WO2025015603A1 - Nanovésicules pour délivrer des biomacromolécules au système nerveux central - Google Patents

Nanovésicules pour délivrer des biomacromolécules au système nerveux central Download PDF

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WO2025015603A1
WO2025015603A1 PCT/CN2023/108370 CN2023108370W WO2025015603A1 WO 2025015603 A1 WO2025015603 A1 WO 2025015603A1 CN 2023108370 W CN2023108370 W CN 2023108370W WO 2025015603 A1 WO2025015603 A1 WO 2025015603A1
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module
nucleic acid
tmd
nptn
acid construct
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王伊
董亚南
高峰
李艳芳
薛苗苗
李俊
王丹枫
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Panexo Biotech Sg PteLtd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present invention relates to the technical field of extracellular vesicles and their application, and in particular to nanovesicles for delivering biomacromolecules to the central nervous system.
  • NV Cell-derived nanovesicles
  • EVs extracellular vesicles
  • SynNVs synthetic nanovesicles
  • the surface and interior of nanovesicles contain a variety of functional protein molecules, and nucleic acid molecules (such as miRNA, lncRNA, etc.) are usually loaded inside. Specific proteins or nucleic acid molecules can be loaded on nanovesicles through recombinant expression or physical and chemical loading methods. Therefore, nanovesicles are ideal carriers for the delivery of biomacromolecule drugs.
  • nanovesicles face a long-standing problem that cannot be solved when delivering drugs in vivo: the vast majority of nanovesicle particles are captured and cleared by the liver, resulting in extremely low bioavailability of nanovesicles for delivering drugs to organs other than the liver. In particular, it is more difficult for natural nanovesicles to penetrate the blood-brain barrier and deliver drugs to brain tissue.
  • the delivery of biomacromolecule drugs to brain tissue by nanovesicles requires the combination of two approaches: 1) improving the targeting of vesicles to brain tissue or specific brain tissue cells and improving the ability to penetrate the blood-brain barrier; 2) prolonging the plasma circulation time of nanovesicles (i.e. reducing the capture and clearance of nanovesicles by the liver).
  • the research and development work reported so far mainly revolves around the first approach, such as obtaining exosomes secreted by neural stem cells (NSC) through culturing them, and using the homing properties of NSC exosomes to improve the blood-brain barrier penetration ability (Robin L. Webb, et al. Stroke. 2018; Vol.
  • NSC neural stem cells
  • the nasal administration method can indeed increase the number of EV particles entering the brain, but on the other hand, because EVs do not have the ability to actively migrate like cells, nor do they have the ability to quickly and freely diffuse like small molecule drugs, they gather near the olfactory center after entering the brain and cannot reach other brain tissue areas.
  • the fixed-point ultrasound method causes transient damage to the local blood-brain barrier and promotes drug penetration. It is not specific to vesicle drugs, and the implementation cost is high and the technical difficulty is great. Therefore, there is still a lack of cost-effective means to achieve efficient delivery of biomacromolecules from vesicles to brain tissue.
  • ApoE protein is a protein molecule that promotes the accumulation and clearance of lipid nanoparticle (LNP) drugs or mRNA vaccines in the liver. It is widely present in human blood circulation. According to existing literature, ApoE not only fails to promote NV entry into the brain, but may also accelerate the clearance of NV by the liver. There are currently no reports on the use of ApoE protein to promote the delivery of drugs to brain tissue.
  • LNP lipid nanoparticle
  • the technical problem to be solved by the present invention is to provide nanovesicles for delivering biomacromolecules to the central nervous system, in order to improve the loading efficiency of drugs in the vesicles and enhance the therapeutic effect.
  • the present invention provides a truncated form of APOE, whose amino acid sequence is shown in any one of SEQ ID NO.1 to 3.
