Determination method of AAV relative infection activity

Abstract

The invention discloses a method for determining the relative infection activity of AAV (adeno-associated virus), which comprises the following steps: cell planking: culturing host cells to form a uniform cell suspension, and adding the cell suspension into a cell culture plate; virus dilution: diluting the virus sample and the reference substance according to a plurality of same gradients to obtain a plurality of groups of sample diluents and reference substance diluents with different concentrations; virus infection: adding the same amount of sample diluent and reference diluent into the same cell culture plate, and incubating; gene extraction: extracting an AAV genome and a host cell housekeeping gene from the infected cells; dPCR or qPCR detection is carried out, and the copy number of the target gene and the copy number of the reference gene are quantified; and calculating the infection activity of the virus sample relative to the reference by using binding regression fitting. The problems of poor precision and large variable coefficient can be solved, the problem of dependence on auxiliary viruses and specific cell lines is solved, meanwhile, the intuition and scientificity of infection ability evaluation can be improved, and the experiment efficiency and flux are improved.

Description

Technical Field

[0001] The present invention relates to the field of quality control in biopharmaceuticals, and particularly to a method for determining the relative infectivity of AAV, specifically to a method for detecting the titer of viral vectors for gene therapy, which is mainly applied to the quality control of recombinant adeno-associated virus products in the biopharmaceutical industry. Background Art

[0002] Recombinant adeno-associated virus (rAAV) is a core vector for gene therapy and vaccine development, and its infectivity titer (i.e., the number of virus particles with infectivity per unit volume) is a key indicator for evaluating the quality of the vector. TCID50 (Tissue Culture Infectious Dose 50), the method of median tissue culture infectious dose, is a classic technique for determining the virus titer by observing the infectivity of the virus on cells, and is the internationally recognized "gold standard" for determining the infectivity titer of rAAV. Its principle is to inoculate cells with virus dilutions, detect whether the cells show cytopathic effects (CPE, i.e., visible changes such as cell swelling and detachment) at different dilution degrees, and combine with real-time quantitative PCR (Quantitative PCR, qPCR) technology to quantitatively analyze the replication of the viral genome, and finally calculate the median tissue culture infectious dose to calculate the infectivity of the virus.

[0003] Currently, the determination process of TCID50 is roughly as follows.

[0004] 1. Experimental Materials and Reagents (1) Cell line: HelaRC32 cells: A human cervical cancer HeLa cell-derived line, which stably expresses the rep / cap genes of AAV2 (responsible for virus packaging and replication) after genetic modification, and at the same time depends on the E1, E2, and E4 genes provided by adenovirus (AdV5) to assist in completing the rAAV replication cycle; (2) Complete medium: Containing DMEM (basic medium), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin mixture (Pen / Strep); (3) Serum-free medium (SFM): Containing DMEM, 25 mM Hepes buffer, and 1% Pen / Strep, used in the virus infection stage to avoid serum interference; (4) rAAV sample: The recombinant AAV vector to be tested (such as AAV2 / 2 serotype); (5) Adenovirus (AdV5): Wild-type adenovirus (ATCC VR-1516), used to provide the cofactors required for rAAV replication; (6)DNA extraction reagent: containing 0.25% sodium deoxycholate (DOC), 0.45% Tween-80, 1×proteinase K buffer (containing 10 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) and 1.2 mg / mL proteinase K.

[0005] (7)Real-time PCR reagent: containing TaqMan MasterMix, SV40 polyA specific primers and probes targeting the AAV genome.

[0006] 2. Experimental procedure (1)Cell preparation and plating 1) Resuscitate the HelaRC32 cell line. Take out the cryopreserved cells from the -80 °C liquid nitrogen tank, place them in a 37 °C water bath for rapid thawing, and then transfer them to a T150 culture flask containing 5 mL of complete medium. 2) Subculture: After the cells cover the bottom of the flask (about 72 hours), digest the cells with 0.25% trypsin (2 mL of trypsin per flask), add 8 mL of complete medium to terminate the digestion, and collect the cell suspension into a 50 mL centrifuge tube. 3) Cell counting: Use a hemocytometer and trypan blue staining method to determine the viable cell density, with the target concentration being 8×10 5 cells / mL; for example, if the initial count is 2×10 6 cells / mL, then it needs to be diluted to the target concentration at a ratio of 1:2.5. 4) Plating in 96-well plates: Distribute the diluted cell suspension (50 μL per well, containing 4×10 4 cells / well) into 96-well plates. 5) Pre-culture: Incubate the plates in a 37 °C, 5% CO2 incubator for 16 - 20 hours to ensure that the cells form a confluent monolayer.

[0007] (2)Virus infection 1) Adenovirus dilution: Place the AdV5 stock solution (stored at -80 °C) on ice for thawing, and dilute it with serum-free medium (SFM) to a final concentration of 3.2×10 8 particles / mL. 2) Serial dilution of rAAV: Take 8 sterile cryotubes and dilute the rAAV sample in a gradient of 10 -2 to 10 -11 (with a final volume of 1 mL per tube). 3) Virus infection well plate layout: Inoculate 50 μL of virus dilution (or virus-free SFM as a negative control) into each well, as shown in Table 1 specifically; Table 1 Virus infection well plate layout Dilution Well position Description <![CDATA[10 －6 > A1 - A12 Highest concentration dilution group <![CDATA[10 －7 > B1 - B12 Second highest concentration dilution group ．．．．．． ．．．．．． ．．．．．． Negative control H1 - H12 Contains only adenovirus diluent 4) Infection: Aspirate and discard the original culture medium in the well plate (use a 200 μL pipette without a tip to aspirate the liquid along the well wall to avoid damaging the cells). After adding the virus dilution, incubate at 37 °C for 2 hours to promote virus adsorption. Add 50 μL of serum-free medium to each well and cover with a breathable membrane (such as Qiagen airtight membrane), and continue to incubate for 72 hours.

