeno-associated virus standards were subjected to quantitative PCR genomic titer detection using specific primers and Taqman probes. The sequences of the specific primers are shown in SEQ ID No. 3-4, and the sequences of the Taqman probes are shown in SEQ ID No. 5.

[0007] According to a specific embodiment of the present invention, the detection method is to detect replicating adeno-associated virus in all adeno-associated virus products, preferably, the detection method is to detect replicating adeno-associated virus in recombinant adeno-associated virus.

[0008] According to a specific embodiment of the present invention, preferably, the positive control is a wild-type adeno-associated virus; preferably, the wild-type adeno-associated virus is a recombinant adeno-associated virus obtained by co-transfecting HEK293 cells with pHelper plasmid, pCap8Rep2 plasmid, and pwtAAV plasmid; more preferably, the pwtAAV plasmid contains a pwtAAV-ITR sequence, the pwtAAV-ITR sequence being shown in SEQ ID No. 1.

[0009] According to a specific embodiment of the present invention, preferably, the sequence of the replicating adeno-associated virus standard is shown in SEQ ID No. 2.

[0010] According to a specific embodiment of the present invention, preferably, the first round of dilution is an 80-100-fold dilution (preferably a 100-fold dilution), and the second and third rounds of dilution are 2-4-fold dilutions (preferably a 2-fold dilution); preferably, in the first, second, and third rounds of amplification, the test sample and the positive control are diluted with HuAd5 diluent, and the negative control is wild-type adeno-associated virus diluted with serum-free culture medium; more preferably, the preparation step of the HuAd5 diluent is: diluting HuAd5 with serum-free culture medium to obtain a HuAd5 diluent with an MOI of 0.01-0.1 (preferably 0.01).

[0011] According to a specific embodiment of the present invention, preferably, in the first round of amplification, the second round of amplification, and the third round of amplification, the number of freeze-thaw cycles is 3, to obtain a freeze-thawed cell suspension; Preferably, in the first, second, and third rounds of amplification, the post-infection culture time is 22-26 hours (preferably 24 hours). Preferably, in the first, second, and third rounds of amplification, the time for changing the culture medium and continuing the culture thereafter is 24-72 hours, more preferably 24 hours; Preferably, the step of inactivating HuAd5 is as follows: incubating the frozen-thawed cell suspension at 50-60℃ (preferably 56℃) for 20-40 min (preferably 30 min) to obtain a cell suspension inactivated with HuAd5; Preferably, the centrifugation conditions are 4000-4500 rpm for 10-15 min (preferably 4200 rpm for 10 min).

[0012] On the other hand, the present invention also provides a nucleic acid molecule comprising sequences encoding adeno-associated virus Rep2 and Cap8 proteins, wherein the sequences are shown in SEQ ID No. 1.

[0013] On the other hand, the present invention also provides a recombinant plasmid comprising the above-described nucleic acid molecules.

[0014] On the other hand, the present invention also provides an adeno-associated virus comprising the above-described recombinant plasmid.

[0015] On the other hand, the present invention also provides a kit for detecting replicating adeno-associated virus, comprising: The aforementioned adeno-associated virus; The replicating adeno-associated virus standard shown in SEQ ID No. 2; The specific primers shown in SEQ ID No. 3-4 and the Taqman probe shown in SEQ ID No. 5.

[0016] Preferably, it further comprises: adeno-associated virus-sensitive somatic cells; more preferably, the adeno-associated virus-sensitive somatic cells are HeLaRC32 cells. More preferably, the kit further includes an auxiliary virus, and even more preferably, the auxiliary virus is HuAd5.

