Ultra-high precision viral vector assay

Abstract

During the production of replication-defective viral gene therapy vectors, random mutations or other events can produce unwanted replication-competent viruses ("RCVs"). Viral gene therapy vector producers therefore assay for the presence of contaminating RCVs by assaying for serial infection, i.e., transducing target cells with a viral vector, then lysing the transduced cells, and then mixing the lysate with live assay cells, and then microscopically observing the assay cells to visually determine if they have been infected with virus. We have tested various alternative approaches, and surprisingly found that droplet digital PCR not only is faster than the prior art approaches, but is an order of magnitude more sensitive, capable of detecting as few as seven (7) replication-competent adenoviruses ("RCs") in, for example, 3 x 1010 assay cells.

Description

[0001] Cross-referencing of related applications:

[0002] This application claims priority from U.S. Patent Application No. 16 / 426124, filed May 30, 2019 and incorporated herein by reference.

[0003] Statement regarding federally funded research or development:

[0004] not applicable

[0005] Names of the parties to the joint research agreement:

[0006] not applicable

[0007] Sequence list reference:

[0008] This application includes, and is incorporated herein by reference, PatentIn... TM document.

[0009] Prior disclosures by the inventors or co-inventors:

[0010] not applicable Background technology:

[0011] Some viral gene therapy vectors are designed to be unable to replicate in patients. For example, to replicate in normal human cells, adenoviruses require functional E1a, E1b, and E3 genomic regions. By deleting or mutating these regions, viral gene therapy vectors can be made to have replication defects.

[0012] Nevertheless, during the production of viral gene therapy vectors, unwanted replicative viruses (“RCVs”) may be formed due to random mutations or other events. For example, an E1a-deficient adenovirus vector can be produced in HEK293 cells, which contain a functional E1a region. Spontaneous recombination theoretically allows the functional E1a region to be reintroduced into the adenovirus, creating a replicative adenovirus (“RCA”).

[0013] Therefore, manufacturers of viral gene therapy vectors test for the presence of contaminating RCVs in replication-defective viral vectors. Regulatory agencies, namely the European Medicines Agency and the U.S. Food and Drug Administration, require this to be done by testing for consecutive infections using a method commonly known as the "rolling bottle" assay.

[0014] In this method, target cells (e.g., HEK cells) are grown in a culture medium. A viral vector sample is then added to transduce the target cells, and the cells are cultured long enough for transduction to complete. The target cells are then pelleted and washed to remove any residual viral vector from the culture medium. The target cells are then lysed, and the lysate is added to a culture of assay cells (e.g., HeLa cells). The assay cells are then grown in the culture medium long enough for an infectious virus (if present) to cause a visible infection of the assay cells. Optionally, these assay cells can be pelleted, washed, lysed again, and the lysate added to a second culture of the assay cells, which are then grown in the culture medium. Visible infection is determined by microscopy, and the assay cells are observed to visually determine whether they have been infected with the virus. This visual examination is an assessment of visible cellular stress; infected cells are noticeably deformed and have a poor appearance, while assay cells appear normal in the absence of an infectious virus. This test is often referred to as the “rolling flask” test because the assay cells are typically cultured in rolling flasks.

[0015] The rolling bottle test is considered sensitive enough to measure 3 × 10⁻⁶. 10 <1 RCA per viral particle. The rolling flask assay is somewhat subjective because it relies on microscopic observation of cell morphology. To find a more objective assay, we tested various alternative methods. When comparing the alternative assays with the industry-standard rolling flask assay, we were surprised to find that, contrary to what is taught in the art, the rolling flask assay is insufficiently sensitive to detect 3 × 10⁻⁶ cells / particles. 10 <1 RCA per viral particle. Conversely, we found that the rolling bottle assay could only detect 3 × 10 10 ≥75 RCAs per viral particle.

[0016] Therefore, we have invested time in developing an alternative approach using digital PCR. This can be faster than existing techniques, provide more objective data, and surprisingly, offer an order of magnitude higher sensitivity, capable of detecting, for example, 3 × 10⁻⁶ cells / day. 10 There are as few as seven (7) RCAs in each viral particle. Summary of the Invention:

[0017] This disclosure describes an assay for detecting replicating viruses (“RCVs”), such as replicating adenoviruses (“RCA”), using digital polymerase chain reaction (dPCR). Preferably, droplet digital PCR (ddPCR) can be used because the device is readily available. Our assay comprises multiple cycles of RCA amplification in cell culture, and detection of the amplified RCA by ddPCR. Attached image description:

[0018] This patent or application contains at least one color drawing. Upon request and payment of the necessary fees, the Patent Office will provide a published copy of this patent or application with one or more color drawings.

[0019] Figure 1 A flowchart overview of our assay method is provided.

[0020] Figure 2 It is a color photograph or reproduction of the QuantaSoft software user interface.

