WO2022217966A1 - Agent de nano-piégeage inhibant le sars-cov -2 - Google Patents

Agent de nano-piégeage inhibant le sars-cov -2 Download PDF

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WO2022217966A1
WO2022217966A1 PCT/CN2021/141286 CN2021141286W WO2022217966A1 WO 2022217966 A1 WO2022217966 A1 WO 2022217966A1 CN 2021141286 W CN2021141286 W CN 2021141286W WO 2022217966 A1 WO2022217966 A1 WO 2022217966A1
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hace2
cells
nano
nanovesicles
ncs
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刘庄
陈倩
张晗
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苏州大学
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4813—Exopeptidases (3.4.11. to 3.4.19)
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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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
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    • 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
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
    • 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
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention relates to the field of functional materials, in particular to a nano-capturer for inhibiting SARS-CoV-2.
  • Severe acute respiratory syndrome coronavirus 2 causes coronavirus disease 2019 (COVID-19), and the SARS-CoV-2 virus has a spike (S) glycoprotein on the surface of the Human angiotensin-converting enzyme II (hACE2) binds, enters cells and infects.
  • S protein is continually mutated and its affinity for the hACE2 receptor increases, enhancing infectivity and transmission.
  • the D614G mutant strain is the main body of the current SARS-CoV-2 virus, and the D614G mutant strain has significantly improved binding efficiency to the hACE2 receptor during virus infection.
  • the novel SARS-CoV-2 variant B.1.1.7 with a large number of mutated genes has a 1000-fold increased affinity of the S protein for the hACE2 receptor, which is 70% more transmissible than previously discovered SARS-CoV-2.
  • SARS-CoV-2 vaccines have received a lot of attention since the outbreak of COVID-19.
  • SARS-CoV-2 vaccines there are more than 160 SARS-CoV-2 vaccines in the research and development stage around the world, of which 4 vaccines have been clinically approved, including mRNA vaccines (BNT162 and mRNA-1273), viral vector vaccines (ChAdOx1-2) and inactivated virus Vaccines (BBIBP-CorV), they bring light in the fight against COVID-19.
  • mRNA vaccines BNT162 and mRNA-1273
  • viral vector vaccines ChAdOx1-2
  • BBIBP-CorV inactivated virus Vaccines
  • the present invention provides a suitable freeze-drying protective agent and a mucoadhesive auxiliary material for the nanovesicles containing hACE2, and provides a safer and more effective storage for the inhalable nano-capturing agent that inhibits SARS-CoV-2.
  • the neutralization efficiency of nanovesicles against pseudoviruses can be significantly improved.
  • a nanometer trapping agent for inhibiting SARS-CoV-2 of the present invention includes nanovesicles containing hACE2, and one or both of a freeze-drying protective agent and a mucoadhesion adjuvant;
  • Nanovesicles containing hACE2 were prepared by the following steps:
  • cells (1) Transfect cells with hACE2-encoding lentivirus, hACE2-encoding plasmid or cationic liposomes carrying hACE2 genetic information to construct cells stably expressing hACE2
  • cells refer to 293T cells, Vero cells, L929 cells, Hela cells, DC2.4 cells, Raw cells, etc.;
  • the SARS-CoV-2 virus infects the host through the hACE2 receptor, and the hACE2-containing nanovesicles of the present invention compete with the host cell to bind the SARS-CoV-2 virus to protect the host cell from infection, instead of producing neutralization on the surface of the S protein Antibodies, therefore not affected by S protein mutation, are effective against different virus mutant strains, thereby improving the neutralization efficiency of nanovesicles against pseudoviruses and achieving the purpose of efficient treatment.
  • the transportation and storage conditions of existing vaccines are harsh, and the productivity of existing vaccines is limited, which cannot meet the requirements of large-scale vaccination in a short period of time.
  • the present invention introduces freeze-drying protective agent and mucoadhesive auxiliary materials for the nano-capturing agent.
  • the freeze-drying protective agent significantly improves the stability of the nano-capturing agent during storage and the convenience of transportation.
  • the neutralization titer of the vesicles remained above 90%, thus greatly increasing the feasibility of its clinical application; the mucoadhesive excipients can significantly prolong the retention of nanocaptures in the lungs and enhance the virus inhibition effect.
  • the lyoprotectant is sucrose, or sucrose and lactose, or sucrose and trehalose, or trehalose and mannitol.
  • the mucoadhesive adjuvant includes hyaluronic acid or polyvinyl alcohol.
