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WO2025102275A1 - Procédé de préparation de vésicule extracellulaire modifiée ciblant des cellules positives au sulfate de chondroïtine oncofoetales, et son utilisation - Google Patents

Procédé de préparation de vésicule extracellulaire modifiée ciblant des cellules positives au sulfate de chondroïtine oncofoetales, et son utilisation Download PDF

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Publication number
WO2025102275A1
WO2025102275A1 PCT/CN2023/131871 CN2023131871W WO2025102275A1 WO 2025102275 A1 WO2025102275 A1 WO 2025102275A1 CN 2023131871 W CN2023131871 W CN 2023131871W WO 2025102275 A1 WO2025102275 A1 WO 2025102275A1
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WO
WIPO (PCT)
Prior art keywords
cells
extracellular vesicle
chondroitin sulfate
positive cells
ofcsbps
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2023/131871
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English (en)
Chinese (zh)
Inventor
张键
于铭
柴德智
蔡金旋
王浩
孙籽宸
肖天霞
李梦霞
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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Priority to PCT/CN2023/131871 priority Critical patent/WO2025102275A1/fr
Publication of WO2025102275A1 publication Critical patent/WO2025102275A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • 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
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention belongs to the field of biomedical technology and specifically relates to an engineered extracellular vesicle targeting carcinoembryonic chondroitin sulfate-positive cells and a preparation method and application thereof.
  • Extracellular vesicles are a general term for various membrane-structured vesicles released by cells. Due to their different diameters and occurrence modes, they are divided into exosomes (diameter 30-150nm), microvesicles (diameter 100-1000nm), apoptotic bodies (diameter 100-5000nm), oncosomes (diameter 1-10 ⁇ m), etc. Extracellular vesicles have a nanoscale lipid inclusion structure, which contains a variety of proteins, nucleic acids and metabolites, regulating the biological functions of target cells to achieve intercellular communication.
  • Extracellular vesicles have many characteristics such as barrier penetration, good biocompatibility, and low immunogenicity, making them ideal drug delivery carriers.
  • the engineered extracellular vesicles secreted by cells obtained by engineering have the advantages of tissue and organ targeting.
  • Oncofetal chondroitin sulfate ofCS was reported by Ali Salanti's research group at the University of Copenhagen in Denmark in the journal Cancer Cell in 2015, and it was found that a common class of glycosaminoglycan molecules exists in most human cancer cells and placental trophoblast cells.
  • VAR2CSA binds to ofCS, and there are several shortest binding peptides (ofCS-Binding Peptides, ofCSbps) ofCS in VAR2CSA.
  • Recombinant VAR2CSA protein or synthetic ofCS-BPs can be used as probes to achieve the diagnosis and treatment of placental diseases and cancer.
  • the purpose of the present invention is to design and provide a method for preparing engineered extracellular vesicles targeting ofCS-positive cells.
  • the present invention uses a lentiviral vector system to stably overexpress ofCSbps-Lamp2b-eGFP in HEK293F cells, collects the cell supernatant, and obtains extracellular vesicles by ultracentrifugation. It has the characteristics of targeting ofCS-positive cells (such as placental trophoblastic cells, tumor/cancer cells, etc.).
  • the diagnosis and treatment related molecules that have been clinically applied or are in preclinical research can be delivered to the target site.
  • the present invention is suitable for basic research and clinical diagnosis and treatment of various diseases such as human and animal placental physiology/pathology and tumor/cancer.
  • the present invention provides an engineered extracellular vesicle targeting carcinoembryonic chondroitin sulfate-positive cells, wherein the engineered extracellular vesicle is obtained by collecting and separating the supernatant of cells stably overexpressing a fusion protein plasmid vector containing ofCSbps;
  • the sequence of ofCSbps is selected from one or more of the combined polypeptide sequences in SEQ ID NO.1-7, or a derivative sequence with one of the polypeptide sequences in SEQ ID NO.1-7 as the backbone or a derivative sequence with multiple combined polypeptide sequences as the backbone.