[0021] This patent or application contains at least one color drawing. Upon request and payment of the necessary fees, the Patent Office will provide a published copy of this patent or application containing one or more color drawings. Detailed implementation method:

[0022] Our assay method is outlined in Figure 1 In. Figure 1 In this study, suspension-cultured HeLa cells were used to expand potential RCA. Cells were cultured at 7.5 × 10⁻⁶ cells / year. 7 Cells were seeded at a density of 100 cells / flask into 500 ml shake flasks. 3 × 10⁻⁶ cells / flask were added to each shake flask. 10 One virus particle (Vp). After incubation (+37°C, 5% CO2, 122 rpm) for three days, cells were collected and lysed using three freeze-thaw cycles. The lysate was clarified by centrifugation.

[0023] Then the lysis buffer was added to cells with fresh assay cells (2 × 10⁻⁶). 7 The cells were placed in 125 ml shake flasks (1 cell / flask). These first assay flasks were treated in the same manner as the target cell cultures.

[0024] Then the lysate from the first assay culture was added to the second assay culture (2 × 10⁻⁶). 7 (cells / flask). After three days of incubation, the second assay cells were collected and lysed. The lysate was clarified by centrifugation and stored in an ultra-low temperature freezer. Multiple RCA amplification cycles were performed to minimize the interfering effects of therapeutic proteins (e.g., transgenes encoding interferon in viral vectors that inhibit the growth of interferon-sensitive assay cells) and maximize RCA yield.

[0025] RCA in lysates was detected using digital polymerase chain reaction (dPCR). We prefer ddPCR and will therefore discuss it below, but other dPCR methods can also be used.

[0026] The lysate was pretreated with proteinase K to release viral DNA encapsulated within the viral particles.

[0027] Pretreated lysis buffer was used as the sample in ddPCR analysis, which uses PCR primers and probes (e.g., TaqMan probes) specific to portions of the viral genome that have been deleted from the virus (thus rendering the virus incapable of replication). For example, we have used this method to determine adenoviruses in which the E1 region has been deleted. The E1 region is essential for adenovirus replication and is therefore deleted from the genomes of various adenovirus gene therapy vectors, but is present in the wild-type (infectious) genome.

[0028] In our ddPCR analysis, we prepared a mixture containing supermix, primers, and probes, and then pipetted it into three parallel wells in a 96-well plate. Lysis sample (from above) was added to each well. We then used an automated droplet generator to produce thousands of tiny droplets. Sample DNA was randomly distributed between the droplets. DNA within the droplets was amplified by PCR. The droplets were read using a reader that counts positive and negative droplets. The results were calculated using a Poisson distribution and expressed as copies / µL.

[0029] We have used this protocol for replication-defective adenovirus gene therapy vectors, but conceptually, it could be used for any other replication-defective virus with a genome suitable for PCR analysis. Similarly, we have tested the system on viral vectors containing transgenes of vascular endothelial growth factor D (“VEGF-D”), but it could be used with vectors containing another transgene.

[0030] As a reference standard (RS) for the aforementioned assay, we use 1.77 × 10⁻⁶. 11 The density was determined using Phase I clinical-grade material at vp / ml. RS samples were processed in a manner similar to that used for test samples (TS). Two parallel RS flasks were prepared. Results from the TS were reported as a comparison with reference standards.

[0031] Amplification was controlled using a negative control (NC). NCs were prepared by “simulating infection” of the cells with cell culture medium. NCs were prepared in single flasks because the expected results were known.

[0032] We used a positive control (PC), which was prepared by infecting target cells with 100 vp of wild-type (replicating) adenovirus reference material (“ARM”) (ATCC catalog number VR-1516). We used the PCs only for trend analysis purposes and prepared them in duplicate.

[0033] ddPCR was controlled using a template-free control (NTC). In the NTC, samples were replaced with the same cell culture medium used to dilute the samples and a positive control containing purified ARM DNA (where cell-free DNA extracted from ARM material was used as the sample). We used a raw ARM DNA concentration of 322.1 ng / μl. The ARM DNA was then diluted to 10 ng / μl and aliquoted into 12 μl aliquots per tube. The aliquots were stored at -20°C. Each aliquot should be thawed no more than five (5) times to minimize freeze-thaw damage to the DNA. We preferred to record or document each thawing on the thawing tube and discard and aliquot after the fifth thawing.

[0034] The assay was performed using HeLaQCWCB2 cells cultured in DMEM supplemented with 10% FBS / Pen / Strep / L-glutamine. Cells were cultured in suspension flasks of various sizes, such as in CO2 incubators equipped with shaker platforms or New Brunswick S41i incubators. TM Incubate in an incubator shaker.

[0035] We found that the process is most efficient when performed by two operators on the days of the first, second, and third infection. One operator inoculates HeLa suspension cells for the assay (7 to 15 flasks for infection, 1 to 5 flasks for further culture). The other operator prepares the virus dilution for the first infection or lyses the infected cells during the second and third infections. Harvesting and dPCR analysis require only one operator.

