WO2024067153A1 - Acide nucléique pour la production de raav dans une cellule d'insecte, mutant de protéine de capside vp1 et utilisation - Google Patents

Acide nucléique pour la production de raav dans une cellule d'insecte, mutant de protéine de capside vp1 et utilisation Download PDF

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WO2024067153A1
WO2024067153A1 PCT/CN2023/119002 CN2023119002W WO2024067153A1 WO 2024067153 A1 WO2024067153 A1 WO 2024067153A1 CN 2023119002 W CN2023119002 W CN 2023119002W WO 2024067153 A1 WO2024067153 A1 WO 2024067153A1
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protein
gene
nucleic acid
intron
associated virus
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肖何
何晓斌
黄刚
胡颖
潘杏
王梦蝶
杜亮
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劲帆生物医药科技（武汉）有限公司
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    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
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    • C12N2750/14011—Parvoviridae
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention belongs to the technical field of genetic engineering, and more specifically, relates to a nucleic acid, a VP1 capsid protein mutant and an application thereof for producing rAAV in insect cells.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the genus Dependoviridae of the family Parvoviridae. It is the simplest single-stranded DNA defective virus discovered so far and requires a helper virus (usually adenovirus) to participate in replication.
  • Recombinant adeno-associated virus rAAV is one of the most promising vectors in the field of gene therapy due to its wide host range, low immunogenicity, high safety, and ability to mediate long-term stable expression of exogenous genes in animals.
  • rAAV adeno-associated virus
  • rAAV recombinant adeno-associated virus
  • mammalian cells such as 293 cells, COS cells, HeLa cells, KB cells, etc.
  • insect cells such as 293 cells, COS cells, HeLa cells, KB cells, etc.
  • the baculovirus/insect cell expression system is one of the main systems for large-scale production of rAAV.
  • the cap gene in the recombinant adeno-associated virus genome encodes the viral VP capsid protein, including three structural proteins, namely VP1, VP2 and VP3.
  • the stoichiometric ratio of VP1, VP2 and VP3 in AAV from wild-type virus is about 1:1:10. Such a stoichiometric ratio is very important for obtaining recombinant AAV.
  • baculovirus/insect cell expression system has been successfully used for rAAV production at various scales, several studies have reported that production in insect cells is superior to that in mammalian cells.
  • the rAAV produced has a reduced VP1 content, resulting in a reduced yield of rAAV. Therefore, to achieve industrial large-scale production of rAAV, the baculovirus/insect cell system still needs to be improved.
  • the purpose of the present invention is to provide a nucleic acid, a VP1 capsid protein mutant and an application for producing rAAV in insect cells, aiming to solve the problem that the wild-type AAV serum type 8 VP1 protein is unstable and easily degraded, thereby leading to a decrease in rAAV yield.
  • the present invention provides a nucleic acid comprising a nucleotide sequence encoding adeno-associated virus VP1, VP2 and VP3 proteins, wherein the amino acid sequence of the VP1 protein comprises mutations at one or more positions 84, 92 and 105 compared to the capsid protein of wild-type AAV serotype 8, and the amino acid sequence of the VP2 protein is not mutated.
  • the amino acid sequence of the VP1 protein comprises one or more mutations selected from the group consisting of: Q84K, R92K and Q105K.
  • the other amino acid sequence of the VP1 protein is identical to the amino acid sequence of the VP1 protein of wild-type AAV serotype 8.
  • the nucleic acid further comprises a promoter capable of driving transcription in insect cells, and the 3' end of the promoter is operably connected to an intron;
  • the start codon ATG of the VP1 protein is located in the sequence of the intron, or the cap gene coding region corresponding to the VP1 protein lacks the start codon and the intron is located between any two adjacent nucleotides in the ATG;
  • the intron does not comprise a promoter capable of driving transcription in insect cells;
  • the nucleic acid can only be transcribed into one precursor mRNA in insect cells, and in the post-transcriptional processing, only through the selective splicing of the intron, the start codon ATG of the VP1 protein is retained or deleted, or the start codon ATG is formed in the cap gene coding region corresponding to the VP1 protein, thereby achieving the regulation of the translation expression of the VP1, VP2 and VP3 proteins.