  • the sequences SEQ ID NO.1-7 are all the shortest binding polypeptides with several segments of ofCS in VAR2CSA.
  • the engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells contains a protein for the expression and localization of the surface of the extracellular vesicles;
  • the protein used for extracellular vesicle surface expression and localization is selected from one of Lamp2b, CD9, CD63, CD47, CD81, APMAP, TSPAN14, TSG101, Alix, Flotillin-1, Syntenin-1, and HSP70.
  • the fusion protein plasmid vector containing ofCSbps contains proteins for cell transfection efficiency detection, screening, purification and tracing;
  • the protein used for cell transfection efficiency detection, screening, purification and tracing is selected from one of eGFP, GFP, eYFP, mRFP1, mCherry, Luciferase, 6*His, Flag, GST and c-Myc.
  • the engineered extracellular vesicles targeting oncofetal chondroitin sulfate-positive cells wherein the cells are selected from one of cell lines, primary cells, organoid cells, single cells, and living cells of plants and animals;
  • the cell line is HEK293F cells.
  • the engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells are transfected by a lentiviral infection method, a liposome transfection method, a calcium phosphate coprecipitation method or an electrotransfection method.
  • the engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells have a size of 30-200 nm, a shape of a saucer or a hemispherical shape with a depression on one side, and the surface of the extracellular vesicles contains ofCSbps or a peptidomimetic of ofCSbps.
  • the present invention provides a method for preparing engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells as described in any one of the above, comprising the following steps:
  • step S2 infecting the plasmid vector obtained in step S1 into cells via lentivirus, and screening to obtain protein-positive cell monoclonal cell lines for cell transfection efficiency detection, screening, purification and tracing;
  • step S3 Collect the cell supernatant obtained in step S2, and obtain engineered extracellular vesicles by separation.
  • the preparation method is characterized in that the screening method is one of flow cytometry or limiting dilution; the separation method is one of ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, tangential flow filtration or immunoaffinity capture.
  • the engineered extracellular vesicles are used as delivery carriers to load components for studying, detecting, preventing or treating tumors, placental-derived physiological or pathological diseases, and uterine-derived physiological or pathological diseases;
  • the component is selected from one of polypeptides, proteins, lipids, carbohydrates, RNA, DNA or small molecule compounds;
  • the placental physiological or pathological disease is one of early miscarriage, recurrent miscarriage, ectopic pregnancy, placental abruption, intrauterine fetal growth restriction, gestational hypertension, preeclampsia, premature birth, gestational diabetes, placenta accreta disease, and gestational trophoblastic disease;
  • the uterine-derived physiological or pathological disease is one of endometriosis, endometrial adenomyosis, intrauterine adhesions, uterine myoma, and uterine fibroids;
  • the loading method is one of genetic engineering, chemical modification, electroporation, liposome transfection, co-incubation, ultrasound, freeze-thaw cycle, extrusion, and surfactant.
  • step S1 The specific construction process of the fusion protein plasmid vector containing ofCSbps in step S1 is as follows:
  • the method of the present invention has the following beneficial effects:
  • the steps of the present invention are simple and the preparation cost is relatively low.
  • the extracellular vesicles prepared by the scheme provided by the present invention have the advantages of low toxicity, low immunogenicity and strong biocompatibility.
  • the engineered extracellular vesicles provided by the present invention are of great significance to the basic research on placental function and tumor/cancer, as well as the clinical diagnosis and treatment of placental diseases and tumor/cancer.
  • drugs can be loaded into the engineered extracellular vesicles to intervene in the placenta in normal physiological state and the placenta in pathological state, respectively, to achieve the purposes of contraception and relief treatment.