[0036] Cell culture

[0037] Cell culture was performed using aseptic techniques. HeLa QCWCB2 cells were cultured in suspension. The number of cell flasks required for the RCA assay depends on the number of TS cells to be analyzed. One flask is used for NC, two for RS, and two for PC. Each TS is analyzed as a parallel sample. Up to five TS cells (15 flasks in total) can be analyzed in a single assay.

[0038] To obtain the required number of flasks, the culture needs to be scaled up appropriately. Table I lists the minimum number of flasks we recommend for inoculating with different numbers of test samples (TS). 250 ml and 500 ml shake flasks are interchangeable, so one 500 ml flask is equivalent to two 250 ml flasks. Note that scale-up needs to begin as early as possible to obtain the required number of cells: for assays with 4–5 TS, this means starting approximately two weeks before the start of the assay, and for assays with 1–3 TS, starting one week before the start of the assay.

[0039]

[0040] The recommended minimum number of flasks to inoculate for initiating assays with varying quantities of test samples (TS) is listed. Scale-up should begin 1–2 weeks prior to the assay. The working days are exemplary and can be adjusted as needed. The flasks required for the assay are indicated; additionally, multiple flasks need to be inoculated simultaneously for further culture.

[0041] Before seeding cells for RCA assays, monitor culture growth and count cells. We prefer to use the following system suitability criteria (SSC) for cells: cell viability ≥80% and RSD% (relative standard deviation) of cell count ≤20%.

[0042] When seeding cells for infection in RCA assays, we prefer to use the seeding parameters listed in Table II.

[0043]

[0044] We prefer to use 1×10 in a 500ml flask. 8 Perform the first infection using 100 ml of cells per flask. Inoculate this cell volume into 100 ml of culture medium. After infection, add another 100 ml of culture medium. The final cell density in the flask should be 5 × 10⁻⁶ cells / flask. 5 Cells / ml. The test sample dose per flask is 3 × 10⁻⁶ cells / ml. 10 1 vp. The dose per cell was 300 vp / cell. The second and third infections were performed in 125 ml flasks at a dose of 2 × 10⁻⁶. 7 One cell per flask was administered in 40 ml of culture medium. The cell density was 5 × 10⁻⁶ cells / flask. 5 Cells / ml. Infect the inoculated cells on the same day.

[0045] First infection / transduction :

[0046] For first infection (or “transduction”, where a transgenic recombinant virus is used), prepare a viral vector dilution in cold medium (removed from the refrigerator). Avoid cross-contamination throughout the protocol. Viral samples are processed in the following order, where applicable: 1) NC, 2) TS, 3) RS, 4) PC.

[0047] a) Label the viral titer (vp / ml).

[0048] b) Calculate the virus dilution.

[0049] c) Synchronize this work with the operator's cell inoculation so that the virus diluent and the cells to be infected are ready approximately at the same time.

[0050] d) Thaw RS, ARM and TS in the refrigerator and refrigerate them before use.

[0051] e) Take the required volume of cold culture medium and place it in a 50 ml tube. The required volumes are as follows: approximately 7.0 ml of initial dilution for PC, approximately 8.5 ml of final dilution for NC, RS, and PC flasks, and approximately 4.5 ml for each TS. (Therefore, one TS requires approximately 20 ml, and five TS require approximately 40 ml).

[0052] f) Prepare the initial diluents of PC (ARM) according to Table III. We found that ARM must be diluted extensively through a series of dilutions to achieve the desired dosage in the appropriate volume. Each diluent needs to be thoroughly mixed before being used to prepare the next diluent.

[0053]

[0054]

[0055] g) Prepare the final dilution. First, pipette the culture medium into all tubes, then pipette only the test sample, RS, and PC (as the last tube). Note that two parallel dilutions should be prepared for RS, PC, and each TS. Use the dilutions within 90 minutes of preparation.

[0056] h) Infect 1×10⁻⁶ cells in 100 ml of medium with NC culture medium and the final dilution in 500 ml shake flasks. 8 Cells. Infect using all dilutions (2 ml).

[0057] i) Approximately 90 minutes (±10 min) after infection, add 100 ml of fresh, preheated culture medium to the flask containing the infected cells.

[0058] j) Incubate the flask with the infected cells for three days (+37°C, 5% CO2, 122 rpm).

[0059] Note that assays can be paused after the first infection. Follow steps a)-h) above. Transfer the supernatant to a clean, sterile 15ml centrifuge tube. Quickly freeze the tube with liquid nitrogen and store it in an ultra-low temperature freezer for up to two months. On the day of the second infection, thaw the frozen supernatant in the freezer and use all the supernatant to infect fresh cells.

