CN1252441A - Recombined-adenovirus accompanying virus production method and application - Google Patents

Abstract

The invention proposes a method of "one vector cell/one helper virus" to produce recombinant AAV virus. The vector cell strain is a stably passaged cell strain integrated with a rescued recombinant AAV DNA obtained by introducing a recombinant AAV vector plasmid containing an exogenous gene expression unit into mammalian cells and obtained through resistance screening. The helper virus is a recombinant HSV-1 virus carrying AAVrep/cap gene. A large amount of infectious recombinant AAV virus can be produced by infecting the carrier cell strain with this full-function helper virus. The invention can be used for large-scale and high-efficiency production of recombinant AAV virus.

Description

Production method and application of recombinant adenovirus-associated virus applicable to large-scale production Production method and application of adenovirus-associated virus applicable to large-scale production

The invention belongs to the field of biotechnology inventions, and relates to the production method of recombinant adenovirus-associated virus and the use of the obtained product, especially the use in the fields of gene therapy and transgenic animals. The present invention is a continuation of Chinese patents 98101753.3 (application number) and 98120033.8 (application number).

Gene delivery system is the core technology of gene therapy research and application, including viral vector system and non-viral vector system. Viral vectors mainly include retroviral vectors, adenoviral vectors, adenoviral-associated viral vectors, herpes simplex viral vectors, and the like. Among them, the adeno-associated virus (AAV) vector has attracted attention because of its safety, stability, ability to infect both dividing cells and non-dividing cells, high infection efficiency, and long-term expression of foreign genes.

AAV virus is a non-pathogenic microvirus with a genome of 4680nt single-stranded DNA. The life cycle of AAV virus has two modes: latent infection and lytic infection. The lytic replication of AAV virus requires the participation of helper virus such as adenovirus or herpes simplex virus (HSV). In the absence of a helper virus, the AAV virus is provirally integrated into the host cell chromosome. There are 145nt ITR sequences at the ends of the AAV genome, which are essential cis-acting elements of the AAV virus and play an important role in the rescue, replication, packaging and integration of the AAV virus genome. Between the two ITRs are the trans-acting rep and cap genes, which are essential for the replication of AAV viral DNA and the production of viral particles. The Rep gene encodes four proteins: Rep78, Rep68, Rep52, and Rep40, which are related to the replication, integration, and rescue of AAV viruses. The Cap gene encodes three coat proteins, VP1, VP2, and VP3, which constitute the 20-hedron coat of AAV.

Samulski RJ et al. (Cloning of adeno-associated virus into pBR322: rescue of intact virus from therecombinant plasmamid in human cells, Proc.Natl Acad.Sci USA, 79:2077-2031, 1982) cloned the complete double-stranded AAV DNA into pBR322 In plasmids, the AAV proviral genome located in the plasmid was also found to be infectious. Therefore, the exogenous gene expression unit is placed between the two ITRs of the AAV virus, and the functions of the rep and cap genes and the helper virus are provided in trans by other means, and the recombinant adenovirus containing the exogenous gene can be obtained in the cell. Virus (recombinantadeno-associated virus, rAAV).

The classic rAAV production method is co-transfection of 293 cells with double plasmids and infection with a helper virus such as adenovirus type 5 (Ad5). One of the double plasmids is a recombinant AAV vector plasmid, and the other is an auxiliary plasmid containing the AAV rep/cap gene. Because this method is complicated to operate, there are many factors that affect the production of rAAV, it is difficult to obtain high-titer rAAV, and it is not easy to expand the production scale. Therefore, many researchers are committed to improving the production method of rAAV. These improvements include the following categories: 1) The rep/cap gene is transduced into the cell line to build a packaging cell line. The expression of rep/cap is controlled by its own promoter, or replaced by other constitutive or inducible promoters. 2) Insert AAV ITRs and the foreign gene expression unit DNA between them into the adenovirus genome to construct a chimeric recombinant adenovirus. 3) AAV ITRs and the exogenous gene expression unit DNA between them are placed in the self-replicating EBV virus vector, transduced into cells, and a cell line carrying a recombinant AAV vector plasmid with self-replicating ability is established. 4) inserting the rep/cap gene into the adenovirus genome to construct a recombinant adenovirus with complete helper functions. However, many experiments have proved that this idea is difficult to realize. It may be that the recombinant adenovirus containing rep/cap gene cannot be produced due to the strong inhibitory effect of rep protein on adenovirus. 5) HSV viral amplicons are used to carry rep/cap genes to produce HSV mixed viruses with complete helper functions.

