CN116622742A

CN116622742A - Nucleic acid for producing rAAV in insect cells, VP1 capsid protein mutant and application

Info

Publication number: CN116622742A
Application number: CN202211181723.7A
Authority: CN
Inventors: 肖何; 何晓斌; 黄刚; 胡颖; 潘杏; 王梦蝶; 杜亮
Original assignee: Ruizheng Pharmaceutical Technology Wuhan Co ltd
Current assignee: Ruizheng Pharmaceutical Technology Wuhan Co ltd
Priority date: 2022-09-27
Filing date: 2022-09-27
Publication date: 2023-08-22
Also published as: WO2024067153A1

Abstract

The invention belongs to the technical field of genetic engineering, and particularly discloses nucleic acid for producing rAAV in insect cells, VP1 capsid protein mutant and application, wherein the nucleic acid comprises nucleotide sequences for encoding adeno-associated virus VP1, VP2 and VP3 proteins, compared with capsid proteins of wild-type AAV serum 8, the amino acid sequence of VP1 protein comprises mutation at one or more sites of 84 th, 92 th and 105 th sites, and the amino acid sequence of VP2 protein is not mutated. According to the invention, the VP1 protein coding sequence is mutated, so that degradation of VP1 protein in insect cells can be reduced, and the expressed VP1, VP2 and VP3 are maintained in a proper proportion so as to be assembled into a capsid correctly, thereby improving the yield and efficacy of rAAV (rAAV) production by utilizing a baculovirus/insect cell expression system.

Description

Nucleic acid for producing rAAV in insect cells, VP1 capsid protein mutant and application

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to nucleic acid for producing rAAV in insect cells, VP1 capsid protein mutant and application thereof.

Background

Adeno-associated virus (AAV), also known as adeno-associated virus, belongs to the genus dependovirus of the family picoviridae, and is the simplest class of structurally single-stranded DNA-deficient viruses currently found, requiring helper virus (typically adenovirus) to participate in replication. The recombinant adeno-associated virus (rAAV) has the characteristics of wide host range, low immunogenicity, high safety, capability of mediating long-term stable expression of exogenous genes in animals, and the like, and is one of the vectors with the most application prospect in the current gene therapy field. With the approval of the first recombinant adeno-associated virus (rAAV) -mediated gene therapy drug, there is an increasing need for large-scale AAV vector manufacturing techniques (Yla-hartturala s.,2012,Mol Ther,20:1831-1832).

The ability to produce large amounts of recombinant adeno-associated virus (rAAV) vectors is an important factor in the development of gene therapy-based drugs. At present, there are two main classes of systems for producing rAAV: one is a conventional production system using mammalian cells (e.g., 293 cells, COS cells, heLa cells, KB cells, etc.); the other is a production system using insect cells. In mammalian cell production systems, single cell rAAV particle yields are low and there is a high risk of contamination in culture, which limits the large-scale production and use of rAAV in mammalian cells. Baculovirus/insect cell expression systems are one of the major systems for large scale production of rAAV. The cap gene in the recombinant adeno-associated virus genome encodes the viral VP capsid protein, comprising three structural proteins, VP1, VP2 and VP3, respectively, and the stoichiometry of VP1, VP2 and VP3 in AAV from wild-type virus is about 1:1:10, which is important for obtaining recombinant AAV.

Although baculovirus/insect cell expression systems have been successfully used for rAAV production at various scales, various studies have reported that rAAV produced in insect cells has reduced VP1 content compared to rAAV produced using mammalian cells, resulting in reduced rAAV yield. Thus, to achieve industrial mass production of rAAV, baculovirus/insect cell systems still need to be improved.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide nucleic acid for producing rAAV in insect cells, VP1 capsid protein mutant and application thereof, and aims to solve the problem that wild-type AAV serum 8-type VP1 protein is unstable and easy to degrade, so that the rAAV yield is reduced.

To achieve the above object, in a first aspect, the present invention provides a nucleic acid comprising nucleotide sequences encoding adeno-associated virus VP1, VP2 and VP3 proteins, the amino acid sequence of the VP1 protein comprising a mutation at one or more of positions 84, 92 and 105, as compared to the capsid protein of wild-type AAV serotype 8, and the amino acid sequence of the VP2 protein is not mutated.

Preferably, the amino acid sequence of the VP1 protein comprises one or more mutations selected from the group consisting of: Q84K, R K and Q105K.

Preferably, the other amino acid sequence of the VP1 protein is identical to the amino acid sequence of VP1 protein of wild-type AAV serotype 8, except for the mutation.

