Ultra-high-precision viral vector assay

Abstract

To provide an ultra-high-precision viral vector assay.SOLUTION: During manufacture of a replication-deficient viral gene therapy vector, random mutations or other events may generate undesirable replication-competent virus ("RCV"). Accordingly, manufacturers of viral gene therapy vectors assay for the presence of contaminating RCV by assaying for serial infection, namely, by transducing target cells with the viral vector, then lysing the transduced cells, then mixing the lysate with live assay cells, and then microscopically observing them to visually determine whether the assay cells have been infected with virus.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS: This application claims priority to U.S. Utility Patent Application Serial No. 16 / 426124, filed May 30, 2019, which is incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT: Not applicable

[0003] Names of parties to the joint research agreement: Not applicable

[0004] Sequence Listing Reference: This application contains a PatentIn™ record file within this application and is incorporated by reference.

[0005] Statement of Prior Disclosure by Inventor or Co-Inventor: Not applicable [Background technology]

[0006] Certain viral gene therapy vectors are, by design, unable to replicate in patients. For example, to replicate in normal human cells, adenovirus requires functional E1a, E1b, and E3 genomic regions. By deleting or mutating these regions, viral gene therapy vectors can be made replication-deficient.

[0007] Nevertheless, during the production of viral gene therapy vectors, unwanted replication-competent viruses ("RCVs") may be formed due to random mutations or other events. For example, E1a-deleted adenoviral vectors can be produced in HEK293 cells containing a functional E1a region. Spontaneous recombination could theoretically restore the functional E1a region to the adenovirus, creating a replication-competent adenovirus ("RCA").

[0008] Therefore, manufacturers of viral gene therapy vectors assay replication-deficient viral vectors for the presence of contaminating RCV, and regulatory authorities, namely the European Medicines Agency and the United States Food and Drug Administration, require this to be done by assaying for serial infection using what is commonly referred to as a "roller bottle" assay.

[0009] In this method, target cells (e.g., HEK cells) are grown in culture medium. A sample of viral vector is then added to transduce the target cells, and the cells are cultured for a time sufficient to complete transduction. The target cells are then pelleted and rinsed to remove any remaining viral vector in 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 culture medium long enough to allow infectious virus, if any, to cause visible infection of the assay cells. Optionally, these assay cells are again pelleted, rinsed, and lysed, and the lysate is added to a second culture of assay cells, which are then grown in culture medium. Visible infection is measured microscopically, and the assay cells are observed to visually determine whether they are infected with virus. This visual inspection is an assessment of visible cellular stress; infected cells will be visibly deformed and disfigured, whereas in the absence of infectious virus, the assay cells will appear normal. This test is often called the "roller bottle" test because the assay cells are usually cultured in roller bottles.

[0010] Roller bottle assay: 3 x 10 10 It is understood to be sensitive enough to detect less than one RCA in a virus particle. The roller bottle test is somewhat subjective because it relies on microscopic observation of the morphology of the assay cells. To find a more objective assay, various alternative approaches were tested. When the alternative assays were compared with the industry standard roller bottle assay, it was surprisingly found that, contrary to the teachings of the art, the roller bottle assay could detect less than 3 x 10 10We found that the roller bottle assay was not sensitive enough to detect less than one RCA in a virus particle. 10 It was found that only more than 75 RCAs could be detected in a virus particle.

[0011] Therefore, we have devoted time to developing an alternative approach using digital PCR, which can be faster, provides more objective data, and surprisingly is 10 times more sensitive than prior art approaches, e.g., 3 x 10 10 Seven RCAs can be detected in the virus particle. Summary of the Invention [Means for solving the problem]

[0012] This disclosure describes an assay for detecting replication-competent viruses ("RCVs"), such as replication-competent adenoviruses ("RCA"), using digital polymerase chain reaction (dPCR). Preferably, droplet digital PCR (ddPCR) may be used because of the readily available equipment. Our assay involves multiple amplification cycles of RCA in cell culture and detection of the amplified RCA by ddPCR.

[0013] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with the drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows a schematic flow chart of the assay. [Figure 2] A color photograph or reprint of the QuantaSoft software user interface.

[0015] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. DETAILED DESCRIPTION OF THE INVENTION

[0016] An outline of the assay is shown in Figure 1, where HeLa cells grown in suspension were used to amplify latent RCA. 7.5 x 10 cells were cultured. 7 Seed 500 ml shake flasks at a density of 3 x 10 cells / flask. 10 Test samples (TS) of viral particles (vp) are added. After 3 days of incubation (+37°C, 5% CO2, 122 rpm), the cells are harvested and lysed by three freeze-thaw cycles. The lysate is clarified by centrifugation.

