Method for preparing recombinant bombyx mori nuclear polyhedrosis virus, and system and method for preparing recombinant adeno-associated virus

Abstract

A method for preparing a recombinant Bombyx mori nuclear polyhedrosis virus, and a system and method for preparing a recombinant adeno-associated virus. The system for preparing a recombinant adeno-associated virus comprises: a) BMN cells cultured in low-serum suspension and / or adherent cells of BMN cells cultured in low-serum suspension, and b) a recombinant Bombyx mori nuclear polyhedrosis virus, wherein the recombinant Bombyx mori nuclear polyhedrosis virus infects a host cell or a host which can at least produce a functional protein required for the packaging of a recombinant adeno-associated virus. The method for preparing a recombinant adeno-associated virus comprises infecting the BMN cells cultured in suspension or suspension to adherent culture using the recombinant Bombyx mori nuclear polyhedrosis virus. A recombinant adeno-associated virus prepared on the basis of a Bombyx mori baculovirus expression system has high titer, low empty capsid rate and good infection activity.

Description

Preparation method of recombinant Bombyx mori nuclear polyhedrosis virus, and preparation system and preparation method of recombinant adeno-associated virus TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a preparation method of recombinant Bombyx mori nuclear polyhedrosis virus (rBmNPV), and a preparation system and a preparation method of recombinant adeno-associated virus (rAAV). BACKGROUND

[0002] Gene therapy (GT) is a new therapy for treating diseases through transduction of therapeutic genetic material such as genes, gene fragments, RNA, gene scissors, etc., transcription or / and translation, indications including genetic diseases, tumors and chronic diseases, etc. At present, the scheme based on virus delivery of therapeutic genetic material accounts for nearly 90% of the total number of gene therapy, and more than 40% of gene therapy is occupied by thousands of clinical trials based on rAAV. So far, several rAAV virus vector gene therapy products have been approved for marketing, but the production cost of these products is high.

[0003] Compared with HEK293T cell / tri-plasmid transfection preparation system, baculovirus-based rAAV production system has the advantages of scalability, low cost and predictable biosafety, and is more suitable for gene therapy drug production. At present, insect cell / baculovirus rAAV preparation system is mainly based on commercialized Bac-to-Bac Autographa californica multiple nucleopolyhedrovirus (AcMNPV) expression system or its improved version, including three baculovirus system, two baculovirus system, one baculovirus system dependent on packaging cell line, one baculovirus system based on shuttle vector independent of packaging cell line (OneBac system). Although the previously authorized invention patent with the publication number CN 109609552 B “Preparation method and system of recombinant adeno-associated virus and recombinant bacmid” and the authorized invention patent with the publication number CN 112553257 B “Preparation method and system of recombinant adeno-associated virus and recombinant bacmid” developed OneBac system solved the industrial bottleneck problems of low yield, poor stability and high cost of traditional insect cell (Sf9) / baculovirus system rAAV preparation, the process of large-scale suspension culture of Sf9 cells using bioreactor is still expensive. As an important economic insect with strong Chinese characteristics, Bombyx mori has the characteristics of convenient feeding and low cost. The live pupae in cocoon can be used as a bioreactor for rAAV production. In particular, the breakthrough of artificial feed lays a foundation for the large-scale, standardized and industrialized feeding of Bombyx mori, making it possible to produce rAAV gene therapy preparations at extremely low cost using SPF silkworm pupae as raw materials.

[0004] However, to carry out the preparation of rAAV gene therapy preparations with extremely low-cost silkworm pupae as raw materials, it is crucial to first produce rAAV with infectivity at the cellular level based on the silkworm baculovirus expression system (BmNPV). The BmNPV expression system is another baculovirus system similar to the AcMNPV expression system. Currently, hundreds of recombinant proteins have been expressed based on this system, including mouse interleukin 3 (IL-3), foot-and-mouth disease subunit vaccine, human granulocyte macrophage colony-stimulating factor (HGM-CSF), hepatitis B surface antigen (HBsAG) vaccine, etc., but the production of rAAV vectors has lagged behind. Currently, three silkworm baculovirus (Lu et al., 2018, Biotechnology Progress, Vol. 8 (No. 1)) and two silkworm baculovirus systems can produce rAAV2 or its chimeric virus (Qian et al., 2021, Viruses, 2021.13.704.), but the yield of rAAV vectors, the full / empty capsid ratio, and the infection efficiency are all unsatisfactory, which cannot meet the production needs of gene therapy drugs. The invention patent "Recombinant adeno-associated virus, its mutant and construction method and application" with publication number CN112961219A successfully prepared rAAV with infectivity using the silkworm baculovirus expression system, but the single recombinant silkworm nucleopolyhedrovirus (rBmNPV) involved was obtained by recombination in silkworm cells, which requires time-consuming and laborious plaque screening to purify rBmNPV, and the rAAV prepared by this method also has the above problems of yield, full / empty capsid ratio, and infection efficiency.

[0005] SUMMARY

[0006] In view of the above defects of the prior art, the present application provides a preparation method of a single recombinant silkworm nucleopolyhedrovirus, a recombinant adeno-associated virus preparation system and a preparation method.

