CN117660532B - Helper plasmid for reducing rcAAV residues in recombinant adeno-associated virus and application - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and discloses an auxiliary plasmid for reducing rcAAV residues in recombinant adeno-associated virus and application thereof. The auxiliary plasmid for reducing rcAAV residues in the recombinant adeno-associated virus comprises a skeleton plasmid and a recombinant sequence; the recombinant sequence comprises: rep, cap protein coding sequence, E2A, E, VA RNA sequence, at least one DA' sequence and at least one promoter sequence. The helper plasmid of the present invention can significantly reduce rcAAV residues, can increase the purity of AAV products, and reduces the therapeutic risk of the product recipients.

Description

Helper plasmid for reducing rcAAV residues in recombinant adeno-associated virus and application

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an auxiliary plasmid for reducing rcAAV residues in recombinant adeno-associated virus and application thereof.

Background

Adeno-associated virus (AAV) is an icosahedral enveloped virus of about 22nm diameter belonging to the genus Paramyxoviridae. The genome is approximately 4.7kb and comprises four Open Reading Frames (ORFs) encoding the Rep protein, capsid protein (VP 1/VP2/VP 3), assembly Activating Protein (AAP) and membrane-associated accessory protein (MAAP), flanked by 145bp inverted terminal repeats, which fold to form a T-hairpin structure. Recombinant adeno-associated virus (rAAV) is the most commonly used delivery vehicle in clinical gene therapy, has been widely used in more than 150 clinical trials, and has good safety.

As increasingly stringent examination of viral formulations for use in transformation medicine has revealed the presence of non-nucleic acid contaminants of interest in rAAV formulations, the introduction of non-therapeutic nucleic acids can increase the immunogenicity or other risk of rAAV therapy. rcAAV contamination of rAAV vectors is a major safety concern, packaging of rAAV can recombinantly produce replication-competent adeno-associated virus (i.e., replication-competent adeno-associated virus, rcAAV), which is a rep and cap genome flanked by ITRs, surrounded by AAV capsid particles, which can replicate in the presence of helper virus, rcAAV possibly promoting immunotoxicity by an incompletely understood mechanism. Rep may induce DNA fragmentation due to its helicase and endonuclease activities, while Cap expression may trigger an immune response. Therefore, it is particularly important to reduce rcAAV to the lowest level achievable in rAAV vectors prepared for clinical trial product development.

Because rcAAV and rAAV vectors are very similar, the inability to separate by purification process steps presents a more difficult challenge to clinical production of rAAV. In systems using AAV/Ad to produce AAV, studies have reported that the production of rcAAV can be reduced by deleting homologous sequences from AAV packaging vectors and replacing promoters, yet the presence of rcAAV is still clearly detectable during mass production. Based on the method, splitting the Rep and the Cap on the helper plasmid into two expression cassettes with opposite directions can reduce the probability that the Rep and the Cap are assembled simultaneously to form rcAAV, but the yield of rAAV is reduced by about 40%. In addition, it has been studied to reduce rcAAV production by limiting the helper genes Rep and Cap to the cytoplasm using poxviruses, and this system uses adenovirus for rAAV production, potentially causing adenovirus contamination to induce an immune response in the body. To inhibit the immune toxicity of rcAAV remaining in rAAV to the body, it was studied to inhibit rcAAV capsid protein production by adding a microRNA-binding cassette to the helper plasmid Cap end. . There is currently no good method of removing rcAAV and therefore there is a need to develop better production systems to reduce rcAAV production.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a helper plasmid for reducing rcAAC residues in recombinant adeno-associated virus and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

In a first aspect, the present invention provides a helper plasmid for reducing replication competent adeno-associated virus residues in a recombinant adeno-associated virus, the helper plasmid comprising a backbone plasmid and a recombinant sequence; the recombinant sequence comprises:

Rep, cap protein coding sequence, E2A, E, VA RNA sequence, at least one DA' sequence and at least one promoter sequence.

The helper plasmid of the present invention can significantly reduce rcAAV residues. Can be used for reducing rcAAV residual AAV production systems, can improve the purity of AAV products and reduce the treatment risk of product acceptors.

As a preferred embodiment of the helper plasmid of the present invention, the E2A, E, VA RNA sequence is located downstream of the Rep, cap protein coding sequence.

