CN110950934B

CN110950934B - Adeno-associated virus capsid protein, vector, construction method and application thereof

Info

Publication number: CN110950934B
Application number: CN201911419650.9A
Authority: CN
Inventors: 凌晨; 郑青云
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2022-11-04
Anticipated expiration: 2039-12-31
Also published as: CN110950934A

Abstract

The invention discloses an adeno-associated virus capsid protein, the amino acid sequence of which is shown as SEQ ID NO:1 is shown in the specification; an isolated nucleic acid molecule encoding the adeno-associated virus capsid protein is disclosed; an adeno-associated virus vector is disclosed, which encodes the adeno-associated virus capsid protein; also discloses a construction method of the adeno-associated virus vector and application of the adeno-associated virus vector in preparation of gene therapy drugs. The invention modifies AAV-DJ capsid protein, which improves transduction efficiency of HEK293, various liver cancer cells, mouse fibroblast and other cell lines and mouse liver compared with wild AAV, avoids immune reaction of receptor caused by high-dose injection of virus vector, optimizes AAV production process, reduces production cost, and promotes AAV gene therapy product to advance clinical process.

Description

Adeno-associated virus capsid protein, vector, construction method and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to adeno-associated virus capsid protein, a vector, a construction method and application thereof.

Background

Currently, gene therapy is a hot spot in research in the biomedical field, and the most commonly used vectors in gene therapy include adenovirus, lentivirus, and adeno-associated virus (AAV, the same below). Wild-type adeno-associated virus (wtAAV, the same below) belongs to parvoviridae and is a single-stranded DNA virus with a diameter of 20nm, and is in an icosahedron shape, a viral genome with inverted terminal repeats (ITRs, the same below) at two ends is coated in capsid protein, and an open reading frame ORF is arranged between the ITRs and comprises capsid protein Cap, replication protein Rep and packaging activation protein AAP. Wild-type adeno-associated viruses are known to integrate into the human chromosomal AAVS1 locus. The recombinant adeno-associated virus (rAAV, the same below) genome sequence only reserves ITRs at two ends necessary for packaging the virus, and the middle is completely replaced by a target gene sequence, so that the virus genome is ensured not to be integrated into a host genome.

Compared with adenovirus and lentivirus, recombinant adeno-associated virus (rAAV) has lower probability of inducing immune response in human body, which is helpful for improving the efficiency of delivering target genes by the vector, and simultaneously, rAAV can reduce the toxicity risk related to immunity; in addition, rAAV can effectively mediate the continuous and stable expression of exogenous genes in vitro and in vivo for a long time, and has become one of the most promising gene therapy vectors. However, the transduction efficiency of rAAV is affected by the length of the target gene sequence, and the serotypes selected for different types of cells and tissues and organs to be transduced are different, and AAV production and packaging costs are relatively high, and high-dose injection can stimulate immune response in humans. Therefore, how to modify AAV, compared with wtAAV, the transduction efficiency of AAV is obviously improved in-vitro and in-vivo experiments of primary cultured cells, bile duct organs, mammalian cells, animal organs and the like; compared with the high-dose viral vector packaged by wtAAV, the low-titer viral vector has the advantages that the efficiency of cell transduction can reach an approximate level, so that the requirement of the traditional gene therapy on the required amount of the viral vector is reduced, and the high-dose injection of the viral vector is avoided to induce the immune response of a receptor; how to improve the targeting of the viral vector by optimizing the AAV capsid protein and provide effective technical support for realizing precise medical treatment; how to optimize the production process of AAV through the optimization and modification of AAV capsid protein, reduce production cost, produce more stable viral vector, and promote the progress of AAV gene therapy to advance clinical is very important.

Disclosure of Invention

In view of the defects of low transduction efficiency and the like in the prior art, the invention provides an adeno-associated virus capsid protein, a vector, a construction method and application thereof.