[0007] The specific technical solutions of the present application are as follows:

[0008] In one aspect, the present application provides a preparation method of a single recombinant silkworm nucleopolyhedrovirus, which is selected from one of methods 1-3:

[0009] Method 1: Transfect the adherent culture cells of low serum suspension cultured BMN cells with a single BmNPV-rAAV recombinant bacmid to rescue the first generation (P1) budded recombinant silkworm nucleopolyhedrovirus (rBmNPV (Budded virus)-rAAV, rBmBEV-rAAV), which is a single recombinant silkworm nucleopolyhedrovirus;

[0010] Method 2: a single BmNPV-rAAV recombinant bacmid is used to transfect adherent culture cells of low serum suspension culture BMN cells to rescue P1 generation rBmBEV-rAAV; the P1 generation rBmBEV-rAAV is used to infect adherent culture cells of low serum suspension culture BMN cells to obtain progeny rBmBEV-rAAV, which is a single recombinant Bombyx mori nuclear polyhedrosis virus;

[0011] Method 3: a single BmNPV-rAAV recombinant bacmid is used to transfect adherent culture cells of low serum suspension culture BMN cells to rescue P1 generation rBmBEV-rAAV; the P1 generation rBmBEV-rAAV is used to infect low serum suspension culture BMN cells to obtain progeny rBmBEV-rAAV, which is a single recombinant Bombyx mori nuclear polyhedrosis virus;

[0012] The single BmNPV-rAAV recombinant bacmid comprises a recombinant adeno-associated virus core expression element ITR-GOI, a Rep gene expression frame and a Cap gene expression frame.

[0013] Further, the low serum suspension culture BMN cells are BMN cells suspended in a culture medium containing 2% fetal bovine serum by volume percentage;

[0014] And / or, the adherent culture cells of the low serum suspension culture BMN cells are low serum suspension culture BMN cells adherently grown in Grace supplemented insect medium containing 10% fetal bovine serum by volume percentage.

[0015] Further, the preparation of the low serum suspension culture BMN cells comprises: the BMN cells are first acclimated to adhere to Grace supplemented insect medium containing 10% fetal bovine serum by volume percentage, then acclimated to adhere to a mixed culture medium composed of insect cell serum-free medium and Grace supplemented insect medium with gradually reduced fetal bovine serum content, and then acclimated to suspend in a mixed culture medium composed of insect cell serum-free medium and Grace supplemented insect medium with gradually reduced fetal bovine serum content, and finally the BMN cells are stably maintained in a mixed culture medium composed of insect cell serum-free medium and Grace supplemented insect medium with 2% fetal bovine serum by volume percentage, and the obtained cells are the low serum suspension culture BMN cells;

[0016] Preferably, the mixed culture medium of insect cell serum-free medium and Grace supplemented insect medium is additionally supplemented with HyPep 1510;

[0017] Preferably, the volume percentage of HyPep 1510 is 0.5%;

[0018] Preferably, the BMN cell adhesion domestication and the suspension domestication fetal bovine serum volume percentage threshold is 5%, that is, when the fetal bovine serum volume percentage in the mixed culture medium is less than 5%, the BMN cells cultured by adhesion are transferred to suspension culture;

[0019] Preferably, the fetal bovine serum volume percentage gradually reduced gradient is 10%, 7.5%, 5%, 3%, and 2%.

[0020] Further, the preparation of the low serum suspension cultured BMN cell adherent cells comprises: adding the low serum suspension cultured BMN cells into the Grace supplemented insect culture medium containing 10% fetal bovine serum for adherent culture, so as to obtain the BMN adherent cells in the logarithmic growth phase, 75%-85% fusion and uniform distribution, and good state, that is, the low serum suspension cultured BMN cell adherent cells.

[0021] Further, the promoter driving the expression of the recombinant adeno-associated virus Cap gene and Rep gene is derived from AcMNPV.

[0022] Preferably, the promoter driving the expression of the Cap gene is Pp10.

[0023] Preferably, the promoter driving the expression of the Rep gene is Pph.

[0024] Preferably, the recombinant adeno-associated virus core expression element ITR-GOI and Cap gene expression frame are located in the same non-essential gene locus of the Bombyx mori nucleopolyhedrovirus genome.

[0025] Further, the single BmNPV-rAAV recombinant bacmid is prepared according to the reference patent "a recombinant bacmid preparation system and BmNPV-rAAV recombinant bacmid, patent application number: 202311779100.4".

[0026] The present application also provides a single recombinant Bombyx mori nucleopolyhedrovirus prepared by the above preparation method.

[0027] The present application also provides a recombinant adeno-associated virus preparation system, which comprises: a) the low serum suspension cultured BMN cells and / or the low serum suspension cultured BMN cell adherent cells, b) the recombinant Bombyx mori nucleopolyhedrovirus, which can infect host cells or hosts to produce at least the functional proteins required for the packaging of the recombinant adeno-associated virus.

[0028] Further, the recombinant Bombyx mori nucleopolyhedrovirus is the single recombinant Bombyx mori nucleopolyhedrovirus prepared by the above preparation method.

