A virus packaging plasmid and its application

Abstract

The present disclosure provides a virus packaging plasmid and applications thereof, and particularly relates to a chimeric sequence and application thereof in enhancing Cap expression, as well as a recombinant adeno-associated virus packaging plasmid comprising the chimeric sequence, a recombinant adeno-associated virus and applications thereof.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a virus packaging plasmid and application thereof. Background Art

[0002] Adeno-associated virus has achieved great success as a gene therapy vector in clinical practice. In recent years, AAV-based gene therapy drugs approved for marketing include Luxturna, Zolgensma, Hemgenix, Elevidys, and Roctavian, covering diseases in ophthalmology, neurology, and blood. Except for Luxtuna for the treatment of congenital amaurosis, the other five approved drugs require large doses (2 × 10 13 to 1.3 × 10 14 vg / kg) is administered systemically by intravenous injection. For adult patients, a single dose of ~10 16 vg. This will pose a severe challenge to the production of viruses. Therefore, maximizing the yield and quality of viruses is what every gene therapy drug development company is working towards. Summary of the invention

[0003] The present disclosure provides a nucleic acid sequence, which comprises a first sequence, a second sequence and a third sequence from the 5' end to the 3' end.

[0004] In some embodiments, the first sequence is shown as SEQ ID NO:13, the second sequence is shown as SEQ ID NO:15, and the third sequence is shown as SEQ ID NO:17.

[0005] In some embodiments, the nucleic acid sequence is as shown in SEQ ID NO:18.

[0006] In some embodiments, the first sequence, the second sequence, and the third sequence are directly connected or connected by a connecting sequence.

[0007] Furthermore, the present disclosure provides use of the aforementioned nucleic acid sequence in enhancing the expression of Cap gene.

[0008] In some embodiments, the Cap gene serotype is selected from AAV9, 8, 1, 2, 3, 3B, 4, 5, 6, 7, 10, 11, 12, 13, rh10 or hu37.

[0009] In some embodiments, the recombinant adeno-associated virus packaging plasmid comprises a Rep gene and a Cap gene, wherein the 5' end of the Cap gene comprises the nucleic acid sequence described above.

[0010] In some embodiments, the nucleic acid sequence of the recombinant adeno-associated packaging plasmid is shown in any one of SEQ ID NOs: 2-4.

[0011] The present disclosure also provides a recombinant adeno-associated virus vector system, characterized in that it comprises the recombinant adeno-associated virus packaging plasmid, the recombinant adeno-associated virus core plasmid and the recombinant adeno-associated virus element helper plasmid as described above.

[0012] The present disclosure also provides a recombinant adeno-associated virus particle, characterized in that it is obtained by co-transfecting a packaging cell line with the recombinant adeno-associated virus packaging plasmid, the recombinant adeno-associated virus core plasmid and the recombinant adeno-associated virus element helper plasmid as described above.

[0013] Furthermore, the present disclosure provides use of the aforementioned recombinant adeno-associated virus packaging plasmid in preparing a recombinant adeno-associated virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present disclosure can be more fully understood with reference to the following drawings.

[0015] Figure 1 The structure of the plasmid is shown.

[0016] Figure 2 Virus titer detection is shown.

[0017] Figure 3 Gel images of coat protein packaging of pEXG100-030RK and pRH85-Cap921 viruses are shown.

[0018] Figure 4 The yields of AAV2 and AAV8 are shown. DETAILED DESCRIPTION

[0019] The following description of the present disclosure is intended only to illustrate various different embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is obvious to those skilled in the art that various different equivalents, changes and modifications can be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments will be included herein. All references cited herein, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0020] In order to enable those skilled in the art to better understand the solutions of the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only embodiments of a part of the present disclosure, rather than all of the embodiments.

[0021] The experimental materials and primers used in the present invention are shown in Table 1 and Table 2, respectively.