  • ApoE or apolipoprotein E (ApoE)
  • ApoE2 has three subtypes: APOE2, APOE3, and APOE4, and their amino acid sequences are: APOE2 is shown in SEQ ID NO: 4, APOE3 is shown in SEQ ID NO: 5, and APOE4 is shown in SEQ ID NO: 6.
  • APOE2 is shown in SEQ ID NO: 4
  • APOE3 is shown in SEQ ID NO: 5
  • APOE4 is shown in SEQ ID NO: 6.
  • Previous studies have shown that ApoE3, in combination with scaffold proteins, can achieve modular loading of therapeutic drugs and improve loading efficiency.
  • the present invention screens and transforms ApoE, and in particular constructs a truncated body of ApoE. The results show that compared with ApoE3, the three truncated bodies constructed can further improve the loading effect.
  • the three truncated bodies provided by the present invention are divided into:
  • ApoE3 The truncated form of ApoE3 consisting of amino acid residues 1-191 is denoted as ApoE3 (1-191), and its amino acid sequence is shown in SEQ ID NO: 1.
  • ApoE4 The truncated form of ApoE4 consisting of amino acid residues 1-191 is denoted as ApoE4 (1-191), and its amino acid sequence is shown in SEQ ID NO: 2.
  • ApoE2 The truncated form of ApoE2 consisting of amino acid residues 1-191 is denoted as ApoE2 (1-191), and its amino acid sequence is shown in SEQ ID NO: 3.
  • the present invention also provides a nucleic acid encoding the aforementioned truncated form.
  • the nucleic acid sequence encoding truncated ApoE3 (1-191) is shown in SEQ ID NO: 7.
  • nucleic acid sequence encoding truncated ApoE4 (1-191) is shown in SEQ ID NO: 8.
  • nucleic acid sequence encoding truncated ApoE2 (1-191) is shown in SEQ ID NO: 9.
  • the present invention also provides a nucleic acid construct, comprising: a brain-entering module, a scaffold module, a transmembrane region module and an intramembrane region anchoring module; the brain-entering module is a nucleic acid encoding the truncation as described above or is a nucleic acid as shown in any one of SEQ ID NOs: 4 to 5.
  • each module may be in the same reading frame or not, and the present invention does not limit this. Similarly, each module may be on the same vector or not, and the present invention does not limit this.
  • the effect verification is carried out by taking each module in the same reading frame as an example, wherein the brain entry module, the scaffold module, the transmembrane region module and the intramembrane region anchoring module are sequentially connected from the 5' end to the 3' end.
  • the nucleic acid construct further comprises a signal labeling module and/or an effector module.
  • the signal labeling module and/or the effector module may or may not be in the same reading frame as the brain entry module, the scaffold module, the transmembrane region module and the intramembrane region anchoring module, and the present invention does not limit this.
  • the signal labeling module and/or the effector module may or may not be on the same vector as the aforementioned brain entry module, the scaffold module, the transmembrane region module and the intramembrane region anchoring module, and the present invention does not limit this.
  • the 5' end of the effector module/detection module is connected to the 3' end of the intramembrane anchoring region module.
  • the signal labeling module and/or the effector module is connected to the intramembrane region anchoring module via a linker.
  • the effector module includes a nucleic acid encoding a protein drug or a nucleic acid drug for treating brain diseases.
  • the signal labeling module includes any one or more of a fluorescent protein module and/or a chemiluminescent module;
  • the fluorescent protein module includes any one or more of green fluorescent protein, red fluorescent protein, blue fluorescent protein or yellow fluorescent protein; the chemiluminescent module includes Nanoluc or Rluc.
  • the modules of the present invention are directly connected or connected through a connecting module, and the connecting module includes a nucleic acid encoding a connecting peptide linker.
  • the connecting peptide is divided into a rigid connecting peptide and a flexible connecting peptide.
  • the amino acid sequence of the connecting peptide of the present invention is (GGGGS) 3 .