[0060] Second infection :

[0061] For secondary infections, cells need to be resuspended in culture medium (taken from the refrigerator). Virus samples should be processed in the following order, where applicable: 1) NC, 2) TS, 3) RS, 4) PC.

[0062] a) Synchronize this work with the operator's cell inoculation so that the supernatant for infection and the cells to be infected are ready almost simultaneously.

[0063] b) Fill the liquid nitrogen container with liquid nitrogen.

[0064] c) Transfer the infected cell suspension (200 ml) from each shake flask to a 50 ml sterile tube (4 tubes / sample).

[0065] d) Centrifuge the cells at +4°C at 1000xg for 10 minutes.

[0066] e) Remove the supernatant.

[0067] f) Resuspend the cell pellet of one sample in 5 ml of fresh, cold culture medium. Add culture medium to one tube and resuspend the pellet by pipetting. Transfer the suspension to a second tube of the same sample, resuspend again, and repeat until all four tubes of the sample have been processed.

[0068] g) Transfer the suspension to a 15 ml sterile centrifuge tube (1 tube / sample).

[0069] h) Lyse the cells using three cycles of freezing in liquid nitrogen (approximately 5 min) and thawing in a water bath at +37°C (approximately 10 min). Place the tubes in metal cages that can be arranged in a row and immersed in liquid nitrogen and warm water. After each freezing, check the tubes for damage before placing them in warm water. After each thawing, reconfirm that there are no cracks in the tubes and vortex the tubes at low speed. Work can be paused during a freezing step. Keep the samples frozen (liquid nitrogen / -80°C) until you are ready to continue.

[0070] i) Centrifuge the lysed cells at +4°C at 2000 x g for 20 minutes to remove cell debris. Retain the supernatant.

[0071] .j) If the HeLa suspension cells to be infected are not yet ready, transfer the supernatant to a clean tube and refrigerate (freeze) until the cells are ready.

[0072] k) Used in 40ml 2×10 7 Infect 125 ml shake flasks with cells and supernatant. Use all supernatant (approximately 5 ml) for infection.

[0073] l) Incubate the flask with the infected cells for three days (+37°C, 5% CO2, 122 rpm).

[0074] The assay can be paused after the second infection. Follow steps a)-h) above. Transfer the supernatant to a clean, sterile 15ml centrifuge tube. Quickly freeze the tube with liquid nitrogen and store it in an ultra-low temperature freezer for up to two months. On the day of the third infection, thaw the frozen supernatant in the freezer and use all the supernatant to infect fresh cells.

[0075] Third infection :

[0076] For a third infection, cold culture medium (removed from the freezer) is used to resuspend the cells. Virus samples are processed in the following order, where applicable: 1) NC, 2) TS, 3) RS, 4) PC.

[0077] a) Synchronize this work with the operator's cell inoculation so that the supernatant for infection and the cells to be infected are ready almost simultaneously.

[0078] b) Fill the liquid nitrogen container with liquid nitrogen.

[0079] c) Transfer the infected cell suspension (40 ml) from each shake flask to a 50 ml sterile tube (1 tube / sample).

[0080] d) Centrifuge the cells at +4°C at 1000xg for 10 minutes.

[0081] e) Remove the supernatant.

[0082] f) Resuspend the cell pellet of one sample in 2 ml of fresh, cold culture medium. If the particles are very dense and difficult to resuspend, the resuspending volume can be increased.

[0083] g) Transfer the suspension to a 15 ml sterile centrifuge tube (1 tube / sample).

[0084] h) Lyse the cells using three cycles of freezing in liquid nitrogen (approximately 5 min) and thawing in a water bath at +37°C (approximately 10 min). Place the tubes in metal cages that can be arranged in a row and immersed in liquid nitrogen and warm water. After each freezing, check the tubes for damage before placing them in warm water. After each thawing, reconfirm that there are no cracks in the tubes and vortex the tubes at low speed. Work can be paused during a freezing step. Keep the samples frozen (liquid nitrogen / -80°C) until you are ready to continue.

[0085] i) Centrifuge the lysed cells at +4°C at 2000 x g for 20 minutes to remove cell debris. Reserve the supernatant.

[0086] j) If the HeLa suspension cells to be infected are not yet ready, transfer the supernatant to a clean tube and refrigerate (freeze) until the cells are ready.

[0087] k) Used in 40ml 2×10 7 Infect 125 ml shake flasks with cells and supernatant. Use all supernatant (approximately 2 ml) for infection.

[0088] 1) Incubate the flask with the infected cells for three days (+37°C, 5% CO2, 122 rpm).

[0089] Harvest :

[0090] For harvesting, sample labels can be printed according to Table IV. Each flask containing infected cells requires four labels: two labels indicating an equal sample volume of 110 μl, and two labels indicating <1000 μl.

[0091]

[0092] Resuspending cells requires cold culture medium (removed from the freezer). Viral samples are processed in the following order, where applicable: 1) NC, 2) TS, 3) RS, 4) PC.