[0022] Table 1. Experimental materials

[0023]

[0024] Table 2. Primer information

[0025]

[0026] Example

[0027] Example 1: Construction of recombinant adeno-associated virus packaging plasmid

[0028] (1) Design and synthesis of vectors, such as Figure 1 As shown, the AAV9 packaging plasmid pRH85-Cap921 was designed, and the specific sequence is shown in Table 3. A nucleic acid sequence was inserted before the Cap9 gene, and the nucleic acid sequence included a Rep8 truncated sequence, a Rep5 truncated sequence, and a Rep2 truncated sequence, and the specific information is shown in Table 2. In some specific embodiments, the recombinant adeno-associated packaging plasmid comprises the following structure: Rep2 gene sequence-Cap truncated sequence-bGH pA-Rep8 truncated sequence-Rep5 truncated sequence-Rep2 truncated sequence-Cap gene sequence.

[0029] Table 3. Sequence Listing

[0030]

[0031] Example 2: Preparation of recombinant adeno-associated virus

[0032] (1) Virus packaging

[0033] Our laboratory used a three-plasmid co-transfection system for virus packaging in adherent HEK293T cells, namely pHelper plasmid (pEXG100-005HK, SEQ ID NO: 7), RepCap plasmid (pEXG100-030RK (SEQ ID NO: 1), pRH85-Cap921 (SEQ ID NO: 2)), and GOI plasmid (pEXG100-042GK, SEQ ID NO: 8), as shown in Table 4.

[0034] a) Cell inoculation: Cells should be plated 24 h before transfection. Cell status should be observed before transfection. If the cell density is between 70% and 80% and the cell status is good and free of contamination, transfection can be performed.

[0035] b) Prepare plasmid-PEI transfection complex with a mass ratio of plasmid:PEI=1:1, add pHelper plasmid, RepCap plasmid, and GOI plasmid (molar ratio 1:1:0.3) as shown in Table 4 to each well of a six-well plate with a total amount of 2.1 μg of plasmid, respectively, to prepare 0.8 ml of transfection complex and let stand for 15 min;

[0036] c) transfection with transfection complex: serum medium = 1:10, the transfection complex is added dropwise to the cells to be transfected for transfection;

[0037] d) After 72 h of incubation, the virus was harvested.

[0038] Table 4. Three-plasmid system

[0039]

[0040] (2) Virus titer detection

[0041] a) Sample information is shown in Table 5:

[0042] Table 5. Sample information

[0043]

[0044] b) Prepare the DNase I treatment system according to Table 6:

[0045] Table 6. DNase Ⅰ treatment system

[0046]

[0047] Samples were pre-diluted to 1E+09 vg / ml to 4E+10 vg / ml, then 40 μL of DNase mix was taken and 5 μL of sample was added to each tube. The samples were placed at 37°C for 30 min and then stopped by adding 5 μL of 50 mM EDTA solution.

[0048] c) Preparation of SSDB: 10 mM Tris-HCl, pH 8.0, containing 2 μg / ml salmon sperm DNA (Invitrogen AM9680), 0.1% F-68.

[0049] Prepare dilutions (SSDB) according to Table 7:

[0050] Table 7. Dilution solution (SSDB) formula

[0051]

[0052] Dilute the sample treated with DNase I 1:10 (20 μL sample + 180 μL SSDB)

[0053] d) Dilute the sample treated with DNase I 1:10 (20 μL sample + 180 μL SSDB)

[0054] e) Prepare the PCR system according to Table 8

[0055] Table 8. PCR reaction system

[0056]

[0057] PCR reaction system: 22.4 µL PCR mix + 2.5 µL template = 24.9 µL.

[0058] f) Set up the reaction program

[0059] The PCR reaction program is shown in Table 9:

[0060] Table 9. PCR reaction program

[0061]

[0062] The heating rate of the PCR instrument should be between 2.5℃ / s, and the reaction volume of the PCR instrument should be set to 40 μL.

[0063] g) Data statistical analysis is shown in Table 10

[0064] Table 10. Statistical analysis of data

[0065]

[0066] The results showed that three independent experiments found that the yield of the newly designed pRH85-Cap921-packaged virus AAV9-100-042GK (pRH85-Cap921) was ~2.5 times higher than that of the commonly used AAV9-100-042GK (pEXG100-030RK) packaged on the market, see Figure 2 .

