WO2023103662A1 - Mutant de virus adéno-associé approprié pour une infection spécifique de cellules u87-mg - Google Patents

Mutant de virus adéno-associé approprié pour une infection spécifique de cellules u87-mg Download PDF

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WO2023103662A1
WO2023103662A1 PCT/CN2022/129386 CN2022129386W WO2023103662A1 WO 2023103662 A1 WO2023103662 A1 WO 2023103662A1 CN 2022129386 W CN2022129386 W CN 2022129386W WO 2023103662 A1 WO2023103662 A1 WO 2023103662A1
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aav2
nucleic acid
cells
mutant
capsid protein
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PCT/CN2022/129386
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Chinese (zh)
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杨佳丽
杨兴林
潘讴东
马佩敏
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和元生物技术（上海）股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to virus packaging and screening, in particular to the screening of adeno-associated virus mutants.
  • Glioma is a common primary malignant brain tumor of the central nervous system. It has the characteristics of high recurrence, poor prognosis, and high mortality. It is one of the deadliest tumors at present. At present, there is no particularly effective treatment strategy for glioma, and the existing core treatment methods are still conventional maximal excision of the tumor, as well as radiotherapy and chemotherapy. However, due to the characteristics of malignant invasive growth of glioma, conventional surgery, chemotherapy and radiotherapy can only slightly improve the quality of life and slightly prolong the survival period of some patients, and have no effect on the five-year survival rate of glioma after diagnosis. Significant improvement, and easy to relapse.
  • Adeno-associated virus is a small, non-enveloped and icosahedral virus. It is the simplest single-stranded DNA defective virus found so far, and it needs helper virus (usually adenovirus or herpes virus) to complete the virus packaging. Due to its good safety, wide range of host cells (dividing and non-dividing cells), low immunogenicity, and long-term expression of foreign genes in vivo, it is regarded as one of the most promising gene transfer vectors and is widely used in the world. It has been widely used in gene therapy and vaccine research.
  • U87-MG is a human brain astroglioblastoma cell, which has been widely used all over the world since it was first established by Uppsala University in Sweden in 1966; U87-MG is derived from malignant glioma, and this cell is naked Subcutaneous inoculation in mice can form tumors, so this cell line is often used as a tool model for human glioblastoma research in vitro, and relevant mechanism research and therapeutic effect testing are carried out on this cell line.
  • the transfection efficiency of this cell is not high, and the infection efficiency of the adeno-associated virus AAV is low, and there is no specific infection serotype at present.
  • AAV2/2 AAV2/2 infects glioma cell line (U87-MG) with low efficiency and poor specificity. Infection works better.
  • the present invention utilizes AAV2/2
  • the AAV serotype mutants that can efficiently and specifically infect U87-MG were obtained through library screening, which provides a powerful tool for studying the mechanism of glioma development in vitro and the development of glioma therapeutic drugs .
  • the invention discloses a heterologous peptide, characterized in that the heterologous peptide is:
  • (b) A polypeptide derived from (a) in which the amino acid sequence in (a) has been substituted, deleted or added with one or several amino acids and has heterologous peptide activity.
  • the invention also discloses the AAV2/2 capsid protein with site-directed mutation, which is characterized in that the heterologous peptide is inserted between amino acids 587-588 of the capsid protein.