[0017] The lysate was then transferred to fresh assay cells (2 × 10 7 These first assay culture flasks are treated in the same manner as the target cell cultures.

[0018] The lysate from the first assay culture was then diluted with the second assay culture (2 × 10 7 After 3 days of incubation, harvest and lyse the second assay culture cells. Clarify the lysate by centrifugation and store it in an ultra-low temperature freezer. Perform multiple RCA amplification cycles to minimize the interference effect of therapeutic proteins (e.g., transgenes in viral vectors encoding interferon (which interfere with the growth of interferon-sensitive assay cells)) and maximize the RCA yield.

[0019] RCA in the lysates is detected by digital polymerase chain reaction (dPCR). We recommend using ddPCR and therefore describe it below, although other dPCR methods may also be used.

[0020] The lysate is pretreated with proteinase K to release the viral DNA encapsulated within the viral particles.

[0021] The precleared lysate is used as a sample in ddPCR analysis with PCR primers and a probe (e.g., TaqMan probe) specific for a portion of the viral genome region that has been deleted from the virus (to render it replication-incompetent). For example, adenoviruses deleted from the E1 region were assayed using this method. The E1 region is essential for adenovirus replication and is therefore deleted from the genomes of various adenoviral gene therapy vectors, but is present in the wild-type (infectious) genome.

[0022] For ddPCR analysis, a mixture containing supermix, primers, and probes was prepared and then pipetted into three wells of a 96-well plate. Lysate samples (from above) were added to each well. An automated droplet generator was then used to generate thousands of small droplets. Sample DNA was randomly partitioned between the droplets. The DNA within the droplets was amplified by PCR. The droplets were read using a reader that counted positive and negative droplets. Results were calculated using a Poisson distribution and given in copies per μl.

[0023] Although we used this protocol with replication-deficient adenoviral gene therapy vectors, it could conceptually be used with other replication-deficient viruses with genomes amenable to PCR analysis. Similarly, we tested this system with a viral vector containing a transgene for vascular endothelial growth factor D ("VEGF-D"), but it could also be used with vectors carrying other transgenes.

[0024] 1.77 × 10 Phase I clinical grade material was used as the reference standard (RS) for the assay. 11 The RS was used at a concentration of vp / ml. The RS was treated similarly to the test sample (TS). Duplicate RS flasks were prepared. TS results were reported relative to the reference standard.

[0025] Amplification is controlled by a negative control (NC), which is prepared by "mock-infecting" cells with cell culture medium. NC is prepared as a single flask, as the expected results are known.

[0026] A positive control (PC) was used, prepared by infecting target cells with 100 vp of wild-type (replication-competent) adenovirus reference material ("ARM") (ATCC catalog number VR-1516). The PC is used for trend analysis purposes only, and the PC is prepared in duplicate.

[0027] The ddPCR was controlled by a no-template control (NTC). For the NTC, the sample was replaced with the same cell culture medium used for diluting the sample and the positive control purified ARM DNA (cell-free DNA extracted from the ARM material was used as the sample). The ARM DNA was used at its original concentration of 322.1 ng / μl. The ARM DNA was then diluted to 10 ng / μl and dispensed into 12 μl aliquots per tube. The aliquots were stored at -20°C. To minimize DNA damage due to freeze-thawing, each aliquot should be thawed no more than five times. We recommend memorizing or recording each thawing of the thawed tube and discarding and aliquoting after the fifth thaw.

[0028] This assay uses HeLa QCWCB2 cells cultured in DMEM supplemented with 10% FBS / Pen / Strep / L-glutamine. Cells are cultured in suspension shaker flasks of different sizes, for example, in a CO2 incubator with a shaker platform or a New Brunswick S41i™ incubator shaker.

[0029] This process was found to be most efficient when performed by two operators on the first, second, and third infection days. One operator seeds HeLa suspension cells for the assay (7–15 flasks for infection and 1–5 more for culture). The other operator prepares virus dilutions for the first infection or lyses infected cells during the second and third infections. Only one operator is required for recovery and dPCR analysis.

[0030] cell culture Cell culture is performed according to sterile technique. HeLa QCWCB2 cells were cultured in suspension. The number of cell flasks required for the RCA assay depends on the number of TSs to be analyzed. Reserve one flask for NC, two flasks for RS, and two flasks for PC. Each TS is analyzed in duplicate. Up to five TSs can be analyzed in one assay (15 flasks total).