[0029] The application also provides a preparation method of the recombinant adeno-associated virus, and the preparation method comprises the following steps: infecting the low-serum suspension culture BMN cells with the recombinant Bombyx mori nucleopolyhedrovirus, or infecting adherent culture cells of the low-serum suspension culture BMN cells with the recombinant Bombyx mori nucleopolyhedrovirus, and then collecting culture supernatant and cell precipitate respectively after the infection is completed, and obtaining the recombinant adeno-associated virus by lysing and purifying the cell precipitate.

[0030] Further, the cell precipitate is purified by iodixanol density gradient centrifugation.

[0031] Preferably, the MOI of the recombinant Bombyx mori nucleopolyhedrovirus is 3-5.

[0032] Preferably, the infection time is 72 hours.

[0033] Further, the preparation method of the recombinant adeno-associated virus comprises the following steps:

[0034] 1) The BMN cells introduced from the American Type Culture Collection (ATCC) are first domesticated in Grace supplemented insect medium with a fetal bovine serum volume percentage of 10%, then adherent domesticated in extra HyPep1510 mixed medium composed of insect cell serum-free medium and Grace supplemented insect medium with a fetal bovine serum volume percentage of 7.5% and 5%, and then suspended domesticated in extra HyPep1510 mixed medium composed of insect cell serum-free medium and Grace supplemented insect medium with a fetal bovine serum volume percentage of 3% and 2%, so as to obtain low-serum suspension culture BMN cells;

[0035] 2) The low-serum suspension culture BMN cells are adherent cultured in Grace supplemented insect medium with a fetal bovine serum volume percentage of 10%, so as to prepare adherent culture cells of low-serum suspension culture BMN cells in the logarithmic growth phase, with 75%-85% fusion and uniform adherent distribution, and in good condition;

[0036] 3) The single BmNPV recombinant bacmid containing the functional protein expression frame and ITR-GOI core expression element required for producing the recombinant adeno-associated virus is transfected into the adherent culture cells of the low-serum suspension culture BMN cells, so as to rescue P1 generation rBmBEV-rAAV, and the prepared P1 generation rBmBEV-rAAV is used to infect the low-serum suspension culture BMN cells, or the adherent culture BMN cells in step 2) are further amplified, so as to obtain progeny rBmBEV-rAAV.

[0037] 4) Infection of low serum suspension-cultured BMN cells or low serum suspension-cultured BMN cells adherent-cultured cells with P1 generation rBmBEV-rAAV or sub-generation rBmBEV-rAAV, and collection of culture supernatant (cell supernatant is sub-generation rBmBEV-rAAV, which can be used for further infection of host cells or preparation of rAAV from host) and cell precipitate 72 hours later, lysis of cell precipitate, and isolation and purification to obtain rAAV vector.

[0038] Compared with the prior art, the present application has the following beneficial effects:

[0039] (1) The present application provides a recombinant adeno-associated virus preparation system and preparation method, which realizes high-efficiency production of rAAV vector with high titer, low empty shell rate and good infection activity based on a silkworm baculovirus expression system by using a single recombinant silkworm nuclear polyhedrosis virus to infect suspension-cultured or suspension-to-adherent-cultured BMN cells.

[0040] (2) The present application provides a single recombinant silkworm nuclear polyhedrosis virus and a preparation method thereof, which rescues P1 generation rBmBEV-rAAV containing a recombinant adeno-associated virus core expression element ITR-GOI and a functional protein expression frame required for production of the recombinant adeno-associated virus in the genome by using a single BmNPV-rAAV recombinant bacmid to transfect adherent-cultured BMN cells, and the P1 generation rBmBEV-rAAV can further infect suspension-cultured or suspension-to-adherent-cultured BMN cells to obtain a larger amount of sub-generation rBmBEV-rAAV, which gets rid of the way of plaque screening and purification of rBmBEV-rAAV based on time-consuming and labor-consuming, realizes convenient, low-cost, high-efficiency and large-scale preparation of rBmBEV-rAAV with lower generation, and lays a foundation for establishment and application of a silkworm larva / pupa-rAAV preparation production platform with Chinese characteristics.

[0041] (3) Compared with traditional BMN cells which can only be adherent-cultured, the low serum suspension-cultured BMN cells provided by the present application can be adherent-cultured in Grace supplemented insect medium with a volume percentage of 10% fetal bovine serum, which makes the rescue of P1 generation budded recombinant silkworm nuclear polyhedrosis virus (rBmNPV (Budded virus), rBmBEV), amplification of rBmBEV seed, and production of recombinant adeno-associated virus or expression of exogenous proteins based on a silkworm baculovirus expression system more convenient. BRIEF DESCRIPTION OF DRAWINGS

[0042] FIG. 1 is a schematic diagram of preparation of rAAV based on a shuttle vector silkworm baculovirus (rBmNPV);

[0043] FIG. 2 is suspension domestication of BMN cells and rescue of sub-generation rBmBEV-rAAV;

[0044] Figure 3 is a single rBmNPV infection of BMN cells to produce rAAV and analysis of the titer, empty capsid rate, and infectious activity of the prepared AAV vectors;

[0045] Figure 4 is the expression of AAV Rep protein and capsid proteins VP1 / VP2 / VP3 in traditional adherent, suspension, and adherent-to-suspension BMN cells at different MOI values;

[0046] Figure 5 is a comparison of the rAAV yield and infectious activity of adherent-to-suspension, suspension, and traditional adherent BMN cells. DETAILED DESCRIPTION

[0047] In order to make the objectives, technical solutions, and advantages of the present application clearer, the present application is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are intended to explain the present application and are not intended to limit the present application.