  • the invention discloses an AAV2/2 serotype mutant, which is characterized in that it includes the capsid protein.
  • the invention discloses a nucleic acid molecule, which is characterized in that it encodes the capsid protein or the mutant.
  • the invention discloses a nucleic acid carrier, which is operably linked with the nucleic acid molecule.
  • the present invention provides a host cell, which is characterized by containing the nucleic acid vector.
  • the present invention provides a composition or a kit containing the capsid protein or the mutant described in 3 or the nucleic acid molecule or the nucleic acid carrier.
  • the invention also discloses the use of the capsid protein or the mutant or the nucleic acid molecule or the nucleic acid carrier in the preparation of drugs for treating tumors.
  • the tumor is human brain astroglioblastoma.
  • the invention discloses the use of the mutant or the capsid protein or the nucleic acid molecule or the nucleic acid carrier for infecting and/or killing U87-MG cells.
  • the present invention has the following beneficial effects:
  • AAV serotype mutants according to the present invention can specifically infect U87-MG cells, while the wild control group has a poor effect on infecting U87-MG cells and a good effect on infecting 293T cells;
  • Fig. 1 is the structural diagram of pAAV-short UBC-mScarlet-polyA-P40-AAV2-Cap-FLEX-SV40 polyA in Example 1 of the present invention
  • Fig. 2 is the fluorescence picture of U87-MG cell infected with library virus in embodiment 2;
  • Fig. 3 is the fluorescence diagram of mutant serotype and AAV2 virus infected cells in Example 3 of the present invention.
  • Fig. 4 is the fluorescence diagram of mutant serotype and AAV2 virus infection 293T and U87-MG in Example 4 of the present invention
  • Fig. 5 is the statistical diagram of the killing experiment results of mutant serotypes and AAV2 virus infection 293T and U87-MG in Example 4 of the present invention
  • Fig. 6 is a curve diagram of tumor killing volume in vivo between mutant serotype and AAV2 virus in Example 5 of the present invention.
  • Figure 7 is a vector map of the shuttle vector pAAV-CAG-mCherry-WPRE in Example 3 of the present invention.
  • Figure 8 is a map of the pAAV-SV40 enhancer-hTERT-EGFP vector in Example 4 of the present invention.
  • Fig. 9 is a map of pAAV-SV40 enhancer-hTERT-TK vector in Example 4 of the present invention.
  • the mutated site is based on the serotype of AAV2/2, and the NCBI number of the wild-type AAV2/2 capsid protein sequence is: NC_001401.
  • AAV peptide mutant library virus packaging Inoculate 1.5*10 ⁇ 7 293AAV packaging cells per dish into a 15cm cell culture dish, culture for 18-24h, and start transfection when the cells adhere to the wall.
  • the pAAV-short UBC-mScarlet-polyA-P40-AAV2-Cap-FLEX-SV40 polyA-insertion expression vector library containing AAV2/2-7mer-NNS insert fragment was packaged with Rep plasmid, and pHelper auxiliary plasmid was transfected.
  • 293AAV cells 72 hours after transfection, count the ratio of vector library cells in AAV-293 cells under a fluorescent microscope to determine the virus packaging efficiency. After the virus packaging is completed, blow the cells repeatedly with the tip of the pipette to completely detach all the cells from the culture dish, and collect all cell samples.
  • the collected cell samples were repeatedly frozen and thawed at -80°C and 37°C, centrifuged, and the cell supernatant was collected, and the cell debris was removed with a 0.45 ⁇ m PVDF filter, and then the AAV purification kit was used to purify the virus.
  • the collected recombinant AAV virus was purified to obtain the recombinant AAV virus.
  • the above sample was diluted and used as a template, and the titer of the recombinant AAV virus was determined by a real-time quantitative PCR detection method.
  • the qPCR reaction system and reaction conditions are: 95°C, 10min; 95°C, 30s; 60°C, 30s, 35 cycles.