  • Targeted delivery is carried out for tumor/cancer tissue lesions to achieve the purpose of treatment.
  • contrast agents can be loaded into the engineered extracellular vesicles to achieve whole-body imaging observation of the placenta and tumor/cancer to achieve the purpose of diagnosis.
  • Figure 1 is a plasmid map and a schematic diagram of fusion protein design
  • Figure 2 is a schematic diagram of the construction process and the monoclonal cell line after eGFP sorting
  • FIG3 is the morphology of extracellular vesicles detected by scanning electron microscopy
  • FIG4 is a graph showing the particle size of extracellular vesicles detected by NTA
  • Figure 5 shows the expression of extracellular vesicle molecular markers detected by Western Blot
  • FIG6 is a distribution diagram of extracellular vesicles in mouse uterine tissue detected by a small animal imaging device
  • FIG7 is a distribution diagram of extracellular vesicles in various organs and tissues of mice detected by small animal imaging
  • FIG8 is a distribution diagram of extracellular vesicles in mouse fetus and placenta detected by small animal imaging instrument
  • FIG. 9 is an immunofluorescence analysis of the enrichment distribution of extracellular vesicles and CK7 in the mouse placenta trophoblast.
  • the primer sequences used are as follows:
  • R1 (SEQ ID NO.9):
  • R2 (SEQ ID NO.11):
  • R3 (SEQ ID NO.13):
  • the three pairs of primers were used to amplify three DNA fragments respectively according to the systems and conditions in Tables 1 and 2 below.
  • reaction products were subjected to agarose gel electrophoresis;
  • the LR reaction conditions were: incubate at 25°C for 3 h. After the reaction, 1 ⁇ L of proteinase K was added and incubated at 37°C for 15 min to terminate the reaction.
  • SnapGene software was used to open the vector map, determine the enzyme cutting scheme, and then the plasmid DNA of the final vector was cut with NEB restriction endonucleases. After the enzyme cutting reaction was completed, agarose gel electrophoresis was performed and analyzed with reference to DNA Ladder. The vector that could only cut out a DNA band of a specific length was the correct final vector.
  • the plasmid map and fusion protein design schematic diagram the amino acid sequence of ofCSbps in Example 1 of the present invention is SEQ ID NO.1.
  • Example 2 Lentivirus packaging and concentration
  • Example 3 Lentivirus infection and eGFP-positive cell sorting
  • Example 4 Extracellular vesicle extraction and identification
  • Extracellular vesicle morphology detection As shown in Figure 3, under a transmission electron microscope, it can be seen that the extracellular vesicles have a saucer-like or hemispherical morphology with one side concave;
  • Extracellular vesicle size detection As shown in Figure 4, the analysis results of the nanoparticle tracking analyzer showed that the average size was 160.4 nm;
  • Detection of extracellular vesicle molecular markers Cells and extracellular vesicle proteins were extracted and Western blot was performed at a loading amount of 30 ⁇ g/well. As shown in Figure 5, compared with cells, CD81, ALIX and TSG101 were highly expressed in extracellular vesicles, while Calnexin was negative. In addition, GFP was positive in HEK293F-ofCSbps-Lamp2b-eGFP cells and extracellular vesicles, indicating that the engineered cells were successfully constructed and the engineered extracellular vesicles secreted by them could be obtained sustainably.
  • Example 5 Placenta-targeted validation of engineered extracellular vesicles
  • Pregnant C57BL/6J mice (E12.5-15.5 days) were purchased. The experiment was divided into four groups: non-treatment group (NT), tail vein injection of DiR dye group (DiR), tail vein injection of HEK293F-Lamp2b-eGFP secreted extracellular vesicles group (L-EVs) and tail vein injection of HEK293F-ofCSbps-Lamp2b-eGFP secreted extracellular vesicles group (ofCSbps-EVs). Before tail vein injection, the extracellular vesicles were stained with DiR dye (Invitrogen, D12731), and the specific method was as follows:
  • mice were killed 24 hours after tail vein injection of extracellular vesicles (50 ⁇ g/mouse), and heart, liver, spleen, lung, kidney, pancreas, brain and uterus (placenta), placenta and fetal tissues were isolated. Fresh tissues were imaged using a small animal in vivo optical imaging system (Caliper Spectrum IVIS), and the specific steps were as follows:
  • the tissues were fixed in 4% paraformaldehyde for subsequent tissue sectioning.