  • 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 connected to a nucleotide sequence encoding a 2A self-cleaving polypeptide. List.
  • the 2A self-cleaving polypeptide is a T2A peptide, a P2A peptide, an E2A peptide or a F2A peptide.
  • the present invention provides an adeno-associated virus VP1 capsid protein mutant, which is encoded by the nucleic acid described in the first aspect of the present invention.
  • the present invention provides an insect cell comprising the nucleic acid according to the first aspect of the present invention, wherein the nucleic acid is a baculovirus vector.
  • the insect cell is a Spodoptera frugiperda cell, a Trichoplusia ni cell, a Drosophila cell or a mosquito cell.
  • the insect cell further comprises another baculovirus vector, wherein the other baculovirus vector comprises an AAV rep gene expression cassette, a foreign gene, and AAV inverted terminal repeat sequences located at both ends of the foreign gene.
  • the other baculovirus vector comprises an AAV rep gene expression cassette, a foreign gene, and AAV inverted terminal repeat sequences located at both ends of the foreign gene.
  • the exogenous gene is a reporter gene
  • the reporter gene is at least one of a chloramphenicol acetyltransferase encoding gene, a ⁇ -galactosidase encoding gene, a ⁇ -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.
  • the exogenous gene is a gene encoding a drug polypeptide
  • the drug polypeptide is at least one of lipoprotein esterase, apolipoprotein, cytokine, interleukin and interferon.
  • the present invention provides a recombinant adeno-associated virus particle, the capsid of which comprises the adeno-associated virus VP1 capsid protein mutant described in the second aspect of the present invention.
  • the present invention further provides a recombinant adeno-associated virus particle, which is produced in the insect cell described in the third aspect of the present invention.
  • the present invention provides a method for producing recombinant adeno-associated virus particles, which is prepared by culturing the insect cells described in the third aspect under conditions capable of producing recombinant adeno-associated virus particles and then recovering them.
  • the present invention reduces the degradation of VP1 protein in insect cells by mutating the amino acid sequence of the unique region of the VP1 protein of wild-type AAV serotype 8 at specific sites (positions 84, 92, and 105) without mutating the existing disclosed protein hydrolysis region, thereby obtaining a more appropriate ratio of VP1, VP2, and VP3 proteins, which are correctly assembled into the capsid of rAAV, thereby improving the yield and efficacy of rAAV production using the baculovirus/insect cell expression system.
  • Figure 1 is a Western Blot detection image of the recombinant baculovirus vector Ac-Cap8 expressing VP protein in Example 2 of the present invention.
  • Figure 2 is a Western Blot detection diagram of the recombinant baculovirus vector Ac-Cap9 expressing VP protein in Example 2 of the present invention.
  • FIG3 is an alignment of the amino acid sequences from position 1 to position 137 of the N-terminus of the VP1 protein encoded by the cap gene of AAV serotypes 8 and 9 provided in Example 3 of the present invention.
  • Figure 4 is a Western Blot detection diagram of the VP protein expressed by the recombinant baculovirus vectors Ac-Cap8, Ac-Cap8-Q84K, Ac-Cap8-R92K and Ac-Cap8-Q105K in Example 3 of the present invention.
  • Figure 5 is a silver staining detection image of SDS-PAGE of the purified recombinant AAV virus particles after the AAV recombinant baculovirus Ac-Rep-ITR co-infected the host cells with Ac-Cap8, Ac-Cap8-Q84K, Ac-Cap8-R92K, and Ac-Cap8-Q105K in Example 6 of the present invention, showing the three capsid proteins VP1, VP2, and VP3.
  • operably linked refers to a polynucleotide (or polypeptide) sequence that is functionally linked. When two nucleotide sequences are placed in a functional relationship, they are "operably linked".
  • a transcriptional regulatory sequence such as a promoter
  • a gene coding sequence if it affects the transcription of the gene coding sequence.
  • coding region means that genes include coding regions and non-coding regions.
  • the coding region can be used as an open reading frame (ORF), starting from the start codon and ending at the stop codon. It may include introns and protein-coding nucleotide sequences; the non-coding region includes cis-acting elements before and after the coding region, including promoters, enhancers, etc.