[0048] The content of the fetal bovine serum and the content of HyPep 1510 in the embodiments of the present application both refer to the volume percentage content.

[0049] Example 1: Recombinant adeno-associated virus (rAAV) preparation system and preparation method

[0050] The recombinant adeno-associated virus preparation system provided in this embodiment includes: a) low serum suspension cultured BMN cells and / or adherent cultured cells of low serum suspension cultured BMN cells, and b) a single recombinant Bombyx mori nuclear polyhedrosis virus, which infects host cells or at least produces functional proteins required for packaging of the recombinant adeno-associated virus. The low serum suspension cultured BMN cells are BMN cells that are cultured in suspension in a culture medium containing 2% fetal bovine serum. The adherent cultured cells of low serum suspension cultured BMN cells are low serum suspension cultured BMN cells that grow adherently in Grace's insect culture medium supplemented with 10% fetal bovine serum.

[0051] The recombinant adeno-associated virus preparation method provided in this embodiment uses the above-described recombinant adeno-associated virus preparation system to prepare a recombinant adeno-associated virus, and includes the following steps: infecting low serum suspension cultured BMN cells with a single recombinant Bombyx mori nuclear polyhedrosis virus, or infecting adherent cultured cells of low serum suspension cultured BMN cells with a single recombinant Bombyx mori nuclear polyhedrosis virus, and collecting the culture supernatant and the cell precipitate after the infection is completed, lysing the cell precipitate, and obtaining the recombinant adeno-associated virus through separation and purification.

[0052] The recombinant adeno-associated virus preparation system and the preparation method are described in detail below.

[0053] 1. Recombinant adeno-associated virus preparation system

[0054] (1) Preparation of low serum suspension-cultured BMN cells

[0055] BMN cells (CRL-8910) were purchased from American Type Culture Collection ATCC. BMN cells were first acclimated to adhere growth in Grace's Insect Medium (Thermo, 11605102) supplemented with 10% fetal bovine serum (FBS, Gibco, 12657-029). After the cells were stable, they were then acclimated to adhere growth in Grace's Insect Medium supplemented with 7.5% FBS, 5% FBS, and 2% FBS, respectively. Sf9 insect cells serum-free medium (Shanghai Youtian Biotech Co., Ltd., H832KJ) and Grace's Insect Medium to form a mixed medium containing 0.5% HyPep1510 (referring to Mi Sun et al., 2005, Biotechnol Appl Biochem, vol. 42, Pt 1). Then, the suspension acclimation (27.5°C / 115 rpm, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., ZWY-2102C) was started in a shake flask (125 mL shake flask, Wuxi Naisi Life Science Co., Ltd., 781111), and the BMN cells were acclimated to suspension growth in a mixed medium containing 0.5% HyPep1510 and supplemented with 3% FBS, 2% FBS, and 0% FBS, respectively. Sf9 insect cells serum-free medium (Shanghai Youtian Biotech Co., Ltd., H832KJ) and Grace's Insect Medium to form a mixed medium containing 0.5% HyPep1510 (referring to Mi Sun et al., 2005, Biotechnol Appl Biochem, vol. 42, Pt 1). Then, the suspension acclimation (27.5°C / 115 rpm, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., ZWY-2102C) was started in a shake flask (125 mL shake flask, Wuxi Naisi Life Science Co., Ltd., 781111), and the BMN cells were acclimated to suspension growth in a mixed medium containing 0.5% HyPep1510 and supplemented with 3% FBS, 2% FBS, and 0% FBS, respectively.

[0056] To determine the division and growth of the low serum suspension-cultured BMN cells obtained above, the acclimated BMN cells were inoculated in a shake flask at a density of 5 x 10 5 cells / mL for suspension culture, and the cells were counted continuously for 10 days, as shown in FIG. 2b. The results showed that the suspension-cultured BMN cells had the maximum growth rate on the 4th day of culture, and the cell density was the highest on the 7th day of culture, close to 3 x 10 6 cells / mL, and the doubling time was about 48 hours.

[0057] (2) Preparation of adherent-cultured cells of low serum suspension-cultured BMN cells

[0058] Based on the fact that adherent-cultured insect cells are an effective method for rescuing recombinant baculovirus, it is still necessary to further verify whether the acclimated low serum suspension-cultured BMN cells can be switched to adherent culture. Therefore, the low serum suspension-cultured BMN cells and the traditional adherent-cultured BMN cells were added to Grace's Insect Medium containing 10% fetal bovine serum at a density of 1 x 10 5The difference between the two was compared by counting the cells every day for 7 days. The results are shown in Figure 2c. The doubling time of the two different types of adherent cultured BMN cells was similar, about 36 hours. The growth of the suspension-adherent cultured BMN cells was similar to that of the traditional adherent cultured BMN cells on the second day before culture. The growth rate of the traditional adherent cultured BMN cells was the fastest on the third day, and the cell density was slightly higher than that of the suspension-adherent cultured BMN cells. Although the growth rate of the suspension-adherent cultured BMN cells was the fastest on the fourth day, the cell density was still lower than that of the traditional adherent cultured BMN cells. On the fifth, sixth and seventh days, the growth rate of the two different types of adherent cultured BMN cells was slow. This indicates that the low serum suspension cultured BMN cells can grow adherently in Grace insect culture medium supplemented with 10% fetal bovine serum, and the growth characteristics of the cells are similar to those of the traditional adherent cultured BMN cells. The suspension-adherent cultured BMN cells in the logarithmic growth phase, 75%-85% confluence and uniform distribution, and in good condition are the adherent cultured cells of the low serum suspension cultured BMN cells.