[0031] To ensure the required number of flasks, cultures must be scaled up appropriately. Table I shows the recommended minimum number of flasks to inoculate for starting assays with various numbers of test samples (TS). Because 250 ml and 500 ml shake flasks are interchangeable, one 500 ml flask corresponds to two 250 ml flasks. Note that scale-up must begin early enough to obtain the required amount of cells: if assaying with 4-5 TSs, this means approximately 2 weeks before the start of the assay; if assaying with 1-3 TSs, this means the week before. [Table 1] The minimum recommended number of flasks to seed for starting the assay with various numbers of test samples (TS) is shown. Scale-up should begin 1-2 weeks prior to the assay. Weekdays are exemplary and can be adjusted as needed. The flasks required for the assay are shown; also, multiple flasks should be seeded for further culture simultaneously.

[0032] Before seeding cells for the RCA assay, monitor the growth of the culture and count the cells. We recommend using the following system suitability criteria (SSC): cell viability ≥ 80% and a cell count RSD% (relative standard deviation) ≤ 20%.

[0033] When seeding cells to be infected in an RCA assay, we recommend using the seeding parameters shown in Table II. [Table 2]

[0034] The first infection was performed in a 500 ml flask with 1 x 10 8 We recommend working with 100 cells / flask. This amount of cells is seeded into 100 ml of medium. After infection, add 100 ml of medium. The final cell density in the flask is 5 x 10 5 The dose of test substance per flask is 3x10 cells / ml. 10 The dose per cell was 300 vp / cell. The second and third infections were performed in 125 ml flasks with 2 x 10 cells in 40 ml of medium. 7 Cells / flask: Cell density is 5 x 10 5 cells / ml. The seeded cells are infected on the same day.

[0035] First infection / transduction: For the first infection (i.e., "transduction" using a recombinant virus containing a transgene), viral vector dilutions are prepared in cold medium (removed from the refrigerator). Cross-contamination is avoided throughout the protocol. Virus samples are processed in the following applicable order: 1) NC, 2) TS, 3) RS, 4) PC.

[0036] a) Record the virus titer (vp / ml). b) Calculate the virus dilution. c) Synchronize the work with the operator who inoculates the cells so that the virus dilutions and the cells to be infected are prepared at approximately the same time. d) Thaw the RS, ARM and TS in the refrigerator and keep them refrigerated until ready to use. e) Add the required amount of cold medium to a 50 ml tube. The required volumes are as follows: approximately 7.0 ml for the PC pre-dilution, approximately 8.5 ml for the final dilution of the NC, RS and PC flasks, and approximately 4.5 ml for each TS. (As a result, approximately 20 ml is required for one TS, and approximately 40 ml for five TSs.) f) Prepare predilutions of PC(ARM) according to Table III. It has been found that the ARM must be serially diluted extensively to achieve the desired dose in the appropriate volume. Each dilution should be thoroughly mixed before being used to prepare the next dilution. [Table 3] g) Prepare the final dilutions. Pipette medium into all tubes first, then the test substance, and finally RS and PC only. Note that duplicate dilutions are prepared for RS, PC, and each TS. Use the dilutions within 90 minutes of preparation. h) 500 ml shake flasks were filled with 1 x 10 cells in 100 ml of medium. 8 Infect cells with medium (NC) and with the final dilution. Use all dilutions (2 ml) for infection. i) Approximately 90 minutes (± 10 minutes) post-infection, add 100 ml of fresh pre-warmed medium to the flask containing infected cells. j) The flask containing the infected cells is incubated for 3 days (+37°C, 5% CO2, 122 rpm).

[0037] Note that the assay can be paused after the first infection. Follow steps a) through h) above. Transfer the supernatant to a clean, 15 ml sterile centrifuge tube. Quickly freeze the tube in liquid nitrogen and store in an ultra-low temperature freezer for up to 2 months. On the day of the second infection, thaw the frozen supernatant in the refrigerator and use all of it to infect fresh cells.

[0038] Secondary infection: For the second infection, media (removed from the refrigerator) is required to resuspend the cells. Virus samples are processed in the following applicable order: 1) NC, 2) TS, 3) RS, 4) PC.