[0008] (3) DNA extraction 1) Centrifuge to collect cells: Centrifuge the well plate at 1500 rpm for 30 seconds to ensure that the liquid is concentrated at the bottom of the well; 2) Addition of lysis solution: Add 85 μL of pre-prepared extraction solution (containing DOC, Tween, proteinase K, and buffer) to each well, vortex and mix well, then seal; 3) Incubation conditions: Incubate in a water bath at 37 °C for 1 hour (to lyse the cell membrane); incubate in a water bath at 55 °C for 2 hours (proteinase K digests proteins); incubate in a metal bath at 95 °C for 30 minutes (inactivate the enzyme and release DNA).

[0009] (4) Real-time PCR amplification 1) Configure the PCR system as shown in Table 2: Each reaction system is 20 μL and contains: Table 2 Configuration of the PCR system TaqMan MasterMix 10 μL (2× concentration) Forward primer / Reverse primer 0.4 μL each (10 μM) Probe 0.2 μL (10 μM) Template DNA 2.5 μL (Well plate extraction solution) Sterile water Make up to 20 μL 2) Amplification program: Pre-denaturation: 95 °C for 10 minutes; Cycle amplification: 40 cycles (95 °C for 15 seconds + 60 °C for 1 minute); 3) Threshold setting and Ct value calculation: Baseline setting: Select the fluorescence signal in PCR cycles 3 - 12 as the background baseline; Threshold setting: Set the threshold to the middle value of the exponential growth phase of the amplification curve (for example, set the Ct value of the standard 10 8 copies to 13.09).

[0010] (5) Data analysis and TCID50 calculation 1) Determination of positive wells: Detection limit: Set the threshold to 740 copies / well (corresponding to a 74-fold dilution of the PCR detection limit of 10 copies); Positive determination: If the input-subtracted value of a well > 740 copies, it is determined as a positive infection; 2) Calculate TCID50 by the Karber method: Positive rate statistics: Count the number of positive wells for 12 replicate wells of each dilution (such as 8 positive wells in the 10 -6 dilution); Determination of the median endpoint: Find the dilution with the first positive rate ≥ 50% (for example, 10-7 Dilution is the positive threshold); Calculated by the formula: TCID50 = (Dilution × Total virus amount) / (Proportion of positive wells × Infection volume) For example: If the threshold dilution is 10 -8 , and the total input virus amount is 1 × 10 10 particles / mL, then: TCID50 = (10 -8 × 1 × 10 10 ) / (0.5 × 0.05 mL) = 4 × 10 6 TCID50 / mL.

[0011] 3. System suitability confirmation (1) Reference product and parallel control Reference product: Each round of experiment includes the AAV2 / 2 reference product (ATCC VR1616), and its expected TCID50 value should be consistent with historical data (such as 1 × 10 8 TCID50 / mL).

[0012] Negative control: Set wells of adenovirus diluent without virus on each plate to ensure no background contamination.

[0013] (2) Verification of data reproducibility Repeated experiment: The same rAAV sample needs to be repeatedly detected 3 times, and the coefficient of variation (CV) should be ≤ 30%.

[0014] Verification of reagent batches: PCR primers, probes, and extraction reagents need to be regularly tested for consistency between batches.

[0015] As the main method for detecting the titer of AAV in the current industry, TCID50 has the following deficiencies and defects: 1. Poor precision and large coefficient of variation.

[0016] First of all, each virus sample needs to occupy a separate 96-well plate, resulting in physical environmental differences - the spatial distribution of the plates in the incubator may cause local temperature and carbon dioxide concentration gradients (such as the microenvironmental differences between the plates near the heat dissipation area and the central area). At the same time, the small errors in cell seeding density (such as the difference in the pipetting volume of the pipette) or the inoculation time interval (such as the difference of several minutes to dozens of minutes between the first and the last plates) in manual operation further exacerbate the discreteness of the cell metabolic state, thereby affecting the infection efficiency.

[0017] Secondly, the time mismatch in the experimental process leads to fluctuations in infection kinetics. During the virus infection step, the processing time for different wells varies due to plate-by-plate operation (for example, the time interval between virus infection operations for the first and the last plates can be 10 - 30 minutes). In the genomic extraction step, since it needs to be processed plate by plate, the time delay is even more significant (for example, the extraction operation interval between the first and the last plates can reach 1 hour or longer), causing cells in different plates to be in different infection stages (some have completed the infection stable stage, while some are still in the early infection stage), thus triggering fluctuations in infection efficiency.

[0018] Moreover, the limitations in the reference product verification logic introduce the risk of systematic errors. Since the reference product and the test samples are not placed in the same well, the effectiveness of the experiment is judged only by whether the "results of the reference product conform to historical experience values" (for example, if the reference product showed a dilution factor of 1:100 as the infection endpoint in previous experiments, then in the current experiment, if the reference product also shows 1:100, it is considered valid). However, this logic does not synchronously compare the differences between plates, leading to potential risks. In a certain round of experiments, the cell state in the reference product well is good, but in the sample wells, due to differences in microenvironments caused by low density, contamination, or position (such as higher temperature in the wells near the exhaust port), the infection efficiency deviates. Experimenters may misjudge the experiment as valid due to the normal results of the reference product and accept incorrect sample data.

[0019] In addition, the limitations of statistical methods exacerbate the uncertainty of the results. The superposition of multiple factors such as between-plate differences, time delays, and defects in the reference product logic leads to the inability of conventional statistical correction methods (such as taking the mean of repeated experiments) to eliminate systematic biases caused by deviations in probability model assumptions (such as the failure of the "linear relationship between infection probability and virus titer" under heterogeneous conditions).

[0020] Finally, the practical challenges of experimental standardization further magnify the problem. For example, time errors in manual operations, cost limitations of equipment (such as the difficulty in popularizing automated equipment), etc., all lead to the difficulty in completely eliminating variable differences.

[0021] The above problems together lead to insufficient precision in the results of the TCID50 method, significantly affecting its reliability and repeatability in virus titer determination.

[0022] 2. It relies on helper viruses or specific cell lines, and the effect is not ideal for different serotypes.

