WO2023103661A1 - Mutant de virus adéno-associé approprié pour une infection spécifique de cellules u251 - Google Patents

Mutant de virus adéno-associé approprié pour une infection spécifique de cellules u251 Download PDF

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WO2023103661A1
WO2023103661A1 PCT/CN2022/129385 CN2022129385W WO2023103661A1 WO 2023103661 A1 WO2023103661 A1 WO 2023103661A1 CN 2022129385 W CN2022129385 W CN 2022129385W WO 2023103661 A1 WO2023103661 A1 WO 2023103661A1
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aav2
cells
nucleic acid
mutant
capsid protein
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杨兴林
杨佳丽
潘讴东
高花
祝翊倩
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
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    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2750/14133—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
    • 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 packaging and screening of the virus vector of the present invention particularly relates to the packaging and screening of AAV 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 (adeno-associated virus, AAV) is a small, non-encapsulated virus with an icosahedral structure. It is currently regarded as one of the most promising gene transfer vectors. widely used in vaccine research.
  • U251 human glioma cells this cell line is often used as a tool model for glioma research in vitro, and related 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.
  • the existing AAV serotype (AAV2/2) infects glioma cell line (U251) with low efficiency and poor specificity. Under the same MOI (multiplicity of infection), the infection effect on commonly used tool cells (293T and NIH3T3) better.
  • the present invention utilizes the peptide of AAV2/2 Segment mutation library, through library screening to obtain AAV serotype mutants that can efficiently and specifically infect U251, 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. Further, the tumor is human glioma.
  • 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 U251 cells.
  • the present invention has the following beneficial effects:
  • AAV serotype mutants according to the present invention can specifically infect U251 cells, while the effect of wild-type control group infecting U251 cells is poor, and the effect of infecting 293T cells is good;
  • Fig. 1 is pAAV-short UBC-mScarlet-polyA-P40-AAV2-Cap-FLEX-SV40polyA structural diagram in embodiment 1;
  • Fig. 2 is the fluorescence picture of U251 cell infected with library virus in embodiment 2;
  • Fig. 3 is the fluorescence picture of mutant serotype and AAV2 virus infection cell
  • Fig. 4 is the fluorescent diagram of mutation serotype and AAV2 virus infection 293T and U251;
  • Figure 5 is a statistical diagram of the results of killing experiments of mutant serotypes and AAV2 virus infection 293T and U251;
  • Fig. 6 Curves of mutant serotypes and AAV2 virus killing tumor volume in vivo.
  • 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 amino acid sequence of the protein is:
  • the site of our mutation is based on the serotype of AAV2/2, and the wild-type AAV2/2 capsid protein sequence comes from the NCBI number: 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.
  • cells after 72 hours of 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 a pipette tip to completely detach all 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.
  • U251 cells were infected with pAAV-short UBC-mScarlet-polyA-P40-AAV2-Cap-FLEX-SV40polyA–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 by adding 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
  • the mutant peptide sequence of AAV2-MT06 and AAV2-MT08 is ARLDITD (SEQ ID NO: 10) or YHAFNIE (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-MT06, and AAV2-MT08 serotypes.
  • AAV2 WT, AAV2-MT06, and AAV2-MT08 as serotypes to package adeno-associated virus vectors pAAV-SV40 enhancer-hTERT-EGFP and pAAV-SV40 enhancer-hTERT-TK, and after infecting 293T and U251 cells for 72 hours, add GCV After drug treatment, the cell viability was detected by CTG method after 72 hours, and the killing effect of the AAV2-SV40 enhancer-hTERT-TK-GCV system packaged with different serotypes AAV2 WT, AAV2-MT06, and AAV2-MT08 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-MT06 and AAV2-MT08 are less effective than AAV2 control group (AAV2 WT) in infecting 293T cells, but they can be compared with Good for infecting U251 cells.
  • the results of the cell killing test are shown in Figure 5.
  • the pAAV-SV40 enhancer-hTERT-TK group and AAV2/2 wild type (control group) have certain killing effects on 293T and U251 cells, and in the 293T cell
  • the survival rate is lower and the killing rate is higher; while AAV2-MT06 and AAV2-MT08 have obvious killing effects on U251, the survival rates are 17.8% and 28% respectively; while the killing effect on 293T is not obvious, the survival rate is higher, the survival The rates were 85.9% and 78.7%, respectively. It shows that AAV2-MT06 and AAV2-MT08 serotypes can specifically infect U251 cells.
  • This example discloses the killing experiment of the pAAV-SV40 enhancer-hTERT-TK-GCV system based on AAV2 WT, AAV2-MT06, and AAV2-MT08 serotypes in an in vivo tumor animal model.