[0093] a) Fill the liquid nitrogen container with liquid nitrogen.

[0094] b) Mark 1ml cryopreservation tubes for equal-sized samples of the harvest.

[0095] c) Transfer the infected cell suspension (40 ml) from each shake flask to a 50 ml sterile tube (1 tube / sample).

[0096] d) Centrifuge the cells at +4°C at 1000xg for 10 minutes.

[0097] e) Remove the supernatant.

[0098] f) Resuspend the cell pellet of one sample in 2 ml of fresh, cold culture medium. If the particles are very dense and difficult to resuspend, the resuspending volume can be increased.

[0099] g) Transfer the suspension to a 15 ml sterile centrifuge tube (1 tube / sample).

[0100] h) Lyse the cells using three cycles of freezing in liquid nitrogen (approximately 5 min) and thawing in a water bath at +37°C (approximately 10 min). Place the tubes in metal cages that can be arranged in a row and immersed in liquid nitrogen and warm water. After each freezing, check the tubes for damage before placing them in warm water. After each thawing, reconfirm that there are no cracks in the tubes and vortex the tubes at low speed. Work can be paused during a freezing step. Keep the samples frozen (liquid nitrogen / -80°C) until you are ready to continue.

[0101] i) Centrifuge the lysed cells at +4°C at 2000 x g for 20 minutes to remove cell debris. Reserve the supernatant.

[0102] j) Transfer the supernatant to a new clean tube and mix.

[0103] k) Distribute the supernatant of each sample equally into pre-labeled 1 ml cryovials: 2 × 110 μl; 2 × < 1000 μl.

[0104] 1) Quickly freeze equal portions of the sample with liquid nitrogen and store them in a freezer until analysis.

[0105] Digital PCR analysis :

[0106] DNA work should be performed under nuclease-free conditions. Sterile, nuclease-free solutions and plastic instruments must be used. Gloves must always be worn when handling DNA samples. DNA-ExitusPlus TM It can be used to remove any DNA residue that may be present after work. After cleaning the laminar flow hood (LFH) specifically designed for DNA samples, the LFH can be inactivated by overnight UV irradiation.

[0107] To pre-treat the harvested samples, thaw them at room temperature for up to one hour. Transfer samples (100 μl / well) to a 96-well plate using a pipette: pipette NC to well A4. Pipette RS_1, RS_2, PC_1, PC_2, TS1_1, and TS1_2 to wells B1-G1 in column 1. Pipette the remaining TS to wells A6-H6 in column 6. Add proteinase K (1 μl / well) to the used wells. Seal the plate with optical sealant. Gently vortex and briefly centrifuge. Run the plate using an AppliedBiosystems 7500 real-time PCR system and the latest version of the SDS template document “Prot K”. The run procedure is as follows: Incubate at +50°C for 60 minutes. Incubate at +95°C for 20 minutes. Cool the samples to +4°C. If no further sample dilution is to be performed, store the plate in a refrigerator.

[0108] For sample dilution, samples need to be diluted to fall within the dynamic range of ddPCR analysis. Dilutions of 1:1000 and 1:10000 are used in the analysis. If these dilutions are unacceptable, higher or lower dilutions can be tested. Pipette 90 μl of cell culture medium (per well) into wells B2-G5 of a 96-well plate containing pretreated harvested samples. Pipette 90 μl of cell culture medium (per well) into wells A7-H10. Pipette 90 μl of cell culture medium (per well) into well H4. This well will be used to prepare NTC for ddPCR analysis. Thoroughly mix the pretreated samples in columns 1 and 6 by pipetting. Prepare dilution series from columns 1 to 5 and from columns 6 to 10. Pipette 10 μl from column 1 to column 2 and thoroughly mix the prepared dilution by pipetting. Use a pipette to transfer 10 μl from column 2 to column 3 and mix. Continue until column 5 is ready and mixed. Repeat the same steps for columns 6-10. If not performing ddPCR analysis directly, store the plate in a refrigerator.

[0109] Prepare ddPCR plate :

[0110] To prepare ddPCR plates, prepare fresh 1 pg / μl ARM DNA dilutions from 10 ng / μl aliquots of each RCA assay targeting product release. See Table V for the ARM DNA dilution series.

[0111] Table V: Dilution series of ARM DNA prepared for use as PC in ddPCR analysis

[0112]

[0113]

[0114] Each dilution should be thoroughly mixed by pipetting before being used to prepare the next. Prepare the dilutions in DNA LFH. Use the last dilution, named ARM DNA (1 pg / μl), in the ddPCR run. The required volume is 5 μl, meaning 5 pg of ARM DNA per reaction.