[0017] Compared with existing technologies, the advantages of this invention are as follows: The detection method provided by this invention features a short experimental cycle and high sensitivity, and can serve as an industry quality control standard to ensure the safety of rAAV products. This method provides a rapid detection method for the rcAAV content in rAAV products. Using this detection system, rcAAV amplification can be successfully achieved within 24 hours of cultivation, significantly reducing the sample detection cycle, alleviating quality control pressure, and offering superior detection efficiency compared to existing technologies. This method has high sensitivity; using this detection system, the detection limit for rcAAV content in rAAV products is as low as 1 / 1E5vg. Furthermore, the DNase pretreatment step designed during viral DNA extraction in this detection method can eliminate the influence of incompletely packaged AAV DNA in the amplification system, detecting only AAV viruses with complete capsid structures, further avoiding false positive results. It can serve as an industry quality control standard to ensure the safety of rAAV products.

[0018] This invention preserves specific regions of the original AAV2 sequence (GenBank ID: AH002785.2, range: 230-405, 792-915) between the ITR and rep2 sequences, and between the cap8 and ITR sequences. First, the preservation of key regulatory sequences enables the pwtAAV-ITR vector to replicate and express more efficiently, and improves the packaging efficiency of the wtAAV virus, thereby increasing detection sensitivity and accuracy. Second, the preservation of the original AAV2 sequence improves the physical stability and functional consistency of the pwtAAV-ITR vector, enhancing both vector stability and reproducibility of detection. The design concept of this invention is based on the detailed analysis and targeted preservation of the original AAV2 sequence. Through systematic bioinformatics analysis of the complete AAV2 genome, cis-acting elements related to important functions such as viral replication, capsid packaging, and gene transcription regulation are accurately identified and preserved. This not only ensures the structural integrity of the core functional elements of the vector but also simulates the natural genomic environment of the virus by preserving its natural flanking sequences and linker regions. This invention promotes more efficient synergy among components and enhances the structural stability and functional reliability of the vector, providing a more structurally optimized and functionally complete viral vector construction scheme. The cells used in this invention are HeLaRC32 cells. In the presence of the helper virus HuAd5, the inserted cap and rep genes in HeLaRC32 cells are overexpressed, further assisting the amplification of AAV virus and greatly improving detection sensitivity. This invention has a HuAd5 MOI of 0.01, requiring less HuAd5 for detection, resulting in amplification efficiency superior to existing technologies. This invention performs detection and amplification on the entire amplified cultured cells and supernatant, avoiding missed detection of rcAAV within the cells, thus ensuring high reliability of the detection results.

[0019] The primer and probe sequences designed in this invention specifically amplify fragments containing ITR terminal sequences and Rep2 sequences. Compared to existing technologies, the amplified fragments targeted by this invention have a more complete structure and function; only genomes containing both ITR and Rep2 can be amplified. This eliminates interference from incomplete genomes and residual plasmid DNA fragments, minimizing the risk of non-specific amplification, increasing sensitivity, and significantly reducing the likelihood of false positives. The upstream and downstream primers and TaqMan probe sequences used in this invention exhibit good detection specificity, making this method both sensitive and specific. Attached Figure Description

[0020] Figure 1 The results are for the enzyme digestion verification of the pwtAAV vector.

[0021] Figure 2 This is a flowchart of the rcAAV content detection experiment.

[0022] Figure 3 The results of rcAAV content detection after 72 hours of amplification culture.

[0023] Figure 4 The results of rcAAV content detection for amplification cultured for 24 hours. Detailed Implementation

[0024] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0025] It should be noted that, unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0027] It should be understood that the terms “comprising,” “including,” and / or “containing” as used herein specify the presence of the stated features, integers, steps, components, or combinations thereof, but do not exclude the presence or addition of one or more other features, integers, steps, components, or combinations thereof.

[0028] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0029] I. Preparation of wtAAV: (1) wtAAV-ITR sequence design The pwtAAV-ITR sequence was constructed by inserting the Rep2 and Cap8 sequences of wild-type AAV between two AAV-ITR sequences, as shown in SEQ ID No. 1.

[0030] (2) Construction and validation of pwtAAV vector

[0031] The pwtAAV-ITR sequence was cloned into the rAAV-ITR vector using the CloneEZ Recombinant Cloning Kit. The synthesized pwtAAV vector was 7234 bp in length. The pwtAAV vector was double-digested with HindIII and MluI. The digestion results of the pwtAAV vector are shown below. Figure 1As shown, M: DNA Marker; 1: pwtAAV vector HindIII and MluI double digestion product; 2: pwtAAV vector.