[0023] The TCID50 method relies on helper viruses (such as wild-type adenovirus AdV5) and specific cell lines (such as Hela-RC32 cells) to activate the replication ability of recombinant AAV (rAAV). Its core principle is that rAAV itself lacks the genes required for autonomous replication (such as viral packaging, DNA replicase, etc.) and must rely on the key genes provided by the helper virus (such as the E1, E2, and E4 genes of adenovirus) or the helper factors already expressed in the cell line (such as the stable helper genes in Hela-RC32 cells) to complete the replication cycle. However, this method has significant limitations: 1. The contradiction between fixed detection conditions and serotype diversity: Different AAV serotypes (such as AAV1, AAV9, etc.) have tissue-specific infection preferences. For example, AAV2 tends to infect hepatocytes, while AAV9 is more likely to infect nerve cells or muscle cells. Since the TCID50 method uses a single cell line (such as Hela-RC32) for detection, its results only reflect the infection ability of this specific cell line and cannot evaluate the actual infection efficiency of rAAV in the target tissue or cell type. For example, if a drug uses the AAV9 serotype to target muscle cells, but the experiment is still based on Hela-RC32 cells (a non-muscle cell line), the detection results cannot truly reflect the infection efficacy of AAV9 in the target tissue.

[0024] 2. The irreplaceability of helper viruses and cell lines: The selection of helper viruses (such as AdV5) and specific cell lines (such as Hela-RC32) is limited by the need for experimental condition standardization. However, different serotypes may require different combinations of helper factors or more suitable host cells (such as using a hepatocyte cell line to detect AAV2 and a nerve cell line to detect AAV9). If fixed conditions are forced, the infection ability of some serotypes may be underestimated or overestimated due to cell type mismatch. For example, the AAVrh10 serotype has a high tropism for retinal cells, but its infection efficiency may be low in Hela-RC32 cells due to the lack of cell membrane receptors, thus misleading the titer assessment.

[0025] 3. The general applicability defect of experimental design: Since the TCID50 method relies on a specific detection system, it cannot be directly extended to cross-cell line comparisons of different serotypes. For example, if it is necessary to evaluate the infection differences of the same rAAV in hepatocytes and nerve cells, multiple sets of helper virus-cell line systems need to be repeatedly established, significantly increasing the experimental complexity and cost, making it difficult for the method to meet the titer detection requirements of diverse serotype drugs in practical applications.

[0026] The above problems together lead to the lack of general applicability of the TCID50 method in evaluating different AAV serotypes. Especially when optimizing viral vectors for specific tissues or cell types in drug development, its results may seriously deviate from the actual infection ability in practical applications.

[0027] 3. The reflected AAV infection ability is not intuitive.

[0028] The fixed use of Hela-RC32 cells as a detection platform leads to cell type-specific limitations. The biological characteristics of this cell line may be significantly different from the tissue cells targeted by the actual drug. For example, if a certain drug uses AAV9 serotype to target muscle cells, but the experiment is still detected based on Hela-RC32 (cervical cancer cell line), the results cannot reflect the actual infection efficiency of AAV9 in muscle cells (such as differences in infection ability due to different expression levels of cell surface receptors on muscle cells). The infection of AAV serotypes to host cells depends on the specific binding of the virus capsid to cell surface receptors (such as AAV2 depends on heparan sulfate proteoglycan, and AAV9 depends on statin-sensitive receptor 2). Hela-RC32 cells may be unable to comprehensively evaluate the infection ability of rAAV in target cells due to limited receptor expression profiles. For example, AAVrh10 serotype has a high tropism for retinal cells, but may have extremely low infection efficiency in Hela-RC32 due to receptor deficiency, resulting in an incorrect determination of its low titer.

[0029] Ultimately, the TCID50 method indirectly evaluates the rAAV infection ability through the synergistic effect of helper virus and fixed cell line. However, its results are affected by helper virus interference, cell line-specific limitations, and the indirectness of experimental design, and cannot intuitively and accurately reflect the true infection efficacy of rAAV in target cells or tissues. Especially when optimizing virus vectors for specific target cells in drug development, its limitations may seriously hinder the R & D process.

[0030] 4. The experiment is time-consuming and has low throughput.

[0031] The TCID50 method has significantly insufficient efficiency due to its long experimental process, high resource occupancy rate, and low throughput, specifically manifested as: From the start of the experiment to obtaining the results, it needs to go through multiple time-consuming links, including the cell preparation stage (it takes about 7 days from the resuscitation of Hela-RC32 cells to being available for the experiment, including subculture and density adjustment; plating requires inoculating cells into 96-well plates 1 day in advance and culturing for 24 hours to ensure the density meets the standard), virus infection and incubation (after virus inoculation, it needs to be cultured for 72 hours to observe the cytopathic effect CPE, and during this period, it needs to be observed and recorded under a microscope multiple times), genomic extraction and qPCR detection (genomic extraction needs to be carried out immediately after infection, and the infection endpoint is confirmed by qPCR, which takes about 1 day). The total process takes about 10 - 15 days; Experimental resources and throughput are limited by equipment and operating conditions. For example, each 96-well plate can only accommodate a single sample, and each round of experiment must include a reference product (for titer calibration). If only one qPCR instrument is equipped, at most 2 samples can be processed in a single round of experiment (1 plate for the sample and 1 plate for the reference product), and qPCR needs to be run in batches (such as the instrument throughput is 96 wells / time, and each plate needs to be detected separately), further reducing the throughput; In addition, the risk of experimental failure is extremely high. If the results are abnormal due to cell contamination, out-of-control adenovirus MOI (such as too high / low titer), misjudgment of CPE, or failure of genomic extraction, it is necessary to start from the cell resuscitation stage and repeat the entire 15-day process, resulting in a significant increase in consumable costs (cell culture medium, adenovirus, reference product, well plates, etc.) and human input; Finally, the TCID50 method is difficult to meet the needs of drug development due to time and throughput limitations. Summary of the Invention

[0032] Aiming at the above defects existing in the prior art, the purpose of the present invention is to provide a method for determining the relative infectivity of AAV, which can solve the problems of poor precision and large coefficient of variation, solve the problems of relying on helper viruses and specific cell lines, and at the same time can improve the intuitiveness and scientificity of infectivity evaluation, and improve experimental efficiency and throughput.