[0039] a) Synchronize the work with the operator who inoculates the cells so that the supernatant for infection and the cells to be infected are prepared at approximately the same time. b) Fill the liquid nitrogen container with liquid nitrogen. c) Transfer the infected cell suspension (200 ml) from each shake flask into a 50 ml sterile tube (4 tubes / sample). d) The cells are centrifuged at 1000 x g for 10 minutes at +4°C. e) Remove the supernatant. f) Resuspend the cell pellet of one sample in 5 ml of fresh cold medium. Add medium to one tube, resuspend the pellet by pipetting, transfer the suspension to the second tube of the same sample, resuspend, and repeat until all four tubes of the sample have been processed. g) Transfer the suspension to sterile 15 ml centrifuge tubes (1 tube / sample). h) Lyse the cells by three cycles of freezing in liquid nitrogen (approximately 5 minutes) and thawing in a water bath at +37°C (approximately 10 minutes). Place the tubes in a metal cage that can be immersed in liquid nitrogen and warm water in a line. After each freeze, check that the tubes are not damaged before placing them in warm water. After each thaw, check again that the tubes are not cracked and vortex the tubes at low speed. You can pause the process between each freezing step. Keep the samples frozen (liquid nitrogen / -80°C) until you are ready to continue. i) Centrifuge the lysed cells at 2000 x g for 20 minutes at +4°C to remove cell debris. Retain the supernatant. j) If you do not have HeLa suspension cells ready for infection, transfer the supernatant to a clean tube and keep it cold (refrigerator) until you are ready to infect the cells. k) 2 x 10 in 40 ml 7 Infect a 125 ml shake flask containing 100 cells with the supernatant. Use all of the supernatant (approximately 5 ml) for the infection. l) The flask containing the infected cells is incubated for 3 days (+37°C, 5% CO2, 122 rpm).

[0040] After the second infection, the assay can be paused. Follow steps a) through h) above. Transfer the supernatant to a clean, 15 ml sterile centrifuge tube. Quickly freeze the tube in liquid nitrogen and store in an ultra-low temperature freezer for up to 2 months. On the day of the third infection, thaw the frozen supernatant in the refrigerator and use all of it to infect fresh cells.

[0041] Third infection: For the third infection, use cold medium (removed from the refrigerator) to resuspend the cells. Virus samples are processed in the following applicable order: 1) NC, 2) TS, 3) RS, 4) PC. a) Synchronize the work with the operator who inoculates the cells so that the supernatant for infection and the cells to be infected are prepared at approximately the same time. b) Fill the liquid nitrogen container with liquid nitrogen. c) Transfer the infected cell suspension (40 ml) from each shake flask into a 50 ml sterile tube (1 tube / sample). d) The cells are centrifuged at 1000 x g for 10 minutes at +4°C. e) Remove the supernatant. f) Resuspend the cell pellet of one sample in 2 ml of fresh cold medium. If the pellet is very dense and difficult to resuspend, the resuspension volume can be increased. g) Transfer the suspension to sterile 15 ml centrifuge tubes (1 tube / sample). h) Lyse the cells by three cycles of freezing in liquid nitrogen (approximately 5 minutes) and thawing in a water bath at +37°C (approximately 10 minutes). Place the tubes in a metal cage that can be immersed in liquid nitrogen and warm water in a line. After each freeze, check that the tubes are not damaged before placing them in warm water. After each thaw, check again that the tubes are not cracked and vortex the tubes at low speed. You can pause the process between each freezing step. Keep the samples frozen (liquid nitrogen / -80°C) until you are ready to continue. i) Centrifuge the lysed cells at 2000 x g for 20 minutes at +4°C to remove cell debris. Save the supernatant. j) If you do not have HeLa suspension cells ready for infection, transfer the supernatant to a clean tube and keep it cold (refrigerator) until you are ready to infect the cells. k) 2 x 10 in 40 ml 7 Infect a 125 ml shake flask containing 100 cells with the supernatant. Use all of the supernatant (approximately 2 ml) for the infection. l) The flask containing the infected cells is incubated for 3 days (+37°C, 5% CO2, 122 rpm).

[0042] collect: For collection, sample labels may be printed according to Table IV. Four labels are required for each flask of infected cells: two for 110 μl aliquot sizes and two for <1000 μl. [Table 4]

[0043] Cold medium (removed from the refrigerator) is required to resuspend the cells. Virus samples are processed in the following applicable order: 1) NC, 2) TS, 3) RS, 4) PC. a) Fill the liquid nitrogen container with liquid nitrogen. b) Label 1 ml cryotubes for collection aliquots. c) Transfer the infected cell suspension (40 ml) from each shake flask into a 50 ml sterile tube (1 tube / sample). d) The cells are centrifuged at 1000 x g for 10 minutes at +4°C. e) Remove the supernatant. f) Resuspend the cell pellet of one sample in 2 ml of fresh cold medium. If the pellet is very dense and difficult to resuspend, the resuspension volume can be increased. g) Transfer the suspension to sterile 15 ml centrifuge tubes (1 tube / sample). h) Lyse the cells by three cycles of freezing in liquid nitrogen (approximately 5 minutes) and thawing in a water bath at +37°C (approximately 10 minutes). Place the tubes in a metal cage that can be immersed in liquid nitrogen and warm water in a line. After each freeze, check that the tubes are not damaged before placing them in warm water. After each thaw, check again that the tubes are not cracked and vortex the tubes at low speed. You may pause the process between each freezing step. Keep the samples frozen (liquid nitrogen / -80°C) until you are ready to continue. i) Centrifuge the lysed cells at 2000 x g for 20 minutes at +4°C to remove cell debris. Save the supernatant. j) Transfer the supernatant to a new clean tube and mix. k) Aliquot the supernatant of each sample into pre-labeled 1 ml cryotubes: 2 x 110 μl; 2 x <1000 μl. l) Quickly freeze aliquots in liquid nitrogen and store in a freezer until analysis.