[0059] (3) Preparation of single recombinant Bombyx mori nucleopolyhedrovirus

[0060] 1) Preparation of single BmNPV-rAAV recombinant bacmid

[0061] To determine the efficiency of the suspension-adherent cultured BMN cells in rescuing rBmBEV, the recombinant bacmid Bm3.3-rAAV2-CMV-eGFP-hGH in Example 3 of the invention patent "A recombinant bacmid preparation system and BmNPV-rAAV recombinant bacmid, application number: CN202311779100.4" was amplified, and Bm3.3-rAAV9-CMV-eGFP-hGH was constructed by reference.

[0062] 2) Rescue of P1 generation rBmBEV-rAAV

[0063] The low serum suspension cultured BMN cells and the traditional adherent cultured BMN cells were inoculated at 1×10 6The cells were inoculated in 6-well plates for adherent culture. When the cell confluence was 75%-85%, 2 μg of recombinant bacmids Bm3.3-rAAV2-CMV-eGFP-hGH and Bm3.3-rAAV9-CMV-eGFP-hGH were transfected into the cells according to the instructions of the Mirus transfection kit (TransIT-Insect Transfection Reagent, MRI6100). No liquid change was performed in the middle. After 3 days of transfection, the supernatant was collected by centrifugation at 2500 rpm for 10 min. An appropriate amount of supernatant was treated at room temperature for 15 min, and then qPCR was performed to determine the BEV titer according to the method in "Alkaline polyethylene glycol-based method for direct PCR from bacteria, eukaryotic tissue samples, and whole blood" (homczynski P et al., Biotechniques. 2006 Apr; 40(4): 454, 456, 458.). The P1 generation rBmBEV-rAAV rescued by transfection of Bm3.3-rAAV2-CMV-eGFP-hGH was recorded as BEV / Bm3.3-rAAV2, and the P1 generation rBmBEV-rAAV rescued by transfection of Bm3.3-rAAV9-CMV-eGFP-hGH was recorded as BEV / Bm3.3-rAAV9.

[0064] The results showed that, whether the BMN cells were suspended and adherent cultured or traditionally adherent cultured, the cells were all green fluorescent protein after transfection of the recombinant bacmids, as shown in FIG. 2d. The fluorescence protein expression abundance of the BMN cells transfected with Bm3.3-rAAV9-CMV-eGFP-hGH was stronger than that of the BMN cells transfected with Bm3.3-rAAV2-CMV-eGFP-hGH. The fluorescence brightness of the suspended and adherent cultured BMN cells was slightly higher than that of the traditionally adherent cultured BMN cells. The rBmBEV titer in the supernatant of the suspended and adherent cultured BMN cells was significantly higher than that of the rBmBEV rescued by the traditionally adherent cultured BMN cells (FIG. 2e). This indicated that the suspended and adherent cultured BMN cells could be used for efficient rescue of rBmBEV.

[0065] 3) Expansion of rBmBEV

[0066] Step 3) is an optional step. The prepared P1 generation rBmBEV-rAAV is used to infect low-serum suspended cultured BMN cells, or the adherent cultured cells of the low-serum suspended cultured BMN cells are further expanded to obtain the progeny rBmBEV-rAAV.

[0067] 2) Preparation method of recombinant adeno-associated virus

[0068] The P1 generation rBmBEV-rAAV or the progeny rBmBEV-rAAV infects the low serum suspension cultured BMN cells or the adherent cultured cells of the low serum suspension cultured BMN cells, and after 72 hours, the culture supernatant (the cell supernatant is the progeny rBmBEV-rAAV, which can be used for further infection of host cells or preparation of rAAV from the host) and the cell precipitate are collected respectively, the cell precipitate is lysed and then purified to obtain the recombinant adeno-associated virus (rAAV).