As a preferred embodiment of the helper plasmid according to the invention, the DA' sequence is located downstream of the E2A, E, VA RNA sequence.

As a preferred embodiment of the helper plasmid of the present invention, the promoter sequence is located downstream of the E2A, E, VA RNA sequence.

As a further preferred embodiment of the helper plasmid according to the invention, the promoter sequence is located downstream of the DA' sequence.

As a preferred embodiment of the helper plasmid of the present invention, the recombinant sequence comprises, in order from 5 'to 3': the Rep, cap protein coding sequence, the E2A, E4, VA RNA sequence, the DA' sequence and the promoter sequence.

In a second aspect, the invention provides a plasmid set for reducing replication competent adeno-associated virus residues in recombinant adeno-associated virus, comprising the helper plasmid and an AAV plasmid. As a preferred embodiment of the plasmid set of the present invention, the AAV plasmid is an AAV plasmid of a fluorescent protein driven by a CAG promoter.

Preferably, the AAV plasmid is AAV plasmid pAAV. CAG. EGFP vector of fluorescent protein driven by CAG promoter.

In a third aspect, the invention provides a low rcAAV residue AAV production system obtained by transforming a host cell with a helper plasmid as described above.

Preferably, the host cell is a HeLa, HEK293 or insect Sf9 cell.

In a fourth aspect, the invention provides a method for producing AAV with reduced rcAAV residues, using the AAV production system described above.

In a fifth aspect, the present invention provides use of the above plasmid set, the above AAV production system, in AAV production.

Compared with the prior art, the invention has the beneficial effects that:

The helper plasmid of the invention can obviously reduce rcAAV residues. Can be used for reducing rcAAV residual AAV production systems, can improve the purity of AAV products and reduce the treatment risk of product acceptors.

Drawings

FIG. 1 is a schematic diagram of the experimental design for studying P5, DA' functions in the examples.

FIG. 2 is a schematic diagram of structural elements of a plasmid backbone in examples.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. It will be appreciated by persons skilled in the art that the specific embodiments described herein are for purposes of illustration only and are not intended to be limiting.

The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.

Example 1: construction of helper plasmid PRCHELPER

By designing combinations of placement positions containing DA' sequences, P5 promoters, rep2 gene sequences (SEQ ID NO: 4), cap gene sequences, E2A gene sequences, E4 gene sequences and VA RNA sequences, PRCHELPER auxiliary vectors (figure 1) are constructed based on skeleton plasmid sequences by a conventional molecular cloning method, and residues of rcAAV in AAV produced by the two auxiliary vectors are compared in parallel.

Wherein, the plasmid skeleton (shown in figure 2) has a sequence shown in SEQ ID NO:1 is shown in the specification; the sequence of the P5 promoter is shown in SEQ ID NO:2 is shown in the figure; DA' has the sequence shown in SEQ ID NO:3 is shown in the figure; the sequence of the Rep2 gene is shown as SEQ ID NO. 4; cap is from type 9 adeno-associated virus, and the sequence of the Cap is shown in SEQ ID NO:5 is shown in the figure; the E2A gene sequence is shown as SEQ ID NO. 6; e4 sequence is shown as SEQ ID NO. 7; the VA RNA sequence is shown as SEQ ID NO. 8. The helper plasmids A and B in FIG. 1 were constructed as follows:

(1) The RepCap gene sequence, DA' sequence, P5 sequence and E2A/E4/VA RNA gene sequence are respectively amplified by taking RepCap gene-containing plasmid as a template.

The PCR reaction system is shown in Table 1:

TABLE 1PCR reaction System

The primer sequences used for plasmid a are shown below:

forward primer 1: ATGGCTGCCGATGGTTATC;

reverse primer 1: ACGTAATCCGTAGATGTACCTGG;

forward primer 2: AGGTACATCTACGGATTACGTAAGCCGAATTCTGCAGATATCC;

Reverse primer 2: GTGACCTCTAATACAGGACCTAGCTCCCCCGATACCGTC;

Forward primer 3: AGGTCCTGTATTAGAGGTCACG;

reverse primer 3: ACCATCGGCAGCCATACCTGATTTAAATCATTTATTGTTCAAAGATG.