[0067] Example 3: Production and purification of viruses

[0068] After packaging a small amount of virus, we determined that the yield of AAV9 packaged with the designed pRH85-Cap921 was higher than that of pEXG100-030RK, so in the next step, we used these two plasmids to produce a batch of virus to compare the activity of the virus and the proportion of viral capsid protein;

[0069] (1) Virus packaging

[0070] a) Cell inoculation: Cells should be plated 24 h before transfection. Cell status should be observed before transfection. If the cell density is between 70% and 80% and the cell status is good and free of contamination, transfection can be performed.

[0071] b) Prepare plasmid-PEI transfection complex: the mass ratio is plasmid: PEI = 1:1, add pHelper plasmid, RepCap plasmid and GOI plasmid (molar ratio 1:1:0.3) to each 150 mm cell culture dish with a total amount of 58 μg of plasmid, prepare 10 ml of transfection complex and let it stand for 15 min;

[0072] c) Transfection was performed with a ratio of transfection complex to serum medium = 1:10, and the transfection complex was added dropwise to a 150 mm cell culture dish to complete the transfection;

[0073] d) After 72 h of incubation, the virus was harvested.

[0074] (2) Virus purification

[0075] a) Use a sterile cell scraper to scrape the cells in a 150 mm cell culture dish and centrifuge at 1500 g for 5 min at room temperature to obtain the virus supernatant and cell pellet;

[0076] b) The cell pellet was resuspended in TMS buffer (50 ml 1 M Tris-HCl pH8, 30 ml 5 M NaCl, 2 ml 1 MMgCl2, add sterile ddH2O to make up to 1000 mL), and 10% TritonX-100 was added to a final concentration of 0.4%; 1 / 4 volume of 5 x PEG8000 (400 g PEG-8000, 146 g NaCl plus ddH2O to make up to 1000 ml) was added to the viral supernatant, and ice bathed for 2 h;

[0077] c) After ice bath, centrifuge the virus supernatant at 4000 g for 30 min at 4°C, discard the supernatant, and resuspend the virus particles in PBS buffer;

[0078] d) Mix the virus particle resuspension with the cell pellet resuspension, add 250 KU / ml nuclease (Benzonase) to a final concentration of 25 U / ml, and place in an air shaker at 220 rpm and 37°C for 120 min;

[0079] e) Add 25% sodium deoxycholate at a ratio of 1:50, shake in a water bath at 37°C for 30 min, centrifuge at 10,000 g for 10 min at 4°C, and collect the supernatant;

[0080] f) Add 5 M NaCl to a final concentration of 250 mM and vortex mix at 500 rpm for 30 min;

[0081] g) Ultracentrifugation was performed using a gradient of 15%, 25%, 40%, and 60% iodixanol, and the 40% layer of virus was taken;

[0082] h) Dilute the virus solution 20 times with buffer C (PBS + 0.005% F68) and filter with a 0.45 μm syringe filter to remove impurities;

[0083] Use 100 kDa ultrafiltration concentrator tubes for ultrafiltration and concentration, use buffer C to replace the buffer, and use a 0.22 μm syringe filter to filter to obtain the final virus solution, and keep a sample for detection.

[0084] (3) The results of virus titer detection are shown in Table 11.

[0085] Table 11. Virus titer test results

[0086]

[0087] (4) Detection of virus shell ratio.

[0088] The denaturation system is prepared as shown in Table 12:

[0089] Table 12. Denaturation system configuration

[0090]

[0091] a) Denaturation: Denature the prepared blank control and virus samples by heating them at 75°C for 10 min.

[0092] b) Prepare 1× electrophoresis buffer: Take 50 ml NuPAGE MES SDS 20× electrophoresis buffer, add it to 950 ml purified water, and mix well. Load the treated NuPAGE 4-12% BT Gel into the vertical electrophoresis tank.

[0093] c) Sample spotting: Take 10 μl of the denatured blank control, virus sample, and 10 μl of Unstained Protein Marker and add them to the sample loading wells respectively.

[0094] d) Electrophoresis: Turn on the power of the electrophoresis instrument and set the voltage to 180 V and the time to 50 min.

