CN112280801A

CN112280801A - Method and kit for rAAV infection titer detection by plasmid assistance

Info

Publication number: CN112280801A
Application number: CN202011196673.0A
Authority: CN
Inventors: 潘杏; 何晓斌; 刘光猛; 张胜; 贾宁; 王梦蝶
Original assignee: Wuhan Brainvta Science And Technology Co ltd
Current assignee: Wuhan Brainvta Science And Technology Co ltd
Priority date: 2020-10-30
Filing date: 2020-10-30
Publication date: 2021-01-29

Abstract

The invention belongs to the field of biology, and particularly relates to a method and a kit for rAAV infection titer detection by plasmid assistance. The method comprises the steps of transiently transfecting HEK cells carrying E1a genes and E1b genes to exogenous plasmids containing gene plasmids encoding Cap and Rep proteins of AAV viruses and adenovirus helper plasmids, and taking the rAAV to be detected as seed viruses to realize the replication and proliferation of the rAAV in the HEK cells transfected with the exogenous plasmids; by detecting the expression of the reporter gene or the copy number of rAAV genome generated by replication and proliferation in the cell, the infection titer of the rAAV to be detected is calculated, and the detection of the rAAV infection titer can be efficiently and simply realized by plasmid assistance without introducing auxiliary virus, thereby solving the technical problems that the rAAV infection titer detection method in the prior art needs to introduce auxiliary virus, has safety risk, needs to be matched with a specific cell line for complex operation, and the like.

Description

Method and kit for rAAV infection titer detection by plasmid assistance

Technical Field

The invention belongs to the field of biology, and particularly relates to a method and a kit for rAAV infection titer detection by plasmid assistance.

Background

Adeno-associated virus (AAV) belongs to the parvoviridae family, and is a small-particle, replication-defective, non-enveloped, single-stranded dna (ssdna) virus that usually requires the assistance of helper viruses such as adenovirus and herpes virus to replicate. The artificially modified recombinant AAV (rAAV) has the advantages of good safety, low immunogenicity, wide tissue tropism, no integration to host cell genome, etc. In recent years, rAAV as a gene therapy vector has become a hotspot of gene therapy research, and Glybera and Luxturna have been approved to be marketed, but rAAV as a gene therapy vector also has obvious defects, firstly, the loading capacity of the rAAV as a gene therapy vector is smaller, the loading upper limit is about 4.7kb, and the integrity of virus particle packaging is reduced along with the increase of the size of the loaded exogenous gene; secondly, in vivo experiments of naturally-occurring multiple serotypes such as AAV1, AAV2, AAV5, AAV7, AAV8 and AAV9 show different infection preferences and infection efficiencies for different tissues and cells, but the overall infection efficiency of in vitro culture cells by various serotypes of AAV is low, researchers try to modify protein capsids determining AAV serotypes to obtain multiple serotypes such as AAVDJ serotypes (Dirk Grimm et al, 2008) capable of infecting multiple cell types, but overall, the infection efficiency of in vitro culture cells by AAV is still low, so that various experiments of AAV at the cell level, such as molecular cell screening, infection titer measurement and the like, are limited.

AAV viruses are generally less efficiently infected for in vitro cultured cells without the addition of helper viruses and other cofactors. The method for promoting AAV in vitro infection is to use adenovirus and herpes simplex virus to assist AAV to infect, mainly because AAV virus genome is single-stranded DNA (ssDNA), and must become double-stranded DNA (dsDNA) after entering into cell to be able to transcribe and translate exogenous gene, under the condition of no helper virus or other helper factors, the process of AAV single-stranded DNA in cell replication to form double-stranded DNA is very slow, and helper virus can obviously promote single-stranded DNA to replicate to double-stranded DNA, thereby the expression level of exogenous gene can be improved by 5-10 times.

Studies have shown that there are also a number of co-factors that can promote AAV infection of cells cultured in vitro. Butyrate is a histone deacetylase inhibitor and can induce the hyperacetylation of chromosomes, thereby leading to the induction of epigenetic levels of silent genes. When AAV infects cells in vitro, 10-50mM sodium butyrate is also often used as an infection promoting agent, so that the expression of exogenous genes can be improved by 5-10 times. Proteasome Inhibitors (PIs) such as zLLL (MG132), LLnL (MG101), bortezomib (bortezomib), and the like, can also promote in vitro cell infection of AAV to some extent by inhibiting degradation of AAV into cells by proteasomes (kriti J et al, 2004; ane M D et al, 2001; Paul E M et al, 2010; Jonathan D F et al, 2010). In addition, Hydroxyurea (HU), Polybrene (Polybrene), siRNA knockdown nucleolin, and the use of microspheres have been reported to promote AAV infection in vitro to some extent, but the promotion effect is limited (Jarrod S J et al, 2009; Cathryn M et al, 2001).

Titer determination of rAAV vectors is generally divided into physical titer and infectious titer. Physical titer viral genome copy number (vg) of rAAV is usually determined by quantitative PCR or by assaying for rAAV capsid content (cp) using ELISA kits of different serotypes. Also commonly used today is the Q-PCR assay. The method for measuring the infectious titer is complicated, and the infectious titer of rAAV cannot be measured by the plaque method because rAAV is defective virus and does not infect cells to generate cytopathic effect (CPE) like adenovirus. The method for measuring the titer of rAAV infection commonly used at present comprises the steps of carrying a fluorescent gene or a luciferase reporter gene by rAAV, carrying out gradient dilution infection under the assistance of adenovirus or herpes virus, and determining the titer of rAAV transduction (sometimes also called the titer of infection) by calculating the expression amount of the reporter gene. However, this method is limited in that the rAAV to be tested must carry a reporter gene to enable detection. To date, international and commonly used assays for AAV infectious titer have been performed mainly on Hela RC32 cells, and the Hela RC32 cell line was constructed by stably transforming the cap and rep genes of AAV2 in Hela cells (Gilliane Chadeuf et al, 2001). AAV was infected with Hela RC32 cells at 10-fold gradient dilutions with the aid of wild-type adenovirus type 5 (wtAd5), AAV genome copy number was calculated by observing fluorescence or RT-PCR, and infectivity titer was determined using TCID50 (Lock M et al, 2010). This approach defines cell lines and wild-type Ad5 aids in infection, presents a safety risk and needs to be performed in a higher safety-level laboratory, thus limiting its widespread use.