[0044] Digital PCR analysis: DNA work should be performed under nuclease-free conditions. Sterile nuclease-free solutions and plasticware must be used. Gloves should always be worn when handling DNA samples. DNA-ExitusPlus™ may be used to remove potential DNA residues after work. After cleaning a laminar flow hood ("LFH") dedicated to DNA samples, the LFH may be inactivated by UV irradiation overnight.

[0045] To pretreat the recovered samples, thaw them at room temperature for up to 1 hour. Pipette the samples (100 μl / well) onto a 96-well plate: NC into well A4; RS_1, RS_2, PC_1, PC_2, TS1_1, and TS1_2 into wells B1-G1, column 1; and the remaining TS into wells A6-H6, column 6. Add Proteinase K (1 μl / well) to the used wells. Tightly seal the plate with an optical adhesive cover. Gently vortex and briefly rotate. Run the plate on an Applied Biosystems 7500 Real-Time PCR System using the latest version of the SDS template document "Prot K." The run program is as follows: Incubate at +50°C for 60 minutes; Incubate at +95°C for 20 minutes; Cool the samples to +4°C. If not continuing directly with sample dilutions, store the plate in the refrigerator.

[0046] When diluting samples, they should be diluted to fit within the dynamic range of the ddPCR analysis. Use dilutions of 1:1000 and 1:10,000 for the analysis. If neither of these dilutions is acceptable, higher or lower dilutions can be tested. Pipette cell culture medium (90 μl / well) into wells B2 through G5 of the 96-well plate containing the pretreated recovered samples. Pipette cell culture medium (90 μl / well) into wells A7 through H10. Pipette cell culture medium (90 μl / well) into well H4. This well will be used to prepare NTCs for ddPCR analysis. Mix the pretreated samples in columns 1 and 6 thoroughly by pipetting. Prepare serial dilutions from columns 1 through 5 and from columns 6 through 10. Pipette 10 μl into columns 1 through 2 and mix thoroughly by pipetting the dilutions. Pipette 10 μl from columns 2 to 3 and mix. Continue until column 5 is ready and mixed. Repeat for columns 6 to 10. If not proceeding directly with ddPCR analysis, store the plate in the refrigerator.

[0047] Preparation of ddPCR plates: To prepare the ddPCR plates, prepare a fresh 1 pg / μl dilution of ARM DNA from the 10 ng / μl aliquot of each RCA assay intended for product release. See Table V for the ARM DNA dilution series. [Table 5]

[0048] Each dilution must be mixed thoroughly by pipetting before being used to prepare the next dilution. The dilutions are prepared in DNA LFH. The last dilution, named ARM DNA (1 pg / μl), is used to perform ddPCR. The volume used is 5 μl, i.e., 5 pg of ARM DNA is used in each reaction.

[0049] Fresh dilutions of forward and reverse primers and TaqMan probes for RCA are prepared for each RCA assay intended for product release. In our experiments, we used E1-deleted adenovirus, so the forward primer for RCA was 5'-AAC CAG TTG CCG TGA GAG TTG-3'; the reverse primer for RCA was 5'-CTC GTT AAG CAA GTC CTC GAT ACA-3'; and the TaqMan probe for RCA was 5'-TGG GCG TCG CCA GGC TGT G-3'.

[0050] Reagents are thawed at room temperature, mixed, and spun down (e.g., by centrifugation / vortexer). See Table VI for dilution instructions. [Table 6]

[0051] Dilutions are prepared in the master mix laminar flow. The final concentrations of primers (6000 nM) and probe (2500 nM) are used in the ddPCR run. 2.5 μl of each reagent is added to a total reaction mixture of 25 μl. The concentrations in the reaction mixture are 600 nM for primers and 250 nM for probes. Previously prepared dilutions stored at -20°C can be used for assay development, in-process samples, and characterization. The master mix is ​​prepared in an LFH dedicated to preparing master mixes. Samples are added to the DNA LFH plate. Separate materials, pipettes, and centrifuge / vortexer are used for master mix and DNA work. Calculate the required well volume and total volume of master mix. Thaw the ddPCR supermix for the probe at room temperature and vortex at high speed once thawed. Mix the diluted forward primer, reverse primer, and TaqMan probe for RCA and spin down the reagents. We have found that a centrifuge / vortexer can be conveniently used for mixing and spinning. Prepare a ddPCR master mix according to Table VII and vortex at high speed. [Table 7]