  • expression cassette refers to a nucleic acid construct comprising a coding sequence and a regulatory sequence that are operably linked when introduced into a host cell, resulting in transcription and/or translation of an RNA or polypeptide, respectively.
  • An expression cassette is understood to include a promoter that allows transcription to begin, a target gene open reading frame, and a transcription terminator. Typically, the promoter sequence is placed upstream of the target gene, and the distance from the target gene is compatible with expression control.
  • intron also known as the intervening sequence, refers to a non-coding fragment in a gene or mRNA molecule, and is an intervening sequence in the DNA of eukaryotic cells.
  • the intron sequence is transcribed in the mRNA precursor, removed by splicing, and ultimately does not exist in the mature mRNA molecule. According to whether the splicing process is spontaneous or requires processing by the spliceosome, introns are divided into self-splicing introns and spliceosomal introns.
  • Self-splicing introns are a special type of intron, a ribozyme that can be removed by its own action to leave the mRNA.
  • the intron involved in the present invention is a spliceosomal intron.
  • the splicing of this type of intron requires the help of the spliceosome.
  • the spliceosome is a ribonucleoprotein complex dynamically composed of small nuclear RNA (snRNA) and protein factors. The spliceosome recognizes the splicing site of the mRNA precursor and catalyzes the splicing reaction, completely splicing the intron, and reconnecting the upstream and downstream RNA sequences.
  • AAV a single-stranded DNA virus with a simple genome structure and a total length of about 4.7kb. Its genome contains the rep gene expression cassette, the cap gene expression cassette, and the AAV inverted terminal repeats (ITRs) at both ends of the genome. ITRs are palindromic structures of 125 nucleotides at both ends of the genome that can form a self-
  • the complementary inverted T-shaped hairpin structure is a cis-acting element required for the initiation of DNA replication and packaging of the recombinant AAV genome into infectious viral particles.
  • AAV As a defective virus, AAV cannot replicate independently without the presence of a helper virus, so AAV can only be site-specifically integrated into the host cell chromosome and is in a latent state. In the presence of a helper virus, the increased expression of the rep gene can rescue the AAV genome integrated into the host cell chromosome, and a large amount of AAV DNA is obtained by replication.
  • the single-stranded rAAV genome is packaged into infectious viral particles under the action of the VP capsid protein.
  • the Cap gene encodes the structural VP capsid protein, which contains 3 overlapping open reading frames, encoding three types of subunits: VP1, VP2, and VP3. VP1, VP2, and VP3 contain different start codons and share a stop codon.
  • VP1 and VP2 share the VP3 sequence.
  • the N-terminus of VP1 has a conserved phospholipase A2 sequence, which is related to the virus's escape from the body and is crucial to its infectivity; the VP2 protein is essential for assembly or infection; the core of the VP3 protein is composed of a conserved ⁇ -barrel motif, which determines the difference between the receptors of different serotypes of AAV and host cells.
  • the correct ratio of the three proteins in wild-type AAV is 3:3:54, which is about 1:1:10.
  • the Rep gene encodes four overlapping multifunctional proteins, Rep78, Rep68, Rep52 and Rep40.
  • Rep78 and Rep68 proteins are involved in the replication and integration of AAV and can bind to specific sequences in ITR; Rep52 and Rep40 proteins have helicase and ATPase activities, and participate in the replication of the single-stranded genome while also participating in the assembly of the virus.
  • the present invention aims to mutate one or more codons of the cap gene so that the VP1 protein encoded by the gene can be more stable during the production process, thereby increasing the yield of rAAV.
  • the present invention provides a nucleic acid comprising a nucleotide sequence encoding adeno-associated virus VP1, VP2 and VP3 proteins, and compared with the capsid protein of wild-type AAV serotype 8, the amino acid sequence of the VP1 protein comprises mutations at one or more sites in positions 84, 92 and 105, and the amino acid sequence of the VP2 protein is not mutated.
  • Chinese patent document CN113728108A proposes to remove the unique region of AAV VP1/VP2 (i.e., the region common to VP1 and VP2 but lacking in VP3) that is easily proteolytically cleaved in insect cells. site, which can lead to rAAV products of higher purity and uniformity and improved capsid proteins. It is also proposed that the unique region of VP1 (i.e., the region present in VP1 but not in VP2 or VP3) contains a PLA2 domain, and mutations in this region can increase the enzymatic activity of the PLA2 domain in the resulting VP1 protein. At the same time, Grirod A et al.