Researchers have stabilized cap and rep into cells as packaging cell lines, such as those most commonly stabilized in Hela cell lines (Clark et al, 1995; Tamayose et al, 1996; Gao et al, 1998; Inoue and Russell, 1998; Chadeuf et al, 2000; Cao et al, 2002; Mathews et al, 2002). The expression products of Human Papilloma Virus (HPV) genes E6 and E7 in Hela cells have a promoting effect on rAAV amplification (Ogston et al, 2000; You et al, 2006; Cao et al, 2011). In view of the safety risk posed by the residual HPV fragment in Hela cells, researchers have developed A549 cell lines (Gao et al, 2002; Farson et al, 2004). However, both the cap and rep stably transfected AAV Hela and A549 cell lines require wild-type Ad5 for helper infection to produce AAV. This is a challenge for the experimental environment and the safety of the operator. To avoid the use of wild-type helper virus, researchers selected HEK293 cells for stable cap and rep transfer, since the cells themselves contained the E1 genes of Ad virus (including E1A and E1B). E1A is a transcription factor of p5 and p19 promoters, and can promote transcription and translation of rep proteins. The rep protein products include rep78, rep68, rep54 and rep40, which inhibit cell growth (Yang et al,1994), are toxic to cells (Schmidt et al,2000) and cannot be overexpressed in HEK293 cells for a long time when stable cell lines are established. Therefore, researchers have prepared stable transgenic cell lines containing rep-cap in HEK293 cells using cre-controlled inducible expression systems (Qiao et al,2002), and also optimized the cloning strategy afterwards (Yuan et al, 2011). However, the addition of multiple helper viruses, cumbersome cell line screening procedures and evaluation of cell line stability still cannot be solved.

The detection of the infectious titer of gene therapy viral vectors is required in "general human gene therapy products" in the Chinese pharmacopoeia, 2020 edition. "establishing the detection indexes of physical quantity and biological quantity of gene therapy products" is also explicitly pointed out in "guiding principles of pharmaceutical research and evaluation techniques (survey papers) of gene therapy products published on 14/9/2020," such as: particle number, infectious titer or infectious particle number, genomic DNA/RNA or plasmid DNA concentration, bacterial number, and the like. Viral vectors should be controlled in the ratio of total particle count to infectious titer or infectious particle count, and viral vector-based formulations are recommended for clinical dosing in terms of physical titer. "the ratio of the total particle number of the viral vector to the infectious titer or the infectious particle number is monitored, mainly because the physical titer does not change much with the change of various conditions, such as the change of the preparation buffer, the preservation temperature, the preservation time, the pH and the like, but the infectious titer (the number of the viral vectors that can effectively infect cells) changes. If the infection titer cannot be effectively detected, the virus produced in the same batch can be stored for more than 1 year or the use effect of the virus is reduced after several times of freeze thawing. This causes considerable trouble to the workers who test the animals. Usually, the time for an animal to be effective is several months or even 1 year, and when the experimental data show that the batch of virus needs to be reused later, whether the infectious activity of the virus is reduced needs to be effectively evaluated. How to determine the identity of the infection performance and the previous batch if another newly produced batch is changed? Therefore, how to simply and rapidly detect the infection titer of the rAAV can solve the problems of influence of the storage time, the freeze-thaw times, the pH value and the like of the rAAV on the virus activity in the prior art. The method needs to establish a universal, simple, practical and credible rAAV infection titer detection method.

The invention aims to develop a novel high-efficiency simple method for AAV infection in vitro culture cells and apply the method.

Disclosure of Invention

Aiming at the defects or the improvement requirements of the prior art, the invention provides a method and a kit for rAAV infection titer detection by plasmid assistance, which are characterized in that HEK cells carrying E1a gene and E1b gene are transiently transfected with exogenous plasmids containing gene plasmids coding AAV (AAV) Cap and Rep proteins and adenovirus helper plasmids, the rAAV to be detected is used as seed virus, and the replication and proliferation of the rAAV are realized in the HEK cells transfected with the exogenous plasmids; the method can efficiently and simply realize the detection of the rAAV infection titer by plasmid assistance without introducing auxiliary viruses by detecting the expression of the reporter gene or detecting the copy number of the rAAV genome generated by replication and proliferation in the cell, thereby solving the technical problems that the rAAV infection titer detection method in the prior art needs to introduce auxiliary viruses to cause safety risks, needs to be matched with a specific cell line to be operated complicatedly, and the like.

To achieve the above object, according to one aspect of the present invention, there is provided a method for performing rAAV infection titer detection using plasmid assist, comprising the steps of:

(1) carrying out transient transfection on HEK cells carrying E1a genes and E1b genes to obtain HEK cells transfected with exogenous plasmids; wherein, the exogenous plasmid contains a gene for coding AAV Cap protein, a gene for coding AAV Rep protein and a gene which plays an auxiliary function for rAAV packaging in adenovirus; the E1a gene and the E1b gene are respectively a gene for coding adenovirus E1a protein and a gene for coding adenovirus E1b protein;

(2) carrying out gradient dilution on rAAV to be detected in the HEK cell transfected with the exogenous plasmid, providing ITR sequences at two ends of a cis-acting element-genome necessary for replication, packaging and integration in a wild AAV genome by the rAAV to be detected, and realizing replication and proliferation of the rAAV in the HEK cell transfected with the exogenous plasmid;

(3) and calculating the infection titer of the rAAV to be detected by detecting the expression of the reporter gene or detecting the copy number of the rAAV genome generated by replication and proliferation in the cell.

Preferably, the genes for assisting rAAV packaging in the adenovirus in the step (1) comprise a gene encoding adenovirus E2a protein, a gene encoding adenovirus E4orf6 protein and a gene encoding adenovirus VA RNA protein.

Preferably, the foreign plasmid is one or more, and the gene encoding the Cap protein of AAV virus, the gene encoding the Rep protein of AAV virus and the gene in adenovirus that assists in rAAV packaging are introduced into the one or more foreign plasmids.

Preferably, the foreign plasmids comprise an RC plasmid and an adenovirus Helper plasmid, wherein the RC plasmid comprises a gene for coding AAV Cap protein and a gene for coding AAV Rep protein; the Helper plasmid contains a gene for coding adenovirus E2a protein, a gene for coding adenovirus E4orf6 protein and a gene for coding adenovirus VA RNA protein.

Preferably, the serotype of said RC plasmid is type 1, type 2, type 3, type 4, type 5, type 6, type 7, type 8, type 9, Rh10, DJ, PHPB, PHPeB, Anc80L65, Retro.

Preferably, a culture medium DMEM and a transfection reagent PEI-MAX are further added in the transient transfection process in the step (1), and when the exogenous plasmid is transiently transfected, the addition mass ratio of the RC plasmid to the Helper plasmid is 1: 0.5-2; the mass ratio of the total added mass of the plasmid to the transfection reagent PEI-MAX is 1:2-1: 3.

Preferably, the HEK cell is HEK293, HEK293F or HEK 293T.

Preferably, the TCID50 infection titer of the rAAV to be detected is calculated in the step (3) according to a Reed-Mench method or a Karber method.

Preferably, the method comprises the following sub-steps:

(1) laying HEK cells carrying E1a gene and E1b gene in a multi-well plate for standing and adherent culture;

(2) after the cells in the step (1) adhere to the wall, instantly transfecting the exogenous plasmid by adopting PEI, and standing and culturing for hours after transfection;

(3) performing 10-fold gradient dilution on rAAV to be detected, adding the rAAV into the cell hole subjected to transient transfection in the step (2), and performing cell culture;

(4) determining the number of positive holes of each gradient by determining the expression quantity of the reporter gene or the gene copy number of the target gene in the rAAV to be detected in each cell hole of each dilution gradient;

(5) and (4) calculating the infection titer of the rAAV to be detected according to the number of the positive holes of each gradient determined in the step (4).