[0033] The present invention provides a method for determining the relative infectivity of AAV, comprising the following steps: Cell seeding, culturing host cells to form a uniform cell suspension, and adding the cell suspension into a cell culture plate; Virus dilution, diluting the virus sample and the reference product according to multiple identical gradients to obtain multiple groups of sample dilutions and reference product dilutions with different concentrations; Virus infection, adding equal amounts of sample dilutions and reference product dilutions into the same cell culture plate and incubating; Gene extraction, extracting the AAV genome and the host cell housekeeping gene from the infected cells; dPCR or qPCR detection, quantifying the copy numbers of the target gene and the internal reference gene; Using constrained linear regression to calculate the infectivity of the virus sample relative to the reference product.

[0034] Furthermore, the host cells of the present invention include one of 293 / 293A cells, C2C12 cells or ARPE-19 cells.

[0035] The 293 / 293A cells serve as a universal host cell, which only needs to support the entry and stable presence of the genomes of most AAV serotypes into the cells (without completing the viral replication cycle). AAV is directly inoculated into 293 / 293A cells at a multiplicity of infection (MOI), without adding adenovirus or other auxiliary factors. After infection, the AAV genome (such as the PolyA sequence) is directly detected by dPCR (digital PCR), without relying on viral replication or AdV5 auxiliary functions. The innovation of this design lies in: the detection logic is changed from "triggering CPE or genome amplification through viral replication" to "directly quantifying the integration efficiency of the AAV genome after infection"; the universality of the cell line, any cell line that can support the entry and stable presence of the AAV genome can be used (such as the muscle cell line C2C12 or the retinal cell line ARPE-19), as long as the experimental conditions are met (such as surface receptor expression, medium adaptability). For example, 293 / 293A cells are selected as the standard protocol because they are easy to culture and can efficiently support AAV infection, but they are not the only choice.

[0036] Furthermore, the host cell described in the present invention is 293 / 293A cells, and the culture is optimized using a complete medium containing 1-10 mM hydroxyurea.

[0037] Furthermore, the step of plating the cells in the present invention further includes adjusting the density of the host cells.

[0038] Furthermore, in the step of viral infection described in the present invention, at least 2 wells are set for each concentration of the sample diluent and the reference product diluent.

[0039] Furthermore, in the step of viral infection described in the present invention, a negative control well is set in the cell culture plate.

[0040] Furthermore, the dPCR detection step described in the present invention includes: Preparing a dPCR reaction system according to the number of samples, and aliquoting the dPCR reaction system into a PCR well plate; Aliquoting the samples, diluting the extracted DNA, and adding the reference product DNA, sample DNA, NTC, and NC DNA into the corresponding wells respectively; Transferring the liquid in the PCR well plate to a nanoplate, adopting the original layout of the PCR well plate; Putting the NanoPlate into a dPCR instrument and running the reaction program.

[0041] Furthermore, the step of calculating the infection activity of the virus sample relative to the reference product described in the present invention includes: Exporting the data after the completion of the dPCR or qPCR run; Calculating the target DNA ratio (target gene / endogenous reference gene) through the following formula: Target DNA ratio = Concentration of target gene (copies / μL) / Concentration of reference gene (copies / μL); Constrained linear regression, and calculate Relative Infectivity through the following formula: Relative Infectivity (%) = antilog[(intercept of the test sample curve - intercept of the AAV control curve) / common slope].

[0042] Furthermore, the present invention also includes quality control. During the dPCR stage, negative control and no-template control are synchronously detected. If the PolyA copy number of NC > 5 copies / μL or a signal appears in NTC, it is determined that there is a risk of contamination in the experiment and the experiment needs to be redesigned; if the data of the reference product does not meet the standard, it is determined that the experimental conditions are not up to standard and the infection and detection steps need to be retested.

[0043] The technical solution provided by the present invention has the following beneficial effects compared with the prior art: 1. Analyze the reference product and the sample on the same well plate to reduce variables; 2. Exclude the participation of wtAdV5, making the experiment simpler and safer; 3. Develop a platform-based in vitro relative infectivity method that can detect differences in AAV vector infectivity as low as 25%. Compared with TCID50, this represents a significant improvement; 4. The relative infectivity method has linearity, accuracy, and precision within the range of 50 - 200% relative infectivity; 5. The relative infectivity method can detect changes in infectivity after forced degradation; 6. In the absence of a quantitative product-specific in vitro potency method, relative infectivity detection is more reliable than TCID50 and can be used to support product comparability assessment and monitor product stability. Description of the Drawings

[0044] Figure 1 It is a linear result diagram of an embodiment of the present invention. Detailed Embodiments

[0045] The following further elaborates on the present invention in detail with reference to embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments.

[0046] This embodiment discloses a method for determining the relative infectivity of AAV, including the following steps: S1. Cell seeding. After culturing host cells to form a uniform cell suspension, add the cell suspension into a cell culture plate; S2. Virus dilution: Dilute the virus sample and the reference product at multiple identical gradients to obtain sample dilutions and reference product dilutions at multiple concentrations. S3. Virus infection: Add equal amounts of the sample dilution and the reference product dilution to the same cell culture plate and incubate. S4. Gene extraction: Extract the AAV genome and the host cell housekeeping gene from the infected cells. S5. dPCR detection: Quantify the copy numbers of the target gene and the internal reference gene. S6. Calculate the infectivity activity of the virus sample relative to the reference product.

[0047] The detection principle is as follows: As a representative vector for gene therapy, the AAV product needs to be evaluated for its biological activity from multiple aspects, such as infectivity activity, protein expression activity, protein function activity, etc. Among them, the infectivity activity can usually be used as a platform and general evaluation index to conduct the first-dimensional activity assessment of similar vectors carrying different genes. The infectivity activity refers to the ability of the AAV vector to infect target cells, enter the target cells, and transfer the target gene into the target cells. The relative infectivity activity is the relative value of the infectivity activity of a batch of products to that of the reference product batch. The relative infectivity activity is between 0 - 100%. During detection, the products of the batch to be tested and the reference product batch are used to infect target cells simultaneously, and each batch of products is subjected to infection experiments with multiple gradient concentrations. Harvest the cells at an appropriate time and extract the DNA or RNA in the cells, and use specific primers to detect the target gene. During data processing, the values of the target genes of the two groups of the products of the batch to be tested and the reference product batch at the same concentration can be obtained. By performing regression analysis on the two groups of data, the relative activity of the two batches of products can be calculated.