  • nucleic acid construct designed by the present invention consists of the following modules:
  • the BBB-penetrating module represents the full-length or truncated ApoE subtype protein loaded outside the membrane;
  • the Scaffold represents the scaffold protein outside the membrane, connected to the C-terminus of the effector protein;
  • the TMD represents the transmembrane region, and the N and C-termini are connected to the EVs membrane and intramembrane sequences respectively;
  • the ICD represents the intramembrane region of the transmembrane protein, contains S-palmitoylcysteine, and can be anchored on the EVs membrane;
  • the Cargo / Detection module represents the effector protein or detection protein loaded inside the membrane.
  • each module is a nucleic acid molecule encoding the corresponding protein / functional region.
  • the brain-entering module is a nucleic acid fragment encoding a brain-entering protein or a functional protein.
  • the brain-entering module includes any one or more of Transferrin, ApoB-RBD, APOE or its truncations, hCD24, ScFv, GDNF or Blr (group B , leucine rich ).
  • the scaffold module comprises any one or more of Fc, Foldon, Fc+NPTN-Ig1-3, ITGB1-I-EGF1-4, Foldon+NPTN-Ig1-3 or NPTN-Ig1-3.
  • the transmembrane region module includes any one or more of EWI-F-TMD, EWI-2-TMD, ITGB1-TMD, TMD_Mut_10, TMD_Mut_13, TMD_Mut_17, and NPTN-TMD.
  • the transmembrane region module includes any one or more of TMD_Mut_10, TMD_Mut_13, TMD_Mut_17 or NPTN-TMD;
  • amino acid sequence encoded by the nucleotide sequence of TMD_Mut_10 is SEQ ID NO.10;
  • amino acid sequence encoded by the nucleotide sequence of TMD_Mut_13 is SEQ ID NO.11;
  • amino acid sequence encoded by the nucleotide sequence of TMD_Mut_17 is SEQ ID NO.12;
  • amino acid sequence encoded by the nucleotide sequence of the NPTN-TMD is SEQ ID NO.13.
  • the intramedullary region anchoring module contains a nucleotide sequence encoding S-palmitoylcysteine, and the intramedullary region anchoring module includes EWI-F-ICD or EWI-2-ICD.
  • the present invention also provides a plasmid, an expression vector or an expression cassette comprising the nucleic acid construct as described above.
  • the present invention also provides a transformant or recombinant cell comprising the plasmid, expression vector or expression cassette as described above.
  • the present invention also provides an engineered EVs, which are prepared using the transformant or recombinant cell as described above.
  • the present invention also provides a protein expressed by the nucleic acid construct as described above.
  • the present invention also provides the use of the nucleic acid construct as described above, the plasmid as described above, the expression vector or expression cassette as described above, the transformant or recombinant cell as described above, the engineered EVs as described above and/or the protein as described above in the preparation of a drug or reagent, wherein the drug has the effector module as an active ingredient.
  • the present invention screens and transforms ApoE, and in particular constructs a truncated body of ApoE.
  • the results show that, relative to ApoE3, the three truncated bodies constructed can further improve the loading effect.
  • ApoE truncated body protein or ApoE protein on the surface of NV to make NV simulate lipoprotein particles, its ability to penetrate the blood-brain barrier is much stronger than the aforementioned brain-entering polypeptide sequences (RVG, Angiopep-2, etc.).
  • the plasma circulation half-life of NV loaded with ApoE in vivo is also significantly improved, showing a decrease in liver clearance, which is impossible for the aforementioned brain-entering polypeptide sequences to achieve.
  • Figure 1 shows the data of NV enzyme activity of each group of plasmids
  • FIG. 1 shows the Transwell results of each group
  • Figure 3 shows the data of brain tissues of mice in each group at different time points
  • FIG4 shows data of mouse liver tissue at different time points
  • Figure 5 shows the characterization of NV enzyme activity corresponding to different truncations
  • Figure 6 shows the data of NV enzyme activity of different truncated forms
  • Figure 7 shows the number of particles in the brain tissue of mice administered with different truncated NVs for 30 minutes
  • Figure 8 shows the enzyme activity data of different NVs
  • Figure 9 shows the number of particles penetrating different NV Transwells at 24 hours
  • Figure 10 shows the number of particles in the brain tissue of mice after 30 minutes of administration of different NVs
  • FIG11 is a schematic diagram of the nucleic acid construct of the present invention.