[0032] (3) wtAAV virus packaging

[0033] HEK293 cells were co-transfected with pHelper plasmid, pCap8Rep2 plasmid, and pwtAAV plasmid. After 24 hours of transfection, the culture medium was changed and the cells were cultured for 72 hours. Cells and culture supernatant were collected and placed in centrifuge tubes. The cells were repeatedly frozen and thawed three times. The cell lysate was clarified, subjected to a first ultrafiltration and concentration, affinity chromatography, ion exchange chromatography, a second ultrafiltration and concentration, and sterile filtration to obtain the wtAAV virus solution.

[0034] (4) Detection of wtAAV (rcAAV) viral genome titer

[0035] The TaqMan probe-based qPCR method was used to detect the viral genomic titer of wtAAV (rcAAV). The sequence of the detection standard is shown in SEQ ID No. 2. The upstream and downstream primers and TaqMan probe sequences are as follows: Upstream primer (SEQ ID No. 3): AGGGGTTCCTGGAGGG; Downstream primer (SEQ ID No. 4): CCGGGCAGATGCTCG; TaqMan probe (SEQ ID No. 5): 5'-FAM-ACGAGATTGTGATTAAGGTCCCCAGCGAC-BHQ1-3'.

[0036] Standard dilution: The concentration of the linearized plasmid from Structure pUC was 2.6 × 10⁻⁶. 8 vg / mL (ST0), followed by sequential 10-fold serial dilutions to a concentration of 2.6 × 10⁻⁶. 7 vg / mL, 2.6×10 6 vg / mL, 2.6×10 5 vg / mL, 2.6×10 4 vg / mL, 2.6×10 3 vg / mL, 2.6×10 2 vg / mL, 2.6×10 1 vg / mL, labeled as ST1, ST2, ST3, ST4, ST5, ST6, ST7.

[0037] Sample preparation: Viral DNA was extracted from wtAAV virus samples using a viral nucleic acid extraction kit (magnetic bead method) (manufacturer: Huzhou Shenke; catalog number: 1506730) or a viral DNA / RNA extraction kit (manufacturer: Trans; catalog number: ER201).

[0038] qPCR reaction system: A 96-well plate was used for the assay, with three technical replicates per sample. The sample consisted of seven standards (ST1-ST7), one wtAAV test sample, and one template-free negative control (NTC). The total reaction volume was 20 μL, including 10 μL of probe-based qPCR premix (manufacturer: TransGen; catalog number: AQ711), 0.4 μL of upstream primer, 0.4 μL of downstream primer, 0.4 μL of probe, 6.8 μL of enzyme-free water (manufacturer: TransGen; catalog number: AQ711), and 2 μL of sample.

[0039] qPCR reaction procedure: Create a new blank program and select the absolute quantification template. Then create a new file, selecting FAM as the reporter fluorescent group and none as the quencher fluorescent group. Create a new detection probe, named IPC, selecting VIC as the reporter fluorescent group and none as the quencher fluorescent group; the detection reference fluorescence is ROX. Set the two-step reaction program: 95℃ pre-denaturation for 10 min; 95℃ for 15 s, 60℃ for 1 min, 40 cycles; reaction volume 20 μL.

[0040] The wtAAV viral genome titer results are shown in Table 1: Table 1

[0041] II. rcAAV content detection

[0042] (1) Experimental materials

[0043] Complete culture medium: 445 mL DMEM (Gibco; catalog number: 11995-065), 50 mL FBS fetal bovine serum (Gibco; catalog number: 10091148), 5 mL double antibiotics against Streptococcus penicillin (100×) (Beyotime; catalog number: C0222). HEPES (1M, pH=8.0): 11.915 g HEPES (Gibco; catalog number: 11344041), added to 40 mL sterile water, adjusted pH to 8.0, and then added sterile water to a final volume of 50 mL. Serum-free culture medium: 482.5 mL DMEM, 12.5 mL HEPES (1M, pH=8.0), 5 mL double antibiotics against Streptococcus penicillin (100×). HeLaRC32 cells (ATCC; catalog number: CRL-2972) were used as assay cells. HuAd5 (human adenovirus bioassay reference, manufacturer: ATCC; catalog number: VR-5) is used as an auxiliary virus.