Preferably, step (1) is specifically: spreading HEK cells carrying E1a gene and E1b gene in 96-well plate, and placing in 3% -8% CO at 35-38 deg.C₂And (3) standing and adherent culture is carried out in an incubator for at least 1 hour, and the cell laying amount is 4E + 4-6E +4 cells/hole.

Preferably, HEK cells carrying the E1a gene and the E1b gene are plated in a 96-well plate and placed in a temperature range of 35-38 ℃ with 3% -8% CO₂And (3) standing in an incubator for adherent culture for 1-2 hours.

Preferably, step (2) is performed by using PEI to transiently transfect the foreign plasmid and then performing 3-8% CO transfection at 35-38 DEG C₂And (5) standing and culturing for 1-2 hours in an incubator.

Preferably, the cell culture in step (3) is specifically: 3-8% CO at 35-38 deg.C₂The incubator is kept still for 3-5 days.

According to another aspect of the invention, a kit for rAAV infection titer detection using plasmid assistance is provided, the kit comprising HEK cells carrying E1a gene and E1b gene and exogenous plasmid;

wherein, the exogenous plasmid contains a gene for coding AAV Cap protein, a gene for coding AAV Rep protein and a gene which plays an auxiliary function for rAAV packaging in adenovirus; the E1a gene and the E1b gene are respectively a gene for coding adenovirus E1a protein and a gene for coding adenovirus E1b protein;

when in use, the exogenous plasmid is used for transiently transfecting HEK cells carrying E1a genes and E1b genes, gradient dilution of rAAV to be detected is carried out in the HEK cells transfected with the exogenous plasmid, the rAAV to be detected provides ITR sequences at two ends of a cis-acting element-genome required by replication, packaging and integration of wild AAV genome, and replication and proliferation of the rAAV are realized in the HEK cells transfected with the exogenous plasmid; and then calculating the infection titer of the rAAV to be detected by detecting the expression of the reporter gene or detecting the copy number of the rAAV genome generated by replication and proliferation in the cell.

Preferably, the genes in the adenovirus that assist in rAAV packaging include the gene encoding adenovirus E2a protein, the gene encoding adenovirus E4orf6 protein, and the gene encoding adenovirus VA RNA protein.

Preferably, the kit also comprises a culture medium and a transfection reagent; the culture medium is preferably DMEM culture medium, and the transfection reagent is preferably PEI-MAX.

Preferably, the kit also comprises rAAV virus with known titer as a positive standard.

In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:

(1) the invention provides a method for detecting rAAV infection titer by plasmid assistance, which comprises the steps of transiently transfecting HEK cells carrying E1a gene and E1b gene with exogenous plasmids containing gene plasmids coding AAV (AAV) Cap and Rep proteins and adenovirus helper plasmids, and detecting the infection titer of rAAV by taking the rAAV to be detected as seed viruses on the basis of realizing the replication and proliferation of rAAV in the HEK cells transfected with the exogenous plasmids.

(2) The invention provides a method for rAAV infection titer detection with plasmid assistance, which adopts HEK cells equipped in most laboratories to carry out transient transfection, does not need to be matched with and package specific cell lines when auxiliary viruses are adopted in the prior art, does not need a fussy cell line screening process and cell line stability evaluation, and is simple, convenient and quick to operate.

(3) Compared with the method for determining AAV infection titer by adenovirus auxiliary infection Hela RC32 which is commonly used at present, the method for detecting rAAV infection titer by plasmid assistance, which is provided by the invention, can obtain the plasmids more easily by performing transient transfection on cap _ rep genes and Helper plasmids of genes related to adenovirus auxiliary infection; the pollution of adenovirus is avoided, the operation is safer, and the requirement on laboratory level is low; meanwhile, the packaging cells and the infection titer determination can be operated in 293T cells, so that the cost and the time for purchasing and culturing Hela RC32 cells are saved.

(4) In the preferred embodiment of the method for detecting the titer of rAAV infection by plasmid assistance, a cap _ rep plasmid (RC plasmid) and an auxiliary plasmid (Helper) are used for assisting the rAAV to infect 293T cells, the 293T cells are transfected by the two plasmids, the rAAV with the titer to be detected is used as a seed virus to realize mass replication and packaging of the rAAV, and experiments show that the same rAAV adopts different serotype RC plasmids, the difference of the virus titers detected by the method is small, the method is suitable for packaging various serotype rAAV, and the two plasmid assistance method has good universality for detection of the titer of the rAAV of different serotypes.

(5) In the embodiment of the invention, it is found that in 293T cells, whether CMV (cytomegalovirus) strong promoter or TTR (TTR) tissue-specific promoter is adopted, the fluorescence of rAAV infection assisted by the plasmid of the invention is strongest, and in the recombinant adenovirus assisted group and non-plasmid assisted group, although the fluorescence is enhanced to a certain extent, the enhancement effect is far different from that of the plasmid assisted group in the embodiment of the invention, and the infection titer is about 3 to 4 orders of magnitude worse. Meanwhile, the recombinant adenovirus has high cytotoxicity, and 293T cells cluster and die 3 days after infection, so that the recombinant adenovirus can be presumed to have high cytotoxicity if used for auxiliary infection of wild type adenovirus. Comprehensive analysis shows that compared with adenovirus helper, the plasmid helper method of the invention has the following advantages: 1) the enhancement of rAAV infection effect is stronger; 2) the experimental operation is safer; 3) the cytotoxicity is less.

(6) In the embodiment of the invention, the repeatability of the virus titer detection method is verified, three different operators simultaneously use plasmid to assist infection to detect the infection titer of the same rAAV virus, the operations are repeated for 3 times respectively, the result is analyzed, and the CV of the three operators is between 9% and 20%, which indicates that the method has better repeatability.

(7) The invention provides a kit for rAAV infection titer detection by plasmid assistance, which comprises HEK cells carrying E1a gene and E1b gene and exogenous plasmids; when the kit is used, the exogenous plasmid is used for transiently transfecting HEK cells carrying E1a genes and E1b genes, gradient dilution of rAAV to be detected is carried out in the HEK cells transfected with the exogenous plasmid, the rAAV to be detected provides ITR sequences at two ends of a cis-acting element-genome required by replication, packaging and integration of wild AAV genome, and replication and proliferation of the rAAV are realized in the HEK cells transfected with the exogenous plasmid; and then calculating the infection titer of the rAAV to be detected by detecting the expression of the reporter gene or detecting the copy number of the rAAV genome generated by replication and proliferation in the cell. In a preferred embodiment, the kit further comprises a culture medium, a transfection reagent and a positive standard substance, so that the kit is convenient to use, and can be suitable for detecting the titer of various serotype rAAV viruses.

Drawings

FIG. 1 is a flow chart of a method for determining rAAV infection titer using plasmid-assisted technology according to the present invention;

FIG. 2 is a comparison of the effect of plasmid-assisted day-and next-day rAAV infection in T cells of example 1293.

FIG. 3 Contents a and b are fluorescence comparisons of same viral plasmid helper and non-helper titer measurements in 293T cells of example 3, respectively.

FIG. 4 is a comparison of plasmid helper and recombinant adenovirus helper infection under strong promoter priming in example 4.