[0069] In order to verify whether the domesticated low serum suspension cultured BMN cells can be used for the production of rAAV, the traditional adherent BMN cells are inoculated at 1 x 10 6 cells per hole in a 6-hole culture plate, and 4 mL of BMN suspension cells are cultured at 1 x 10 6 cells / mL in a 50 mL centrifuge tube, and then the P1 generation BEV / Bm3.3-AAV2 is used to infect the BMN cells in the above different culture modes at an MOI of 10 for comparison. The results show that both types of BMN cells are lit by green fluorescent protein after 3 days of infection (Figure 3a), and the titer of P2 generation BEV / Bm3.3-AAV2 in the supernatant of the suspension cultured BMN cells is higher than that of the traditional adherent cultured BMN cells (Figure 3b). The expression of AAV Rep protein and capsid proteins VP1 / 2 / 3 in the cell precipitate (2500 rpm centrifugation of 1 hole of traditional adherent BMN cells without infection, 1 hole of traditional adherent BMN cells infected with rBmBEV, and 1 mL of suspension BMN cells infected with rBmBEV) is quantitatively analyzed by Western Blot, and GAPDH is used as an internal reference protein, as shown in Figure 3c, all the functional proteins required for AAV production are detected in the BMN cells infected with BEV / Bm3.3-AAV2, in which the expression amount of Rep52 is higher than that of Rep78, and the expression abundance of capsid protein VP2 is lower; compared with the traditional adherent BMN cells, the expression abundance of Rep52 in the suspension cultured BMN cells is higher, the expression abundance of Rep78 is lower, and the ratio of VP1, VP2, and VP3 is closer to 1:1:10. Therefore, it is more conducive to the production of rAAV to infect the low serum suspension cultured BMN cells with rBmBEV.

[0070] Further, 2 x 10 6BMN cells were seeded at a density of 1 x 105cells / mL in 50 mL of BMN suspension cells, and P1 generation BEV / Bm3.3-AAV2 was added to the flask at an MOI of 10 for 3 days of infection. The BMN cell pellet was collected and purified by iodixanol density gradient centrifugation to obtain rAAV2-CMV-eGFP-hGH. The same method as described above (P1 generation BEV / Bm3.3-AAV9 infection of BMN suspension cells) was used to produce rAAV9-CMV-eGFP-hGH. AAV9 produced by 293T cells was used as a positive control, and the above-mentioned AAV from BMN suspension cells was quantitatively detected by silver staining and qPCR. The results showed that whether AAV2 or AAV9 produced by BMN suspension cells, their capsid proteins VP1, VP2, and VP3 could be detected and the ratio was close to 1:1:10 (Figure 3d, e), and the silver staining results were consistent with the AAV titer determined by qPCR (Figure 3f). In addition, transmission electron microscopy (TEM, Southern University of Science and Technology Public Analysis Testing Center) was used to image the AAV prepared based on BMN suspension cells, and it was observed that the viral particles of AAV2 (Figure 3g) and AAV9 (Figure 3h) were spherical in shape with a diameter of 20-25 nm.

[0071] To test the infection activity of BMN suspension cell-derived AAV, in vivo and in vitro experiments were carried out, including 1) 3 μL of AAV was added to a 96-well plate seeded with 293T cells; 2) 300 nL of AAV was injected into the mouse vlPAG brain area using a brain stereotactic injection system (Figure 3k). The results showed that after 48 hours of infection, 293T cells were illuminated by green fluorescent protein (Figure 3i, j); although the fluorescence expression abundance of 293T cells infected with AAV2 (BMN) was much higher than that of 293T cells infected with AAV9 (BMN), the infection efficiency of these two AAVs (BMN) on mouse vlPAG brain cells was similar (Figure 3k, l), except that the inflammation induced by injection of AAV9 (BMN) in the vlPAG area was more obvious. This indicates that the use of rBmBEV to infect suspension-cultured BMN cells can produce rAAV with high titer and infection activity. In summary, the present application successfully established a BMN cell / rBmNPV-AAV preparation system and preparation method, and Figure 1 is a schematic diagram of rAAV preparation based on a shuttle vector of Bombyx mori baculovirus (rBmNPV).

[0072] Example 2: MOI value of rBmNPV infection of BMN cells for rAAV preparation

[0073] The multiplicity of infection (MOI) of rBmNPV is a key parameter to determine the yield of AAV and the yield of rBmBEV progeny, and therefore it is necessary to determine the optimal MOI value of the BMN cell / rBmNPV-AAV preparation system in Example 1 of the present application.

[0074] The conventional adherent BMN cells and the suspension BMN cells were inoculated in 6-well culture plates at 1 × 10 6 cells / well, respectively, and cultured to 80% confluence (about 1.5 × 10 6 cells / well), and 4 mL of the BMN suspension cells were cultured in a 50 mL centrifuge tube at 1 × 10 6 After the P1 generation of BEV / Bm3.3-AAV2 and BEV / Bm3.3-AAV9 was determined by qPCR, the above three kinds of BMN cells were infected at MOI = 0, 0.5, 1, 3, 5, and 10, respectively, and the cell precipitates were collected after 3 days (1 mL of the cell precipitate was collected for the suspension cultured BMN cells), and the expression of the AAV Rep protein and the capsid proteins VP1 / 2 / 3 in the BMN cells at different MOI values was detected by Western Blot. As shown in FIG. 4, when the BMN cells were infected with BEV / Bm3.3-AAV2, the expression abundance of the AAV Rep protein and the capsid proteins VP1 / 2 / 3 in the conventional adherent and suspension cultured BMN cells was the highest at MOI = 10, and the expression abundance of the AAV Rep protein and the capsid proteins VP1 / 2 / 3 in the suspension to adherent cultured BMN cells was close at different MOI values of 3-10 (FIG. 4a); when the BMN cells were infected with BEV / Bm3.3-AAV9, the expression abundance of the AAV Rep protein and the capsid proteins VP1 / 2 / 3 in the suspension and suspension to adherent cultured BMN cells was close at different MOI values of 3-10, and the expression abundance of the AAV Rep protein and the capsid proteins VP1 / 2 / 3 in the conventional adherent BMN cells at MOI = 5 was close to that at MOI = 10 (FIG. 4b). Regardless of whether the BMN cells were infected with BEV / Bm3.3-AAV2 or BEV / Bm3.3-AAV9, the suspension cultured BMN cells could detect the functional proteins required for AAV production at different MOI values, and the expression abundance of the AAV Rep protein and the capsid proteins VP1 / 2 / 3 was not significantly different at different MOI values, which indicated that the suspension cultured BMN cells were more beneficial to the production of AAV. Considering the dosage of BEV / Bm3.3-AAV and the expression abundance of the AAV Rep protein and the capsid proteins VP1 / 2 / 3 in the corresponding BMN cells, it was concluded that the optimal MOI value for preparing rAAV by infecting the BMN cells with rBmNPV was 3-5.