[0095] e) Preparation of fixative: Prepare the fixative with ultrapure water at a ratio of 50% methanol and 7% acetic acid.

[0096] f) Gel stripping: After electrophoresis is completed, turn off the power, take out the gel, place the gel in a container containing purified water, rinse and pour out the purified water.

[0097] g) Gel fixation: Add 50 rpm fixative to the container containing the gel and place on an orbital shaker at 70 rpm for 30 min. Repeat once.

[0098] h) Staining: Pour off the fixative solution, add 60 ml SYPRO Ruby protein gel stain, place on an orbital shaker, and run at 70 rpm overnight.

[0099] i) Rinse: Pour out the staining solution and gently rinse the gel with 100 ml of purified water. Discard the purified water.

[0100] j) Preparation of washing solution: Prepare washing solution with ultrapure water at a ratio of 10% methanol and 7% acetic acid.

[0101] k) Washing: Add 100 ml of washing solution to the container, place it on an orbital shaker, and run it at 70 rpm for 30 min.

[0102] l) Rinse: Pour out the washing solution, rinse the gel gently with purified water, discard the purified water, and then add purified water to preserve the gel.

[0103] The results are shown in Figure 3,It can be seen from the gel image that the ratio of the capsid protein packaged by both pEXG100-030RK and pRH85-Cap921 viruses is consistent, close to VP1:VP2:VP3=1:1:10, indicating that the newly designed pRH85-Cap921 has no effect on the ratio of the viral capsid.

[0104] Example 4: Detection of viral infection activity (TCID50)

[0105] The experiment is to dilute the rAAV-GOI sample with known DNaseⅠ resistance (DRP) titer (ie, physical titer) but unknown infection titer in a series of gradients, and then co-infect HeLaRC32 cells (HeLa cells expressing AAV2Rep and Cap, ATCC number CRL-2972) in a 96-well plate with a saturating concentration of Ad5 (ATCC number VR-1516). The infected cells continue to be cultured at 37°C. After 72 h, the cells in each well are lysed and a small amount of lysate is analyzed by real-time quantitative PCR (qPCR). The amount of vector is calculated based on the Cq value output by qPCR and the dilution in each well. In each dilution, the wells with a vector amount higher than the initial input value are scored positive (yes), and the wells with a vector amount lower than the initial input value are scored negative (no). The TCID50 / mL is then calculated using the Karber method.

[0106] (1) Day 1: HeLaRC32 cells are plated into 96-well plates

[0107] 20-24 h before infection, HeLaRC32 cells in good condition were digested and counted, with a concentration of 1.2×10 4 Cells were plated at 1.2 × 10 cells / well using complete medium (DMEM + 10% FBS). 5 cells / ml, add 100 μl of cell suspension to each well. Place the plated cells in a CO2 incubator and continue culturing.

[0108] (2) Day 2: Virus infection of HeLaRC32 cells

[0109] a) Reagent preparation: Incubate the culture medium in a 37°C water bath for 20 min and thaw Ad5 on ice;

[0110] b) Dilute Ad5 to 2 × 10 8 / ml for standby use;

[0111] c) Dilution of the virus to be tested: dilute the virus to be tested to 1.0×10 12 / ml, and then dilute to 1.0×10 11 / ml for standby use.

[0112] d) Cell virus infection: Aspirate the supernatant from the cells and add 50 μl of the virus to be tested at an appropriate dilution to infect the cells. Each virus is infected with 5 dilutions and 1 Ad5 group, and each dilution is infected with 8 wells. This batch of viruses is 1.0×10 6 / ml to 1.0×10 10 / ml concentration for infection, and then placed in a CO2 incubator for further culture;

[0113] (3) Day 5: qPCR detection

[0114] a) Cell lysis and DNA extraction: Add 80 μl of 0.2% TritonX-100 to each well and mix by pipetting at least 10 times, then incubate at 37°C for 2 h.