Although various improvements in rAAV production methods have improved the yield of rAAV viruses to varying degrees or simplified their preparation methods, these methods still have not solved the problem of large-scale and efficient production of rAAV viruses. At present, the cost (including time cost and capital cost) of preparing enough rAAV virus for human trials is still extremely high. Therefore, it is necessary to invent a method for large-scale and high-efficiency production of rAAV virus.

The purpose of the present invention is to propose a production method of a high-efficiency recombinant adeno-associated virus (rAAV) vector that can be used for large-scale preparation, that is, a production method of "one vector cell/one helper virus". The method has the characteristics of high yield, simple and convenient operation, and easy large-scale and technological production. The invention can be used for mass production of recombinant AAV virus, and the produced recombinant AAV virus can be used for gene therapy of various diseases.

The traditional method of preparing rAAV virus is to co-transfect 293 cells with double plasmids and infect the cells with a helper virus (adenovirus type 5). The dual plasmids include recombinant AAV vector plasmids such as pAB11 and auxiliary plasmids containing AAV rep/cap genes such as pAd8. Because this method needs to transfect cells, it has the disadvantages of low or unstable transfection efficiency, complicated operation, and difficulty in scale-up and process. Although after improvement, the transfection efficiency of 293 cells can be reached or close to 100% with the calcium phosphate co-precipitation method, the transfection method is difficult to be used for a large number of cells and the requirements for the 293 cells themselves (generations below 50 generations, cells It is flat and has a doubling time of 36hr to 48hr) which limits the use of this method to obtain large quantities of rAAV virus.

In view of the disadvantages of current rAAV production, such as many influencing factors, complex operation, unfavorable large-scale production, and difficulty in obtaining high-yield rAAV viruses, the present invention proposes a production strategy of "one carrier cell/one helper virus": use The recombinant HSV-1 virus (HSV1-rc) carrying the AAV virus rep/cap gene infects the production cell line stably integrated with the recombinant AAV vector DNA, and a large amount of rAAV virus can be obtained from the diseased cells. The HSV1-rc virus can simultaneously provide the helper virus HSV-1 necessary for rAAV packaging and the trans-acting protein Rep and coat protein. These two functions are usually produced by wild-type helper viruses such as Ad5 or HSV-1 and contain rep/cap genes A helper plasmid such as pAd8 is provided. The recombinant AAV genomic DNA (single strand) packaged into the recombinant AAV virus particle is derived from the recombinant AAV vector cell line. One or more copies of the recombinant AAV vector DNA are stably integrated in the genome of the recombinant AAV vector cell, which contains two ITR sequences of the AAV virus and a therapeutic gene expression unit located therebetween. The therapeutic gene expression unit consists of a eukaryotic expression promoter (such as HCMV IE promoter, SV40 promoter, β globin gene promoter, inducible promoter, various tissue-specific promoters, etc.), therapeutic gene, mRNA tailing signal Composition, the length does not exceed 5.0kb.

When the rHSV-rc virus infects the carrier cell strain, the DNA of the HSV1-rc virus enters the cell and its DNA replicates in large quantities and finally produces progeny viruses. The rep/cap gene carried during HSV1-rc viral DNA replication is also replicated synchronously, thereby producing a high copy of the rep/cap gene. The rep gene encodes four Rep proteins (Rep78, Rep68, Rep52, Rep40), which rescue the recombinant AAV vector DNA from the cell genome and replicate in large quantities, and finally become a single strand and be packaged into the AAV capsomere; the cap gene encodes three kinds of shells Proteins VP1, VP2, and VP3 assemble into capsomeres in the nucleus.