[0048] The experimental materials and instruments used in this example are shown in Table 3.

[0049] Table 3 Functions and operation key points of materials and instruments Material / Instrument name Use Explanation 293A cells Simulate virus host Provide target cells for AAV infection HU Culture medium additive Promote the ability of AAV to infect cells dPCR instrument Statistical gene copy number By physically dividing the reaction system, directly count the number of virus genes and host genes Primer Specifically recognize target genes Precisely recognize DNA fragments of virus genes (PolyA) and host genes (HK) DNeasy kit Extract DNA Purify DNA fragments of virus genes (PolyA) and host genes (HK) In this example, the AAV sample to be detected has a virus titer of vg = 2E+12 vg / mL. The specific experimental procedure is as follows: S1. Cell seeding: The purpose of cell seeding is to provide host cells for virus infection, specifically including: S11. Culture the host cells to form a uniform cell suspension.

[0050] In this example, the host cells are 293A cells (human embryonic kidney cell line, with the E1A helper factor) in the logarithmic growth phase, which directly support the replication cycle of AAV9 and other serotypes and do not require dependence on adenovirus. Such a design eliminates the risk of adenovirus contamination and at the same time avoids the problem of overestimation of the infectivity ability caused by the synergistic effect of AdV5 and AAV serotypes in the prior art.

[0051] The host cells were cultured by resuspending the cells in complete medium (DMEM + 10% FBS) containing 4 mM hydroxyurea (HU). DMEM: Dulbecco's Modified Eagle Medium, which provides nutrients such as glucose, amino acids, and vitamins required for cell growth. 10% FBS: 10% fetal bovine serum is added, which provides growth factors, hormones, and proteins to help the cells adhere and proliferate.

[0052] The precipitated 293A cells after centrifugation were dispersed into the medium to form a uniform cell suspension.

[0053] S12. Unified control of inoculation density.

[0054] The cell density was adjusted to the same standard. In this example, the density of 293A cells was controlled at 4×10 5 cells / mL to ensure that the number of cells in each well was the same, eliminating fluctuations in infection efficiency caused by differences in inoculation density, such as different virus infection efficiencies and gene expression levels.

[0055] For example: If the original concentration of the cell suspension was 2×10 6 cells / mL, 2 mL of the cell suspension was taken and added to 8 mL of medium for dilution to obtain a cell suspension with a density of 293A cells of 4×10 5 cells / mL.

[0056] Of course, the selection of host cells and medium in this example is not limited to the above methods. By changing the host cell line (such as C2C12 muscle cell line, ARPE-19 retinal cell line) and optimizing the medium composition (such as containing 4 mM hydroxyurea to inhibit non-specific proliferation), the detection requirements of different AAV serotypes can be adapted. For example: For muscle-targeted AAV9, C2C12 cells (highly expressing ST6GalNAc2 receptor) were used instead to ensure that the infection efficiency matched the clinical target cells.

[0057] For retinal-targeted AAVrh10, ARPE-19 cells (expressing retinal-related receptors) were used instead to avoid the receptor expression limitations of Hela-RC32.

[0058] In this example, only by adjusting the host cell type and medium formula, it can be quickly extended to the evaluation of multiple serotypes such as AAV2, AAV9, and AAVrh10, significantly reducing the experimental complexity and cost.

[0059] This embodiment can enable the detection of infection titer to get rid of the dependence on AdV5 and Hela-RC32 cells, directly reflect the infection ability of the target serotype in clinically relevant cells, and at the same time achieve multi-serotype compatibility through cell line replacement, significantly improving the universality and clinical relevance of the detection. For example, for the AAV9 muscle-targeted vector, after changing to C2C12 cells, its infection efficiency is 10-20 times higher than that of Hela-RC32 (due to receptor matching), and there is no need for adenovirus assistance, avoiding the risk of cross-contamination.

[0060] S13. Plating operation, add the cell suspension into the cell culture plate.

[0061] This embodiment uses a 48-well cell culture plate, add 500 μL of the adjusted cell suspension to each well; gently shake the culture plate to evenly spread the cells, and pay attention to avoiding the generation of bubbles during the process; then place the cell culture plate in an incubator at 37 °C and 5% CO2 for 22-26 hours. The layout of the culture plate is shown in Table 4.

[0062] Table 4 48-well cell culture plate 1 2 3 4 5 6 7 8 A 293A 293A 293A 293A 293A 293A 293A 293A B 293A 293A 293A 293A 293A 293A 293A 293A C 293A 293A 293A 293A 293A 293A 293A 293A D 293A 293A 293A 293A 293A 293A 293A 293A E 293A 293A 293A 293A 293A 293A 293A 293A F 293A 293A 293A 293A 293A 293A 293A 293A S2. Virus dilution, dilute the virus sample and the reference product according to multiple identical gradients to obtain sample dilutions and reference product dilutions with multiple concentrations. Specifically, it includes: S21. Reference product dilution Assume the reference product titer vg = 2.7×10 13 vg / mL. To detect whether the cells show lesions at different dilutions, 5 dilution gradients are prepared (such as 4×10 10 ~2.5×10 9 vg / mL) and added to the culture plate. The dilution scheme is shown in Table 5.

[0063] Table 5 Gradient dilution of reference product

[0064] Dilution 1: Take 2 μL of the reference product + 1348 μL of DMEM → total volume 1350 μL → the titer after 1 dilution is 4×10 10 vg / mL, and the dilution factor is 675 times.

[0065] Dilution 2: Take 100 μL of the reference product after 1 dilution + 100 μL of DMEM → total volume 200 μL → the titer after 2 dilutions is 2×10 10 vg / mL, and the dilution factor is 2 times, with a total dilution factor of 1350 times.