[0044] (2) Detection of rcAAV content

[0045] This method is used to detect rcAAV content in adeno-associated virus (AAV) products (e.g., rAAV samples). The experimental flowchart for rcAAV content detection is shown below. Figure 2 As shown, the specific steps include the following: 1) First round of amplification Cell plating: Cells were prepared using complete culture medium at a density of 5 × 10⁶ cells / year. 5 Add 2 mL of HeLaRC32 cell suspension per well to a 6-well cell culture plate and incubate overnight at 37°C in a 5% CO2 cell culture incubator.

[0046] Preparation of HuAd5 dilution solution: Take 10 μL of solution with a titer of 8 × 10⁸ μL. 6 CCID 50 HuAd5 was added at a concentration of 1 / mL to 7990 μL of serum-free culture medium to obtain a diluted HuAd5 solution.

[0047] Preparation of amplification systems for test samples, positive controls, and negative controls: Add 40 μL of rAAV test sample with a known genomic titer and 40 μL of wtAAV with a known genomic titer to 3960 μL of HuAd5 dilution buffer to obtain the test sample solution and positive control sample solution with known genomic titers. Take 40 μL of genomic titer 2×10... 8 Adding vg / mL wtAAV to 3960 μL of serum-free culture medium yielded a genomic titer of 2×10⁻⁶. 6 The negative control sample solution was prepared at a concentration of vg / mL. The cell density was 1×10⁻⁶. 6Seed HeLaRC32 cells per well into 6-well cell culture plates, discard the supernatant, and add 2 mL of the test sample solution, positive control sample solution, and negative control sample solution per well. Each sample is repeated in 2 wells.

[0048] rcAAV amplification: Incubate at 37℃ in a 5% CO2 cell culture incubator for 24 hours, then replace with complete culture medium and continue culturing for 24-72 hours.

[0049] Sample collection: Adherent cells were pipetted to suspension, and the cell suspension was collected into a 15 mL centrifuge tube. The tube was then subjected to three freeze-thaw cycles. If HuAd5 was present in the amplification system, it was incubated at 56°C for 30 min to inactivate HuAd5. The cells were centrifuged at 4200 rpm for 10 min. 1 mL of the supernatant was used for the second round of amplification, and 100 μL of the supernatant was used for subsequent viral DNA extraction and rcAAV genome titer detection.

[0050] 2) Second round of amplification

[0051] Cell plating and HuAd5 dilution preparation: Same as the preparation method for the first round of amplification.

[0052] Preparation of amplification systems for test samples, positive control samples, and negative control samples: Take 1 mL of the supernatant from the first round of amplification of the test sample and 1 mL of the supernatant from the positive control sample, and add 1 mL of HuAd5 diluent to each to obtain the test sample solution and the positive control sample solution. Take 1 mL of the supernatant from the first round of amplification of the negative control sample, and add 1 mL of serum-free culture medium to obtain the negative control sample solution. The cell density should be 1×10⁻⁶ cells / mL. 6 For a 6-well cell culture plate containing HeLaRC32 cells per cell / well, discard the supernatant and add 2 mL of the test sample solution, positive control sample solution, and negative control sample solution per well, with each sample containing 2 replicate wells.

[0053] rcAAV amplification: The steps are the same as in the first round of amplification.

[0054] Sample collection: Adherent cells were pipetted to suspension, and the cell suspension was collected into a 15 mL centrifuge tube. The tube was then subjected to three freeze-thaw cycles. If HuAd5 was present in the amplification system, it was incubated at 56°C for 30 min to inactivate HuAd5. The cells were centrifuged at 4200 rpm for 10 min. 1 mL of the supernatant was used for the third round of amplification, and 100 μL of the supernatant was used for subsequent viral DNA extraction and rcAAV genome titer detection.