FIG. 5 is a comparison of plasmid helper and recombinant adenovirus helper infection under strong promoter priming in example 4.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

AAV virus has a weak ability to infect in vitro cultured cells compared with viruses such as adenovirus and lentivirus, so that the determination of the infection titer is difficult, and adenovirus is required to assist, and currently, Hela RC32 cells are mainly used for the determination of the AAV infection titer, and Hela RC32 cell lines are cell lines constructed by stably transferring cap and rep genes of AAV2 in Hela cells (Gilliane C et al, 2001). AAV was subjected to 10-fold gradient dilution with the aid of adenovirus to infect Hela RC32 cells, AAV genome copy number was calculated by observing fluorescence or RT-PCR, and infection titer was determined using the method of TCID50 (Lock M et al, 2010). This approach limits the safety issues associated with cell line and adenovirus helper infection, thereby limiting its range of application.

The method for detecting rAAV infectious titer by plasmid assistance, which is provided by the invention, is shown in figure 1 and comprises the following steps:

(1) carrying out transient transfection on HEK cells which are attached to walls or suspended and carry the E1a gene and the E1b gene to obtain the HEK cells transfected with the exogenous plasmids; wherein, the exogenous plasmid contains a gene for coding AAV Cap protein, a gene for coding AAV Rep protein and a gene which plays an auxiliary function for rAAV packaging in adenovirus; the E1a gene and the E1b gene are respectively a gene for coding adenovirus E1a protein and a gene for coding adenovirus E1b protein;

In some embodiments, the genes that assist in rAAV packaging in the adenovirus of step (1) include a gene encoding adenovirus E2a protein, a gene encoding adenovirus E4orf6 protein, and a gene encoding adenovirus VA RNA protein.

The foreign plasmid is one or more, and the gene for coding AAV Cap protein, the gene for coding AAV Rep protein and the gene for assisting rAAV packaging in adenovirus are introduced into the foreign plasmid or the foreign plasmids. The present invention introduces the gene coding AAV Cap protein, the gene coding AAV Rep protein and the gene in adenovirus with auxiliary rAAV packing function into exogenous plasmid, and may introduce all these genes into the same plasmid or into several different plasmids.

The genes provided by the foreign plasmids of the invention can be provided by at least one plasmid, and the arrangement of the genes on the plasmid is not sequentially required. In a preferred embodiment, the cap and rep genes of AAV are placed on the same plasmid, and adenovirus E2a, E4orf6, VA RNA are placed on the same plasmid. That is, in some embodiments, the foreign plasmid comprises an RC plasmid comprising a gene encoding an AAV virus Cap protein and a gene encoding an AAV virus Rep protein, and an adenovirus Helper plasmid; the Helper plasmid contains a gene for coding adenovirus E2a protein, a gene for coding adenovirus E4orf6 protein and a gene for coding adenovirus VA RNA protein. In some embodiments, the cap _ rep plasmid and the adenoviral helper plasmid in a rAAV triplasmid packaging process are used to facilitate rAAV infection of 293T cells. The AAV cap and AAV rep are provided by the same RC plasmid, the adenovirus E2a, adenovirus E4orf6 and adenovirus VA RNA are provided by another plasmid Helper, and E1a and E1b are provided by HEK cells.

In some embodiments, the RC plasmid pAAV2_2 is purchased from addgene, cat # 104963, which constructs sequences between ITRs at both ends of wild-type AAV2/2 into a plasmid vector for expression of the CAP and REP proteins of AAV 2. The RC plasmid can also be prepared by self, and the specific construction method comprises the following steps:

1) selecting a proper vector and a proper multiple cloning site, and performing enzyme digestion by using restriction enzyme to obtain a vector framework;

2) amplifying a cap _ rep sequence by a PCR method, and adding restriction enzyme sites which are the same with enzyme digestion vectors or adding a section of homologous arms on a vector skeleton when designing an amplification primer;

3) connecting the vector and a cap _ rep sequence amplified by PCR in a digestion connection mode or a homologous recombination mode;

4) and transforming the connecting product or the recombinant product into escherichia coli competence, coating a resistant LB solid plate, selecting monoclonal shake bacteria to extract plasmids, and performing sequencing verification to obtain correct plasmids.

In a preferred embodiment, Helper plasmid pAdDeltaF6 was purchased from adddge, cat # 112867, i.e., wild-type adenovirus Ad5E2a gene, E4orf6 gene, VA RNA gene were constructed into vectors expressing E2a protein, E4orf6 protein, and VA RNA protein. The Helper plasmid can also be prepared by itself, and the specific construction method is the same as that of the RC plasmid.

The method for determining rAAV infection titer by using plasmid assistance needs to use AAV serotype plasmids to assist AAV viruses to be detected to replicate, proliferate and release from cells to a supernatant and infect peripheral cells, so that an expression signal of a reporter gene is amplified, and the genome copy number of AAV in the cells is increased. The ability of different AAV serotypes to infect cells in vitro has been reported in the literature to be different, with AAVDJ and AAV2 being the best for infecting cells in vitro. It is generally believed that the serotype that best infects cells in vitro will be the serotype that is more suitable for titer-assisted detection of rAAV infection. In the embodiment of the invention, the influence of different serotype plasmids of RC2, RCDJ, RC8 and RC 94 on the detection of the same virus titer is compared, and the fact that when the method is used for virus titer detection, the different serotype plasmids assist in virus packaging and virus titer measurement results is not greatly different shows that the plasmid assist method disclosed by the invention adopts different serotype Cap plasmids to assist in virus titer detection results, so that the virus titer detection method disclosed by the invention has serum universality. Therefore, the RC plasmid (Cap-Rep) in the helper plasmid used in the rAAV infection titer determination method provided by the invention has no serotype requirement and limitation. The invention can select corresponding AAV cap and rep genes according to requirements, and the corresponding coded AAV serotype can be type 2, and can also be various serotypes such as type 1, type 3, type 4, type 5, type 6, type 7, type 8, type 9, Rh10, DJ, PHPB, PHPeB, Anc80L65, Retro and the like. Accordingly, the serotypes of the RC plasmids employed in some embodiments are type 1, type 2, type 3, type 4, type 5, type 6, type 7, type 8, type 9, Rh10, DJ, PHPB, PHPeB, Anc80L65, Retro, and the like.

The gene for coding AAV Cap protein and the gene for coding AAV Rep protein can be Rep genes corresponding to different serotype AAV, and can also be Rep genes modified based on wild type AAV Cap and Rep genes. The gene for coding adenovirus E2a protein, the gene for coding adenovirus E4orf6 protein and the gene for coding adenovirus VA RNA protein can be various genes corresponding to different serotypes of adenovirus, such as the genes corresponding to common

human adenovirus type

2 and 5.