The process of producing rAAV virus in the present invention is very similar to the process of lytic infection and propagation of wild-type AAV virus when HSV-1 is the helper virus, the difference is that the ITRs of the wild-type AAV virus genome are rep/cap Gene, the helper virus is wild-type HSV-1; and the present invention inserts the rep/cap gene into the genome of the helper virus HSV-1 to obtain recombinant HSV-1 virus rHSV1-rc with complete helper functions; AAV ITRs and The therapeutic gene expression unit is provided by the carrier cell strain. Using rHSV1-rc to infect the monoclonal cell line with high rAAV virus yield screened from the carrier cell clone, under certain conditions, the rAAV virus production efficiency similar to that of the wild-type HSV-1 assisted wild-type AAV production efficiency can be obtained. When BHK cells are used as production cells, the rate of rAAV virus production can reach 10 ^{4 to 5} particles rAAV/cell.

The mass production of rAAV virus is carried out in 4 steps: a) mass production of HSV1-rc virus. Because the virus has the ability to replicate, it is easy to be cultured in large quantities on BHK cells or other sensitive cells, and the infected cells will have obvious lesions, and there are a large amount of infectious HSV1-rc viruses in the supernatant and cells. The titer of HSV1-rc virus in the supernatant can reach 10 ⁷ pfu/ml; the cells can be lysed by freeze-thaw method to obtain 10 ⁸ pfu/ml HSV1-rc virus. The obtained virus liquid can be used in the next step after low-speed centrifugation to remove cell debris; b) mass culture of vector cell strains. Can adopt spinner bottle culture, cell fermenter culture and other high-density cell culture equipment and methods; c) use HSV1-rc to infect a large number of cultured carrier cell strains. After the cells are completely damaged, the cells are lysed to obtain a cell lysate, which contains a large amount of rAAV virus. d) Separation and purification of rAAV virus.

The invention relates to preparation of recombinant HSV-1 virus HSV1-rc carrying AAV virus rep/cap gene and establishment of production cell strain carrying recombinant AAV vector. The preparation method and application of HSV1-rc virus have been discussed in Chinese patent 98120033.8 (application number). The production of HSV1-rc is a set of cosmids (Set C cosmids, including cos6, cos14, cos28, cos48, cos56) inserted into 5 large fragments (the end sequences of which have partial overlaps in turn) of the whole genome of HSV1 virus implemented on an operational basis. First, use in vitro recombinant DNA technology to insert the rep/cap gene of AAV-2 virus into the HSV-1 genome of one of the cosmids, for example, insert it into the HSV1 UL2 gene of cos6 to construct cos6-rcΔUL2; insert it into the HSV1 UL44 gene of cos56 Constructed into cos56-rcΔUL44. The rep/cap genes can also be inserted elsewhere in the HSV-1 genome. The above process was carried out in Escherichia coli. Digest the recombinant cosmid DNA with rep/cap inserted and the corresponding remaining 4 cosmid DNAs with PacI (cut off the cosmid backbone part), extract and purify the digested DNA with phenol/chloroform, and use liposome method or other transfection methods to transfect it into BHK-21 cells or other cells sensitive to HSV1 virus infection, and lesions and plaques can be observed in the cells after 5 to 7 days, indicating that the five HSV1 fragments have the same occurrence in the cells. Source recombination produces recombinant HSV1. A single plaque was picked for detection, including detection of rep gene and cap gene fragments by PCR method, and detection of its function of packaging rAAV. The positive test result is the recombinant HSV1 virus (HSV1-rc) inserted with the rep/cap gene. The obtained HSV1-rc was subjected to two plaque purifications to ensure its purity.

The establishment of the production cell line carrying the recombinant AAV vector, that is, the vector cell line: the plasmid containing the recombinant AAV vector DNA (AAVITRs and the therapeutic gene expression unit between them) is introduced into the cell by liposome method or other transfection methods, and then passed through a new Resistant cell clones were obtained by selection culture of mycin analogue G418. Monoclonal cells were picked and expanded for culture, and stable passage cell lines capable of producing high-titer rAAV virus under HSV1-rc infection were screened out, and a large number of them were frozen for future use. The selection gene neo can be on the recombinant AAV vector plasmid, located between or outside the AAV ITRs on the plasmid backbone; neo can also be on another plasmid such as pSV2neo, and it can be used to co-transfect cells with the recombinant AAV vector plasmid.