[0066] And so on, Dilution 2-5: Dilute by a 2-fold gradient to the lowest concentration of 2.5×10 9vg / mL, with a total dilution factor of 10,800 times.

[0067] S22. Dilution of virus samples Synchronize the dilution gradient of the reference product and adjust the initial dilution volume according to the corresponding virus sample titer (vg / mL). For example, if the virus sample titer vg = 2×10 12 vg / mL, then the corresponding initial dilution volume is: Take 10 μL of the sample + 490 μL of DMEM to obtain a 4×10 10 vg / mL dilution, with a dilution factor of 50. Subsequently, dilute it in a 2-fold gradient until it is diluted to 2.5×10 9 vg / mL as the lowest concentration virus diluent. See Table 6 for the specific dilution scheme.

[0068] Table 6. Gradient dilution of virus samples

[0069] S3. Virus infection. Add equal amounts of the sample diluent and the reference product diluent to the same cell culture plate.

[0070] Specifically, add 50 μL of the diluted virus liquid to each well. In this example, a 48-well culture plate (6 rows × 8 columns) is used, and virus samples, reference products, and negative controls are synchronously detected within the same plate. For example, for 3 AAV9 samples and 1 reference product (such as AAV2 / 2 control), set 5 dilution gradients (such as 4×10 10 ~2.5×10 9 vg / mL) on each plate, with 2 wells set for each gradient (replicated across two plates or in duplicate rows within the same plate), and simultaneously include the reference product dilution series and the negative control. See Table 7 for details.

[0071] Table 7. Control design for the culture plate 1 2 3 4 5 6 7 8 A Sample 1 dilution 1 Sample 1 dilution 1 Sample 2 dilution 1 Sample 2 dilution 1 Sample 3 dilution 1 Sample 3 dilution 1 Reference product dilution 1 Reference product dilution 1 B Sample 1 dilution 2 Sample 1 dilution 2 Sample 2 dilution 2 Sample 2 dilution 2 Sample 3 dilution 2 Sample 3 dilution 2 Reference product dilution 2 Reference product dilution 2 C Sample 1 dilution 3 Sample 1 dilution 3 Sample 2 dilution 3 Sample 2 dilution 3 Sample 3 dilution 3 Sample 3 dilution 3 Reference product dilution 3 Reference product dilution 3 D Sample 1 dilution 4 Sample 1 dilution 4 Sample 2 dilution 4 Sample 2 dilution 4 Sample 3 dilution 4 Sample 3 dilution 4 Reference product dilution 4 Reference product dilution 4 E Sample 1 dilution 5 Sample 1 dilution 5 Sample 2 dilution 5 Sample 2 dilution 5 Sample 3 dilution 5 Sample 3 dilution 5 Reference product dilution 5 Reference product dilution 5 F NC - 1 NC - 2 NC - 3 NC - 4 NC - 5 NC - 6 Set 2 wells for each dilution degree to reduce systematic errors during data analysis. For example, A1 and A2 are the same sample at the same concentration. At the same time, set a negative control NC (containing only DMEM) for each experiment to verify that the experiment is not contaminated.

[0072] After adding the virus liquid, place the 48-well culture plate back into the incubator and incubate for 44 - 52 hours.

[0073] In this example, a within-plate control design is adopted, which allows 3 samples and 1 reference product to share the same 48-well culture plate, completely eliminating differences in temperature, CO2 concentration, etc. between plates, and can solve the problems of poor precision and large coefficient of variation.

[0074] Meanwhile, only one 48-well culture plate is needed to complete the detection of all dilution gradients (4×10 10 ~2.5×10 9 vg / mL), and the throughput can be increased by 4 times. Compared with TCID50, only one sample can be detected with one 96-well plate, and four 96-well plates are needed to process the same number of samples and reference products.

[0075] S4. Gene extraction: Extract the AAV genome (GOI) and host cell housekeeping gene (HK) from infected cells. The specific steps are as follows: S41. Cell lysis and DNA release 1) Aspirate the culture medium, resuspend the cells with 1 mL of PBS, and pipette up and down 8 - 10 times with a 1 mL pipette tip to ensure that most cells are collected. Transfer the liquid to a 1.5 mL centrifuge tube. 2) Centrifuge at 10000 rpm for 1 minute using a centrifuge tube. Carefully aspirate the supernatant with a pipette tip and retain the cell pellet.

[0076] S42. DNA purification and elution 1) Sequentially add 200 μL of PBS, 20 μL of proteinase K (20 mg / mL), 4 μL of RNase A (100 mg / mL), and 200 μL of Buffer AL to the pellet. Mix well in a timely manner and then place the centrifuge tube in a 56°C water bath for 10 minutes. 2) After the water bath, take out the centrifuge tube, add 200 μL of absolute ethanol, vortex for 15 seconds in a timely manner, centrifuge quickly, collect the liquid that has been vortexed onto the bottle cap, and transfer the liquid to a DNeasy spin column in a timely manner. 3) Centrifuge at 8000 rpm for 1 minute and replace the 2 mL collection tube with a new one. 4) Add 500 μL of Buffer AW1 to the spin column, centrifuge at 8000 rpm for 1 minute, discard the filtrate, and replace the 2 mL collection tube with a new one. 5) Add 500 μL of Buffer AW2 to the spin column, centrifuge at 13300 rpm for 3 minutes, discard the filtrate, and replace the 1.5 mL centrifuge tube. 6) Add 50 μL of Buffer AE to the center of the spin column, incubate the spin column at room temperature for 1 minute, centrifuge at 8000 rpm for 1 minute, discard the spin column (if a higher concentration is required, the filtrate can be collected and passed through the spin column again), retain the filtrate, and obtain the DNA solution.

[0077] S43. Measure the DNA concentration Use NanoDrop to measure the concentration of the extracted DNA, and at the same time pay attention to A260 / 280 to evaluate the quality of the extracted DNA in this experiment.

[0078] S5. dPCR detection to quantify the copy numbers of the target gene (GOI) and the reference gene (HK). The specific steps are as follows: S51. Preparation of the dPCR reaction system 1) Prepare the dPCR reaction system according to the number of samples. Table 8 shows the dPCR reaction system formulation required for a single sample well.