  • the present invention provides nanovesicles for delivering biomacromolecules to the central nervous system.
  • Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.
  • amino acid sequence of APOE2 is (SEQ ID NO.4):
  • amino acid sequence of APOE3 is (SEQ ID NO.5):
  • amino acid sequence of APOE3 (1-191) is (SEQ ID NO.1):
  • the nucleic acid sequence encoding the truncated ApoE3 (1-191) is (SEQ ID NO: 7):
  • amino acid sequence of APOE2 (1-191) is (SEQ ID NO.3):
  • the nucleic acid sequence encoding the truncated ApoE2 (1-191) is (SEQ ID NO: 9):
  • amino acid sequence of APOE4 is (SEQ ID NO.6):
  • amino acid sequence of APOE4 (1-191) is (SEQ ID NO.2):
  • the nucleic acid sequence encoding the truncated ApoE4 (1-191) is (SEQ ID NO: 8):
  • amino acid sequence of ApoE (130-149) is (SEQ ID NO.14):
  • amino acid sequence of Angiopep-2 is (SEQ ID NO.15):
  • RVG The amino acid sequence of RVG is (SEQ ID NO.16):
  • amino acid sequence of the linker is (SEQ ID NO.17):
  • amino acid sequence of Ig3(NPTN) is (SEQ ID NO.18):
  • TMD The amino acid sequence of TMD (NPTN) is (SEQ ID NO.13):
  • amino acid sequence of ICD (EWI-F) is (SEQ ID NO.19):
  • Nanoluc The amino acid sequence of Nanoluc is (SEQ ID NO.20):
  • TMD_Mut_10 The amino acid sequence of TMD_Mut_10 is SEQ ID NO.10;
  • TMD_Mut_13 The amino acid sequence of TMD_Mut_13 is SEQ ID NO.11:
  • TMD_Mut_17 The amino acid sequence of TMD_Mut_17 is SEQ ID NO.12:
  • test materials used in the present invention are all common commercial products and can be purchased in the market.
  • Example 1 Construction of NV loaded with full-length human ApoE2, ApoE3 or ApoE4 protein
  • Expression plasmid ApoE2-Ig3 (NPTN-Ig3)-TMD (NPTN-TMD)-ICD (EWI-F-ICD)-linker-Nanoluc,
  • a control plasmid without the loading module was also constructed:
  • the above plasmids were transfected into Expi HEK293F cells by front loading method.
  • the cell density was 1.0 ⁇ 10 6 /ml and the mass ratio of plasmid to PEI was 1:1.5.
  • the cells were cultured for 72h.
  • the cells and supernatant were collected for NV preparation.
  • the prepared NV was loaded.
  • the cell supernatant after culture was used to prepare NV by combining tangential flow filtration (TFF) with size exclusion chromatography (SEC), that is, the collected cell supernatant was first filtered through a 0.45 ⁇ m filter, then washed and concentrated into 1*PBS buffer through a 300KD membrane package and a hollow fiber column, and the concentrate was further purified through a Capto Core700 chromatography column to remove impurities, and the flow-through was collected and concentrated through a 10KD concentration tube.
  • TNF tangential flow filtration
  • SEC size exclusion chromatography
  • the cells were first resuspended in 0.1*PBS buffer containing 10U/mL nuclease, stirred at 120rpm for about 5min for hypotonic swelling, and then the cell suspension was filtered through a 0.45 ⁇ m filter for 4 times, restored to 1*PBS salt concentration, nuclease was added, digested at 4°C for about 16h, and concentrated by washing and filtration through a 300KD hollow fiber column. After the final volume of the concentrate was 50mL, the concentrate was further purified by Capto Core700 chromatography column to remove impurities, and the flow-through was collected and concentrated through a 10KD concentrator. Cell-derived NV and supernatant-derived NV can be used together.