  • mutations in the unique region of the VP1 protein can significantly reduce the infectivity of the mutant virus (J Gen Virol. 2002, 83 (5): 973-978).
  • mutations at certain sites in the unique region of VP1 can improve the resistance of VP1 protein to protein degradation.
  • the inventor unexpectedly discovered that one or more of the 84th, 92nd and 105th amino acids in the unique region of VP1 of AAV serotype 8 (corresponding to residues 1 to 137 of SEQ ID No.
  • the capsid proteins VP1, VP2 and VP3 of the wild-type AAV serotype 9 have a suitable ratio.
  • one or more of the amino acids at positions 84, 92 and 105 of the VP1 protein of AAV serotype 8 are mutated into the same amino acids as AAV serotype 9, that is, the amino acid sequence of the mutated VP1 protein contains one or more mutations selected from the following: Q84K, R92K and Q105K.
  • the amino acid sequence of the VP1 protein undergoes mutations at only one or more of the above three sites (positions 84, 92, and 105), and no other sequences are mutated.
  • an intron selective splicing strategy can also be introduced (refer to the patent CN113897396B previously applied by the inventor) to regulate the expression of VP1, VP2 and VP3 proteins in the overlapping open reading frame of the AAV cap gene according to the correct stoichiometry.
  • the nucleic acid of the present invention comprises a complete cap gene expression cassette, that is, the nucleic acid also comprises a promoter capable of driving transcription in insect cells, and the 3' end of the promoter is operably connected to an intron; the start codon ATG of the VP1 protein is located in the sequence of the intron, or the cap gene coding region corresponding to the VP1 protein lacks the start codon and the intron is located between any two adjacent nucleotides in ATG; the intron does not contain a promoter capable of driving transcription in insect cells;
  • the nucleic acid can only be transcribed into one precursor mRNA in insect cells, and in the post-transcriptional processing, only through the selective splicing of the intron, the start codon ATG of the VP1 protein is retained or deleted, or the start codon ATG is formed in the cap gene coding region corresponding to the VP1 protein, thereby achieving the regulation of the translation expression of the VP1, VP2 and VP3 proteins.
  • 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 connected to a nucleotide sequence encoding a 2A self-cleaving polypeptide.
  • this embodiment adopts the intron regulation strategy, it is necessary to mutate the intron splicing site at the N-terminus of the wild-type AAV serum type 8 VP1 protein coding sequence itself, so that the splicing body in the insect cell cannot recognize the splicing site.
  • the splicing body in the insect cell recognizes the splicing site of the intron and catalyzes the splicing reaction, the front-end AUG-start site of the mRNA is removed, and when the ribosome recognizes the start codon of the VP2 protein from 5' to 3', the VP2 protein is translated and expressed.
  • the VP2 start codon is the suboptimal codon ACG, it will cause ribosome scanning leakage, so the VP3 protein is translated and expressed; if the intron splicing site is not recognized and the AUG-start site is not removed, the mRNA is translated from the first AUG, and the VP1 protein is expressed.
  • the relative expression levels of the capsid proteins VP1, VP2 and VP3 are controlled by the splicing effect of the intron with a certain probability.
  • the coding sequence of 2A self-cleaving peptides is introduced into the cap gene expression cassette provided by the present invention.
  • 2A self-cleaving peptides are a class of peptide fragments with 18-22 amino acid residues in length, which can induce self-cleavage of recombinant proteins containing 2A peptides in cells.
  • This type of peptide has a sequence motif, which often prevents the ribosome from connecting at the last glycine (G) and proline (P) connection, resulting in a "cleavage" effect, causing the C-terminus of the 2A self-cleaving peptide to disconnect from the N-terminus of the VP1 protein to obtain a normal VP1 protein.
  • G glycine
  • P proline
  • the present invention provides an adeno-associated virus VP1 capsid protein mutant, which is encoded by the nucleic acid provided by the present invention.