The primer sequences used for plasmid B are shown below:

forward primer 1: CCCCCTCGATCGAGGATGCCGGGGTTTTACGAGAT;

Reverse primer 1: CCTCCCACCAGATCACCATC;

forward primer 2: AGGTACATCTACGGATTACGTACCTGCAAGGAACCCCTAGT;

Reverse primer 2: GAGTTGGGTACCGGATCCGTTCAACTGAAACGAATCAACCG;

forward primer 3: GGATCCGGTACCCAACTCCA;

Reverse primer 3: ACGTAATCCGTAGATGTACCTGG.

The PCR reaction conditions are shown in Table 2:

TABLE 2PCR reaction conditions

(2) The PCR amplified bands were detected by agarose gel electrophoresis, and the target fragment was recovered using a gel recovery kit.

(3) Multi-fragment ligation was performed using a seamless cloning kit.

The seamless cloning reaction system is shown in table 3:

TABLE 3 reaction system

Reaction components	Volume (mu L)
		2×Assembly Mix	5
Linearized vector	1
		Inserts	n
Nuclease-free Water	to 10

The reaction conditions were 50℃for 1h.

E.coli is transformed into the product after the reaction, the product is coated on a plate with kana resistance, colony PCR identification is carried out by picking clones every other day, positive clones are sent to Guangzhou Jinwei corporation for sequencing, and the required correct plasmid is selected.

Example 2: AAV production by double plasmid transfection

(1) Laying 293T cells in a cell culture dish of about 5E+06 to 15cm, culturing the cells in a culture dish of 5% CO ₂ at 37 ℃ for about 48 hours by using a high-sugar DMEM culture medium containing 10% of new born calf serum and 1% of Penicillin/Streptomycin, wherein the cell density is about 60 to 70% when in transfection;

(2) The helper plasmids A and B (pRCHelper) constructed in example 1 and AAV plasmid pAAV. CAG. EGFP vector of fluorescent protein driven by CAG promoter were added to 0.5mL DMEM medium at 1. Mu.g/0.5. Mu.g, followed by adding PEI 3. Mu.L (1. Mu.g/. Mu.L), vortexing, standing at room temperature for 10 min, and then adding to 25mL transfection medium, vortexing. The medium in the dish was aspirated, the transfection mixture medium was added, and the mixture was returned to the 37℃cell incubator (5% CO2 concentration) for cultivation.

(3) After 72 hours of culture, 25. Mu.L of cell lysate was added and the cells and supernatant were collected in a centrifuge bottle. AAV virus is purified by iodixanol gradient ultra-high speed centrifugation, the virus titer is measured to be proper titer between 1E+12GC/mL and 1E+13GC/mL, and the AAV virus is placed at the temperature of minus 80 ℃ for standby.

Example 3 replication-complete AAV (rcAAV) detection

(1) 293T cells were plated at approximately 1E+07 in T75 cell flasks and incubated overnight in a 5% CO ₂ cell incubator at 37℃using high-sugar DMEM medium with 10% neo-calf serum and 1% Penicillin/Streptomycin.

(2) The test pieces (AAV produced by transfection of helper plasmids A and B in example 2) were diluted to 1E+11vg/mL with DMEM, respectively, wtAAV2 work (ATCC-derived AAV2 standard: ATCC VR-680) was diluted to 1E+13IU/mL, and the Ad5 work (TCC-derived Ad5 standard: ATCC VR-1516) was diluted 4-fold.

(3) The cells were removed from the incubator, the medium was aspirated, rinsed once with PBS, and then the prepared working solution was added and incubated for 4h at 37 ℃. The loading system is shown in Table 4:

TABLE 4 sample addition System

(4) After 4h, 7mL of 2 Xcomplete medium was added per flask, and the culture was continued overnight in a 5% CO ₂ cell incubator at 37 ℃. Then, the whole culture medium is used for liquid exchange, 14mL of the culture medium is placed into a 37 ℃ and 5% CO ₂ cell culture box for culture overnight. After the culture, the cells were collected in 15mL centrifuge tubes, centrifuged at 3000g for 3min, the supernatant was aspirated, and 1mL PBS was added to resuspend the cells and transferred to 1.5mL centrifuge tubes, frozen at-80℃for 2h, and thawed at 37℃for 10min. The virus was inactivated in a water bath at 56℃for 60min after repeated freeze thawing. The whole inactivated supernatant was collected by centrifugation at 10000g for 5min in a1.5 mL centrifuge tube as a second round of infection samples.