  • control plasmid-transfected cells and supernatant were prepared using the same method to obtain the control Nanoluc NV.
  • NanoFCM was used to detect the particle concentration of NV.
  • the cell supernatant and cell-derived NV were combined and added to the Nanoluc substrate.
  • the Nanoluc enzyme activity was detected by an ELISA instrument to verify the expression of Nanoluc.
  • Mouse bEND.3 cells were inoculated into a Transwell culture plate with a pore size of 0.1 ⁇ m at a rate of 5E5 cells/well, and cultured in a cell culture incubator at 37°C and 5% carbon dioxide until the cell confluence reached 80%-90%.
  • the culture medium in the Transwell wells was carefully discarded, and different NVs were diluted to the same number of particles with cell culture medium.
  • the particles were then added into the Transwell wells and cultured in a cell culture incubator at 37°C and 5% carbon dioxide for 2 h, 4 h, 6 h, 8 h, and 24 h.
  • the liquid in the lower chamber of the Transwell was taken to detect the Nanoluc enzyme activity, and the enzyme activity detected by NV and the corresponding number of particles were used as a standard curve to calculate the number of NV particles in each tissue.
  • the drug was administered via the tail vein of mice, and samples were collected at 0.5h, 2h, 4h and 6h after administration: the eyeballs were removed to collect blood, and the blood was centrifuged at 4°C and 3000g for 20mins. The supernatant was taken as plasma, and the Nanoluc enzyme activity in the plasma was detected; the liver and brain were taken after perfusion, and PBS was added to the liver and brain tissues respectively. After homogenization, they were centrifuged at 12000rpm for 10min, and the supernatant was taken to detect the Nanoluc enzyme activity in the supernatant. The enzyme activity detected by NV and the corresponding number of particles were used as a standard curve to calculate the number of NV particles in each tissue.
  • Transwell results (Table 2 + Figure 2): Compared with the control NV, ApoE2 NV, ApoE3 NV, and ApoE4 NV can be significantly secreted into the lower chamber by bEND.3 cells. After 24 hours of culture, the number of ApoE4 NV particles detected in the lower chamber of Transwell was as high as about 2.3E9 particles, while the number of particles in the lower chamber of the control NV after 24 hours of culture was only 7E7 particles.
  • mice Results in mice (Table 3+ Figure 3, Figure 4): ApoE2 NV, ApoE3 NV, and ApoE4 NV all had significant ability to penetrate the blood-brain barrier compared with the control NV. NV reached a peak in the brain tissue 0.5 h after administration, and the number of particles in the brain tissue gradually decreased 2 h and 2 h later. ApoE2 NV, ApoE3 NV, and ApoE4 NV were all able to resist liver clearance. After 2 h, the control NV was almost completely cleared from the liver, while the experimental group NV still had >1E9 particles.
  • Example 2 Construction of NV loaded with different truncations of human ApoE2, ApoE3 or ApoE4 protein
  • the inventors designed different truncated sequences of ApoE, ApoE3 (1-191), ApoE4 (1-191), ApoE2 (1-191), ApoE (130-149), and constructed the following using pHEK293_Ultra_Expression_I (Takara) as the vector:
  • the loading method is the same as in Example 1.
  • the preparation method is the same as that of Example 1.
  • NanoFCM was used to detect the particle concentration of NV; NV was added to the Nanoluc substrate, and the Nanoluc enzyme activity was detected by an ELISA instrument to verify the expression of Nanoluc.