  • the present invention also provides an insect cell, which comprises the nucleic acid provided by the present invention, wherein the nucleic acid is a baculovirus vector.
  • the insect cell can be any insect cell, such as but not limited to a Spodoptera frugiperda cell, a Trichoplusia cell, a Drosophila cell or a mosquito cell, preferably a Spodoptera frugiperda cell sf9.
  • the concepts of the present invention can be applied to any existing suitable baculovirus/insect cell expression system to produce rAAV, including but not limited to the triple baculovirus system (Urabe M et al., Hum Gene Ther, 2002, 13(16):1935-1943), the double baculovirus system (Chen et al., Mol Ther, 2008, 16(5):924-930; Smith R H et al., Mol Ther, 2009, 17(11):1888-1896), the One Bac system (Mietzsch M et al., Hum Gene Ther, 2014, 25(3):212-222) and the Monobac system (Galibert L et al., Abstract at the ESGCT Meeting, Madrid/Spain, 2013, P074).
  • the triple baculovirus system Urabe M et al., Hum Gene Ther, 2002, 13(16):1935-1943
  • the double baculovirus system Choen et al., Mol Ther, 2008,
  • mutations are introduced into a dual baculovirus system, wherein the insect cells contain two baculovirus vectors, one containing a mutated cap gene expression cassette, and the other containing an AAV rep gene expression cassette, an exogenous gene, and AAV inverted terminal repeat sequences located at both ends of the exogenous gene.
  • the exogenous gene can be at least one nucleotide sequence encoding a gene of interest (GOI) product
  • the gene of interest product can be a therapeutic gene product, specifically a polypeptide, an RNA molecule (siRNA) or other gene products, such as but not limited to lipoprotein esterase, apolipoprotein, cytokine, interleukin or interferon; it can also be a reporter protein for evaluating vector transformation and expression, such as but not limited to fluorescent protein (green fluorescent protein GFP, red fluorescent protein RFP), chloramphenicol acetyltransferase, ⁇ -galactosidase, ⁇ -glucuronidase, Renilla luciferase, firefly luciferase or alkaline phosphatase.
  • fluorescent protein green fluorescent protein GFP, red fluorescent protein RFP
  • chloramphenicol acetyltransferase ⁇ -galactosidase, ⁇ -glucuronidase, Ren
  • the present invention provides a recombinant adeno-associated virus particle, wherein the capsid of the recombinant adeno-associated virus particle comprises
  • the invention contains the adeno-associated virus VP1 capsid protein mutant provided by the invention, and the VP1 capsid protein mutant is encoded by the mutated VP1 protein coding sequence of the invention.
  • the recombinant adeno-associated virus particles provided by the present invention can be produced in the insect cells provided by the present invention using the baculovirus/insect cell expression system.
  • the present invention also provides a method for producing recombinant adeno-associated virus particles, which is prepared by culturing the insect cells provided by the present invention under conditions capable of producing recombinant adeno-associated virus particles and then recovering the particles.
  • cap gene expression cassettes Referring to the method described in Example 1 of Chinese patent CN113897396B, cap gene expression cassettes of AAV serotype 8 and AAV serotype 9 were constructed respectively.
  • the cap gene expression cassettes included, from 5' to 3', p10 promoter, intron, nucleotide sequence encoding T2A peptide and nucleotide sequence encoding AAV serotype 8 or 9 VP protein lacking only the VP1 protein translation start codon ATG.
  • the fragments were connected by direct artificial synthesis or overlap extension PCR amplification to obtain constructs Cap8 and Cap9, respectively, whose nucleotide sequences are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.
  • the recombinant baculovirus vectors Ac-Cap8 and Ac-Cap9 prepared in Example 1 were respectively transfected into host cell lines to obtain recombinant baculoviruses, and the expression of VP proteins (VP1, VP2, VP3) was detected.
  • the specific operation steps are as follows:
  • the cell culture fluid was centrifuged at 1000g for 5 minutes, and the culture supernatant and cell pellet were collected separately. The supernatant was marked as the 1st generation BEV-P1.
  • the cell culture fluid was centrifuged at 1000 g for 5 min, and the cell pellets were collected for Western Blot examination of the expression of VP proteins (VP1, VP2, VP3).