In the method of the present invention, BHK-21 cell is a good rAAV production cell line. Other cells such as 293 cells, KB cells, HeLa cells can also be used to produce rAAV.

Various target genes can be carried in the rAAV virus produced by the present invention. These rAAV viruses can be used for gene therapy of genetic diseases, tumors, cardiovascular diseases, and infectious diseases.

The advantages that the present invention has:

The method provided by the invention can be used for large-scale production of rAAV virus. The strategy of "one carrier cell strain/one helper virus" adopted in the present invention overcomes the disadvantages of complex operation, many influencing factors or difficulty in amplifying production in the traditional method and various improved methods, and greatly simplifies the production of rAAV. factors and production process. The vector cell strain and the full-function helper virus HSV1-rc all have the characteristics of stable passage and amplification, which is conducive to production amplification. During the production of rAAV virus, the rep/cap gene of AAV virus can be replicated into a high copy along with the replication of helper virus HSV1-rc, simulating the process of wild-type AAV virus replication, which is conducive to the production of high titer rAAV. The microbial bacterial classification that the present invention relates to:

The present invention relates to the microbial strains used in the preparation of HSV1-rc, including DH5α/cos6-rcΔUL2 and DH5α/cos56-rcΔUL44, which were preserved in the General Microorganism Center of China Microbiological Culture Collection Committee on September 24, 1998, registered The registration numbers are CGMCC No.0361-1 and CGMCC No.0360-2 respectively.

Examples: The following examples describe in detail the production method and application of the adenovirus-associated virus that can be used in large-scale production of the present invention, but are not meant to limit the content of the present invention. Example 1 Construction of recombinant AAV vector plasmid carrying reporter gene GFP 1) Construction of pSNAV-1/GFP

The GFP (Green Fluorescent Protein) gene was derived from pGreen Lantern-1 (GIBCO BRL, #10642-015). The general-purpose AAV vector plasmid pSNAV-1 was constructed by our laboratory (Chinese patent application number:          ].

The pGreen Lantern-1 plasmid DNA was digested with NotI, and the GFP fragment was recovered and inserted into the corresponding site of the pcDNA2.1 plasmid (Invitrogen Company) to construct pcDNA2.1/GFP (+/-). The recombinant plasmid whose transcription direction of GFP is opposite to that of T7 promoter was named pcDNA2.1/GFP(-). Digest pcDNA2.1/GFP(-) with EcoR I and Xho I, recover the GFP fragment, insert it into the EcoRI and SalI sites of pSNAV-1, and construct pSNAV-1/GFP. The plasmid includes the following elements in order: ITR-CMV-GFP-SV40 polyA-ITR-SV40 promoter-neo-polyA-amp ^R -E.coli ori. 2) Construction of pSNAV-2/GFP

The GFP (Green Fluorescent Protein) gene was derived from pGreen Lantern-1 (GIBCO BRL, #10642-015). The general-purpose AAV vector plasmid pSNAV-2 was constructed in our laboratory (Chinese patent application number:          ].

pcDNA2.1/GFP(-) was digested with KpnI and XhoI, the GFP fragment was recovered, inserted into the KpnI and Sal I sites of pSNAV-2, and pSNAV-2/GFP was constructed. The plasmid comprises the following elements in order: ITR-CMV-GFP-SV40 polyA-SV40 promoter-neo-polyA-ITR-amp ^R -E.coli ori.

The main difference between this plasmid and pSNAV-1/GFP is that in pSNAV-1/GFP, the expression unit of neo gene is located within two ITR sequences; while in pSNAV-2/GFP, the expression unit of neo gene is located in outside of the two ITR sequences. Example 2 Establishment of AAV-GFP virus vector cell strain

BHK cells were transfected with pSNAV-1/GFP and pSNAV-2/GFP plasmids respectively, and two strains of AAV-GFP virus vector cell lines BHK/pSNAV-1/GFP and BHK/pSNAV-2/GFP were obtained through selection and culture.