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Abstract

La présente invention concerne un mutant de virus adéno-associé approprié pour une infection spécifique de cellules U251. Selon la présente invention, un mutant de sérotype de VAA capable d'infecter de manière efficace et spécifique U251 est obtenu au moyen d'un criblage de bibliothèque à l'aide d'une bibliothèque de mutations de fragments peptidiques de VAA2/2. Une expérience d'élimination in vitro prouve que le mutant de sérotype de VAA obtenu par criblage selon la présente invention peut infecter et détruire de manière spécifique des cellules U251, et présente de larges applications dans la recherche in vitro sur l'occurrence et le mécanisme de développement du gliome et le développement de médicaments pour le traitement du gliome.
PCT/CN2022/129385 2021-12-10 2022-11-03 Mutant de virus adéno-associé approprié pour une infection spécifique de cellules u251 WO2023103661A1 (fr)

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CN202111507708.2A CN114195859B (zh) 2021-12-10 2021-12-10 一种适用于特异感染u251细胞的腺相关病毒突变体

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104592364A (zh) * 2013-10-30 2015-05-06 北京大学 定点突变和定点修饰的腺相关病毒、其制备方法及应用
CN108347932A (zh) * 2015-02-20 2018-07-31 衣阿华大学研究基金会 用于治疗遗传性眼病的方法和组合物
CN110461368A (zh) * 2017-06-30 2019-11-15 加利福尼亚大学董事会 具有变异衣壳的腺相关病毒病毒体及其使用方法
CN111808175A (zh) * 2020-09-01 2020-10-23 和元生物技术（上海）股份有限公司 重组腺相关病毒颗粒及其应用
CN111825772A (zh) * 2020-07-30 2020-10-27 中国科学院精密测量科学与技术创新研究院 具有变异衣壳蛋白的腺相关病毒及其应用
CN112813037A (zh) * 2021-01-08 2021-05-18 中国科学院动物研究所 一种高效感染原代小胶质细胞的重组突变腺相关病毒及其相关生物材料
WO2021168509A1 (fr) * 2020-02-25 2021-09-02 Children's Medical Research Institute Polypeptides et vecteurs capsidiques de virus adéno-associés

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4219806A3 (fr) * 2017-04-11 2023-09-27 Ruprecht-Karls-Universität Heidelberg Banque de virus adéno-associés

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104592364A (zh) * 2013-10-30 2015-05-06 北京大学 定点突变和定点修饰的腺相关病毒、其制备方法及应用
CN108347932A (zh) * 2015-02-20 2018-07-31 衣阿华大学研究基金会 用于治疗遗传性眼病的方法和组合物
CN110461368A (zh) * 2017-06-30 2019-11-15 加利福尼亚大学董事会 具有变异衣壳的腺相关病毒病毒体及其使用方法
WO2021168509A1 (fr) * 2020-02-25 2021-09-02 Children's Medical Research Institute Polypeptides et vecteurs capsidiques de virus adéno-associés
CN111825772A (zh) * 2020-07-30 2020-10-27 中国科学院精密测量科学与技术创新研究院 具有变异衣壳蛋白的腺相关病毒及其应用
CN111808175A (zh) * 2020-09-01 2020-10-23 和元生物技术（上海）股份有限公司 重组腺相关病毒颗粒及其应用
CN112813037A (zh) * 2021-01-08 2021-05-18 中国科学院动物研究所 一种高效感染原代小胶质细胞的重组突变腺相关病毒及其相关生物材料

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