[0075] Further, the AAV2 and AAV9 yields of suspension-adherent cells (s-a), suspension cells (s), and traditional adherent cells (a) BMN cells were analyzed at MOI = 3. The same number of s-a, s (2 x 105cells / mL, 50 mL), and a BMN cells (1 x 105) were infected with P1 BEV / Bm3.3-AAV2 and BEV / Bm3.3-AAV9 at MOI = 3, respectively. After 3 days, it was observed that the above-mentioned BMN cells were labeled with eGFP, and the s-a cells had the highest fluorescence protein expression (Figure 5a). The cell precipitates were collected, and rAAV2-CMV-eGFP-hGH (AAV2) and rAAV9-CMV-eGFP-hGH (AAV9) were obtained by purification using iodixanol density gradient centrifugation, and silver staining and qPCR detection (293T cell-produced AAV9 as a positive control) were performed, as shown in Figures 5b and 5c. Different culture methods of BMN cells can be used to produce AAV2 and AAV9, and the titer of AAV9 is generally higher than that of AAV2. Suspension culture of BMN cells is more conducive to the production of high-titer AAV. The results of 293T cell infection showed that the eGFP expression intensity of 293T cells infected with AAV2 was generally higher than that of 293T cells infected with AAV9, and the eGFP expression intensity of 293T cells infected with AAV2 (s) was the highest. Compared with traditional adherent culture of BMN cells, the AAV produced by domesticated suspension BMN cells, whether adherent (s-a) or suspension (s), had good infectivity to 293T cells (Figures 5d and 5e). 6 8 Further, the AAV2 and AAV9 yields of suspension-adherent cells (s-a), suspension cells (s), and traditional adherent cells (a) BMN cells were analyzed at MOI = 3. The same number of s-a, s (2 x 105cells / mL, 50 mL), and a BMN cells (1 x 105) were infected with P1 BEV / Bm3.3-AAV2 and BEV / Bm3.3-AAV9 at MOI = 3, respectively. After 3 days, it was observed that the above-mentioned BMN cells were labeled with eGFP, and the s-a cells had the highest fluorescence protein expression (Figure 5a). The cell precipitates were collected, and rAAV2-CMV-eGFP-hGH (AAV2) and rAAV9-CMV-eGFP-hGH (AAV9) were obtained by purification using iodixanol density gradient centrifugation, and silver staining and qPCR detection (293T cell-produced AAV9 as a positive control) were performed, as shown in Figures 5b and 5c. Different culture methods of BMN cells can be used to produce AAV2 and AAV9, and the titer of AAV9 is generally higher than that of AAV2. Suspension culture of BMN cells is more conducive to the production of high-titer AAV. The results of 293T cell infection showed that the eGFP expression intensity of 293T cells infected with AAV2 was generally higher than that of 293T cells infected with AAV9, and the eGFP expression intensity of 293T cells infected with AAV2 (s) was the highest. Compared with traditional adherent culture of BMN cells, the AAV produced by domesticated suspension BMN cells, whether adherent (s-a) or suspension (s), had good infectivity to 293T cells (Figures 5d and 5e).

[0076] It is obvious that the above embodiments are only examples for the purpose of clarity, and are not intended to limit the embodiments. Based on the above description, those skilled in the art can make other different forms of changes or modifications. It is not necessary or possible to exhaust all embodiments. The obvious changes or modifications derived therefrom are still within the protection scope of the present application.​

Claims

1. A method for preparing a single recombinant Bombyx mori nucleopolyhedrovirus, characterized by, The preparation method is selected from one of methods 1-3: Method 1: single BmNPV-rAAV recombinant bacmid is transfected into adherent culture cells of low serum suspension cultured BMN cells to rescue the first generation of rBmBEV-rAAV, and the first generation of rBmBEV-rAAV is denoted as P1 generation rBmBEV-rAAV, which is a single recombinant Bombyx mori nucleopolyhedrovirus; Method 2: single BmNPV-rAAV recombinant bacmid is transfected into adherent culture cells of low serum suspension cultured BMN cells to rescue P1 generation rBmBEV-rAAV; P1 generation rBmBEV-rAAV is used to infect adherent culture cells of low serum suspension cultured BMN cells to obtain the progeny rBmBEV-rAAV, which is a single recombinant Bombyx mori nucleopolyhedrovirus; Method 3: single BmNPV-rAAV recombinant bacmid is transfected into adherent culture cells of low serum suspension cultured BMN cells to rescue P1 generation rBmBEV-rAAV; P1 generation rBmBEV-rAAV is used to infect low serum suspension cultured BMN cells to obtain the progeny rBmBEV-rAAV, which is a single recombinant Bombyx mori nucleopolyhedrovirus; The single BmNPV-rAAV recombinant bacmid comprises a recombinant adeno-associated virus core expression element ITR-GOI, a Rep gene expression frame and a Cap gene expression frame.