Preferably, the nucleic acid further comprises a promoter capable of driving transcription in an insect cell, the 3' end of the promoter being operably linked to an intron; the initiation codon ATG of the VP1 protein is positioned in the sequence of the intron, or the initiation codon is deleted in the cap gene coding region corresponding to the VP1 protein and the intron is positioned between any two adjacent nucleotides in the ATG; the intron does not comprise a promoter capable of driving transcription in an insect cell;

the nucleic acid can be transcribed into only one kind of precursor mRNA in insect cells, and in the post-transcriptional processing process, the initiation codon ATG of VP1 protein is reserved or deleted only through the alternative splicing action of the intron, or the initiation codon ATG is formed in a cap gene coding region corresponding to VP1 protein, so that the translational expression of VP1, VP2 and VP3 proteins is regulated.

Further preferably, the start codon ATG of the VP1 protein is located within the sequence of the intron, and the 3' -terminus of the intron is operably linked to a nucleotide sequence encoding a 2A self-cleaving polypeptide.

Further preferred, the 2A self-cleaving polypeptide is a T2A peptide, a P2A peptide, an E2A peptide or an F2A peptide.

In a second aspect, the invention provides an adeno-associated virus VP1 capsid protein mutant, encoded by a nucleic acid according to the first aspect of the invention.

In a third aspect, the invention provides an insect cell comprising a nucleic acid according to the first aspect of the invention, said nucleic acid being a baculovirus vector.

Preferably, the insect cell is a spodoptera frugiperda cell, a drosophila cell or a mosquito cell.

Further preferably, the insect cell further comprises another baculovirus vector comprising a rep gene expression cassette of AAV, a foreign gene, and AAV inverted terminal repeats located at both ends of the foreign gene.

Further preferably, the exogenous gene is a reporter gene which is at least one of a chloramphenicol acetyl transferase-encoding gene, a beta-galactosidase-encoding gene, a beta-glucuronidase-encoding gene, a renilla luciferase-encoding gene, an alkaline phosphatase-encoding gene, a firefly luciferase-encoding gene, a green fluorescent protein-encoding gene, and a red fluorescent protein-encoding gene.

Further preferably, the exogenous gene is a gene encoding a pharmaceutical polypeptide that is at least one of lipoprotein esterase, apolipoprotein, cytokine, interleukin, and interferon.

In a fourth aspect, the present invention provides a recombinant adeno-associated virus particle whose capsid comprises the adeno-associated virus VP1 capsid protein mutant of the second aspect of the invention.

In a fifth aspect, the invention also provides a recombinant adeno-associated virus particle produced in an insect cell according to the third aspect of the invention.

In a sixth aspect, the present invention provides a method for producing recombinant adeno-associated virus particles, which comprises culturing the insect cells of the third aspect under conditions that allow production of recombinant adeno-associated virus particles, and recovering the resulting recombinant adeno-associated virus particles.

In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art: according to the invention, through carrying out mutation of specific sites (84 th, 92 th and 105 th sites) on the amino acid sequence of the VP1 protein unique region of wild-type AAV serotype 8, and without carrying out mutation on the protein hydrolysis region disclosed in the prior art, the degradation of VP1 proteins in insect cells can be reduced, and VP1, VP2 and VP3 proteins with more proper proportion are obtained, so that a capsid of rAAV is correctly assembled, and the yield and efficacy of rAAV production by utilizing a baculovirus/insect cell expression system are improved.

Drawings

FIG. 1 is a Western Blot detection of VP protein expressed by recombinant baculovirus vector Ac-Cap8 in example 2 of the present invention.

FIG. 2 is a Western Blot detection of VP protein expressed by recombinant baculovirus vector Ac-Cap9 in example 2 of the invention.

FIG. 3 is an alignment of amino acid sequences from 1 st to 137 th at the N-terminus of VP1 protein encoded by AAV serum type 8 and type 9 cap genes provided in example 3 of the present invention.

FIG. 4 is a Western Blot detection of VP proteins expressed by recombinant baculovirus vectors Ac-Cap8, ac-Cap8-Q84K, ac-Cap8-R92K and Ac-Cap8-Q105K in example 3 of the present invention.

FIG. 5 is a silver staining detection chart of SDS-PAGE of purified recombinant AAV virions after the AAV recombinant baculovirus Ac-Rep-ITR in example 6 of the present invention is co-infected with Ac-Cap8, ac-Cap8-Q84K, ac-Cap8-R92K, ac-Cap8-Q105K, respectively, showing three capsid proteins VP1, VP2, VP 3.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Interpretation of the terms

As used herein, the term "operably linked" refers to the linkage of polynucleotide (or polypeptide) sequences in a functional relationship. Two nucleotide sequences are "operably linked" when they are placed into a functional relationship. For example, a transcriptional regulatory sequence (e.g., a promoter) is operably linked to a gene coding sequence if it affects the transcription of the gene coding sequence.