(5) 293T cells were plated at approximately 1E+07 in T75 cell flasks and incubated overnight in a 5% CO ₂ cell incubator at 37℃using high-sugar DMEM medium with 10% neo-calf serum and 1% Penicillin/Streptomycin.

(6) The cells were removed from the incubator, the medium was aspirated, rinsed once with PBS, and then the prepared working solution was added and incubated for 4h at 37 ℃. The loading system is shown in Table 5:

TABLE 5 sample addition System

Group of	DMEM(mL)	Ad5 work (mu L)	Inactivating the supernatant
				Blank control	6	4	All inactivated supernatant
Test article	6	4	All inactivated supernatant
				Reference control	6	4	All inactivated supernatant
Positive control	6	4	All inactivated supernatant

(7) After 4h, 7mL of 2 Xcomplete medium was added to each flask, and the culture was continued in a 5% CO ₂ cell incubator at 37℃for 44h.

After the culture is finished, collecting cells into a 15mL centrifuge tube, centrifuging for 3min at 3000g, sucking the supernatant, adding 2mL PBS to resuspend the cells, equally dividing into 2 tubes and 1.5mL centrifuge tubes, centrifuging for 3min at 3000g, sucking the supernatant, and harvesting cell sediment.

(8) Cell DNA was extracted and DNA concentration was determined using Nanodrop.

(9) The fluorescent quantitative PCR detection is carried out on each group of samples, 11+6n reaction systems are prepared, n is the number of the samples to be detected, 15 μl of reaction solution is subpackaged in PCR tubes, 5 μl of reaction templates are added into each tube, and the reaction templates are respectively blank control DNA, sample DNA, reference control DNA, positive control DNA and H ₂ O (NTC), and 3 holes are respectively formed, wherein the reaction solution systems are shown in the following table 6:

TABLE 6 reaction liquid System

10 After the sample addition is completed, a matched cover is covered, the mixture is mixed by slight shaking, and the mixture is rapidly centrifuged for 10 seconds and then is placed into a fluorescent quantitative PCR instrument. The reporter fluorophore is FAM and the probe is REP. The procedure for the set-up reaction is shown in table 7 below:

TABLE 7 reaction liquid System

RcAAV the test results are shown in Table 8:

TABLE 8 detection results

Comparison of rcAAV residues in the purified viruses packed with the two plasmids detected above shows that rcAAV residues can be reduced when the promoter sequence is placed after the Rep/Cap protein coding sequence and E2A/E4/VA RNA and DA' sequences are added, compared with the P5 promoter before Rep2 and compared with the P5 promoter before Rep 2.

Helper plasmids of a particular serotype (Rep 2Cap 9) are employed in the examples, and it will be understood by those skilled in the art that the invention is not limited to this particular serotype, but may be practiced with other serotypes of helper plasmids that are currently known and that may continue to be discovered in the future.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims (4)

1. Use of a helper plasmid for reducing replication competent adeno-associated virus residues in a recombinant adeno-associated virus, wherein the helper plasmid comprises a backbone plasmid and a recombinant sequence;

The recombination sequence comprises the following steps in sequence from 5 'to 3': rep, cap protein coding sequences, E2A, E4, VA RNA sequences, DA' sequences and promoter sequences;

The promoter sequence is shown in SEQ ID NO:2 is shown in the figure;

The DA' sequence is shown in SEQ ID NO:3 is shown in the figure;

The Rep and Cap protein coding sequences are shown as SEQ ID NO. 4 and SEQ ID NO:5 is shown in the figure;

the sequences of the E2A, E and the VA RNA are sequentially shown as SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8.

2. Use of a plasmid set comprising the helper plasmid of claim 1 and an AAV plasmid for reducing replication competent adeno-associated virus residues in a recombinant adeno-associated virus.

3. The use according to claim 2, wherein the AAV plasmid is an AAV plasmid of a CAG promoter-driven fluorescent protein.

4. Use of an AAV production system for reducing replication competent adeno-associated virus residues in a recombinant adeno-associated virus, wherein the AAV production system is obtained by transforming a host cell with a helper plasmid according to claim 1.