In some embodiments of the invention, the ratio and amount of the RC plasmid and the Helper plasmid introduced may be optimized and selected according to the cell type selected in the system. For example, when HEK cells carrying the E1a gene and the E1b gene are laid in a 96-well plate and are subjected to static adherent culture for at least 1 hour, the laying amount of the cells is 4E + 4-6E +4 cells/well, the addition amount of RC plasmid in each well is 0.05-0.5ug RC, and the addition mass ratio of the RC plasmid to the Helper plasmid is 1:0.5-2, preferably 1: 1-2. And a culture medium DMEM and a transfection reagent PEI-MAX are also added in the transient transfection process, and when the exogenous plasmid is transiently transfected, the mass ratio of the total added mass of the RC plasmid and the Helper plasmid to the mass of the transfection reagent PEI-MAX is 1:2-1: 3. In some embodiments, the amount of each raw material added to each well of a 96-well plate during transient transfection is: 20-100ul DMEM, 0.05-0.5ug RC plasmid, 0.05-0.5ug Helper plasmid, and 0.2-2ul transfection reagent PEI-MAX (1 mg/ml). If 24-well plate, 12-well plate, 6-well plate and the like are used, the system can be adjusted according to the corresponding proportion of the cell usage.

The system for promoting AAV to infect in vitro culture cells provided by the invention is not only limited to the 293T cells in the examples, but also can be applied to other HEK cells. Suitable cells may be cell lines of human, rat, mouse, hamster origin, or isolated primary cells, which are commonly used in laboratories.

The HEK cells of the invention may be suspended or adherent HEK cells, including but not limited to HEK293, HEK293F, or HEK 293T.

In some embodiments of the invention, the TCID50 infection titer of the rAAV to be detected is calculated in step (3) according to a Reed-Mench method or a Karber method.

In some embodiments of the present invention, the method comprises the following sub-steps:

In some embodiments, step (1) is specifically: spreading HEK cells carrying E1a gene and E1b gene in 96-well plate, and placing at 35-38 deg.C for 3-8%CO₂And (3) standing and adherent culture in an incubator for at least 1 hour, preferably 1-2 hours, wherein the cell laying amount is 4E + 4-6E +4 cells/hole.

In some embodiments, step (2) is performed by transiently transfecting the foreign plasmid with PEI at 35-38 ℃ with 3-8% CO₂And (5) standing and culturing for 1-2 hours in an incubator.

In some embodiments, the cell culture of step (3) is specifically: 3-8% CO at 35-38 deg.C₂The incubator is kept still for 3-5 days.

It will be appreciated by those skilled in the art that, in particular, when different cells are used, and different transfection reagents are used, the amount of plasmid can be optimally adjusted as required to achieve better transfection efficiency without causing greater toxicity to the cells.

In some embodiments of the present invention, a Reed-Muench two-law method is adopted to calculate TCID50, and the specific calculation method is as follows: lgTCID50 is distance scale x difference between log of dilutions + dilutions above 50% disease rate, distance scale is (percentage above 50% disease rate-50%)/(percentage above 50% disease rate-percentage below 50% disease rate).

Compared with the currently most commonly used rAAV packaging system three-plasmid transfection HEK293T cell method, the plasmid assisted method disclosed by the invention comprises three parts, namely a cap _ rep plasmid (RC plasmid) and a Helper plasmid (Helper) and HEK293T cells, compared with the three-plasmid transfection method, and the difference between the two parts is that the method disclosed by the invention replaces a core plasmid in a three-plasmid system with a small amount of existing rAAV virus serving as seed virus (which can be purified or unpurified rAAV virus). Compared with the traditional three-plasmid transfection method, the method greatly reduces the consumption of the core plasmid, replaces the core plasmid with the seed virus, only needs a small amount of the core plasmid for seed virus packaging, saves the time and the cost for greatly increasing the core plasmid, and considers that the types of RC plasmid and Helper are only limited in production, the core plasmids are different, the method saves the large increase of the core plasmid, and the cost and the time are saved considerably.

On the other hand, the existing three-plasmid transfection packaging method considers that AAV8 type and AAV9 type are packaged most easily, the packaged virus titer is also highest, and AAV2 is difficult to package, and the virus is mostly distributed in cells and is not secreted into a supernatant; however, the plasmid auxiliary method provided by the invention replaces the core plasmid in the three-plasmid system with the existing small amount of rAAV virus as seed virus (which can be purified or unpurified rAAV virus), so that the use amount and the large extraction time of the core plasmid are greatly reduced, and experiments prove that the method is not limited to the packaging of viruses such as AAV8 type and AAV9 type which are easy to package in the traditional three-plasmid method, but is suitable for the packaging and titer determination of all serotype rAAV viruses, and has no serum preference.

The method for determining the rAAV infection titer by using the plasmid assistance is suitable for rAAV to be detected produced by various methods or system packages, such as the rAAV virus packaged by using a HEK293T three-plasmid system, an sf9/bac system or an rHSV or rAd auxiliary system.

The invention also provides a kit for rAAV infection titer detection by plasmid assistance, which comprises HEK cells carrying the E1a gene and the E1b gene and exogenous plasmids; wherein, the exogenous plasmid contains a gene for coding AAV Cap protein, a gene for coding AAV Rep protein and a gene which plays an auxiliary function for rAAV packaging in adenovirus; the E1a gene and the E1b gene are respectively a gene for coding adenovirus E1a protein and a gene for coding adenovirus E1b protein; when in use, the exogenous plasmid is used for transiently transfecting HEK cells carrying E1a genes and E1b genes, gradient dilution of rAAV to be detected is carried out in the HEK cells transfected with the exogenous plasmid, the rAAV to be detected provides ITR sequences at two ends of a cis-acting element-genome required by replication, packaging and integration of wild AAV genome, and replication and proliferation of the rAAV are realized in the HEK cells transfected with the exogenous plasmid; and then calculating the infection titer of the rAAV to be detected by detecting the expression of the reporter gene or detecting the copy number of the rAAV genome generated by replication and proliferation in the cell.

In some embodiments, the genes in the adenovirus that contribute to rAAV packaging include a gene encoding adenovirus E2a protein, a gene encoding adenovirus E4orf6 protein, and a gene encoding adenovirus VA RNA protein.

The foreign plasmid can be one or more, and the gene coding AAV Cap protein, the gene coding AAV Rep protein and the gene in adenovirus which plays a role in assisting rAAV packaging are introduced into the one or more foreign plasmids. The present invention introduces the gene coding AAV Cap protein, the gene coding AAV Rep protein and the gene in adenovirus with auxiliary rAAV packing function into exogenous plasmid, and may introduce all these genes into the same plasmid or into several different plasmids.

In some embodiments, the kit further comprises a culture medium and a transfection reagent; the culture medium is preferably a DMEM culture medium, and the transfection reagent is preferably PEI-MAX, so that the transfection reagent is convenient for a user to directly use.

In a preferred embodiment, the kit further comprises a known titer of rAAV virus, and the rAAV virus is used as a positive standard for a user to perform determination of the titer of rAAV to be detected under conditions that ensure that the kit is effective.

The kit provided by the invention comprises HEK cells and exogenous plasmids, and can be preserved according to conventional cell and plasmid preservation methods, such as preservation at-80 ℃. When used, the detection method can be used. The specific plasmid species, composition and construction methods are described in the specification. In addition, experiments prove that the determination of the rAAV to be detected is not influenced when the serotype of the exogenous plasmid is different from the serotype of the rAAV to be detected. For example, the kit of the present invention contains the type 2 RC plasmid, and the kit is suitable for measuring rAAV titer of different serotypes.