[0115] For each RCA assay targeting product release, fresh dilutions of the forward and reverse primers and the TaqMan probe for RCA were prepared. In our experiments, we used E1-deficient adenovirus, therefore using 5′-AAC CAG TTGCCG TGA GAG TTG-3′ as the forward primer for RCA; 5′-CTC GTTAAG CAAGTC CTC GAT ACA-3′ as the reverse primer for RCA; and 5′-TGG GCG TCG CCAGGC TGT G-3′ as the TaqMan probe for RCA.

[0116] Thaw the reagent at room temperature, mix, and centrifuge (e.g., using a centrifuge / vortex mixer). See Table VI for dilution instructions.

[0117]

[0118] The diluent was prepared in a master mix laminar flow blotting system. Final concentrations of primers (6000 nM) and probes (2500 nM) were used in the ddPCR run. 2.5 μl of each reagent was added to the reaction mixture, for a total volume of 25 μl. The primer concentration in the reaction mixture was 600 nM, and the probe concentration was 250 nM. For analytical development, in-process sample preparation, and characterization purposes, earlier prepared diluents stored at -20°C can be used. The master mix was prepared in a dedicated LFH for master mix preparation. Samples were added to the DNA LFH on the plate. Separate materials, pipettes, and centrifuges / vortex mixers were used for the master mix and DNA work. The required well volume and total volume of master mix were calculated. The ddPCR master mix for the probes was thawed at room temperature and vortexed at high speed during thawing. The diluted forward and reverse primers and TaqMan probes of RCA were mixed and centrifuged. We found that centrifuges / vortex mixers were convenient for mixing and centrifugation. Prepare the premixed solution for ddPCR according to Table VII and vortex it at high speed.

[0119]

[0120] Use a pipette to transfer the premix (20 μl / well) to the wells of a 96-well plate. Optionally, the premix can be poured onto a reagent container, and a multichannel pipette can be used to transfer the mixture onto the plate. Add the ddPCR Buffer Control Kit (BC, 25 μl / well) to wells H4-H6 and wells where samples are not needed. For the maximum number of test samples, ensure that all wells in each column are filled. The ddPCR Buffer Control Kit (“BC”) is used in wells where samples are not needed. We prefer to use wells containing, for example, 1:1000 and 1:10000 dilutions.

[0121] We prefer to vortex the premixed samples and diluent into a 96-well plate and then centrifuge the samples. Add the sample (5 μl / well) to a ddPCR plate containing the premix (20 μl / well). After use, store the plate in a refrigerator. The samples can be used for repeated ddPCR analyses.

[0122] For the control, the ARM DNA dilution was mixed by pipetting and added (5 μl / well) to ddPCR plates (wells A4, A5, and A6).

[0123] Seal the plate with heatfoil at +180°C for 5 seconds using plate sealer. Briefly vortex the plate and centrifuge the sample mixture. Proceed with droplet generation and PCR run.

[0124] Droplet generation, PCR reading and droplet reading :

[0125] We prefer to use AutoDG TM The equipment performs automated droplet generation. To do this, we seal the droplet-containing plates with heatfoil at +180°C for 5 seconds using plate sealant. Then, we proceed with the PCR run. We prefer to use C1000. TM The Touch thermal cycler was used to run the plate under the PCR conditions shown in Table VIII.

[0126]

[0127]

[0128] After the PCR run, the droplets are stable. The plate can be stored in the refrigerator overnight. Read the droplets using a droplet reader with the latest version of the QuantaSoft template "RCA ddPCR". The QuantaSoft software will automatically create a folder for the run and save it as a QuantaSoft plate file. After the run, manually set the threshold for all wells to 2000 as follows: Click the "Analyze" button in the left-hand menu; select all wells from the plate layout in the upper right corner; select the "One-Dimensional Amplitude" display; at the bottom of the left-hand menu, activate the button marked in yellow (Multi-Well Tool). This allows you to set the threshold for all wells simultaneously. Set the threshold by entering 2000 in the Set Threshold box (press Enter). For a more detailed explanation of the visualization of threshold settings and the software user interface, please refer to [link to relevant documentation]. Figure 2Close the QuantaSoft software and click "Yes" when prompted to "Save board information?". Copy the created folder to the server, ensuring it is named after the test run number.

[0129] The ddPCR run data was automatically analyzed using QuantaSoft software. The completion status of SSC was evaluated, and the results were read using the software.

[0130] Data Analysis :

[0131] In ddPCR, sample DNA is randomly distributed among thousands of droplets. The more droplets present, the better the accuracy of the analysis. The QX200 ddPCR system can generate and read more than 20,000 droplets per well. To ensure the required accuracy, a standard of ≥8,000 accepted droplets is set for each analysis well. However, even if individual wells do not meet this standard, the assay is not considered unqualified. Unqualified wells are ignored in further analysis. In ddPCR analysis, each sample is analyzed in three parallel wells. If at least two of the three parallel wells have ≥8,000 accepted droplets, the result for that sample can be read. To check the number of accepted droplets, follow these steps: Open the plate document (threshold 2000) saved on the server. Click the "Analyze" button on the left menu. Select all wells from the plate layout in the upper right corner. Select the "Events" display. Check the "total" box on the right. For clarity, the other boxes (positive / negative) should not be checked. Check that each well has ≥8000 qualified droplets based on the values ​​on the histogram.