  • the freeze-dried protective agent is mixed with the nanovesicles containing hACE2, the obtained mixed solution is freeze-dried, and finally the mucoadhesive auxiliary material is added to the freeze-dried powder. , to obtain nanometer collectors.
  • step (1) puromycin was used to select 293T cells stably expressing hACE2.
  • step (2) nanovesicles containing hACE2 are prepared by a micro-liposome extruder.
  • the particle size of the hACE2-containing nanovesicles is 100-400 nm.
  • the dosage form of the nano-collecting agent is a powder dosage form or an aerosol dosage form.
  • the lyophilized powder reconstituted in ultrapure water can be used to prepare an inhalable aerosol dosage form via a nasal spray bottle.
  • the mass ratio of the hACE2-containing nanovesicles and the lyoprotectant is 0.2-5:1, wherein the mass concentration ratio of the hACE2 transmembrane protein and the lyoprotectant is 1:25-100.
  • the mass ratio of the hACE2-containing nanovesicles and the mucoadhesive excipients is 0.2-5:1-5.
  • the present invention also claims the application of the above-mentioned nano-capturing agent in the preparation of a medicine for protecting lung tissue from SARS-CoV-2 virus infection.
  • Lung tissue can be non-invasively and efficiently protected from SARS-CoV-2 pseudovirus infection by inhaling nano-capturing agent/lyophilized protective agent/mucoadhesive excipient aerosol.
  • the present invention has at least the following advantages:
  • the nano-capturing agent of the invention has high safety, is easy to store and transport, has low cost, and can be mass-produced quickly, so as to timely and effectively deal with frequently occurring mutant strains, and has good clinical transformation prospects.
  • Figure 1 shows the results of Western blotting of hACE2-293T cells prepared in Example 1 and negative control 293T cells (1a) and the transmission electron micrograph (1b) of NCs;
  • Figure 2 is a graph showing the neutralization curves of nanovesicles extracted from hACE2-293T cells and nanovesicles extracted from negative control 293T cells to pseudoviruses;
  • Figure 3 is a comparison chart of the retention time and retention site of NCs after mixing different mucoadhesive excipients with NCs;
  • Figure 4 is the particle size and potential diagram of NCs containing different lyoprotectant powder formulations after reconstitution
  • Figure 5 is a graph showing the neutralization effect of different lyophilized protective agents and NCs mixed solutions on pseudoviruses
  • Figure 6 is a graph showing the expression of hACE2 in mouse lung tissue
  • Figure 7 is a graph showing the expression of LUCI in mouse lung tissue
  • Figure 8 is a graph showing the biosafety results of NCs/HA/sucrose in vivo
  • Fig. 9 is the transmission electron microscope image of the NCs prepared in Example 2.
  • Figure 10 shows the results of the reconstituted nanovesicle lyophilized powder obtained by lyophilization after adding HA.
  • NCs nanovesicles
  • human embryonic kidney epithelial cell 293T cells were transfected with lentivirus encoding the transmembrane protein hACE2, and incubated with 2 ⁇ g/ml puromycin to construct 293T (hACE2-293T) cells stably expressing hACE2.
  • hACE2-293T cells were harvested by trypsinization and resuspended in homogeneous medium containing 0.25M sucrose, 1 mM EDTA, 10 mM Hepes (pH 7.4) and protease inhibitors. Cells were disrupted with a sonicator with a power of 200W in an ice bath.
  • Figure 1a shows the Western blot results of hACE2-293T cells and negative control 293T cells (with ⁇ -actin protein as an internal reference to ensure the same amount of protein loaded), the figure shows that hACE2-293T cells express a large amount of hACE2 on the surface, with neutralization
  • Figure 1b is a transmission electron micrograph of the nanovesicles prepared in Example 1. It can be seen from the figure that NCs containing hACE2 on the surface and having a particle size of about 200 nm were successfully prepared.
  • Hela cells were transfected with lentivirus encoding the transmembrane protein hACE2, and incubated with 2 ⁇ g/ml puromycin to construct Hela (hACE2-Hela) cells stably expressing hACE2.
  • hACE2-Hela cells were harvested by trypsinization and resuspended in homogeneous medium containing 0.25M sucrose, 1 mM EDTA, 10 mM Hepes (pH 7.4) and protease inhibitors. Cells were disrupted with a sonicator with a power of 200W in an ice bath. Then, the suspension was centrifuged at 3000 rpm for 10 min to remove nuclei and cytoplasm.
  • the resulting cell membranes were washed twice with cold homogeneous medium. After that, cell membranes were collected by centrifugation at 14800 rpm for 30 min. Finally, the obtained suspension was sequentially extruded through a 400-nm and 200-nm polycarbonate porous membrane for 10 times using a micro-liposome extruder to obtain hACE2-containing nanovesicles. Transmission electron microscope image.