[0055] 3) Third round of expansion

[0056] Cell plating and HuAd5 dilution preparation: Same as the preparation method for the first round of amplification.

[0057] Preparation of amplification systems for test samples, positive control samples, and negative control samples: Take 1 mL of the supernatant from the second round of amplification of the test sample and 1 mL of the supernatant from the positive control sample, and add 1 mL of HuAd5 diluent to each to obtain the test sample solution and the positive control sample solution. Take 1 mL of the supernatant from the second round of amplification of the negative control sample, and add 1 mL of serum-free culture medium to obtain the negative control sample solution. The cell density should be 1×10⁻⁶ cells / mL. 6 For a 6-well cell culture plate containing HeLaRC32 cells per cell / well, discard the supernatant and add 2 mL of the test sample solution, positive control sample solution, and negative control sample solution per well, with each sample containing 2 replicate wells.

[0058] rcAAV amplification: The steps are the same as in the first round of amplification.

[0059] Sample collection: Adherent cells were pipetted to suspension, and the cell suspension was collected into a 15 mL centrifuge tube. The tube was then subjected to three freeze-thaw cycles. If HuAd5 was present in the amplification system, it was incubated at 56 °C for 30 min to inactivate HuAd5. The cells were centrifuged at 4200 rpm for 10 min, and 100 μL of the supernatant was used for subsequent viral DNA extraction and rcAAV genome titer detection.

[0060] 4) Viral DNA extraction

[0061] Preparation of DNase reaction system: Take 2 μL of DNase I (RNase-free) (manufacturer: NEB; catalog number: M0303LVIAL), 10 μL of 10×DNase I Reaction Buffer (manufacturer: NEB; catalog number: B0303SVIAL), and 8 μL of enzyme-free water (manufacturer: Beyotime; catalog number: ST876) to prepare a total volume of 20 μL of DNase reaction system.

[0062] DNase treatment: Take 80 μL of supernatant and add it to 20 μL of DNase reaction system, incubate at 37℃ for 15 min, and inactivate at 80℃ for 10 min.

[0063] Viral DNA extraction: Viral DNA was extracted from the DNA-treated samples using a viral nucleic acid extraction kit (magnetic bead method) or a viral DNA / RNA extraction kit.

[0064] 5) rcAAV genome titer detection

[0065] The detection standard, probe, primer and experimental operation are the same as those in "wtAAV viral genome titer detection" above. Take 2 μL of viral DNA extract and perform rcAAV genome titer detection by Taqman probe qPCR. The detection method is the same as "(4) wtAAV (rcAAV) viral genome titer detection".

[0066] III. Detection kit for rcAAV content

[0067] A kit for detecting rcAAV content in adeno-associated virus (aAV) products is provided, specifically comprising the following components: detection cells, namely HeLaRC32 cells; helper virus, namely HuAd5; control sample, namely the wtAAV sample designed in this protocol; the rcAAV standard, the sequence of which is shown in SEQ ID No. 2; and upstream and downstream primers and TaqMan probes for rcAAV genomic titer detection, the sequences of which are shown in SEQ ID Nos. 3-5. The above kit enables the implementation of the method of the present invention for detecting rcAAV content in rAAV products. IV. Detailed Implementation