[0052] Pipette the master mix onto the wells of a 96-well plate (20 μl / well). Optionally, pour the master mix into a reagent reservoir and use a multichannel pipette to pipette the mixture onto the plate. Add the ddPCR Buffer Control Kit (BC, 25 μl / well) to wells H4-H6 and any wells that do not require samples. Note that for the maximum number of samples to be tested, all wells in each column must be filled. Use the ddPCR Buffer Control Kit ("BC") in wells that do not require samples. For example, we recommend using wells at 1:1000 and 1:10,000 dilutions.

[0053] We recommend vortexing the 96-well plate containing the pretreated samples and dilutions and spinning down the samples. Add the samples (5 μl / well) to the ddPCR plate containing the master mix (20 μl / well). Store the sample plate in the refrigerator after use. The samples can be used to repeat the ddPCR analysis.

[0054] For the control, mix the ARM DNA dilutions by pipetting and add (5 μl / well) to the ddPCR plate (wells A4, A5 and A6).

[0055] Seal the plate with heat foil using a plate sealer at +180°C for 5 seconds. Vortex the plate briefly and spin down the sample mixture. Proceed to droplet generation and PCR run.

[0056] Droplet generation, PCR reading and droplet reading: We recommend using an AutoDG™ instrument to perform automated droplet generation. To do so, we seal the plate containing the droplets with heat foil using a plate sealer at +180°C for 5 seconds. PCR is then continued. We recommend running the plate using a C1000™ Touch Thermal Cycler with the PCR conditions shown in Table VIII. [Table 8]

[0057] After the PCR run, the droplets are stable. The plate can be stored overnight in a refrigerator. Read the droplets using the Droplet Reader with the latest version of the QuantaSoft template "RCA ddPCR." The QuantaSoft software automatically creates a folder for the run and saves the run results as a QuantaSoft plate document. After the run, manually set the threshold for all wells to 2000 by clicking the "Analyze" button on the left menu; selecting all wells from the plate layout in the upper right corner; selecting the "1D Amplitude" display; and activating the yellow-marked button (multi-well tool) at the bottom of the left menu. This allows you to set the threshold for all wells simultaneously. Enter 2000 in the Set Threshold box to set the threshold (press Enter). See Figure 2 for details on the visualization of threshold settings and the software user interface. Close the QuantaSoft software and click "Yes" when the software prompts you to select "Save Plate Information?" Copy the created folder to the server, ensuring that the folder name includes the assay run number.

[0058] The ddPCR run data is automatically analyzed by QuantaSoft software. SSC performance is assessed and the results are read by the software.

[0059] Data Analysis: In ddPCR technology, sample DNA is randomly divided into thousands of droplets. The more droplets present, the better the analytical accuracy. The QX200 ddPCR system can generate and read over 20,000 droplets per well. To ensure the desired accuracy, a criterion of 8,000 or more acceptable droplets is set for each well analyzed. However, the assay does not fail if individual wells do not meet this criterion. Wells that do not meet the criterion are excluded from further analysis. Each sample is analyzed in triplicate in the ddPCR analysis. The sample result can be read if at least two of the three wells contain 8,000 or more acceptable droplets. To verify the number of acceptable droplets, follow these steps: Open the plate document (threshold 2,000) saved on the server. Click the "Analysis" button in the left menu. Select all wells from the plate layout in the upper right corner. Select the "Events" view. Check the "Total" box on the right. For clarity, the other boxes (pos / neg) should not be checked. From the histogram values, confirm that there are 8,000 or more acceptable droplets in each well.

[0060] The system acceptance criteria for NC, NTC, and ARM DNA is that at least two of the three wells have 8,000 or more acceptable droplets. Check whether the criteria are met. If the criteria are not met, the ddPCR analysis must be repeated. For further SSC consideration, only wells with 8,000 or more acceptable droplets are considered.

[0061] In ddPCR analysis, samples containing five or fewer positive droplets are considered negative. Samples containing 6 to 34 positive droplets are not considered negative, but may be contaminated or contain very low amounts of target DNA. Samples with 35 or more positive droplets are considered clearly positive. Check the number of positive and negative droplets in each well by following steps a) to e) above, but check the positive or negative box instead of the total.

[0062] The assay SSC for NC, NTC, and ARM DNA is as follows: any of the acceptable wells for NC samples has 5 or fewer positive droplets, or any of the acceptable wells for NTC has 5 or fewer positive droplets, or at least two of the triplicate wells for ARM DNA show 35 or more positive droplets.