The term "coding region" includes coding and non-coding regions, the coding region may act as an open reading frame (Open Reading Frame, ORF), starting with a start codon and ending with a stop codon, which may include introns and nucleotide sequences encoding proteins; the non-coding region includes cis-acting elements, including promoters, enhancers, and the like, before and after the coding region.

The term "expression cassette" refers to a nucleic acid construct comprising a coding sequence and a regulatory sequence operably linked when introduced into a host cell, resulting in the transcription and/or translation of an RNA or polypeptide, respectively. Expression cassette is understood to include a promoter that allows transcription to begin, a gene open reading frame of interest, and a transcription terminator. Typically, the promoter sequence is placed upstream of the gene of interest, at a distance from the gene of interest that is compatible with expression control.

The term "intron" also known as spacer sequence, refers to a fragment of a gene or mRNA molecule that has no coding effect and is an intervening sequence in eukaryotic cellular DNA. Intronic sequences are transcribed in the pre-mRNA, removed by splicing, and eventually are not present in the mature mRNA molecule. Introns are divided into self-splicing introns and spliced-body introns, depending on whether the splicing process is spontaneous or is to be processed by the spliceosome. Self-splicing introns are a special intron, a ribozyme, that can be excised by itself to leave the mRNA. The introns referred to in the present invention are splice introns, the excision of which is aided by a splice, and the intron sequences are flanked by splice donor and splice acceptor sequences, which are sequences flanking the splice and reclose sites. The spliceosome is a ribonucleoprotein complex dynamically composed of small nuclear RNAs (small nuclearRNA, snRNA) and protein factors, recognizes splice sites of mRNA precursors and catalyzes a splicing reaction, completely cleaves introns, and religates upstream and downstream RNA sequences.

The genomic structure of all known adeno-associated virus serotypes is very similar, AAV is a single stranded DNA virus, the genome is simple in structure and is about 4.7kb in length, and the genome contains a rep gene expression cassette, a cap gene expression cassette and AAV inverted terminal repeats (inverted terminal repeats, ITR) at both ends of the genome. ITR is a 125 nucleotide palindromic structure at both ends of the genome, which can form a self-complementary inverted T-shaped hairpin structure, and is a cis-acting element required for DNA replication initiation and packaging of recombinant AAV genomes as infectious viral particles. AAV, which is a defective virus, cannot replicate independently in the absence of helper virus, and therefore, AAV is only site-specific for integration into the host cell chromosome, and is thus latent. In the presence of helper virus, increased rep gene expression can rescue AAV genomes integrated in host cell chromosomes, replication of large amounts to AAVDNA, and packaging of single-stranded rAAV genomes into infectious virions under the action of VP capsid proteins. The Cap gene encodes a structural VP capsid protein comprising 3 overlapping open reading frames, encoding three types of subunits, VP1, VP2, VP3, respectively, VP1, VP2, and VP3, contain different start codons, share a stop codon, and VP1 and VP2 share the VP3 sequence. The N-terminus of VP1 has a conserved phospholipase A2 sequence which is involved in viral escape from the body and is critical for its infectivity; VP2 protein is not necessarily less than necessary for assembly or infection; the core of VP3 protein consists of a conserved beta-barrel motif, which determines the difference between AAV of different serotypes and the host cell acting receptor. The correct ratio of the three proteins in wild-type AAV is 3:3:54, approximately 1:1:10. The Rep gene codes four overlapped multifunctional proteins of Rep78, rep68, rep52 and Rep40, and the Rep78 and Rep68 proteins participate in the replication and integration of AAV and can be combined with a specific sequence in ITR; rep52 and Rep40 proteins have helicase and ATPase activities and are involved in replication of the single stranded genome as well as in viral assembly.

The inventors found in the study that VP1 protein produced by wild-type AAV serotype 8 expression was readily degraded in insect cells, resulting in a significant reduction in relative content. The invention aims to mutate one or more codons of a cap gene, so that VP1 protein encoded by the gene can be more stable in the production process, thereby improving the yield of rAAV. The invention provides a nucleic acid comprising nucleotide sequences encoding adeno-associated virus VP1, VP2, and VP3 proteins, wherein the amino acid sequence of VP1 protein comprises mutations at one or more of positions 84, 92, and 105, and wherein the amino acid sequence of VP2 protein is not mutated, as compared to a capsid protein of wild-type AAV serotype 8.