The technical scheme of the invention is as follows:

the invention provides in a first aspect an adeno-associated virus capsid protein which is mutated from serine to threonine at amino acid 269 of the DJ serotype capsid protein (S269T), the amino acid sequence of which is as set forth in SEQ ID NO:1 is shown.

In a second aspect, the present invention provides an isolated nucleic acid molecule encoding the adeno-associated virus capsid protein (SEQ ID NO: 1) described above.

In a third aspect, the present invention provides an adeno-associated viral vector, which encodes the adeno-associated viral capsid protein (SEQ ID NO: 1) described above.

In a fourth aspect, the present invention provides an adeno-associated viral vector comprising the isolated nucleic acid molecule described above.

In a fifth aspect, the present invention provides a method for constructing the above-described adeno-associated viral vector, comprising the steps of:

step 1: selecting a plasmid expressing an adeno-associated virus DJ serotype capsid protein as a DNA template for PCR amplification;

and 2, step: designing a primer of site-directed mutagenesis, wherein the 269 th amino acid of the wtAVDJ capsid protein VP1 is mutated from the original serine to threonine by site-directed mutagenesis (S269T);

and 3, step 3: introducing the site-directed mutagenesis through PCR amplification, and carrying out enzyme digestion on a DNA template after the PCR is finished;

and 4, step 4: purifying and screening to obtain the adeno-associated virus capsid mutation plasmid.

In a preferred embodiment, the primer sequence of the primer is shown in SEQ ID NO:2, the sequence of the reverse primer is shown as SEQ ID NO:3, respectively.

The invention provides a preparation method of the adeno-associated virus mutant in a sixth aspect, which comprises the following steps:

step 1: mixing the adeno-associated virus capsid mutation plasmid obtained by the method with helper plasmid and target plasmid, and adding the mixture into a packaging cell to package adeno-associated virus;

step 2: purifying the packaged adeno-associated virus to obtain adeno-associated virus mutant.

In a preferred embodiment, the packaging cell used is HEK293.

The invention provides an application of the adeno-associated virus vector in preparing a gene therapy medicament in a sixth aspect, in particular an application in gene therapy of liver cancer.

The invention provides an application of the adeno-associated virus mutant obtained by the preparation method in preparing gene therapy medicines, in particular an application in gene therapy of liver cancer.

The invention has the advantages that: the AAV-DJ capsid protein is reformed, so that the transduction efficiency of the AAV-DJ capsid protein is remarkably improved in cell lines such as HEK293 and liver cancer cells, mouse fibroblasts and the like and mouse liver compared with wtAAV. On the other hand, the transduction efficiency under the condition of lower titer can reach approximate transduction efficiency compared with the high-dose vector packaged by wtAAV, the dosage of the virus vector required by the traditional gene therapy is greatly reduced, and the immune response of a receptor caused by high-dose injection of the virus vector is avoided; in addition, by optimizing the AAV capsid protein, the targeting of the viral vector to tissues is improved, and effective technical support is provided for realizing accurate treatment; finally, through the optimization and modification of the AAV capsid protein, the production process of the AAV can be optimized, the production cost is reduced, the produced virus vector is more stable, and the progress of the AAV gene therapy product in clinic is promoted.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space. It is therefore contemplated to cover by the present invention, equivalents and modifications that fall within the scope of the invention, and that fall within the scope of the invention.

The present invention will be further described with reference to the accompanying drawings to fully illustrate the objects, technical features and technical effects of the present invention.

Drawings

FIG. 1 shows fluorescence plots of GFP expression after infection of cells with viruses packaged with mutated capsid protein particles in a preferred embodiment of the invention;

FIG. 2 is a graph showing fluorescence of GFP expression 30 days after tail vein injection of a purified viral vector into mice in a preferred embodiment of the present invention;

FIG. 3 shows the Western Blot detection result of liver extraction for detecting the expression of target gene in purified virus vector tail vein injection mouse in the preferred embodiment of the present invention, wherein FIG. 3a is the expression of GFP in scAAV-CMV-eGFP, and FIG. 3b is the expression of SOX9 in ssAAV-CMV-SOX 9;

FIG. 4 shows the results of mouse in vivo imaging detection of fluc expression in a preferred embodiment of the present invention.