The following are examples:

example 1

Infection with AAV virus was performed after the 293T cells were plated, on the day after helper plasmid transfection, or the next day, and confirmation was required. Experiments were designed, and following transfection of helper plasmids, the first set of experiments were infected with virus the day and the second set of experiments were infected with virus the next day. The specific operation is as follows:

1) AAV2/9 serotype scAAV-CBh-GFP-BGHpolyA virus (code PB2-623, batch number RC9-623-Z20190420) is packaged by using three plasmids of an HEK293T system, and a finished product AAV virus is obtained by an iodixanol density gradient centrifugation purification method. And Q-PCR quantification was performed by ITR primers, with a physical titer of 3.03E +13 vg/ml. The weak infection ability can better show whether the adjuvant has effect, therefore, the AAV2/9 serotype which infects 293T cells and is lower than other serotypes is selected in the experiment; 2) 2E +6 293T cells are paved in each hole of the 6-hole cell plate; 3) after the cells are attached to the wall, 2 6-well plates are used, a No. 1 plate is selected for plasmid transfection, the transfection system RC9 plasmid (serotype AAV9, addendum, cat # 112865) and Helper plasmid (addendum, cat # 112867) of each well are 0.8ug, the transfection reagent PEI-max (Polyscience, molecular weight 40KDa,1mg/ml) is 3.2ul, the DMEM culture medium is 250ul, and the cells are added after shaking, mixing and standing for 15 min. Put in 5% CO at 37 DEG C₂Standing and culturing for 2h in an incubator; 4) PB2-623 virus was added to 3 wells of plate No. 1 and virus was added to 3 wells of plate No. 2 according to MOI ═ 1E + 5. Returning the culture plate to the incubator; 5) the next day, the plates were removed and PB2-623 virus was added at MOI ═ 1E +5 to 3 additional wells of plate No. 1. The plate was returned to the incubator. And (5) continuing culturing. Observations were made under a fluorescent microscope and photographed every other day.

FIG. 2 is a comparison of the effect of plasmid-assisted day-and next-day rAAV infection in 293T cells. scAAV 2/9-CBh-GFP-bghppolya virus infected 293T cells at MOI ═ 1E +5 without plasmid help, with dim green fluorescence. After the plasmid assistance is added, the fluorescence brightness is obviously enhanced. The addition of the virus after plasmid-assisted day was brighter and the cell status was better than the addition of the virus on the next day, with the virus added first on the same day. From the results in fig. 2, day-of-day infection and alternate-day infection AAV had little effect on the state of the cells. From the aspect of fluorescence, the fluorescence intensity of the infection every other day is weaker than that of the infection on the same day when the fluorescence comparison is performed at the same time. In view of the ease of handling and the reduction of the time for detection, subsequent experiments were performed to select AAV infected the day after transfection.

Example 2

Selection of helper plasmid serotype plasmids

This example compares the effect of several different Cap helper plasmids on the assay results. The following experiments were designed: the effect of different serotype plasmids RC2, RCDJ, RC8, RC 94 on the same virus titer test was compared. The specific operation is as follows:

(1) AAV2/9 serotype scAAV-CBh-GFP-BGHpolyA virus packaged by sf9/bac system (number 1-9KC-623, batch number: 9KC-623-Z20191202 MIX). 96-well cell plates were plated with 4E +4 293T cells per well, 4 plates in total, 2 serotype plasmids per plate, 4 serotype plasmids per plate, and dilutions from-5 to-10. 1 operator tested 4 serotypes simultaneously, and the test was repeated 2 times.

(2) After the cells are attached to the wall, 0.1ug of each of the RC plasmid and the Helper plasmid (adddge, cat # 112867) of each hole of the transfection system is respectively AAV2 (adddge, cat # 104963), AAVDJ (gift acquisition), AAV8 (addge, cat # 112864) and AAV9 (addge, cat # 112865); the transfection reagent PEI-max 0.4ul, DMEM medium 50ul, shaking, mixing evenly, stewing for 15min, adding into the cell. Put in 5% CO at 37 DEG C₂The culture was left to stand in the incubator for 2 hours.

(3) Diluting the virus to be detected by using a 10-fold gradient of a 2% DMEM culture medium, diluting 6ul virus stock solution to 60ul and recording as a gradient-1, diluting 50ul virus of the gradient-1 to 500ul and recording as-2, diluting 50ul virus of the gradient-2 to 500ul and recording as-3, and so on, recording as gradients of-4, -5, -6, -7, -8, -9, -10 and the like, wherein each dilution gradient has 8 holes, and 50ul diluted virus is added into each hole. A total of 4 plates, corresponding to different helper RC plasmids. Placing into a 5% CO2 incubator at 37 ℃ for standing culture.

(4) Counting the number of the cells with positive fluorescence on the 4 th day, and calculating TCID50 according to a Reed-Muench two-handed method, wherein the specific calculation method comprises the following steps: lgTCID50 is distance scale x difference between log of dilutions + dilutions above 50% disease rate, distance scale is (percentage above 50% disease rate-50%)/(percentage above 50% disease rate-percentage below 50% disease rate). The results are shown in Table 1.

From the results in table 1, when different serotypes of plasmids were used for helper infection, the differences of the TCID50 titer detection results of the same viruses were small, and were basically within 3 times. Thus, the range of choice for the RC plasmid includes plasmids of each serotype.

TABLE 1 detection results of different serotype plasmid helper infection titers

Example 3

Plasmid (RC plasmid and Helper) assisted and unassisted rAAV infection titer detection

The infection titer of rAAV is determined by using a plasmid-assisted method, an experiment 1) is designed, an AAV2/9 serotype scAAV-CBh-GFP-BGHpolyA virus (number is 1-1-9KC-623, batch number is 9KC-623-Z20190529) is packaged by using an Sf9/bac system, Q-PCR quantification is carried out by an ITR primer, the physical titer is determined to be 5E +12vg/ml, and the experiment selects AAV2/9 serotype which infects 293T cells and is lower than other serotypes; 2) laying 4E +4 293T cells in each well of a 96-well cell plate, diluting 1-1-9KC-623 virus by using a 10-fold gradient of a 2% DMEM culture medium, diluting 6ul virus stock solution to 60ul, recording the gradient-1, diluting 50ul virus of the gradient-1 to 500ul, recording the gradient-2, diluting 50ul virus of the gradient-2 to 500ul, recording the gradient-3, and so on, recording the gradients of-4, -5, -6, -7, -8, -9, -10 and the like, wherein 8 wells are formed in each dilution gradient, and 50ul diluted virus is added in each well; 3) rAAV infection was simultaneously performed with plasmid-assisted group plasmid-assisted rAAV infection was added as described in example 2, and only 50ul DMEM medium was added to the control group. Fluorescence observation and photographing were performed 3 days after infection (fig. 3). From the fluorescence results, in the absence of plasmid-assisted, -2 gradient, most cells expressed fluorescent protein, but the overall fluorescence intensity was weak, and as the dilution gradient increased, the fluorescence intensity was almost indistinguishable. And after plasmid assistance, the fluorescence brightness is obviously enhanced, even if only one cell shows fluorescence when diluted to-8 and-9, the fluorescence brightness is very bright, and when positive cell holes are calculated by TCID50, the fluorescence brightness is easily distinguished from negative cell holes. The dilution gradient positive wells were counted and the TCID50 calculated using Reed-Muench two-wire method. Results of the RC2 plasmid-assisted TCID50 titer calculations are shown in table 2:

TABLE 2 number of plasmid-assisted TCID50 fluorescence wells

Distance ratio (percentage above 50% rate of illness-50%)/(percentage above 50% rate of illness-percentage below 50% rate of illness) — (81.8-50)/(81.8-30) — 0.614

lgTCID50 distance scale x difference between log of dilution + log of dilution above 50% disease rate 0.614 x (-1) + (-8) — 8.614

TCID50＝10^8.614/50ul＝8.2E+9/mL，GC/TU＝610

The results of TCID50 titer calculations without plasmid assist are shown in table 3:

TABLE 3 number of fluorescence wells without plasmid-assisted TCID50

Distance ratio (100-50)/(100-31.33) 0.728

lgTCID50＝0.728×(-1)+(-6)＝-6.728

TCID50＝10^6.728/50ul＝1.07E+8/mL，GC/TU＝4.67E+4

From the GC/TU ratios of the 1-1-9KC-623 viruses detected by adding the helper plasmid and not adding the helper plasmid, the GC/TU ratio of the plasmid-assisted virus is only 610, which is far smaller than the GC/TU ratio of the virus detected by not adding the plasmid-assisted virus, namely 4.67E + 4.

FIG. 3, Contents a and b are fluorescence comparisons of same viral plasmid helper and non-helper titer detection in 293T cells, respectively. After plasmid assistance is carried out on the same virus, the green fluorescence brightness is obviously enhanced, and under a high dilution multiple, the brightness of single fluorescence is still very strong, so that positive and negative holes can be easily distinguished. Without the plasmid-assisted wells, fluorescence was still very dim at low dilutions, making it difficult to distinguish between positive and negative wells.

Example 4

Differences in the Effect of recombinant adenovirus helper and plasmid helper infection

In the previous embodiment, purified finished rAAV is used, the promoter is also a broad-spectrum strong promoter, and the titer difference between the virus rAAV2/DJ-CMV-mCherry-BGHpA (No. PB2-0948) and the rAAV2/DJ-TTR-mCherry-BGHpA (No. GT-0065, physical titer 1.57E +12vg/mL) purified to the intermediate product (chloroform treatment method) is detected. The specific operation is as follows: 1) selecting a broad-spectrum strong promoter CMV and a liver specific promoter TTR, and constructing rAAV-CMV-mCHERRY-bGHpA and rAAV-TTR-mCHERRY-bGHpA vectors; 2) packaging AAVDJ serotype rAAV-TTR-mCHERRY-bGHpA virus and rAAV-CMV-mCHERRY-bGHpA virus by using 293T system; 3) helper plasmids RC2 and helper, adenovirus used rAd5-mCMV-EGFP (batch no: 20180328, TCID50 Titers: 3.16E + 9/mL); 4) 96-well cell plates were plated with 4E +4 293T cells per well, PB2-0948 virus infected cells with two MOIs, 2E +4 and 2E +3, GT-0065 virus infected cells with MOI 4E +3, infection while plasmid helper groups were plasmid-helped as described in example 2, recombinant adenovirus helper groups were helper-infected with rAd5-mCMV-EGFP MOI of 0.2 at plasmid transfection, 5% CO at 37 ℃₂Standing and culturing in an incubator for 1-2 hours, and then infecting rAAV. Fluorescence was observed by fluorescence microscope 3 days after infection and photographed (FIG. 3).

FIGS. 4 and 5 are comparisons of plasmid helper and recombinant adenovirus helper infections. From the fluorescence condition, in 293T cells, whether CMV (CMV) as a strong promoter or TTR (FIG. 5) as a tissue-specific promoter, plasmid-assisted rAAV infection has the strongest fluorescence, and the recombinant adenovirus helper has a certain enhancement effect compared with the non-helper group, but the enhancement effect is far different from that of the plasmid helper group, and the infection titer is about 3 to 4 orders of magnitude worse. Moreover, with the aid of recombinant adenovirus, the fluorescence intensity of rAAV is enhanced, but the enhancement effect is reduced with weak promoter. Meanwhile, the recombinant adenovirus has high cytotoxicity, and 293T cells cluster and die 3 days after infection, so that the recombinant adenovirus can be presumed to have high cytotoxicity if used for auxiliary infection of wild type adenovirus. Comprehensive analysis shows that compared with adenovirus helper, the plasmid helper method of the invention has the following advantages: 1) the enhancement of rAAV infection effect is stronger; 2) the experimental operation is safer; 3) the cytotoxicity is less.

Example 5

In order to investigate whether rAAV viruses which do not express fluorescence can carry out infection titer determination by the plasmid-assisted infection method, the invention improves the method (Lock et al,2010) for determining rAAV infection titer by Taqman TCID50, and the specific method comprises the following steps:

four replicate wells of each gradient dilution of plasmid-assisted infection in example 3 were picked for virus lysis by the method of Taqman TCID50 assay for rAAV infectious titer; diluting the virus lysate by 10 times, and quantifying the genome copy number of the rAAV virus by using an EGFP primer through an RT-PCR method after dilution, because the prior art method of the laboratory can not reach the sensitivity as low as 10 copies, the Cq mean value measured basically tends to be stable and approaches to negative control ddH along with the increase of the dilution gradient₂And measuring the Cq mean value by using O, selecting the lowest Cq mean value when the Cq mean value is basically kept unchanged as a detection lower limit, selecting the detection lower limit Cq mean value at this time as 29.63, and marking the hole lower than the value as a positive hole, wherein the specific measurement result is shown in Table 4.

TABLE 4Q-PCR detection of EGFP copy number of target gene

TABLE 5 plasmid-assisted number of positive wells for TCID50 Gene copy number

TCID50 was measured by the Reed-Muench two-wire method described above, and the distance ratio was (100-50)/(100-0) ═ 0.5, and lgTCID50 was 0.5 × (-1) + (-8) ═ 8.5, and found to be 6.31E +9 TU/ml.

The result of observing the positive hole by EGFP fluorescence and calculating rAAV TCID50 by determining the genome copy number of rAAV by Q-PCR is analyzed, the difference between the positive hole and the rAAV is small, namely 8.20E +9TU/ml and 6.31E +9TU/ml, which shows that the two have better consistency, and the method for determining the infection titer by the plasmid-assisted rAAV can be used for determining the infection titer of the rAAV virus expressing fluorescence and can also be used for determining the infection titer of the rAAV expressing without fluorescence.

Compared with the method for determining AAV infection titer by adenovirus assisted infection Hela RC32 which is commonly used at present, the plasmid assisted rAAV infection method is characterized in that the plasmid is more easily obtained by transiently transfecting a cap _ rep gene and some genes related to adenovirus assisted infection with RC plasmid and Helper plasmid in the rAAV three-plasmid packaging process; the pollution of adenovirus is avoided, the operation is safer, and the requirement on laboratory level is low; meanwhile, the packaging cells and the infection titer determination can be operated in 293T cells, so that the cost and the time for purchasing and culturing RC32 cells are saved.