[0069] Example 1: This embodiment provides a method for detecting rcAAV content, wherein the rcAAV amplification process is as follows: cultured in a 37℃, 5% CO2 cell culture incubator for 24 hours, then replaced with complete culture medium and cultured for another 72 hours. Specific conditions are as follows: According to the protocol for "II. rcAAV content detection", the genomic titer of the target gene was taken as 2×10. 8 Add vg / mL of rAAV test sample to HuAd5 dilution buffer to prepare a target gene genomic titer of 2×10⁻⁶. 6 vg / mL of rAAV test sample solution. Take a genomic titer of 2×10 8 vg / mL, 2×10 7 vg / mL, 2×10 6 vg / mL, 2×10 5 vg / mL, 2×10 4 vg / mL, 2×10 3 wtAAV vg / mL was diluted with HuAd5 dilution buffer to prepare genomic titers of 2×10⁻⁶. 6 vg / mL, 2×10 5 vg / mL, 2×10 4 vg / mL, 2×10 3 vg / mL, 2×10 2 vg / mL, 2×10 1 Vg / mL wtAAV positive control sample solution. Take 2×10⁻⁶ genomic titers. 8 Add vg / mL wtAAV to serum-free culture medium to prepare a genomic titer of 2×10⁻⁶. 6The negative control sample solution was prepared at a concentration of vg / mL. In each round of rcAAV amplification, the complete culture medium was replaced, and the culture was continued for 72 hours before detecting the rcAAV genomic titer. The rcAAV content detection results are shown in Table 2. Table 2

[0070] This invention utilizes the ITR-Rep on the wtAAV genome to characterize the genomic titer of rcAAV. The rcAAV content detection results after 72 hours of amplification culture are as follows: Figure 3 As shown, when the positive control sample contains 2×10 1 When rcAAV was applied at a concentration of vg / mL, its expression was enriched after three rounds of amplification. The genomic titer of the target gene was 2 × 10⁻⁶. 6 The rAAV test sample at vg / mL did not show any rcAAV presence in 1-3 rounds of amplification. Therefore, the rcAAV content in the rAAV test sample after 72h of amplification and incubation was less than 20 / 2E6, or 1 / 1E5. Specific raw data are as follows: The rcAAV genomic titers of the test samples in Example 1 are shown in Table 3: Table 3

[0071] Example 1: The rcAAV genomic titers of the control sample in the first round of amplification are shown in Tables 4-5 (Table 5 is a continuation of Table 4): Table 4

[0072] Table 5

[0073] The rcAAV genomic titers of the control sample in the second round of amplification in Example 1 are shown in Tables 6-7 (Table 7 is a continuation of Table 6): Table 6

[0074] Table 7

[0075] The rcAAV genomic titers of the control sample in the third round of amplification in Example 1 are shown in Tables 8-9 (Table 9 is a continuation of Table 8): Table 8

[0076] Table 9

[0077] Example 2: This embodiment provides a method for detecting rcAAV content, wherein the rcAAV amplification process is as follows: cultured in a 37℃, 5% CO2 cell culture incubator for 24 hours, then replaced with complete culture medium and cultured for another 24 hours. Specific conditions are as follows: According to the protocol for "II. rcAAV content detection", the genomic titer of the target gene was taken as 2×10. 8 Add vg / mL of rAAV test sample to HuAd5 dilution buffer to prepare a target gene genomic titer of 2×10⁻⁶. 6 vg / mL of rAAV test sample solution. Take a genomic titer of 2×10 8 vg / mL, 2×10 7 vg / mL, 2×10 6 vg / mL, 2×10 5 vg / mL, 2×10 4 vg / mL, 2×10 3 wtAAV vg / mL was diluted with HuAd5 dilution buffer to prepare genomic titers of 2×10⁻⁶. 6 vg / mL, 2×10 5 vg / mL, 2×10 4 vg / mL, 2×10 3 vg / mL, 2×10 2 vg / mL, 2×10 1 Vg / mL wtAAV positive control sample solution. Take 2×10⁻⁶ genomic titers. 8 Add vg / mL wtAAV to serum-free culture medium to prepare a genomic titer of 2×10⁻⁶. 6 The negative control sample solution was prepared at a concentration of vg / mL. After replacing the complete culture medium in each round of rcAAV amplification, the culture was continued for 24 hours, and the rcAAV genomic titer was detected. The rcAAV content detection results are shown in Table 10. Table 10

[0078] This invention utilizes the ITR-Rep on the wtAAV genome to characterize the genomic titer of rcAAV. The rcAAV content detection results after 24 hours of amplification culture are as follows: Figure 4 As shown, when the positive control sample contains 2×10 1 When rcAAV was applied at a concentration of vg / mL, its expression was enriched after three rounds of amplification. The genomic titer of the target gene was 2 × 10⁻⁶. 6 In the rAAV test sample with vg / mL, rcAAV was not detected in any of the 1-3 rounds of amplification. Therefore, the rcAAV content in the rAAV test sample after 24h of amplification and culture was less than 20 / 2E6, or 1 / 1E5.