Chinese patent document CN113728108A proposes that removal of sites in the AAVVP1/VP2 unique region (i.e. the region common to VP1 and VP2 but lacking in VP 3) that are prone to proteolytic cleavage in insect cells can result in rAAV products of higher purity and uniformity, as well as improved capsid proteins. It is also proposed that the VP1 unique region (i.e., the region that is present in VP1 but not in VP2 or VP 3) contains a PLA2 domain, and that mutations in this region can increase the enzymatic activity of the PLA2 domain in the resulting VP1 protein. Meanwhile, grirodA et al also suggested that mutation of the VP1 protein unique region significantly reduced the infectivity of the mutant virus (JGen virol.2002,83 (5): 973-978). However, none of the prior art suggests that mutations at certain sites in the VP1 unique region can increase the resistance of VP1 protein to protein degradation. The inventors have unexpectedly found that mutations in one or more of amino acids 84, 92 and 105 in the VP 1-unique region of AAV serotype 8 (corresponding to residues 1 through 137 of SEQ ID No. 3) are absent from the VP2 protein, which reduces degradation of the VP1 protein during production, and allows for a more suitable ratio of VP1, VP2 and VP3 proteins, thereby increasing the packaging rate and titre of the rAAV.

The inventors found that capsid proteins VP1, VP2 and VP3 of wild-type AAV serotype 9 have a suitable ratio compared to wild-type AAV serotype 8, preferably by mutating one or more of amino acids 84, 92 and 105 of VP1 protein of AAV serotype 8 to the same amino acid of AAV serotype 9, i.e. the amino acid sequence of the mutated VP1 protein comprises one or more mutations selected from the group consisting of: Q84K, R K and Q105K.

In some embodiments, the amino acid sequence of the VP1 protein is mutated only at one or more of the above three positions (positions 84, 92 and 105), and none of the other sequences are mutated.

Based on the inventive concept, an intron alternative splicing strategy (see patent CN113897396B filed by the inventor before) can also be introduced to regulate VP1, VP2 and VP3 proteins in the overlapping open reading frame of the AAVcap gene to be expressed in correct stoichiometry. Specifically, the nucleic acid of the invention comprises an intact cap gene expression cassette, i.e., the nucleic acid further comprises a promoter capable of driving transcription in insect cells, the 3' end of the promoter being operably linked to an intron; the initiation codon ATG of the VP1 protein is positioned in the sequence of the intron, or the initiation codon is deleted in the cap gene coding region corresponding to the VP1 protein and the intron is positioned between any two adjacent nucleotides in the ATG; the intron does not comprise a promoter capable of driving transcription in an insect cell;

In some embodiments, the start codon ATG of the VP1 protein is located within the sequence of the intron, and the 3' end of the intron is operably linked to a nucleotide sequence encoding a 2A self-cleaving polypeptide.

In this example, when an intron regulation strategy is adopted, it is necessary to mutate the intron splice site at the N-terminal of the wild-type AAV serotype 8 VP1 protein coding sequence itself, so that the splice site cannot be recognized by the spliceosome in insect cells. In the post-transcriptional processing, if the spliceosome in the insect cell recognizes the splice site of the intron and catalyzes the splicing reaction, the AUG-initial site at the forefront end of mRNA is removed, and when the ribosome recognizes the initial codon of VP2 protein from 5 'to 3', VP2 protein is expressed in a translation mode, since the VP2 initial codon is the suboptimal codon ACG, ribosome scanning leakage can be caused, and VP3 protein is expressed in a translation mode; if the intron splice site is not recognized and the AUG-start site is not removed, the mRNA is translated from the first AUG and VP1 protein expressed. The relative expression of the capsid proteins VP1, VP2 and VP3 is controlled by the splicing action of the intron with a certain probability. However, when VP1 protein is expressed in a translation mode, an extra sequence (namely a part of an intron sequence) from the first AUG start codon of mRNA to the N-terminal sequence of VP1 protein is also translated to influence the normal expression of VP1 protein, so that the coding sequence of the 2A self-cleaving polypeptide (2A self-cleaving peptides) is introduced into the cap gene expression cassette provided by the invention.

The 2A self-cleaving polypeptide is a peptide fragment with 18-22 amino acid residues in length, and can induce the self-cleaving of recombinant proteins containing 2A peptide in cells. The peptides all have a sequence motif, which often results in a ribosome incapable of being linked at the junction of the last glycine (G) and proline (P), thus causing a "cleavage" effect, such that the C-terminus of the 2A self-cleaving polypeptide is cleaved from the N-terminus of the VP1 protein to obtain the normal VP1 protein. There are currently four general 2A peptides depending on the source of the virus: T2A, P2A, E A and F2A, the above four 2A peptides can be applied to the technical scheme of the invention, and T2A is taken as an example in the embodiment of the invention.

The invention provides an adeno-associated virus VP1 capsid protein mutant, which is obtained by encoding the nucleic acid provided by the invention.