Detailed Description

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Example 1 construction of adeno-associated Virus vector

1. Site-directed mutagenesis on adeno-associated virus capsid proteins

1.1, determining the capsid proteins necessary for packaging adeno-associated virus by the plasmid used for site-directed mutagenesis, in this example, the capsid proteins of serotype DJ of adeno-associated virus are selected;

1.2, site-directed mutagenesis is carried out on the adeno-associated virus VP1, the 269 th amino acid is mutated from the original serine to threonine (S269T), a mutation primer is designed for the site, about 20bp reading length is selected before and after the mutation site, the codon triplet sequence of the amino acid at the original position is replaced by the codon triplet sequence of the target amino acid, verification is carried out on primer design software, and deletion is carried out slightly according to the requirements of GC proportion, tm value, primer dimer or hairpin structure and the like, and in the embodiment, the most preferable primer sequence is as follows:

forward primer-agcacatctggaggatctacaaatgacaacgcctacttcg(SEQ ID NO：2)；

Reverse primer-cgaagtaggcgttgtcatttgtagatcctccagatgtgct (SEQ ID NO: 3);

1.3 using ddH ₂ O dilution template plasmid DJ serotype capsid protein plasmidAnd a primer, wherein 50ng of template plasmid and 125ng of primer are selected;

1.4, selecting site-directed mutagenesis reagent, adding 5 μ l of 10xreaction buffer, dNTPmix, template plasmid and primer into the PCR tube, and using ddH ₂ Supplementing O to 50 μ l, and finally adding 1 μ l Pfu polymerase to operate on ice;

1.5, vortex oscillation PCR tube, setting PCR program: pre-denaturation at 95 ℃ for 3 min; 10 cycles; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 1min, extension at 68 ℃ for 2min/kb, performing PCR for 18 cycles, and introducing mutation of the required amino acid in the amplification process;

1.6, after the PCR is finished, adding DpnI enzyme in the site-directed mutagenesis kit, and incubating for 1h at 37 ℃ to cut off a template DNA chain;

and 1.7, purifying the digestion product by using a DNA purification kit to obtain a high-purity concentrated product.

2. Screening for the desired mutant product

And transforming the purified and concentrated product into escherichia coli, coating a plate with corresponding resistance on a shaking table, culturing overnight, selecting monoclonal sequencing for verification, and screening to obtain the target capsid protein mutant plasmid.

Example 2 preparation of adeno-associated virus mutants

1. Small scale packaging of viruses

1.1, mixing an adeno-associated virus capsid mutation plasmid with correct sequencing, an auxiliary plasmid and a target plasmid (pAAV-CMV-EGFP is selected in the embodiment) which are necessary for packaging the adeno-associated virus according to the proportion of 1;

1.2, filtering and purifying the packed virus vector by using a 0.22 mu m needle filter for primary impurity removal;

1.3, quantitatively infecting HEK293, huh7, hepG2 and NIH3T3 cells respectively by using the small-package purified adeno-associated virus, screening, and detecting GFP expression for 72h to observe whether HEK293 and liver cancer cell lines can be efficiently infected;

2. large-scale packaging of viruses

2.1, packaging the adeno-associated virus capsid mutant plasmid which is verified to be packaged in a small scale and can efficiently transduce cells, and auxiliary plasmids and target plasmids (in the embodiment, pAAV-CMV-eGFP, pAAV-CMV-SOX9 and pAAV-CMV-fluc are selected for use) which are necessary for packaging the adeno-associated virus, mixing and packaging the adeno-associated virus according to a certain proportion, wherein the cells used for packaging are HEK293, each 20 disks (15 cm disks), observing the transfection condition of the three plasmids by using a fluorescence microscope after 72 hours, and harvesting the virus;