[0063] If these SSCs do not meet the criteria, we recommend repeating the ddPCR. If the NCs still do not meet the criteria, we recommend repeating the entire RCA assay starting from the first infection. If the reason for the failure to meet the criteria is NTC or ARM DNA, the RCA amplification may have been successful, but there is some problem with the ddPCR analysis. If NTC or ARM DNA repeatedly fails to meet the criteria, the reason for the failure to meet the criteria must be investigated.

[0064] To read a result for a particular sample, the sample must have both positive and negative droplets. A small amount of negative droplets will reduce the accuracy of the analysis. A well should show more than 100 negative droplets; otherwise, the well is considered "saturated" and the result for that well cannot be read.

[0065] The dynamic range of ddPCR analysis is narrow. RS and TS are analyzed at two dilutions: 1:1000 and 1:10000. At least one of the dilutions must be within range. The criteria for an acceptable RS dilution are that at least two of the three wells show 8000 or more acceptable droplets, more than 100 negative droplets (well is non-saturated), and 35 or more positive droplets (well shows a positive result).

[0066] Evaluate the assay SSC at acceptable dilutions. The assay SSC for RS is as follows: both RS_1 and RS_2 show positive results (35 or more positive droplets) at acceptable dilutions on acceptable wells. The SSC passes if both RS_1 and RS_2 have at least one acceptable dilution. Failure to meet the SSC criteria may be due to improper dilution. The dilution used may be too low (wells saturated) or too high (negative result). In this case, ddPCR can be repeated with an adjusted dilution. Dilutions of 1:10 and / or 1:100 from the pretreatment plate can be used as appropriate dilutions, or an additional dilution of 1:10,000 can be prepared. Consult an expert to determine how to proceed. If one of the duplicate flasks (RS_1 or RS_2) still shows a negative result while the other is within the dynamic range, the entire RCA assay, starting from the first infection, must be repeated.

[0067] The criteria for an acceptable TS dilution are that at least two of the triplicate wells show 8,000 or more acceptable droplets and more than 100 negative droplets (wells are non-saturated). Evaluate the sample SSC at the acceptable dilution(s). Note that the TS does not need to pass the criterion of 35 or more positive droplets, as the TS may not contain RCA.

[0068] The sample SSC is as follows: both TSX_1 and TSX_2 have at least one acceptable dilution. If a sample does not meet the SSC criteria, the result cannot be reported and the sample must be reanalyzed. However, the results of other samples can be read and reported. The reason for not meeting the SSC criteria may be an inappropriate dilution. The dilution used may be too low (wells saturated). In this case, the ddPCR can be repeated with an adjusted dilution. Further dilutions can be prepared starting from 1:10,000.

[0069] RCA assay results: The results of the ddPCR analysis are given as copies / μl. The QuantaSoft software reports this value for each well in the results table in the upper left corner. If there are no positive droplets in the well, the value is 0; if there are no negative droplets in the well, the value is 1,000,000 (saturation). To calculate the RS and TS results, record the concentration (copies / μl) reported in the QuantaSoft software results table for each well. Record only the concentrations of wells and dilutions that were accepted; otherwise, record N / A. Then, calculate the adjusted concentration (copies / μl) by multiplying the reported concentration by the dilution factor. Then, calculate the average of the adjusted concentrations (copies / μl). Record this as an integer without decimals. Note that if one of the duplicate flasks (TSX_1 or TSX_2) for a particular TS shows negative results at both dilutions, calculate the average from the positive flask. For RS, calculate the RS range as the mean ± 20%; the lower limit of the range is 0.8 x mean and the upper limit of the range is 1.2 x mean. Compare the TS results to this RS range. For each TS, compare the mean to the RS range. If the mean is below the RS range, the result is "less RCA than RS." If the mean is within the RS range, the result is "same amount of RCA as RS." If the mean is above the RS range, the result is "more RCA than RS."

[0070] If one of the duplicate flasks for a particular TS (TSX_1 or TSX_2) shows negative results at both dilutions and the other clearly shows positive results, the average (copies / µl) is based on the positive flask. If the comparison with the RS range yields a result of "more RCA than RS," the ddPCR analysis should be repeated. If the results are still the same, the entire RCA assay should be repeated, starting from the first infection.

[0071] Assay results may be trended in an Excel™ file. The following parameters are recommended for trending: RCA assay run number Assay SSC pass / fail Possible reasons for SSC rejection ARM DNA result (copies / μl) Allowable dilution ratios for RS and PC RS and PC mean (copies / μl) (Note that PC mean is only calculated in Excel for trend analysis.)