The invention also provides an insect cell, which comprises the nucleic acid provided by the invention, wherein the nucleic acid is a baculovirus vector. The insect cell may be any insect cell, such as, but not limited to, spodoptera frugiperda cells (Spodoptera frugiperda cell), spodoptera frugiperda cells, drosophila cells or mosquito cells, preferably spodoptera frugiperda cells sf9.

The inventive concepts may be applied to any suitable baculovirus/insect cell expression system available for the production of rAAV, including, but not limited to, the three baculovirus system (UrabeM et al, hum Gene Ther,2002,13 (16): 1935-1943), the two baculovirus (Chen et al, mol Ther,2008,16 (5): 924-930; smithRH et al, mol Ther,2009,17 (11): 1888-1896), the OneBac system (Mietzsch M et al, hum Gene Ther,2014,25 (3): 212-222) and the Monobac system (GalibertL et al, abstract at the ESGCT Meeting, madrid/Spain,2013, P074). The embodiment of the invention introduces mutation into a double-rod virus system, wherein insect cells comprise two baculovirus vectors, one of which comprises a mutated cap gene expression cassette, and the other comprises a rep gene expression cassette of AAV, an exogenous gene and AAV inverted terminal repeated sequences positioned at two ends of the exogenous gene.

The exogenous Gene may be at least one nucleotide sequence encoding a Gene of Interest (GOI) product, which may be a therapeutic Gene product, and in particular may be a polypeptide, RNA molecule (siRNA) or other Gene product, such as but not limited to lipoprotein esterase, apolipoprotein, cytokine, interleukin or interferon; also useful are reporter proteins that evaluate vector transformation and expression, such as, but not limited to, fluorescent proteins (green fluorescent protein GFP, red fluorescent protein RFP), chloramphenicol acetyl transferase, beta-galactosidase, beta-glucuronidase, renilla luciferase, firefly luciferase, or alkaline phosphatase.

The invention provides a recombinant adeno-associated virus particle, wherein the capsid of the recombinant adeno-associated virus particle comprises the adeno-associated virus VP1 capsid protein mutant provided by the invention, and the VP1 capsid protein mutant is obtained by encoding the mutated VP1 protein coding sequence of the invention.

The recombinant adeno-associated virus particle provided by the invention can be produced in the insect cell provided by the invention by utilizing a baculovirus/insect cell expression system.

In addition, the present invention also provides a method for producing recombinant adeno-associated virus particles, which comprises culturing the insect cells provided by the present invention under conditions that allow production of recombinant adeno-associated virus particles, and recovering the resulting insect cells.

The following describes the above technical scheme in detail with reference to specific embodiments.

EXAMPLE 1 construction of recombinant baculovirus vectors comprising AAV serum 8 and 9 cap gene expression cassettes, respectively

(1) Constructing a cap gene expression cassette: the cap gene expression cassettes of AAV serotype 8 and AAV serotype 9 were constructed as described in example 1 with reference to chinese patent CN113897396B, and included, in order from 5 'to 3', a p10 promoter, an intron, a nucleotide sequence encoding a T2A peptide, and a nucleotide sequence encoding AAV serotype 8 or type 9 VP protein lacking only the VP1 protein translation initiation codon ATG. The fragments are connected through artificial direct synthesis or overlap extension PCR amplification to respectively obtain constructs Cap8 and Cap9, and the nucleotide sequences of the constructs are respectively shown as SEQ ID No.1 and SEQ ID No. 2.

(2) Construction of recombinant baculovirus vectors: cloning the construct Cap8 onto a pFastBacDual vector to prepare a transfer plasmid; the plasmids are respectively transformed into DH10Bac strain, and the recombinant baculovirus vector Ac-Cap8 is obtained through Tn7 transposition. Similarly, recombinant baculovirus vector Ac-Cap9 containing Cap9 was prepared.

Example 2 detection of VP protein expression of recombinant baculovirus vectors Ac-Cap8, ac-Cap9

The recombinant baculovirus vectors Ac-Cap8 and Ac-Cap9 prepared in example 1 are respectively transfected into a host cell line for culturing to obtain recombinant baculovirus, and the expression condition of VP proteins (VP 1, VP2 and VP 3) is detected, wherein the specific operation steps are as follows:

and (3) extracting the recombinant baculovirus vector DNA to transfect Sf9 insect cells, and preparing the recombinant baculovirus BEV. The transfected Sf9 insect cells successfully produced BEV, and further infection with a large number of replicative BEV resulted in a significant cytopathic effect of Sf9 cells (cytopathic effect, CPE). The culture supernatant of Sf9 cells with CPE was collected and contained a large amount of BEV, i.e. the generation 0 BEV (P0), while Sf9 cells containing a large amount of rAAV were collected. The prepared BEV-P0 is used for infecting suspension-cultured Sf9 cells with a multiplicity of infection (MOI=1), the activity of the cells is reduced to below 50% after 72 hours of infection, 1000g of the cell culture solution is centrifuged for 5min, culture supernatant and cell sediment are respectively collected, and the supernatant is marked as the 1 st generation BEV-P1. Continuing the expansion culture, the prepared BEV-P1 was infected with Sf9 cells cultured in suspension at a multiplicity of infection (MOI=1), after 72 hours of infection, the cell activity was reduced to 50% or less, 1000g of the cell culture broth was centrifuged for 5min, and cell pellets were collected for Western Blot examination of the expression of VP proteins (VP 1, VP2, VP 3).

FIGS. 1 and 2 are Western Blot detection diagrams of VP proteins (VP 1, VP2, and VP 3) of recombinant baculovirus vectors Ac-Cap8 and Ac-Cap9, respectively, comprising Cap gene expression cassettes. From the results, it was found that both recombinant baculovirus vectors were successful in producing VP1, VP2, and VP3; wherein the ratio of VP1 and VP2 proteins produced by recombinant baculovirus vector Ac-Cap9 is close to 1:1, and the content of VP1 protein produced by recombinant baculovirus vector Ac-Cap8 is too low. Theoretically, because Ac-Cap8 and Ac-Cap9 expressed VP proteins using the same regulatory strategy and using the same intron regulatory sequences, the ratio of VP proteins (VP 1, VP2, and VP 3) produced by both should be identical. The inventors speculate that VP1 protein is likely to be degraded easily in sf9 cells due to the structural instability of AAV serotype 8 VP1 protein, resulting in too low a detection of VP1 protein.

EXAMPLE 3 construction of recombinant baculovirus vector containing AAV serum 8-type cap Gene mutant expression cassette and detection of VP protein (VP 1, VP2, VP 3) expression

From the conclusion of example 2, the abnormal VP1 protein expression content of the recombinant baculovirus vector Ac-Cap8 is obtained, so that the VP1 protein amino acid sequences encoded by AAV serum 8 type and 9 type Cap genes are compared, the VP1 protein amino acid sequence of the AAV serum 8 type is shown as SEQ ID No.3, the VP1 protein amino acid sequence of the AAV serum 9 type is shown as SEQ ID No.4, the difference between the N-terminal amino acid sequences of the AAV serum 8 type and 9 type VP1 proteins is found, and the fact that the difference between the N-terminal amino acid sequences of the VP1 proteins leads to the abnormal VP1 protein expression content of the recombinant baculovirus vector Ac-Cap8 is deduced.

To verify the above conclusions, this example mutated the 84 th, 92 th and 105 th amino acids of the N-terminal of VP1 protein encoded by AAV serotype 8 cap gene, respectively, into amino acids corresponding to the N-terminal of AAV serotype 9 VP1 protein, respectively, and FIG. 3 shows the amino acid sequences 1 st to 137 th amino acids of the N-terminal of VP1 protein encoded by AAV serotype 8 and 9 cap genes, wherein the mutation sites (Q84K, R K and Q105K) are marked with black boxes.

Referring to example 1, recombinant baculovirus vectors Ac-Cap8-Q84K, ac-Cap8-R92K and Ac-Cap8-Q105K containing AAV serotype 8 Cap gene mutant expression cassettes were constructed, respectively. The glutamine at 84 th position in VP1 protein amino acid sequence coded by Cap gene of the recombinant baculovirus vector Ac-Cap8-Q84K is mutated into lysine, and the corresponding codon CAG is mutated into AAG; arginine at 92 th position in VP1 protein amino acid sequence coded by Cap gene of the recombinant baculovirus vector Ac-Cap8-R92K is mutated into lysine, and corresponding codon CGG is mutated into AAG; the 105 th glutamine in the VP1 protein amino acid sequence coded by the Cap gene of the recombinant baculovirus vector Ac-Cap8-Q105K is mutated into lysine, and the corresponding codon CAA is mutated into AAA.

Referring to example 2, recombinant baculovirus vectors Ac-Cap8-Q84K, ac-Cap8-R92K and Ac-Cap8-Q105K prepared in this example were transfected into host cell lines, respectively, and cultured to obtain recombinant baculoviruses, and VP protein (VP 1, VP2, VP 3) expression was examined.