BHK cells were cultured at 37°C with RPMI1640 medium containing 10% fetal bovine serum. The pSNAV-1/GFP and pSNAV-2/GFP plasmids were respectively transfected into BHK cells with lipofectamine (GIBCO BRL), digested after 24 hours, and passaged at a ratio of 2 to 5. Add G418 800μg/ml for selection culture. After 10 days, obviously resistant cell clones can be formed. The cell clones were observed under an inverted fluorescent microscope, and 20 single cell clones with green fluorescence were picked for expansion and culture respectively, and were frozen and preserved. The cloned cells of each strain were infected with the full-function helper virus HSV1-rc, and it was found that the cells of each strain produced recombinant AAV-GFP virus to varying degrees. The titers of the recombinant AAV-GFP virus were measured respectively. The cell line with high titer of recombinant AAV virus was selected as the AAV-GFP virus vector cell line. The genomic DNA (single strand) in the recombinant AAV virus particle packaged by the vector cell strain BHK/pSNAV-1/GFP is about 1600nt in length, consisting of ITRs at both ends and CMV-GFP-SV40 polyA in between. The genomic DNA (single strand) in the recombinant AAV virus particle packaged by the vector cell strain BHK/pSNAV-2/GFP is about 3700nt long, consisting of ITRs at both ends and CMV-intron-GFP-SV40 polyA-SV40 promoter- Neo-polyA composition. Example 3 Preparation of recombinant AAV-GFP virus

Cells were cultured at 37°C with RPMI1640 medium containing 10% fetal bovine serum. 5×10 ⁶ carrier cells BHK/pSNAV-1/GFP were inoculated in a 15-cm culture plate, and cultured in a 5% CO2 incubator at 37°C for 48 hours. Add 1ml of full-function helper virus HSV1-rc (moi=0.5～1), and absorb for 1hr. Add RPMI1640 culture solution containing 2% fetal bovine serum to 30ml/culture plate. Incubate at 37°C for 36-48 hours. Collect the culture supernatant and cells, freeze and thaw repeatedly 4 times to lyse the cells, centrifuge at 1500rpm for 10min, take the supernatant, and inactivate the helper virus at 56°C. The rAAV-GFP virus was concentrated by ammonium sulfate salting-out method and purified by cesium chloride gradient centrifugation. Desalt was dialyzed against PBS buffer. Store at 4°C or add 5% glycerol or sucrose at -70°C.

Claims (16)

The invention belongs to the field of biotechnology inventions, and particularly relates to the preparation and application of recombinant adenovirus-associated virus vector (rAAV) for gene therapy. 1. The feature of the present invention is to adopt the method of "one strain of cell/one strain of virus" to prepare rAAV virus. 2. Use the method of "one vector cell/one fully functional helper virus" to prepare rAAV virus, including: (a) establishment of recombinant AAV vector cell strain; (b) Production and preparation of a fully functional helper virus; (c) Infect the vector cell line with the full-function helper virus to produce rAAV virus. 3. A recombinant AAV vector cell strain that is stably passed down is obtained by introducing the recombinant AAV vector plasmid into mammalian cells and culturing through resistance selection. 4. The vector cell line contains AAV ITRs and therapeutic gene expression units stably integrated in the chromosome. 5. Inserting the AAV virus rep/cap gene into the herpes simplex virus genome to form a fully functional helper virus. 6. The produced rAAV virus carrying the target gene is used for gene therapy of genetic diseases, tumors, cardiovascular diseases, and infectious diseases. 7. According to claim 4, the carrier cells are derived from BHK cells. 8. According to claim 4, the carrier cells are derived from KB cells, 293 cells, HeLa cells. 9. According to claim 4, the antibiotic resistance gene is provided by a recombinant AAV vector plasmid. 10. According to claim 4, the resistance gene is provided by another plasmid. 11. According to claim 4, the resistance gene is a neo gene. 12. According to claim 4, the resistance gene is the hph gene. 13. According to claim 6, AAV viral rep/cap genes are inserted into the genome of herpes simplex virus type I (HSV-1). 14. According to claim 6, AAV viral rep/cap genes are inserted into the herpes simplex virus type II (HSV-1) genome. 15. According to claim 6, AAV virus rep/cap gene is inserted in UL2 and/or UL44 of I herpes simplex virus (HSV-1) genome. 16. According to claim 6, the AAV virus rep/cap gene is inserted into any site of the herpes simplex virus carrying genome.