  • Figures 1 and 2 are Western Blot detection images of VP proteins (VP1, VP2 and VP3) of recombinant baculovirus vectors Ac-Cap8 and Ac-Cap9 containing cap gene expression cassettes, respectively.
  • both recombinant baculovirus vectors can successfully produce VP1, VP2 and VP3; the ratio of VP1 and VP2 proteins produced by the recombinant baculovirus vector Ac-Cap9 is close to 1:1, while the recombinant baculovirus vector Ac-Cap8 produces too low a VP1 protein content.
  • the ratio of VP proteins (VP1, VP2 and VP3) produced by the two should be consistent.
  • the inventors speculate that the low detection amount of VP1 protein may be due to the unstable structure of AAV serum type 8 VP1 protein, which is easily degraded in sf9 cells.
  • Example 3 Construction of a recombinant baculovirus vector containing an AAV serotype 8 cap gene mutant expression cassette and detection of VP protein (VP1, VP2, VP3) expression
  • the recombinant baculovirus vector Ac-Cap8 has an abnormal VP1 protein expression content. Therefore, the amino acid sequences of the VP1 proteins encoded by the cap genes of AAV serotypes 8 and 9 were compared.
  • the amino acid sequence of the VP1 protein of AAV serotype 8 is shown in SEQ ID No.3, and the amino acid sequence of the VP1 protein of AAV serotype 9 is shown in SEQ ID No.4. It was found that the amino acid sequence of the VP1 protein of AAV serotype 8 was There is a difference in the N-terminal amino acid sequence of the VP1 protein of type 9. It is inferred that the difference in the N-terminal amino acid sequence of the VP1 protein leads to the abnormal expression level of the VP1 protein in the recombinant baculovirus vector Ac-Cap8.
  • the 84th, 92nd and 105th amino acids at the N-terminus of the VP1 protein encoded by the cap gene of AAV serotype 8 were mutated into amino acids corresponding to the N-terminus of the VP1 protein of AAV serotype 9, respectively.
  • Figure 3 shows the amino acid sequences of positions 1 to 137 at the N-terminus of the VP1 protein encoded by the cap gene of AAV serotypes 8 and 9, wherein the mutation sites (Q84K, R92K and Q105K) are marked with black boxes.
  • this example respectively constructs recombinant baculovirus vectors Ac-Cap8-Q84K, Ac-Cap8-R92K and Ac-Cap8-Q105K containing AAV serotype 8 cap gene mutant expression cassettes.
  • the glutamine at position 84 in the amino acid sequence of the VP1 protein encoded by the cap gene of the recombinant baculovirus vector Ac-Cap8-Q84K is mutated to lysine, and the corresponding codon CAG is mutated to AAG;
  • the arginine at position 92 in the amino acid sequence of the VP1 protein encoded by the cap gene of the recombinant baculovirus vector Ac-Cap8-R92K is mutated to lysine, and the corresponding codon CGG is mutated to AAG;
  • the glutamine at position 105 in the amino acid sequence of the VP1 protein encoded by the cap gene of the recombinant baculovirus vector Ac-Cap8-Q105K is
  • the recombinant baculovirus vectors Ac-Cap8-Q84K, Ac-Cap8-R92K and Ac-Cap8-Q105K prepared in this example were transfected into host cell lines to culture and obtain recombinant baculoviruses, and the expression of VP proteins (VP1, VP2, VP3) was detected.
  • Figure 4 is a Western Blot detection diagram of VP proteins (VP1, VP2 and VP3) of recombinant baculovirus vectors Ac-Cap8, Ac-Cap8-Q84K, Ac-Cap8-R92K and Ac-Cap8-Q105K containing cap gene expression cassettes.
  • the VP1 protein content produced by Ac-Cap8-Q84K, Ac-Cap8-R92K and Ac-Cap8-Q105K is significantly increased. It can be seen that the site-directed mutation of the N-terminal amino acid of AAV serotype 8 VP1 protein can improve the stability of VP1 protein, thereby significantly increasing the relative content of VP1 protein.
  • the present invention adopts the Two-bac system for AAV packaging, wherein one bacmid contains the Cap expression cassette of AAV, and the other bacmid contains the Rep gene expression cassette and the ITR core element, and the two recombinant baculoviruses are used to co-infect sf9 cells for AAV packaging.