FIG. 4 is a Western Blot detection of VP proteins (VP 1, VP2 and VP 3) of recombinant baculovirus vectors Ac-Cap8, ac-Cap8-Q84K, ac-Cap8-R92K and Ac-Cap8-Q105K comprising a Cap gene expression cassette. As can be seen from FIG. 4, the content of VP1 protein produced by Ac-Cap8, ac-Cap8-Q84K, ac-Cap8-R92K and Ac-Cap8-Q105K was significantly improved compared with that produced by Ac-Cap8, ac-Cap8-Q105K, which had no mutation at the N-terminus of VP1 protein. Thus, the site-directed mutagenesis of the N-terminal amino acid of the VP1 protein of AAV serum type 8 can improve the stability of the VP1 protein, thereby remarkably increasing the relative content of the VP1 protein.

EXAMPLE 4 construction of recombinant baculovirus vector Ac-Rep-ITR containing the essential AAV packaging element Rep Gene expression cassette and the ITR core element (ITR-GOI)

In order to test the effect of recombinant baculovirus vectors Ac-Cap8, ac-Cap8-Q84K, ac-Cap8-R92K and Ac-Cap8-Q105K for producing AAV respectively, the invention adopts a Two-bac system for AAV packaging, wherein one rod particle contains a Cap expression cassette of AAV, the other rod particle contains a Rep gene expression cassette and an ITR core element, and Two recombinant baculovirus co-infected sf9 cells are used for AAV packaging.

First, the essential AAV packaging elements, the Rep gene expression cassette and the ITR core element (ITR-GOI), were constructed. The Rep gene expression cassette is constructed by referring to the method described in the example 2 of the Chinese patent CN113897396B, and the nucleotide sequence of the Rep gene expression cassette is shown as SEQ ID No. 5; the nucleotide sequence of the ITR-GOI is shown as SEQ ID No.6, and the GOI in the ITR core element in the embodiment adopts a red fluorescent protein mcherry gene expression cassette, namely, the mcherry expression is controlled by a miniEf1a promoter.

Then cloning the Rep gene expression cassette and the ITR-GOI element to a shuttle vector pFastBacDual to prepare a transfer plasmid; the plasmids are respectively transformed into DH10Bac strain, and the recombinant baculovirus vector Ac-Rep-ITR containing the Rep gene expression cassette and ITR-GOI elements necessary for rAAV production is finally obtained through Tn7 transposition.

Example 5 preparation of AAV recombinant baculovirus

The recombinant baculovirus vectors Ac-Cap8, ac-Cap8-Q84K, ac-Cap8-R92K, ac-Cap8-Q105K and Ac-Rep-ITR prepared in examples 1, 3 and 4 were transfected into host cell lines, respectively, and cultured to obtain AAV recombinant baculovirus.

The recombinant bacmid DNA was extracted and transfected into Sf9 insect cells to prepare AAV recombinant Baculovirus (BEV). The transfected Sf9 insect cells successfully produced BEV, and further infection with a large number of replicative BEV resulted in significant cytopathic effects (CPE) of Sf9 cells. The culture supernatant of Sf9 cells, which had undergone CPE, was collected and contained a large amount of BEV, namely, the 0 th generation BEV (P0). The prepared BEV-P0 is used for infecting suspension-cultured Sf9 cells with a multiplicity of infection (MOI=1), the activity of the cells is reduced to below 50% after 72 hours of infection, 1000g of the cell culture solution is centrifuged for 5min, culture supernatant and cell sediment are respectively collected, and the supernatant is marked as the 1 st generation BEV-P1.

Example 6 purification of recombinant AAV virions and detection of their packaging Rate and viral titre

The expansion culture was continued according to the procedure of example 5 until rAAV was packaged with Sf9 cells of BEV-P2 seed virus Ac-Rep-ITR, which had been co-infected with Ac-Cap8, ac-Cap8-Q84K, ac-Cap8-R92K, ac-Cap8-Q105K, respectively, according to the multiplicity of infection (MOI=1), in suspension culture, the packaging volume being 300 mL-400 mL. Monitoring cell activity after 3 days of infection, centrifuging to obtain cell sediment and supernatant, purifying the cell sediment and supernatant, repeatedly freezing and thawing the cells for 3 times, centrifuging at 5000rpm for 10min, collecting supernatant, adding nuclease (Benzonase) into the supernatant, treating in water bath at 37deg.C for 60min, and centrifuging at 5000rpm for 10min. The collected cell lysates and the collected supernatant PEG were precipitated and purified by iodixanol density gradient centrifugation after resuspension (see Aslanidi et al 2009, proc. Natl. Acad. Sci. USA, 206:5059-5064). The final purified finished virus was resuspended in 80. Mu.L-190. Mu.L LPBS and 10. Mu.L of purified finished virus was run on SDS-PAGE gels and silver stained.