[0079] The detection sensitivity of the positive controls from 72h and 24h amplification cultures in Examples 1 and 2 was 20 vg / mL, and the lowest detectable concentration of rcAAV was 20 vg / mL. That is, if the concentration of rcAAV in the rAAV test sample is ≥20 vg / mL, it can be detected, but the rAAV test sample (target gene genomic titer 2×10⁻⁶) has a lower detection threshold. 6 rcAAV was not detected in the sample (vg / mL), therefore the concentration of rcAAV in the sample is < 20 vg / mL, i.e., the detection limit is 20 / 2E6, or 1 / 1E5. The amplification system of this invention uses an amplification culture time as low as 24 hours, compared to the 72-hour amplification culture time used in the existing technology. This invention has a shorter experimental cycle and better detection efficiency than the existing technology.

[0080] The specific raw data is as follows: The rcAAV genomic titers of the samples to be tested in Example 2 are shown in Table 11: Table 11

[0081] Example 2: The rcAAV genomic titers of the control samples in the first and second rounds of amplification are shown in Tables 12 and 13 (Table 13 is a continuation of Table 12): Table 12

[0082] Table 13

[0083] The rcAAV genomic titers of the control samples in the third round of amplification in Example 2 are shown in Tables 14-15 (Table 15 is a continuation of Table 14): Table 14

[0084] Table 15

[0085] Example 3: This embodiment provides a method for detecting rcAAV content, used to verify the specificity of primers and probes. Screened rcAAV standards, upstream and downstream primers, and Taqman probes were used to detect pwtAAV-ITR plasmid, pHelper plasmid, pCap8Rep2 plasmid, pGOI01 plasmid (target gene 1 plasmid), pGOI02 plasmid (target gene 2 plasmid), HeLaRC32 cell DNA, and template-free control NTC, verifying the detection specificity of the upstream and downstream primers and Taqman probe sequences. The detection method is the same as in "(4) wtAAV (rcAAV) viral genome titer detection". The detection results of the downstream primers and Taqman probes for specificity verification are shown in Tables 16-17 (Table 17 is a continuation of Table 16): Table 16

[0086] Table 17

[0087] As the results show, the positive control pwtAAV-ITR plasmid amplification reaction was positive, while the negative control pGOI01 plasmid, pGOI02 plasmid, pHelper plasmid, HeLaRC32 cell DNA and template-free control NTC all had no CT value. Moreover, when the negative control pCap8Rep2 plasmid had a CT value, it was not lower than the CT value of the lowest concentration of the standard solution. This fully demonstrates that the upstream and downstream primers and Taqman probe sequences used in this invention have good detection specificity.

[0088] Compared with existing technologies, this invention has made significant progress in specificity, sensitivity and experimental efficiency, providing a better solution for the detection of reproducible AAV in adeno-associated virus products.

[0089] The above embodiments illustrate and describe the main features and advantages of the present invention in detail. However, the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of this application.