Example 6

According to the plasmid-assisted method of embodiment 3, three different operators simultaneously use plasmid-assisted infection to detect the infection titer of the same rAAV virus, repeat the steps for 3 times respectively, and analyze the results, which are shown in Table 6. CV of three operators is between 9% and 20% and is less than 30%. The requirement of variation coefficient of in vitro cell assay in the 'Chinese pharmacopoeia' 2020 edition is within 30%.

TABLE 6 precision analysis of the Virus titre test method (TCID50/ml)

Example 7

The rAAV packaging system most commonly used at present is the HEK293T cell method transfected by three plasmids, namely, an rAAV transfer vector (core plasmid), a cap _ rep plasmid (RC plasmid) and a Helper plasmid (Helper), wherein E1a and E1b are provided by HEK293T cells. The three-plasmid transfection HEK293T cell method has the advantages of rapidness, effectiveness and avoidance of the use of helper virus, but has the disadvantages of large plasmid and cell use amount and time and labor consumption in large-scale rAAV preparation. The present embodiment provides a new rAAV virus packaging method by using a plasmid-assisted infection method, and the general idea is to obtain a small amount of rAAV virus seed viruses by using a prior art method (such as three-plasmid packaging, sf9 systematic packaging, and the like), and then to use a plasmid-assisted infection method to inoculate the seed viruses with a suitable MOI, where the MOI is generally 1E +3 to 1E +4 when packaging different serotypes, and to harvest the viruses after 72 hours. The specific embodiment comprises the following steps:

(1) packaging rAAV-CMV-EGFP-pA viruses of four serotypes, namely AAV2/2, AAV2/8, AV2/9 and AV2/DJ by using a three-plasmid transfection HEK293T cell method, collecting supernatant and performing titer determination by using an ITR primer RT-PCR method;

(2) the collected virus supernatant was re-infected with HEK293T cells at MOI 1E +4 and 1E +3, and simultaneously transfection of RC plasmid and Helper plasmid was performed for assistance, and the control group was packaged by three plasmid transfection, and compared with the control group, the plasmid-assisted infection group was only packaged by replacing the core plasmid with the virus supernatant, each of which was packaged in 2 10cm plates.

(3) And collecting supernatant liquid for 72h respectively, combining two dishes of the same group, and performing titer determination by using an ITR primer RT-PCR method. The results are shown in Table 7.

TABLE 7 measurement of the physical titer of the viral packaging supernatants

Note: MOI-0 stands for three plasmid transfection control group

According to RT-PCR quantitative titer data analysis, four serotypes can assemble rAAV by virus assembly through a plasmid-assisted infection method, and the rAAV virus packaging mode is feasible, and in a whole view, the titer of a high-MOI (1E +4) packaging virus is higher than that of a low-MOI (1E +3), the ratio of the titer of the high-MOI packaging virus to that of a control group is 0.48-3.08, the ratio of the titer of the low-MOI packaging virus to that of the control group is 0.39-1.98, and the difference between the titer of the high-MOI packaging virus and that of the low-MOI packaging virus is basically controlled within a range of 2 times, so that the virus receiving MOI has a relatively large range space, and the possibility is provided for omitting the quantification of the virus receiving titer and carrying out blind connection according to experience (namely, the virus receiving titer is controlled to be more than 1E +3 according to estimate the experience for carrying out the virus receiving). Calculating by using the high MOI 1E +4 and a plasmid transfection control group, wherein the number of cells per 10cm dish is 6E +6, collecting 10ml of supernatant seed virus, using the virus supernatant of the plasmid transfection control group for subsequent plasmid auxiliary infection rAAV virus packaging, and the number of the infected dishes of the supernatant of each dish seed virus is as follows: 14.43 dishes (AAV2/DJ), 9.73 dishes (AAV2/9), 46.85 dishes (AAV2/8), 1.07 dishes (AAV2/2), the AAV2/2 serotype is removed, the single dish supernatant seed virus of other three serotypes can secondarily infect 10-50 dishes, but the single dish yield of plasmid auxiliary infection is higher and the advantage is more obvious when the AAV2/2 serotype three-plasmid transfection package is considered to be more difficult than other serotypes.

Taken together, plasmid-assisted infection is a viable method for packaging rAAV viruses and can be used for packaging of a variety of serotypes, including AAV2/2 serotype, which is more difficult to package using a three-plasmid transfection packaging method. Therefore, compared with the traditional three-plasmid transfection method, the method greatly reduces the using amount of the core plasmid, replaces the core plasmid with the seed virus, only needs a small amount of the core plasmid for seed virus packaging, saves the time and the cost for greatly increasing the core plasmid, considers that the types of RC plasmid and Helper are only limited in production, and the core plasmids are different, saves the large increase of the core plasmid, and has considerable saving on the cost and the time. Based on the method, the rAAV virus titer determination method using the plasmid-assisted method can be also suitable for various serotype viruses, and the method has good universality.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A method for rAAV infection titer detection by plasmid assistance is characterized by comprising the following steps:

2. The method of claim 1, wherein the genes that assist in rAAV packaging in the adenovirus of step (1) comprise a gene encoding adenovirus E2a protein, a gene encoding adenovirus E4orf6 protein, and a gene encoding adenovirus VA RNA protein.

3. The method of claim 1, wherein the exogenous plasmid is one or more and the gene encoding the Cap protein of the AAV virus, the gene encoding the Rep protein of the AAV virus, and the gene in the adenovirus that contributes to rAAV packaging are introduced into the one or more exogenous plasmids.

4. The method of claim 1, wherein the exogenous plasmids comprise an RC plasmid and an adenovirus Helper plasmid, the RC plasmid comprising a gene encoding an AAV viral Cap protein and a gene encoding an AAV viral Rep protein; the Helper plasmid contains a gene for coding adenovirus E2a protein, a gene for coding adenovirus E4orf6 protein and a gene for coding adenovirus VARNA protein.

5. The method according to claim 1, wherein a culture medium DMEM and a transfection reagent PEI-MAX are further added during the transient transfection in the step (1), and when the exogenous plasmid is transiently transfected, the mass ratio of the added RC plasmid to the added Helper plasmid is 1: 0.5-2; the mass ratio of the total added mass of the plasmid to the transfection reagent PEI-MAX is 1:2-1: 3.

6. The method of claim 1, comprising the steps of:

7. A kit for rAAV infection titer detection with plasmid assistance is characterized by comprising HEK cells carrying E1a gene and E1b gene and exogenous plasmids;

8. The kit of claim 7, wherein the genes in the adenovirus that contribute to rAAV packaging comprise a gene encoding adenovirus E2a protein, a gene encoding adenovirus E4orf6 protein, and a gene encoding adenovirus VA RNA protein.

9. The kit of claim 7, further comprising a culture medium and a transfection reagent; the culture medium is preferably DMEM culture medium, and the transfection reagent is preferably PEI-MAX.

10. The kit of claim 7, further comprising a known titer of rAAV virus as a positive standard.