FIG. 5 provides SDS-PAGE silver staining of purified virions after co-infection of AAV recombinant baculovirus Ac-Rep-ITR with Ac-Cap8, ac-Cap8-Q84K, ac-Cap8-R92K, ac-Cap8-Q105K, respectively. From the results, the ratio of VP1 to VP2 protein incorporated in AAV particles produced from Ac-Cap8-Q84K, ac-Cap8-R92K and Ac-Cap8-Q105K was close to 1:1, as compared to Ac-Cap8, which had no mutation at the N-terminus of VP1 protein.

The packaging rate and supernatant titers of harvested recombinant adeno-associated virus (rAAV) were also measured using Q-PCR, and the titer units are expressed in VG/L (VG, viruses genome). The rAAV titer was detected using a pair of primers targeting the ITR sequence (Q-ITR-F: GGAACCCCTAGTGATGGAGTT and Q-ITR-R: CGGCCTCAGTGAGCGA) or the WPRE sequence (Q-WPRE-F: CCGTTGTCAGGCAACGTG and Q-WPRE-R: AGCTGACAGGTGGTGGCAAT). The test results are shown in Table 1.

TABLE 1 packaging Rate and supernatant titres of different recombinant adeno-associated viruses

As can be seen from Table 1, the cell packing rate and yield were higher for rAAV produced from Ac-Cap8-Q84K, ac-Cap8-R92K and Ac-Cap8-Q105K than for Ac-Cap8 without mutation at the N-terminus of VP1 protein.

It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. A nucleic acid, characterized in that: comprising nucleotide sequences encoding adeno-associated virus VP1, VP2 and VP3 proteins, wherein the amino acid sequence of the VP1 protein comprises a mutation at one or more of positions 84, 92 and 105, and wherein the amino acid sequence of the VP2 protein is not mutated, as compared to a capsid protein of wild-type AAV serotype 8.

2. The nucleic acid of claim 1, wherein the amino acid sequence of VP1 protein comprises one or more mutations selected from the group consisting of: Q84K, R K and Q105K.

3. The nucleic acid of claim 1, wherein: the other amino acid sequences of the VP1 protein are identical to the amino acid sequence of VP1 protein of wild-type AAV serotype 8, except for the mutation.

4. A nucleic acid according to any one of claims 1 to 3, further comprising a promoter capable of driving transcription in an insect cell, the 3' end of the promoter being operably linked to an intron; the initiation codon ATG of the VP1 protein is positioned in the sequence of the intron, or the initiation codon is deleted in the cap gene coding region corresponding to the VP1 protein and the intron is positioned between any two adjacent nucleotides in the ATG; the intron does not comprise a promoter capable of driving transcription in an insect cell;

5. The nucleic acid of claim 4, wherein: the start codon ATG of the VP1 protein is located in the sequence of the intron, and the 3' end of the intron is operably linked with a nucleotide sequence encoding a 2A self-cleaving polypeptide.

6. The nucleic acid of claim 5, wherein: the 2A self-cleaving polypeptide is a T2A peptide, a P2A peptide, an E2A peptide or an F2A peptide.

7. An adeno-associated virus VP1 capsid protein mutant characterized by: encoded by a nucleic acid according to any one of claims 1 to 6.

8. An insect cell, characterized in that: comprising the nucleic acid of any one of claims 1-6, which is a baculovirus vector.

9. The insect cell of claim 8, wherein: the insect cells are spodoptera frugiperda cells, drosophila cells or mosquito cells.

10. The insect cell of claim 8 or 9, wherein: and the recombinant strain also comprises another baculovirus vector, wherein the other baculovirus vector comprises an expression cassette of a rep gene of AAV, an exogenous gene and AAV inverted terminal repeated sequences positioned at two ends of the exogenous gene.

11. The insect cell of claim 10, wherein: the exogenous gene is a reporter gene, and the reporter gene is at least one of chloramphenicol acetyl transferase coding gene, beta-galactosidase coding gene, beta-glucuronidase coding gene, renilla luciferase coding gene, alkaline phosphatase coding gene, firefly luciferase coding gene, green fluorescent protein coding gene and red fluorescent protein coding gene.

12. The insect cell of claim 10, wherein: the exogenous gene is a gene encoding a pharmaceutical polypeptide, and the pharmaceutical polypeptide is at least one of lipoprotein esterase, apolipoprotein, cytokine, interleukin and interferon.

13. A recombinant adeno-associated virus particle, characterized in that: the capsid of the recombinant adeno-associated virus particle comprises the adeno-associated virus VP1 capsid protein mutant of claim 7.

14. A recombinant adeno-associated virus particle, characterized in that: produced in an insect cell according to any one of claims 8-12.

15. A method for producing recombinant adeno-associated virus particles, characterized by: the insect cell of any one of claims 8-12 is cultured under conditions that produce recombinant adeno-associated virus particles, and then recovered.