[0115] b) Collect DNA samples: After incubation, transfer 45 μl of lysate from each well to a new PCR plate;

[0116] c) Proteinase K treatment: Add 5 μl of 10 mg / mL proteinase K working solution to each well in b), mix well, and then treat according to the following procedures: After treatment, place the sample at 4°C for use as a reaction template for qPCR;

[0117] •50°C for 1 h

[0118] • 95°C for 20 min

[0119] •Store at 12°C

[0120] d) Standard curve configuration: The standard is a linearized plasmid DNA containing the AAV standard reference genome. The standard is serially diluted to obtain a dilution of 2×10 6 to 2×10 1 The standard curve of ds copies / μl is reserved.

[0121] e) qPCR detection: The reaction system and reaction procedure are shown in Tables 13 and 14, respectively, and are set as follows: the template loading volume is 1 μl; after 2 h, 50 μl of fresh culture medium is added to the experimental wells, and the wells are placed in a CO2 incubator for continued cultivation.

[0122] Table 13. qPCR reaction system

[0123]

[0124] Table 14. qPCR reaction conditions

[0125]

[0126] (4) Result analysis:

[0127] a) Calculate the initial vector copy number added to each well based on the physical titer of the virus to be tested, the volume of virus added to the 96-well plate, and the dilution;

[0128] b) The number of vector copies detected by qPCR is multiplied by the dilution factor of 160, which is the total number of vector copies in the target well. Subtract the initial vector copy number in a) to get the number of copies generated by amplification after infection. Calculate the copy value of each well.

[0129] c) Determine the positive wells for each dilution: For this assay, the quantitative detection limit of qPCR was set at 20 vector copies. Multiplying by the dilution factor of 160, this is 3200 vector copies. Therefore, in b), copy values ​​<3200 are considered background, i.e., negative wells, and >3200 are positive wells.

[0130] d) TCID50 / mL of the tested virus was calculated by the Spearman-Karber method. The infectious activity is shown in Table 15:

[0131] Table 15. Infection activity

[0132]

[0133] The experimental results showed that there was no significant difference in the infectious activity of AAV9 packaged by the new and old RepCaps.

[0134] Example 5: Capsid ID Detection

[0135] Different serotypes of viruses contain different coat proteins. Differential scanning fluorescence (DSF) uses the process of heating the sample on the qPCR instrument. As the temperature continues to rise, the protein surface denatures, and the fluorescent dye SYPRO™ Orange can bind to the exposed hydrophobic groups until the protein is fully unfolded and the fluorescence signal reaches a maximum. After that, the fluorescence signal decreases due to thermal quenching and / or protein aggregation. Therefore, the temperature at which the fluorescence signal changes is considered to correspond to the transition temperature (T m Each serotype has a specific T m Therefore, differential scanning fluorimetry can be used to identify the capsid ID of AAV virus.

[0136] (1) Virus and dye preparation (to be performed in the Viral BSC)

[0137] a) Virus treatment: Take out the virus and thaw it on ice, vortex thoroughly for 30 seconds, centrifuge at 3000 rpm for 10 seconds, and place it on ice until use. If the titer of the sample is greater than 1E+13GC / mL, dilute the sample to 1E+13GC / mL with PBS. If the titer is less than 1E+13GC / mL, load the sample directly without dilution;

[0138] b) Dye dilution: Take out SYPRO™ Orange dye (stock solution is 5000×) and dilute it 100 times to 50× with PBS;

[0139] (2) qPCR reaction setup

[0140] a) In the BSC, add the test solution and sample to the qPCR 96-well plate in sequence according to Table 16.

[0141] Table 16. qPCR reaction system

[0142]

[0143] Note: When the sample titer is 1E+13 GC / mL, the loading volume is 10 μL, and when the sample titer is less than 1E+13 GC / mL, the loading volume is 15 μL. NC is 2 μL 50 × SYPRO™ Orange dye + 18 μL PBS.

[0144] b) Seal the plate with samples with quantitative PCR sealing film, vortex, and centrifuge in a microplate centrifuge.

[0145] (3) qPCR detection

[0146] a) Program design: Click File → New → Protocol, change the Sample Volume to 20 μL, select Insert Melt Curve for the qPCR reaction program, set the temperature to 45℃-90℃ for 2 min, and increase the temperature by 0.4℃.