2. The production method according to claim 1, characterized by, The low serum suspension cultured BMN cells are BMN cells suspended in a culture medium containing 2% fetal bovine serum by volume percentage; And / or, the adherent culture cells of the low serum suspension cultured BMN cells are low serum suspension cultured BMN cells adhered and grown in Grace supplemented insect medium containing 10% fetal bovine serum by volume percentage.

3. The preparation method according to claim 2, characterized in that, The preparation of the low serum suspension cultured BMN cells comprises: the BMN cells are first acclimated to adhere and grow in Grace supplemented insect medium containing 10% fetal bovine serum by volume percentage, then acclimated to adhere in a mixed culture medium composed of insect cell serum-free medium and Grace supplemented insect medium with gradually reduced fetal bovine serum content, and then acclimated to suspend in the mixed culture medium composed of insect cell serum-free medium and Grace supplemented insect medium with gradually reduced fetal bovine serum content, and finally the BMN cells are stably in the mixed culture medium composed of insect cell serum-free medium and Grace supplemented insect medium with 2% fetal bovine serum by volume percentage, and the obtained cells are the low serum suspension cultured BMN cells.

4. The production method according to claim 3, characterized by, The mixed culture medium of the insect cell serum-free medium and Grace supplemented insect medium is additionally added with HyPep 1510, and the volume percentage of the HyPep 1510 is 0.5%.

5. The preparation method according to claim 3, characterized in that, The threshold value of the volume percentage of fetal bovine serum for adherent acclimation and suspension acclimation of the BMN cells is 5%, that is, when the volume percentage of fetal bovine serum in the mixed culture medium is lower than 5%, the adherently cultured BMN cells are transferred to suspension culture.

6. The preparation method according to claim 3, characterized in that, The gradient of the gradually reduced volume percentage of fetal bovine serum is 10%, 7.5%, 5%, 3% and 2%.

7. The preparation method according to claim 2, characterized in that, The preparation of the adherent culture cells of the low serum suspension culture BMN cells comprises: adding the low serum suspension culture BMN cells into Grace supplemented insect medium containing 10% fetal bovine serum by volume percentage for adherent culture, to obtain BMN adherent cells in logarithmic growth phase, 75%-85% fusion and uniform distribution, and good state, i.e. the adherent culture cells of the low serum suspension culture BMN cells.

8. The method of claim 1, wherein, The promoter driving the expression of the recombinant adeno-associated virus Cap gene and Rep gene is derived from AcMNPV.

9. The method of claim 1, wherein, The promoter driving the expression of the Cap gene is Pp10. The promoter driving the expression of the Rep gene is Pph.

10. The method of claim 1, wherein, The recombinant adeno-associated virus core expression element ITR-GOI and Cap gene expression frame are located in the same non-essential gene locus of the Bombyx mori nucleopolyhedrovirus genome.

11. The single recombinant Bombyx mori nucleopolyhedrovirus prepared by the preparation method of claim 1.

12. A recombinant adeno-associated virus production system, characterized in that, The recombinant adeno-associated virus preparation system comprises: a) the low serum suspension culture BMN cells of claim 1 and / or the adherent culture cells of the low serum suspension culture BMN cells of claim 1, b) the recombinant Bombyx mori nucleopolyhedrovirus, which infects host cells or hosts to produce at least the functional proteins required for the packaging of the recombinant adeno-associated virus.

13. The recombinant adeno-associated virus production system of claim 12, wherein, The recombinant Bombyx mori nucleopolyhedrovirus is the single recombinant Bombyx mori nucleopolyhedrovirus of claim 11.

14. A method for preparing recombinant adeno-associated virus, characterized in that, The preparation method of the recombinant adeno-associated virus using the preparation system of claim 12 comprises the following steps: infecting the low serum suspension culture BMN cells of claim 1 with the recombinant Bombyx mori nucleopolyhedrovirus, or infecting the adherent culture cells of the low serum suspension culture BMN cells of claim 1 with the recombinant Bombyx mori nucleopolyhedrovirus, and collecting the culture supernatant and the cell precipitate respectively after the infection, lysing the cell precipitate to obtain the recombinant adeno-associated virus through separation and purification.

15. The method for preparing recombinant adeno-associated virus according to claim 14, characterized in that, The cell precipitate is purified by iodixanol density gradient centrifugation.

16. The method for preparing recombinant adeno-associated virus according to claim 14, characterized in that, The MOI of the recombinant Bombyx mori nucleopolyhedrovirus is 3-5.

17. The method for preparing recombinant adeno-associated virus according to claim 14, characterized in that, The infection time is 72 hours.

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