  • the essential elements for AAV packaging namely the Rep gene expression cassette and the ITR core element (ITR-GOI) were constructed.
  • the Rep gene expression cassette was constructed with reference to the method described in Example 2 of Chinese patent CN113897396B, and its nucleotide sequence is shown in SEQ ID No.5; the nucleotide sequence of the ITR-GOI is shown in SEQ ID No.6.
  • the GOI in the ITR core element adopts the red fluorescent protein mcherry gene expression cassette, that is, the mcherry expression is controlled by the miniEf1a promoter.
  • 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 respectively transfected into host cell lines for culture to obtain AAV recombinant baculovirus.
  • the above-mentioned recombinant bacmid DNA was extracted and transfected into Sf9 insect cells to prepare AAV recombinant baculovirus (BEV).
  • BEV AAV recombinant baculovirus
  • the transfected Sf9 insect cells successfully produced BEV, and the massively replicated and proliferated BEV further infected Sf9 cells, causing obvious cytopathic effect (CPE).
  • CPE cytopathic effect
  • the culture supernatant of Sf9 cells with CPE was collected, which contained a large amount of BEV, namely the 0th generation BEV (P0).
  • the cell culture fluid was centrifuged at 1000g for 5 minutes, and the culture supernatant and cell pellet were collected respectively. The supernatant was marked as the 1s
  • Example 6 Purification of recombinant AAV virus particles and detection of their packaging efficiency and virus titer
  • MOI multiplicity of infection
  • Figure 5 provides the SDS-PAGE silver staining detection of purified virus particles after co-infection of host cells with AAV recombinant baculovirus Ac-Rep-ITR and Ac-Cap8, Ac-Cap8-Q84K, Ac-Cap8-R92K, and Ac-Cap8-Q105K, respectively.
  • the results show that the ratio of VP1 to VP2 proteins incorporated into AAV particles produced by Ac-Cap8-Q84K, Ac-Cap8-R92K, and Ac-Cap8-Q105K is close to 1:1, compared with Ac-Cap8 without mutation at the N-terminus of VP1 protein.
  • This example also uses Q-PCR to detect the packaging rate and supernatant titer of the harvested recombinant adeno-associated virus (rAAV), and the titer unit is expressed in VG/L (VG, virus genomes).
  • the detection of rAAV titer uses a pair of primers targeting the ITR sequence (Q-ITR-F: GGAACCCCTAGTGATGGAGTT and Q-ITR-R: CGGCCTCAGTGAGCGA) or a pair of primers targeting the WPRE sequence (Q-WPRE-F: CCGTTGTCAGGCAACGTG and Q-WPRE-R: AGCTGACAGGTGGTGGCAAT).
  • the test results are shown in Table 1.
  • rAAV produced by Ac-Cap8-Q84K, Ac-Cap8-R92K and Ac-Cap8-Q105K has higher cell packaging rate and yield.

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Abstract

L'invention concerne un acide nucléique pour la production de rAAV dans une cellule d'insecte, un mutant de protéine de capside VP1, et l'utilisation. L'acide nucléique comprend des séquences nucléotidiques codant pour des virus adéno-associés VP1, VP2 et des protéines VP3. Par comparaison avec la protéine de capside du sérotype 8 de l'AAV de type sauvage, la séquence d'acides aminés de la protéine VP1 comprend une mutation sur un ou plusieurs sites parmi les sites 84, 92 et 105, et la séquence d'acides aminés de la protéine VP2 n'est pas mutée. La séquence codante de la protéine VP1 est mutée, afin de réduire la dégradation de la protéine VP1 dans une cellule d'insecte, et les protéines VP1, VP2 et VP3 exprimées peuvent conserver un rapport adéquat pour assembler correctement une capside, améliorant ainsi le rendement et l'efficacité de la production de rAAV à l'aide d'un système d'expression baculovirus/cellule d'insecte.
PCT/CN2023/119002 2022-09-27 2023-09-15 Acide nucléique pour la production de raav dans une cellule d'insecte, mutant de protéine de capside vp1 et utilisation WO2024067153A1 (fr)

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