Claims

1. A method for detecting replicating adeno-associated virus, comprising the following steps: First round of amplification: The test sample, positive control and negative control were diluted to obtain the first round of diluted sample. The first round of diluted sample was used to infect HeLaRC32 cells. After infection, the culture medium was changed after a period of time. After a period of further culture, the cell suspension was collected and subjected to freeze-thaw, HuAd5 inactivation and centrifugation. After centrifugation, the supernatant of the first round of amplification of the test sample, positive control and negative control sample was collected. Second round of amplification: The supernatant from the first round of amplification was diluted a second time to obtain the second round of diluted samples. The second round of diluted samples were used to infect HeLaRC32 cells. After infection, the culture medium was changed after a period of culture. After a period of culture, the cell suspension was collected and subjected to freeze-thaw, HuAd5 inactivation, and centrifugation. After centrifugation, the supernatant from the second round of amplification of the test sample, positive control sample, and negative control sample was collected. Third round of amplification: The supernatant of the second round of amplification was diluted in the third round to obtain the third round of diluted sample. The third round of diluted sample was used to infect HeLaRC32 cells. After infection, the culture medium was changed after a period of culture. After a period of culture, the cell suspension was collected and subjected to freeze-thaw, HuAd5 inactivation, and centrifugation. After centrifugation, the supernatant of the third round of amplification of the test sample, positive control sample, and negative control sample was collected. Detection: Viral DNA was extracted from the supernatant of the first round of amplification, the supernatant of the second round of amplification, and / or the supernatant of the third round of amplification. The viral DNA and replicating adeno-associated virus standards were subjected to quantitative PCR genomic titer detection using specific primers and Taqman probes. The sequences of the specific primers are shown in SEQ ID No. 3-4, and the sequences of the Taqman probes are shown in SEQ ID No.

5.

2. The detection method according to claim 1, wherein, The positive control is wild-type adeno-associated virus; preferably, the wild-type adeno-associated virus is a recombinant adeno-associated virus obtained by co-transfecting HEK293 cells with pHelper plasmid, pCap8Rep2 plasmid and pwtAAV plasmid; more preferably, the pwtAAV plasmid contains the pwtAAV-ITR sequence, and the pwtAAV-ITR sequence is shown in SEQ ID No.

1.

3. The detection method according to claim 1, wherein, The sequence of the replicating adeno-associated virus standard is shown in SEQ ID No.

2.

4. The detection method according to claim 1, wherein, The first round of dilution is an 80-100-fold dilution, and the second and third rounds of dilution are 2-4-fold dilutions. Preferably, in the first, second, and third rounds of amplification, the test sample and the positive control are diluted with HuAd5 diluent, and the negative control is wild-type adeno-associated virus diluted with serum-free culture medium. More preferably, the preparation steps of the HuAd5 diluent are: diluting HuAd5 with serum-free culture medium to obtain a HuAd5 diluent with an MOI of 0.01-0.1, wherein the MOI is preferably 0.

01.

5. The detection method according to claim 1, wherein, In the first, second, and third rounds of amplification, the freeze-thaw cycle was repeated three times to obtain a freeze-thawed cell suspension. Preferably, in the first, second, and third rounds of amplification, the post-infection culture time is 22-26 hours; Preferably, in the first, second, and third rounds of amplification, the time for changing the culture medium and continuing the culture thereafter is 24-72 hours, more preferably 24 hours; Preferably, the step of inactivating HuAd5 is as follows: incubating the frozen-thawed cell suspension at 50-60℃ for 20-40 min to obtain a cell suspension inactivated with HuAd5; Preferably, the centrifugation conditions are 4000-4500 rpm for 10-15 min.

6. A nucleic acid molecule, said nucleic acid molecule comprising sequences encoding adeno-associated virus Rep2 and Cap8 proteins, wherein, The sequence is shown in SEQ ID No.

1.

7. A recombinant plasmid comprising the nucleic acid molecule of claim 6.

8. An adeno-associated virus comprising the recombinant plasmid of claim 7.

9. A kit for detecting replicating adeno-associated virus, comprising: The adeno-associated virus as described in claim 8; The replicating adeno-associated virus standard shown in SEQ ID No. 2; The specific primers shown in SEQ ID No. 3-4 and the Taqman probe shown in SEQ ID No.

5.

10. The kit according to claim 9, further comprising: adeno-associated virus-sensitive somatic cells, preferably, the adeno-associated virus-sensitive somatic cells being HeLaRC32 cells; Preferably, the kit further comprises an auxiliary virus; more preferably, the auxiliary virus is HuAd5.

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