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  • General Engineering & Computer Science (AREA)
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  • Animal Behavior & Ethology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

L'invention concerne un procédé de préparation d'une vésicule extracellulaire modifiée ciblant des cellules positives au sulfate de chondroïtine oncofoetales, et son utilisation, appartenant au domaine technique des biomédicaments. La présente invention concerne une vésicule extracellulaire ingénierisée ciblant les cellules positives au sulfate de chondroïtine oncofœtales, obtenue en recueillant un surnageant de cellules qui surexpriment de manière stable une protéine contenant des ofCSbp et en la séparant, la séquence des ofCSbp étant l'une des séquences polypeptidiques suivantes ou une séquence polypeptidique combinée de plus d'une des séquences SEQ ID NO. 1 à 7, ou une séquence dérivée prenant une séquence polypeptidique de SEQ ID NO. 1 à 7 en tant que chaîne principale, ou une séquence dérivée prenant une séquence polypeptidique combinée de plus d'un des SEQ ID NO. 1 à 7 en tant que chaîne principale. La vésicule extracellulaire ingénierisée de la présente invention présente la caractéristique de cibler les cellules ofCS-positives. Étant donné que les vésicules extracellulaires présentent des structures d'encapsulation de liposomes à l'échelle nanométrique, la présente invention convient à la recherche fondamentale et au diagnostic clinique ainsi qu'au traitement de diverses maladies, telles que les maladies physiologiques/pathologiques et tumorales/cancéreuses liées au placenta, chez les êtres humains et les animaux.
PCT/CN2023/131871 2023-11-15 2023-11-15 Procédé de préparation de vésicule extracellulaire modifiée ciblant des cellules positives au sulfate de chondroïtine oncofoetales, et son utilisation Pending WO2025102275A1 (fr)

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PCT/CN2023/131871 WO2025102275A1 (fr) 2023-11-15 2023-11-15 Procédé de préparation de vésicule extracellulaire modifiée ciblant des cellules positives au sulfate de chondroïtine oncofoetales, et son utilisation

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Citations (4)

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Publication number Priority date Publication date Assignee Title
CN109568289A (zh) * 2017-09-28 2019-04-05 中国科学院深圳先进技术研究院 胎盘样硫酸软骨素a靶向传输系统及其制备方法和应用
CN109589416A (zh) * 2017-09-28 2019-04-09 中国科学院深圳先进技术研究院 胎盘样硫酸软骨素a靶向纳米投递系统及其制备方法和应用
CN109589413A (zh) * 2017-09-28 2019-04-09 中国科学院深圳先进技术研究院 靶向胎盘样硫酸软骨素a的多肽、靶向纳米颗粒及其制备方法和应用
US20230203532A1 (en) * 2019-11-28 2023-06-29 Mcmaster University Recombinant polypeptides for programming extracellular vesicles

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
CN109568289A (zh) * 2017-09-28 2019-04-05 中国科学院深圳先进技术研究院 胎盘样硫酸软骨素a靶向传输系统及其制备方法和应用
CN109589416A (zh) * 2017-09-28 2019-04-09 中国科学院深圳先进技术研究院 胎盘样硫酸软骨素a靶向纳米投递系统及其制备方法和应用
CN109589413A (zh) * 2017-09-28 2019-04-09 中国科学院深圳先进技术研究院 靶向胎盘样硫酸软骨素a的多肽、靶向纳米颗粒及其制备方法和应用
US20230203532A1 (en) * 2019-11-28 2023-06-29 Mcmaster University Recombinant polypeptides for programming extracellular vesicles

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JIANG JIN-LU , PAN HAI-FENG ,YU SI-YUAN , LI TING-DONG ,GE SHENG-XIANG: "Recombinant Malaria Protein rVAR2 and Its Application in Tumor-targeted Therapy", CHINA BIOTECHNOLOGY, vol. 43, no. 6, 25 June 2023 (2023-06-25), pages 69 - 75, XP093316536, DOI: 10.13523/j.cb.2301003 *

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