[0072] Based on the disclosure of the present inventors, those skilled in the art can easily modify this. For example, the present inventors have actually developed an improved assay using a recombinant adenovirus carrying a transgene for vascular endothelial growth factor D, but this assay can also identify replication-competent contaminating viruses in vectors carrying other transgenes (e.g., p53, interferon, etc.).

[0073] "Infection" refers to a virus replicating and forming progeny within a target cell. In contrast, "transfection" refers to the delivery of foreign DNA or RNA to a target cell via a viral vector. Transfection does not require viral replication within the target cell.

[0074] Similarly, although the inventors have indeed tested the assay with viral vectors intended to not replicate at all in human patients, the assay can readily be used with viral vectors intended to replicate conditionally, e.g., only in cancerous human cells and not in normal human cells, and therefore the phrase "incapable of replicating in normal human cells" is used in the appended claims to indicate this.

[0075] Similarly, although our experiments were performed with adenovirus, this methodology readily lends itself to other types of gene therapy viral vectors.

[0076] Accordingly, it is intended that the legal scope of our patent be defined by the appended claims and their permissible equivalents, rather than by the specific laboratory work described above. The present invention provides, for example, the following items. (Item 1) 1. A method for identifying viruses capable of replicating in normal human cells in a sample containing a viral gene therapy vector that is incapable of replicating in normal human cells, said method comprising: a. obtaining a sample comprising a viral gene therapy vector that is incapable of replicating in normal human cells, said viral vector comprising a transgene and a viral genome that has been genetically modified from said wild-type viral genome by having a modification or deletion in a region of the wild-type viral genome that is essential for viral replication in normal human cells, whereby the resulting viral gene therapy vector is incapable of replicating in normal human cells, said method also comprising then: b) mixing the sample with live target cells that can be transduced by the viral gene therapy vector to form a transduction mixture; and c) maintaining the transduction mixture for a time and under conditions sufficient to allow the viral gene therapy vector to transduce the target cells; and d) separating the target cells from any remaining sample, and then e) lysing the target cells to release the intracellular contents, and then f) mixing the intracellular contents of the lysed target cells with viable assay cells that can be infected with the virus to form an infection mixture; and g) maintaining the infection mixture for a time and under conditions sufficient to allow the virus, if present, to infect the assay cells; and h) lysing the assay cells to release their intracellular contents, and then i) isolating nucleic acids from the intracellular contents of said assay cells, and then j) evaluating the isolated nucleic acid by digital PCR using a probe that hybridizes to the modified or deleted region of the viral genome that is essential for viral replication; This method of identification comprises determining the approximate amount of virus in the sample that is capable of replicating in normal human cells. (Item 2) 2. The method of claim 1, wherein the virus is an adenovirus and the probe comprises a DNA probe having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. (Item 3) The method comprises administering to a subject a 3×10 10 2. The method of item 1, wherein as few as 25 viruses capable of replicating in normal human cells are detected per viral gene therapy vector particle. (Item 4) The method comprises administering to a subject a 3×10 10 4. The method of item 3, wherein as few as 7 viruses capable of replicating in normal human cells are detected per viral gene therapy vector particle. (Item 5) 2. The method of claim 1, wherein the transgene expresses a polypeptide selected from the group consisting of interferon and p53. (Item 6) 1. A pharmaceutical finished dosage form comprising a pharmaceutically acceptable excipient and a viral gene therapy vector that is unable to replicate in normal human cells and that contains a transgene, wherein said dosage form comprises 3×10 10 The pharmaceutical finished dosage form comprises no more than about 25 viral particles capable of replicating in normal human cells per viral particle. (Item 7) 3 × 10 unable to replicate in normal human cells 10 7. The method according to claim 6, wherein the virus particles contain no more than about 7 virus particles capable of replicating in normal human cells per 1000 virus particles. Pharmaceutical finished dosage form. (Item 8) 7. The pharmaceutical finished dosage form according to item 6, wherein the virus is an adenovirus. (Item 9) 7. The pharmaceutical finished dosage form according to item 6, wherein the transgene expresses a polypeptide selected from the group consisting of interferon and p53. (Item 10) 10. The pharmaceutical finished dosage form according to item 9, wherein the transgene expresses an interferon. (Item 11) 3 × 10 unable to replicate in normal human cells 10 11. The pharmaceutical finished dosage form according to item 10, comprising no more than about 7 viral particles capable of replicating in normal human cells per viral particle. (Item 12) 10. The pharmaceutical finished dosage form according to item 9, wherein the transgene expresses p53. (Item 13) 3 × 10 unable to replicate in normal human cells 10 13. The pharmaceutical finished dosage form according to item 12, comprising no more than about 7 viral particles capable of replicating in normal human cells per viral particle.

Claims

[Claim 1] The invention described in the specification.

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