2.2, carrying out gradient centrifugation on iodixanol, passing through an ion exchange chromatography column to obtain a purified virus mutant, and carrying out ultrafiltration concentration to obtain 0.5ml of purified virus mutant;

2.3, injecting the purified virus mutants into C57 mice by tail vein injection of 10^11vgs respectively, and killing livers to be sliced one month later to detect GFP expression;

2.4, injecting the purified virus vectors into C57 mice by tail vein injection of 10^11vgs respectively, and killing livers one month later and detecting SOX9 expression by Western;

2.5, injecting the purified virus vectors into C57 mice by tail vein injection of 10^11vgs respectively, and observing fluc expression by a small animal living body imager after one month.

3. Results of the experiment

GFP expression of mutant capsid granulosa packaging viruses

The following table shows the results of comparison of the transduction efficiencies of the non-mutated and S269T mutant capsid protein particle packaging viruses in HEK293 cells, which can be seen to have higher transduction efficiencies than the wild type.

FIG. 1 shows fluorescence of GFP expression after infection of cells with mutant capsid protein plasmid-packaged viruses, which indicates that S269T mutant capsid protein plasmid-packaged viruses have the strongest GFP expression in HEK293, NIH3T3, hepG2 and Huh7 cells.

FIG. 2 shows fluorescence of GFP expression 30 days after tail vein injection of purified viral vector into mice, from which it is clear that S269T mutant capsid protein particle-packaged virus has the strongest GFP expression.

FIG. 3 shows the Western Blot assay results of the expression of the target gene of the purified viral vector, wherein FIG. 3a is the expression of GFP in scAAV-CMV-eGFP and FIG. 3b is the expression of SOX9 in ssAAV-CMV-SOX 9; from the figure, it is clear that the S269T mutant capsid protein particle packaging virus has the strongest expression for both of the genes of interest.

FIG. 4 shows the results of mouse in vivo imaging to detect fluc expression. As can be seen from the figure, the S269T mutant capsid protein particle packaging virus has the strongest fluorescence expression, shows stronger targeting to the liver, can be applied to the preparation of liver-targeted gene therapy drugs, and provides effective technical support for realizing precise medical treatment.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the prior art according to the concepts of the present invention should be within the scope of protection determined by the claims.

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Claims

1. An adeno-associated virus capsid protein, wherein the adeno-associated virus capsid protein is mutated from serine to threonine at amino acid 269 of the DJ serotype capsid protein and the amino acid sequence is as set forth in SEQ ID NO:1 is shown.

2. An isolated nucleic acid molecule encoding the adeno-associated virus capsid protein according to claim 1.

3. An adeno-associated viral vector, wherein the adeno-associated viral vector encodes the adeno-associated viral capsid protein according to claim 1.

4. An adeno-associated viral vector comprising the isolated nucleic acid molecule according to claim 2.

5. The method for constructing an adeno-associated virus vector according to claim 3 or 4, wherein the method comprises the steps of:

step 2: designing a primer of site-directed mutagenesis, wherein the 269 th amino acid of the primer is mutated from serine to threonine on the wtAVDJ capsid protein VP1 by the site-directed mutagenesis;

and step 3: introducing the site-directed mutagenesis through PCR amplification, and carrying out enzyme digestion on a DNA template after the PCR is finished;

6. The construction method according to claim 5, wherein the forward primer sequence of the primer is as shown in SEQ ID NO:2, the sequence of the reverse primer is shown as SEQ ID NO:3, respectively.

7. A preparation method of an adeno-associated virus mutant is characterized by comprising the following steps:

step 1: mixing the adeno-associated virus capsid mutation plasmid obtained in claim 5 with helper plasmid and objective plasmid, and adding into packaging cells to package adeno-associated virus;

8. The method of claim 7, wherein the packaging cell is HEK293.

9. Use of the adeno-associated virus vector according to claim 3 or 4 in the preparation of a gene therapy medicament.

10. Use of the adeno-associated virus mutant obtained by the method according to claim 7 in the preparation of a gene therapy drug.