[0079] Table 8 dPCR reaction system formulation Reagent 1× reaction volume 4× Probe PCR Master Mix (4× probe - based PCR premix) 3 μL Target primer F (20 μM) 0.48 μL Target primer R (20 μM) 0.48 μL Target probe (20 μM) 0.24 μL Reference primer F (20 μM) 0.48 μL Reference primer R (20 μM) 0.48 μL Reference probe (20 μM) 0.24 μL EcoRI endonuclease 0.25 μL Sterilized water 1.35 μL Total volume 7 μL 2) Aliquot the dPCR reaction system into clean PCR well plates using a 10 μL pipette tip, adding 7 μL to each well.

[0080] S52. Sample aliquoting 1) Uniformly dilute the extracted DNA to 10 ng / μL using Buffer AE. The specific dilution scheme is adjusted according to the sample concentration. Add the reference DNA, sample DNA, NTC (negative control, using AE Buffer), and NC DNA to the corresponding wells, each adding 5 μL. See Table 9 for details.

[0081] Table 9 PCR well plate control design 1 2 3 4 5 6 7 8 A Reference product 1 DNA dilution 1 Reference product 1 DNA dilution 1 Reference product 2 DNA dilution 1 Reference product 2 DNA dilution 1 Sample 1DNA dilution 1 Sample 1DNA dilution 1 Sample 2DNA dilution 1 Sample 2DNA dilution 1 B Reference product 1 DNA dilution 2 Reference product 1 DNA dilution 2 Reference product 2 DNA dilution 2 Reference product 2 DNA dilution 2 Sample 1 DNA dilution 2 Sample 1 DNA dilution 2 Sample 2 DNA dilution 2 Sample 2 DNA dilution 2 C Reference product 1 DNA dilution 3 Reference product 1 DNA dilution 3 Reference product 2 DNA dilution 3 Reference product 2 DNA dilution 3 Sample 1 DNA dilution 3 Sample 1 DNA dilution 3 Sample 2 DNA dilution 3 Sample 2 DNA Dilution 3 D Reference Standard 1 DNA Dilution 4 Reference Standard 1 DNA Dilution 4 Reference Standard 2 DNA Dilution 4 Reference Standard 2 DNA Dilution 4 Sample 1 DNA Dilution 4 Sample 1 DNA Dilution 4 Sample 2 DNA Dilution 4 Sample 2 DNA Dilution 4 E Reference Standard 1 DNA Dilution 5 Reference Standard 1 DNA Dilution 5 Reference Standard 2 DNA Dilution 5 Reference Standard 2 DNA Dilution 5 Sample 1 DNA Dilution 5 Sample 1 DNA Dilution 5 Sample 2 DNA Dilution 5 Sample 2 DNA Dilution 5 F G NC-1 NC-1 NTC NTC H NC-2 NC-2 2) Seal the PCR well plate with a sealing film, transfer it to a PCR shaker, and mix the samples and the dPCR reaction system for 30 seconds at 2000 rpm.

[0082] 3) Place the PCR well plate in a well plate centrifuge and centrifuge for 10 seconds to concentrate the liquid at the bottom of the well.

[0083] S53. Preparation of the NanoPlate 1) Transfer 11 μL of the liquid in the PCR well plate to an 8.5k nanoplate, maintaining the original layout of the PCR well plate; 2) Seal the 8.5k nanoplate with a sealing film.

[0084] S54. Loading the NanoPlate onto the instrument 1) Set the PCR reaction program in the instrument, as shown in Table 10 for details.

[0085] Table 10 PCR reaction program settings

[0086] 2) Set the instrument signal collection channels (detecting GOI with the FAM channel and HK with the HEX channel), as shown in Table 11 for details.

[0087] Table 11 Instrument Signal Collection Channels Channel Exposure Time [ms] Gain Green Channel Green (FAM) 500 6 Yellow Channel Yellow (HEX) 500 6 3) Place the NanoPlate into the dPCR instrument and run the reaction program.

[0088] In this example, dPCR absolute quantification is used to achieve absolute quantification of copy number at the single-molecule level through the QIAcuity One system, eliminating the amplification efficiency differences of qPCR. For example, dPCR distributes the amplification signals of the target gene (PolyA sequence) and the internal reference gene (HK, such as the housekeeping gene GAPDH) to independent droplets, and directly reflects the copy number by the "number of positive droplets", and the CV can be reduced to <20%. At the same time, by calculating the PolyA / HK Ratio (the ratio of the copy number of the target gene to the copy number of the housekeeping gene of the host cell), the errors caused by differences in cell density and lysis efficiency are corrected (such as the constant copy number of the HK gene can eliminate the influence of cell number fluctuations).

[0089] S6. Data processing, calculating the infectivity of the virus sample relative to the reference sample. The specific steps are as follows: S61. Data export 1) After the run is completed, open the QIAcuity software suite, click on the "..." in the upper right corner of the nanoplate icon, and then select "Analysis".

[0090] 2) To set the plate layout, define the reaction mixture, samples, and controls. The plate layout can be set before or after the nanoplate run. After the run is completed, the raw data will be automatically transferred to the QIAcuity software suite.

[0091] 3) Select the "target wells" to be analyzed, click "Show Results" after selecting the detection target.

[0092] 4) Click "Export as CSV" to export the raw data and save the file to a secure folder.

[0093] S62. Data calculation 1) The reportable results and system / sample suitability criteria (such as coefficient of variation %CV and recovery rate %) can be automatically calculated using formulas in the Excel worksheet template, and data outside the measurement range can be excluded.

[0094] 2) Open the exported CSV file in Microsoft Excel and copy and paste the data into the corresponding Excel worksheet template.