[0132] The system suitability criterion for NC, NTC, and ARM DNA is that at least two of the three parallel wells have ≥8000 qualifying droplets. Verify that this criterion is met. If it is not met, the ddPCR analysis needs to be repeated. When considering further SSC, only wells with ≥8000 qualifying droplets should be considered.

[0133] In ddPCR analysis, samples with ≤5 positive droplets are considered negative. Samples with 6–34 positive droplets cannot be considered negative, but may be contaminated or represent a very small amount of target DNA. Samples with ≥35 positive droplets are considered definitively positive. The number of positive and negative droplets on each well is checked using the procedures a)–e) above, but by checking the positive or negative boxes instead of the total number.

[0134] The SSC for NC, NTC, and ARMDNA assays is: no more than 5 positive droplets in any qualified well of an NC sample, or no more than 5 positive droplets in any qualified well of an NTC sample, or at least two out of three parallel wells of an ARMDNA sample showing ≥35 positive droplets.

[0135] If these SSCs are invalid, we recommend repeating the ddPCR. If the NC is still invalid, we suggest repeating the entire RCA assay from the first infection. If the invalidity is due to NTC or ARM DNA, the RCA amplification may have been successful, but there are some issues with the ddPCR analysis. In cases of repeated invalidity with NTC or ARM DNA, the cause of the invalidity needs to be investigated.

[0136] To read the results for a sample, the sample should contain both positive and negative droplets. If the number of negative droplets is low, the accuracy of the analysis will be affected. The well should show >100 negative droplets. Otherwise, the well will be considered "saturated" and the results cannot be read.

[0137] ddPCR analysis has a narrow dynamic range. RS and TS are analyzed at two dilutions: 1:1000 and 1:10000. At least one dilution needs to be within this range. The criteria for an acceptable RS dilution are that at least two out of three parallel wells show ≥8000 qualified droplets, >100 negative droplets (wells unsaturated), and ≥35 positive droplets (wells showing a positive result).

[0138] Evaluate the SSC assay using one or more acceptable dilutions. An SSC for RS assays is defined as follows: both RS_1 and RS_2 show positive results (≥35 positive droplets) in acceptable wells at one or more acceptable dilutions. An SSC passes if both RS_1 and RS_2 have at least one acceptable dilution. An unsuccessful SSC may be due to improper dilution. The dilution used may be too low (well saturation) or too high (negative result). In this case, ddPCR can be repeated with an adjusted dilution. 1:10 and / or 1:100 dilutions from the pretreatment plate can be used as appropriate, or further dilutions can be prepared from 1:10000. Consult an expert to determine how to proceed. If one of the parallel flasks (RS_1 or RS_2) still shows a negative result, while the other flask is within the dynamic range, the entire RCA assay needs to be repeated from the first infection.

[0139] The acceptable TS dilution is defined as at least two out of three parallel wells showing ≥8000 positive droplets and >100 negative droplets (wells unsaturated). Evaluate the sample SSC using one or more acceptable dilutions. Note that because TS may not meet RCA, the ≥35 positive droplet criterion is not required for TS.

[0140] The SSC for samples is: both TSX_1 and TSX_2 must have at least one acceptable dilution. If a sample fails the SSC, its results cannot be reported, and the sample needs to be reanalyzed. However, results for other samples can be read and reported. An SSC failure may be due to improper dilution. The dilution used may be too low (well saturation). In this case, ddPCR can be repeated with an adjusted dilution. Further dilutions can be prepared from 1:10000.

[0141] RCA measurement results :

[0142] ddPCR analysis results are expressed as copies / µL. QuantaSoft software reports this value for each well in the results table in the upper left corner. The value is 0 if there are no positive droplets in the well; and 1,000,000 (saturated) if there are no negative droplets. To calculate the RS and TS results, record the concentration (copy / µL) for each well reported in the QuantaSoft software results table. Only record the concentrations of qualified wells and diluents; otherwise, mark N / A. The adjusted concentration (copy / µL) is then calculated by multiplying the reported concentration by the dilution factor. The average of the adjusted concentrations (copy / µL) is then calculated. This is recorded as an integer without decimal places. Note that if one of the parallel flasks (TSX_1 or TSX_2) of a TS produces a negative result at both dilutions, the average is calculated from the positive flask. For RS, the RS range is calculated as the average ± 20%: the lower limit of the range is 0.8 × the average, and the upper limit is 1.2 × the average. Compare the TS results to this RS range. For each TS, compare the average to the RS range. If the average is below the RS range, the result is "RCA is less than RS". If the average is within the RS range, the result is "RCA is equal to RS". If the average is above the RS range, the result is "RCA is more than RS".