  • the drug was administered via the tail vein of mice, and samples were collected 0.5 hours after administration: the eyeballs were removed to collect blood, and the blood was centrifuged at 4°C and 3000g for 20 minutes. The supernatant was taken as plasma, and the Nanoluc enzyme activity in the plasma was detected; the liver and brain were taken after perfusion, and PBS was added to the liver and brain tissues respectively. After homogenization, they were centrifuged at 12000rpm for 10 minutes, and the supernatant was taken to detect the Nanoluc enzyme activity in the supernatant. The enzyme activity detected by NV and the corresponding number of particles were used as a standard curve to calculate the number of NV particles in each tissue.
  • NV enzyme activity (Table 4 + Figure 5): The different truncated forms of ApoE3 (1-191) NV and ApoE4 (1-191) NV prepared had slightly higher loading efficiency and slightly higher unit enzyme activity than the control NV. The loading efficiency of ApoE2 (1-191) NV and ApoE (131-149) NV was slightly lower.
  • mice Results in mice (Table 6+ Figure 7): The test was performed 30 minutes after administration. Compared with the control NV, different truncated forms of ApoE3(1-191)NV and ApoE4(1-191)NV had significant ability to penetrate the blood-brain barrier, while ApoE2(1-191)NV had relatively weak ability to penetrate the blood-brain barrier, and ApoE(131-149)NV had basically no ability to penetrate the blood-brain barrier.
  • Example 3 ApoE-NV has a stronger ability to penetrate the blood-brain barrier than EVs loaded with Angiopep-2, RVG and other brain-entering peptides reported previously
  • RVG-linker-Ig3 (NPTN-Ig3)-TMD(NPTN-TMD)-ICD(EWI-F-ICD)-linker-Nanoluc expression plasmid
  • the loading method is the same as in Example 1
  • the preparation method is the same as that of Example 1.
  • NanoFCM was used to detect the particle concentration of NV.
  • NV was added to the Nanoluc substrate, and the Nanoluc enzyme activity was detected by an ELISA instrument to verify the expression of Nanoluc.
  • Example 1 The method was the same as in Example 1. After culturing for 24 hours, the liquid in the lower chamber of the Transwell was taken to detect the Nanoluc enzyme activity. At the same time, various ApoE NVs in Examples 1 and 2 were compared with Angiopep-2 NV and RVG NV.
  • the drug was administered via the tail vein of mice, and samples were collected 30 minutes after administration: the eyeballs were removed to collect blood, and the blood was centrifuged at 4°C and 3000g for 20mins. The supernatant was taken as plasma, and the Nanoluc enzyme activity in the plasma was detected; the liver and brain were taken after perfusion, and PBS was added to the liver and brain tissues respectively. After homogenization, they were centrifuged at 12000rpm for 10min, and the supernatant was taken to detect the Nanoluc enzyme activity in the supernatant. The enzyme activity detected by NV and the corresponding number of particles were used as a standard curve to calculate the number of NV particles in each tissue.
  • Transwell results (Table 8 + Figure 9): Compared with the control NV, ApoE NV, RVG NV and Angiopep-2 NV can be significantly secreted into the lower chamber through Transwell cells. After 24 hours of culture, the number of ApoE NV particles detected in the lower chamber of Transwell was larger than that of RVG NV and Angiopep-2 NV, especially ApoE4 NV and ApoE4(1-191) NV.
  • mice In vivo results in mice (Table 9 + Figure 10): The test was performed 30 minutes after administration. Compared with the control NV, ApoE NV, RVG NV and Angiopep-2 NV all had the ability to penetrate the blood-brain barrier, and the ability of various ApoE NV to penetrate the blood-brain barrier was stronger than that of RVG and Angiopep-2.

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Abstract

L'invention concerne des vésicules extracellulaires et leur utilisation, et des nanovésicules pour délivrer des biomacromolécules au système nerveux central. L'invention concerne également un ApoE tronqué.
PCT/CN2023/108370 2023-07-20 2023-07-20 Nanovésicules pour délivrer des biomacromolécules au système nerveux central Pending WO2025015603A1 (fr)

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