[0095] 3) According to the "Concentration" column (unit: copies / μL) in the exported CSV file, calculate the target DNA ratio (PolyA / HK) using the following formula: Target DNA ratio = PolyA concentration (copies / μL) / HK concentration (copies / μL) 4) Constrained linear regression: Using the built-in program, with the logarithm of the vg concentration of the reference standard (ref Std) and the dilution series of the test samples as the abscissa, fit the relationship curve between the logarithm of the PolyA / HK ratio and it. Calculate the relative infectivity through the following formula: Relative infectivity (%) = antilog[(intercept of the test sample curve - intercept of the AAV control curve) / common slope] In this example, linearity was evaluated by serially diluting the AAV sample and testing 6 dilution points separately using the AAV8 reference standard. The measured results of relative infectivity were compared with the expected results using linear regression. The expected results were recalculated based on the average precision results. Calculate the coefficient of determination R 2 = 0.9902, slope = 0.8892, and y-intercept = 0.091. The coefficient of determination (R 2 ) value was reported as the linearity result, see Figure 1 . For a summary of the results, see Table 12.

[0096] Table 12 Results of relative infectivity experiment

[0097] It can be seen that in this example, the method for measuring relative infectivity can detect differences in AAV vector infectivity as low as 25%, and the method is linear, accurate, and precise in the range of relative infectivity of 50 - 200%.

[0098] The recovery rate can be calculated by the measured values (relative infectivity percentage) and the expected values (relative infectivity percentage) at each dilution point. The lowest and highest recovery rates within the reported range are used as the accuracy results. For a summary of the specific results, see Table 13.

[0099] Recovery rate % = measured value (relative infectivity %) / expected value (relative infectivity %) × 100 Table 13 Results of recovery rate experiment Test Number Expected Result Test Result % Recovery Test 1 184% 171% 93% Test 2 184% 172% 93% Test 3 123% 118% 96% Test 4 123% 112% 91% Test 5 102% 109% 107% Test 6 102% 104% 102% Test 7 82% 84% 103% Test 8 82% 80% 98% Test 9 61% 67% 109% Test 10 61% 63% 102% Test 11 41% 39% 95% Test 12 41% 45% 110% The recovery rate range is 93% to 110%, meeting the accuracy acceptance criteria (recovery rate = 50% to 200%), and the accuracy is good.

[0100] S7. Quality control confirmation. The specific steps are as follows: S71. Confirm whether the experimental system is used in this experiment and whether the relevant indicators meet the quality control requirements, as shown in Table 14 specifically.

[0101] Table 14 Experimental Standards Quality Control Acceptance Criteria Effective Droplet Number of AAV Reference Standard ≥8000 Negative Droplet Proportion of AAV Reference Standard 3%-94% Copy Number of Target Gene in Negative Control <5 copies / μL Copy Number of No-Template Control NTC <5 copies / μl Coefficient of Variation of AAV Reference Standard <30% <![CDATA[Standard curve linear coefficient R 2 > ≥ 0.95 S72. Confirm whether the data of this sample is available, as shown in Table 15 specifically.

[0102] Table 15 Sample Data Standards Quality Control Acceptance Criteria Effective Droplet Number of AAV Sample ≥8000 Negative Droplet Proportion of AAV Sample 3%-94% Coefficient of Variation of AAV Sample <30% During the dPCR stage, synchronously detect the negative control and the no-template control. If the PolyA copy number of NC > 5 copies / μL or a signal appears in NTC, it is determined that there is a risk of experimental contamination and the experiment needs to be redesigned; if the reference product data does not meet the standards, it is determined that the experimental conditions are not up to standard and the infection and detection steps need to be retested.

[0103] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

1. A method for determining the relative infectivity of AAV, characterized in that, It includes the following steps: Cell seeding: After culturing the host cells to form a uniform cell suspension, add the cell suspension into a cell culture plate; Virus dilution: Dilute the virus sample and the reference product according to multiple identical gradients to obtain multiple groups of sample dilutions and reference product dilutions with different concentrations; Virus infection: Add equal amounts of the sample dilution and the reference product dilution into the same cell culture plate and incubate; Gene extraction: Extract the AAV genome and the host cell housekeeping gene from the infected cells; dPCR or qPCR detection: Quantify the copy numbers of the target gene and the internal reference gene; Use constrained regression fitting to calculate the infectivity of the virus sample relative to the reference product.

2. The method for measuring the relative infectivity of AAV according to claim 1, wherein: The host cell is one of 293A cells, C2C12 cells or ARPE-19 cells.

3. The method for determining the relative infectivity of AAV according to claim 1 or 2, characterized in that: The host cell is 293 / 293A cells, and the culture is optimized using a complete medium containing 1-10 mM hydroxyurea.

4. The method for determining the relative infectivity of AAV according to claim 1, characterized in that: The step of cell seeding further includes adjusting the density of the host cells.

5. The method for determining the relative infectivity of AAV according to claim 1, characterized in that: In the step of virus infection, at least 2 wells are set for the sample dilutions and reference product dilutions of each concentration.

6. The method for determining the relative infectivity of AAV according to claim 1, wherein: In the step of virus infection, a negative control well is set in the cell culture plate.

7. The method for determining the relative infectivity of AAV according to claim 1, characterized in that: The dPCR detection step includes: Configure the dPCR reaction system according to the number of samples, and aliquot the dPCR reaction system into a PCR well plate; Sample aliquoting: Dilute the extracted DNA, and add the reference product DNA, sample DNA, NTC and NC DNA into the corresponding wells respectively; Transfer the liquid in the PCR well plate to a nanoplate, adopting the original layout of the PCR well plate; Put the NanoPlate into a dPCR instrument and run the reaction program.

8. The method for determining the relative infectivity of AAV according to claim 1 or 7, characterized in that: The step of calculating the infectivity of the virus sample relative to the reference product includes: Export the data after the completion of dPCR or qPCR run; Calculate the target DNA ratio; Constrained linear regression to calculate the relative infectivity.

9. The method for measuring the relative infectivity activity of AAV according to claim 1, wherein: It also includes quality control. At the dPCR stage, the negative control and the no-template control are synchronously detected. If the PolyA copy number of NC > 5 copies / μL or a signal appears in NTC, it is determined that there is a risk of contamination in the experiment and the experiment needs to be redesigned; if the data of the reference product do not meet the standard, it is determined that the experimental conditions are not up to standard and the infection and detection steps need to be retested.

Images