  • U87-MG cells were infected with pAAV-short UBC-mScarlet-polyA-P40-AAV2-Cap-FLEX-SV40 polyA-insertion expression vector library virus.
  • Amplification primers :
  • AAV2-F AACCAATCCCGTGGCTACGGAGC (forward primer on vector, SEQ ID NO: 3)
  • AAV2-R CCAGACCATGCCTGGAAGAACGC (reverse primer on vector, SEQ ID NO: 4)
  • the primers used for high-throughput sequencing with adapters and index sequences are as follows:
  • NGS-AAV2-F TTACTATGCCGCTGGTGGCTCTAGATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGAAACCAATCCCGTGGCTACGGAGC (SEQ ID NO: 5)
  • sequence sequence expected to be obtained is:
  • Figure 7 is a vector map of the shuttle vector pAAV-CAG-mCherry-WPRE
  • AAV2/2-GLxx and other viruses of pAAV-CAG-mCherry-WPRE were respectively infected with 293T, NIH3T3 and U87-MG cells at an MOI (multiplicity of infection) of 1*10 5 , and the fluorescence images of infection for 72 hours are shown in Figure 3.
  • MOI multiplicity of infection
  • AAV2-GL03 and AAV2-GL05 are less effective in infecting 293T and NIH3T3 cells than the AAV2 control group (AAV2 WT), but have a good infection effect in the target cell U87-MG, indicating that AAV2-GL03 and AAV2-GL05 serotype can specifically infect U87-MG cells.
  • the mutant peptide sequence of AAV2-GL03 and AAV2-GL05 is KENDTKG (SEQ ID NO: 10) or LHANKLE (SEQ ID NO: 11).
  • This example discloses the in vitro killing experiment of the pAAV-SV40 enhancer-hTERT-TK-GCV system based on AAV2 WT, AAV2-G03, and AAV2-GL05 serotypes.
  • AAV2 WT, AAV2-GL03, and AAV2-GL05 as serotypes to package adeno-associated virus vectors pAAV-SV40 enhancer-hTERT-EGFP and pAAV-SV40 enhancer-hTERT-TK, after infecting 293T and U87-MG cells for 72 hours, respectively.
  • the cell viability was detected by CTG method after 72 hours, and the killing effect of AAV2-SV40 enhancer-hTERT-TK-GCV system packaged with different serotypes AAV2 WT, AAV2-GL03, and AAV2-GL05 on in vitro cultured cells was evaluated.
  • Figure 8 is a map of the pAAV-SV40 enhancer-hTERT-EGFP vector
  • Figure 9 is a map of the pAAV-SV40 enhancer-hTERT-TK vector.
  • AAV2-GL03 and AAV2-GL05 are less effective than AAV2 control group (AAV2 WT) in infecting 293T cells, but It can well infect U87-MG cells.
  • pAAV-SV40 enhancer-hTERT-TK group, AAV2/2 wild type (control group) have a certain killing effect on 293T and U87-MG cells, and in 293T
  • the cell survival rate is lower and the killing rate is higher; it shows that the AAV2/2 wild type is non-specific for the infection of U87-MG cells; while AAV2-GL03 and AAV2-GL05 have obvious killing effects on U87-MG, and the survival rate is 15.8 % and 24.4%; while the killing of 293T was not obvious, the survival rate was higher, the survival rate was 80.0% and 77.2%, respectively.
  • AAV2-GL03 and AAV2-GL05 serotypes can specifically infect U87-MG cells.
  • This example discloses the killing experiment of the pAAV-SV40 enhancer-hTERT-TK-GCV system based on AAV2 WT, AAV2-G03, and AAV2-GL05 serotypes in an in vivo tumor animal model.

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Abstract

L'invention concerne un mutant de virus adéno-associé approprié pour l'infection spécifique de cellules U87-MG. Un mutant de sérotype d'AAV capable d'infecter spécifiquement U87-MG est obtenu au moyen d'un criblage de banque à l'aide d'une banque de mutations de fragments peptidiques de AAV2/2. Une expérience d'élimination in vitro prouve que le mutant de Sérotype d'AAV obtenu par criblage peut spécifiquement infecter et tuer des cellules U87-MG.
PCT/CN2022/129386 2021-12-10 2022-11-03 Mutant de virus adéno-associé approprié pour une infection spécifique de cellules u87-mg WO2023103662A1 (fr)

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CN202111507712.9A CN114213505B (zh) 2021-12-10 2021-12-10 一种适用于特异感染u87-mg细胞的腺相关病毒突变体
CN202111507712.9 2021-12-10

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CN114213505B (zh) * 2021-12-10 2022-09-20 和元生物技术（上海）股份有限公司 一种适用于特异感染u87-mg细胞的腺相关病毒突变体

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