[0143] If one of the parallel flasks (TSX_1 or TSX_2) of a certain TS produces negative results at both dilutions, and the other flask produces a clearly positive result, then the mean (copy / µL) is based on the positive flask. If the comparison with the RS range shows "RCA is higher than in RS," then the ddPCR analysis needs to be repeated. If the results are still the same, then the entire RCA assay needs to be repeated from the first infection.

[0144] The trend analysis of the measurement results can be converted into Excel. TM Documents. We prefer to perform trend analysis on the following parameters:

[0145] ·RCA Measurement Operation Number

[0146] • SSC test pass / fail

[0147] • Reasons why SSC may fail

[0148] • Results of ARM DNA (copy / µL)

[0149] Acceptable dilutions for RS and PC

[0150] • Average of RS and PC (copies / µL) (Note that the average of PC is only calculated in the trend Excel).

[0151] According to our disclosure, technicians can easily modify it. For example, although we have actually developed our modified assay using recombinant adenoviruses carrying vascular endothelial growth factor D, our assay can also identify contaminated viruses with replication capabilities in vectors containing other transgenes (e.g., p53, interferon, etc.).

[0152] "Infection" refers to the replication of a virus in a target cell, resulting in offspring. Conversely, "transfection" refers to the delivery of foreign DNA or RNA into a target cell via a viral vector. Transfection does not require viral replication in the target cell.

[0153] Similarly, while we have actually tested our assay on viral vectors that are not expected to replicate at all in human patients, our assay can also be readily used on viral vectors that are expected to replicate conditionally, for example, only in human cancer cells but not in normal human cells. Therefore, we use the phrase "cannot replicate in normal human cells" in the accompanying legal claims to indicate this.

[0154] Similarly, although our experiments were conducted on adenoviruses, our method is equally useful for other types of gene therapy viral vectors.

[0155] Therefore, we anticipate that the legal scope of our patent is not limited by the specific laboratory work described above, but by our accompanying legal claims and their permissible equivalents. sequence list <110> Trizell Ltd. <120> Ultra-high precision viral vector assay <130> US16 / 426124 <150> US16 / 426124 <151> 2019-05-30 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Adenovirus type 5 <400> 1 aaccagttgc cgtgagagtt g 21 <210> 2 <211> twenty four <212> DNA <213> Adenovirus type 5 <400> 2 ctcgttaagc aagtcctcga taca 24 <210> 3 <211> 19 <212> DNA <213> Adenovirus type 5 <400> 3 tgggcgtcgc caggctgtg 19

Claims

1. A method for identifying adenoviruses capable of replicating in normal human cells in a sample, said sample containing an adenovirus gene therapy vector that cannot replicate in normal human cells, said method comprising: a. Obtaining a sample containing an adenovirus gene therapy vector that cannot replicate in normal human cells, said adenovirus gene therapy vector comprising a transgene and an adenovirus genome, said adenovirus genome being genetically modified from the wild-type adenovirus genome by modifying or deleting regions of the wild-type adenovirus genome that are crucial for the replication of said adenovirus in normal human cells, thus the resulting adenovirus gene therapy vector is expected to be unable to replicate in normal human cells, and then... b. Mix the sample with live target cells capable of being transduced by the adenovirus gene therapy vector to prepare a transduction mixture, and then... c. Maintain the transduction mixture under conditions sufficient to enable the adenovirus gene therapy vector to transduce the target cells for a certain period of time, and then... d. Isolate the target cells from any residual sample, and then e. Lyse the target cells to release their intracellular contents, and then f. Mixing the intracellular contents of the lysed target cells with live assay cells capable of being infected by the adenovirus to prepare an infection mixture, and then... g. Maintain the infection mixture under conditions sufficient to enable the adenovirus to infect the assay cells for a period of time, and then h. Lyse the assay cells to release their intracellular contents, and then i. Isolate nucleic acids from the intracellular contents of the cells being measured, and then j. Evaluate the isolated nucleic acids by digital PCR using probes that hybridize to the modified or deleted regions of the adenovirus genome that are crucial for adenovirus replication. This allows for the measurement of the approximate number of adenoviruses in the sample that can replicate in normal human cells.

2. The method of claim 1, wherein the probe comprises a DNA probe having a sequence selected from the following: SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO.

3.

3. The method of claim 1, wherein the method is capable of detecting every 3 × 10 10 The adenovirus gene therapy vector particle contains as few as 25 adenoviruses that can replicate in normal human cells, even though they cannot replicate in normal human cells.

4. The method of claim 3, wherein the method is capable of detecting every 3 × 10 10 The adenovirus gene therapy vector particle contains as few as seven adenoviruses that can replicate in normal human cells, even though they cannot replicate in normal human cells.

5. The method of claim 1, wherein the transgenic expression is a polypeptide selected from the group consisting of interferon and p53.

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