[0147] b) Plate layout: Click Next→Create New Plate, hold down the left mouse button to select all sample areas, click User Preferences, set Plate Type to BR White, and check Cal Gold540 in the Fluorophores option.

[0148] c) Data export: Select Negative for Peak Type, export the data to Excel, and analyze the results.

[0149] (4) Data analysis is shown in Tables 17 and 18:

[0150] Table 17. Capsid IDs of different serotypes m Value reference range

[0151] Table 18. Test data

[0152]

[0153] The experimental tests were all within the reference range, and the T m This further indicates that the newly designed pRH85-Cap921 has no effect on the physical properties of the virus.

[0154] Example 6: Effects on other serotypes

[0155] In order to test the effect of the pRH85-Cap921 plasmid structure on the yield of other AAV serotypes, we replaced Cap9 of pRH85-Cap921 with Cap2 and Cap8 to form pRH85-Cap201 and pRH85-Cap801, respectively. The pRH85-Cap201 and pRH85-Cap801 plasmids were constructed according to the method described in Example 1, the virus was prepared according to the method described in Example 2, and the production and purification were carried out according to the method described in Example 3. The yield of AAV2 and AAV8 was tested according to the AAV9 yield test method described in Examples 4 and 5. The statistical results are shown in Figure 4 , it can be seen that the viruses AAV2-100-042GK (pRH85-Cap201) and AV8-100-042GK (pRH85-Cap801) packaged by the newly designed plasmid structure have higher yields than the common plasmid viruses on the market, so our new plasmid structure is also suitable for other serotypes. Therefore, we have invented a structure of AAV packaging plasmid that can increase the yield of all AAV serotypes without affecting the quality of the virus.

[0156] Incorporated by Reference

[0157] Each patent and scientific article mentioned herein is incorporated by reference in its entirety for all purposes.

[0158] Equivalence

[0159] The present disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be regarded as illustrative in all cases, rather than limiting the invention described herein. Therefore, the scope of the present disclosure is indicated by the appended claims rather than by the above description, and all changes within the equivalent meaning and scope of the claims are intended to be covered therein.

Claims

1. A nucleic acid, wherein the sequence of the nucleic acid comprises a first sequence, a second sequence and a third sequence from the 5' end to the 3' end, wherein the first sequence is as shown in SEQ ID NO: 13, the second sequence is as shown in SEQ ID NO: 15, and the third sequence is as shown in SEQ ID NO:

17.

2. The nucleic acid according to claim 1, wherein the sequence of the nucleic acid is shown in SEQ ID NO:

18.

3. The nucleic acid of claim 1, wherein the first sequence, the second sequence and the third sequence are directly connected or connected by a connecting sequence.

4. Use of the nucleic acid according to any one of claims 1 to 3 for enhancing the expression of a Cap gene.

5. The use according to claim 4, wherein the Cap gene serotype is selected from AAV9, 8, 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13 or hu37.

6. The use according to claim 4, wherein the Cap gene serotype is selected from AAV3B or AAVrh10.

7. A recombinant adeno-associated virus packaging plasmid, characterized in that: The recombinant adeno-associated virus packaging plasmid comprises a Rep gene and a Cap gene, wherein the 5' end of the Cap gene comprises the nucleic acid according to any one of claims 1 to 3.

8. The recombinant adeno-associated virus packaging plasmid according to claim 7, wherein the sequence of the nucleic acid of the recombinant adeno-associated virus packaging plasmid is shown in any one of SEQ ID NOs: 2-4.

9. A recombinant adeno-associated virus vector system, characterized in that: It comprises the recombinant adeno-associated virus packaging plasmid, the recombinant adeno-associated virus core plasmid and the recombinant adeno-associated virus element helper plasmid as described in claim 7 or 8.

10. A recombinant adeno-associated virus particle, characterized in that: The method is obtained by co-transfecting a packaging cell line with the recombinant adeno-associated virus packaging plasmid, the recombinant adeno-associated virus core plasmid and the recombinant adeno-associated virus element helper plasmid as described in claim 7 or 8.

11. Use of the recombinant adeno-associated virus packaging plasmid according to claim 7 or 8 in preparing recombinant adeno-associated virus.

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