  • the inventors also tried other cells to express hACE2 through lentivirus transfection, and then prepared nanovesicles containing different hACE2 on the surface by a similar method.
  • the efficiency of lentivirus transfecting different cells is different, and the 293T cell in Example 1 is preferred, and its transfection efficiency is higher.
  • the nanovesicles in the present invention can be derived from different cells to obtain specific nanovesicles accordingly.
  • NCs nanovesicles
  • VSV vesicular stomatitis virus
  • LUCI luciferase reporter gene
  • Neutralization efficiency (%) [1–[(fluorescence intensity value of sample – average value of background fluorescence intensity value)/(average value of fluorescence intensity value of control virus only – average value of background fluorescence intensity value)]] ⁇ 100 %.
  • TCID 50 refers to half the tissue culture infectious dose. The results are shown in Figure 2.
  • Figure 2 is the neutralization curve of nanovesicle NCs extracted from hACE2-293T cells and nanovesicle NVs extracted from negative control 293T cells against pseudoviruses.
  • the axis is for neutralizing efficiency. It can be seen that compared with NVs, NCs showed strong pseudovirus neutralization ability, and its half-inhibitory concentration IC 50 value was 9.8 ⁇ g/ml.
  • Figure 3a is a graph showing the fluorescence intensity statistics of lung tissue at 6 and 24 h after inhaling a mixture of different excipients and NCs in mice.
  • the lung tissue of mice inhaled with NCs/HA showed the strongest fluorescence intensity values at different time points, and its lung treatment effect was the best. It is also significantly better than inhaling NCs/PVP and NCs/CD;
  • Figure 3b shows the biodistribution of NCs in various major organs at different time points after inhaling the mixture of different excipients and NCs in mice.
  • Figure 3c further shows the biodistribution of NCs in lung, liver, spleen, kidney, heart and other organs at different time points after mice inhaled a mixture of different excipients and NCs, and the fluorescence intensity statistics
  • the figure shows that, compared with other organs, the lung tissue has the strongest fluorescence signal at different time points, which further indicates that HA, PVA, CS and PLL all prolong the lung retention time of NCs and improve the bioavailability; The survival rate of mice after the drug is completed.
  • the figure shows that the cationic CS and PLL are highly toxic. Although they have a good therapeutic effect, they are not suitable for practical application. In conclusion, HA has a good therapeutic effect and is safe, followed by PVA.
  • the lyophilized protective agents sucrose, trehalose, mannitol, lactose and their mixtures were selected to prepare the NCs powder dosage form.
  • a mixed solution of NCs (2 mg/ml) and lyoprotectant (2.5 mg/ml) was prepared.
  • the mixed solution (1 ml) was quickly lyophilized in liquid nitrogen for 10 min and lyophilized using a lyophilizer for 24 h.
  • HA powder 2.5 mg was added to the lyophilized powder and stored at 4°C.
  • dissolve with molecular biological grade ultrapure water, and install the matching nozzle to spray the aerosol containing NCs The related results are shown in Figure 4.
  • Figure 4a shows the particle size and potential diagram of nanovesicles after lyophilization and reconstitution with different lyoprotectants.
  • the size of the NCs in the control group and the mannitol group increased after being dissolved in water, and obvious aggregation occurred
  • the size of the NCs in the lactose group decreased after being dissolved in water, and the size of the other groups did not change significantly, indicating that The homogeneity of vesicles after lyophilization and reconstitution with these lyophilized protective agents was better; in terms of potential, the size and zeta potential of NCs in other groups remained basically unchanged
  • Figure 4b is a schematic diagram of the samples used in lyophilized powder. Dissolve the lyophilized powder containing NCs with water, install the nose tip and spray the aerosol. The lyophilized powder dosage form facilitates long-term storage and transportation.
  • the lyophilized powder of nanovesicles can only be obtained by lyophilization After mixing the HA solid powder and dissolving it at the time of use, the ideal spray formulation effect can be achieved. This is different from the conventional preparation of freeze-dried powder preparations. In actual production, freeze-dried powder preparations are freeze-dried and packaged in the final step, which is more conducive to controlling parameters such as water content in freeze-dried powder. Applicable to the present invention, in order to better obtain a lyophilized powder formulation, the nanovesicles and the lyophilized protective agent must be mixed and lyophilized, and then mixed with dried auxiliary materials to obtain a usable formulation.
  • Example 4 the lyophilized powder prepared in Example 4 was dissolved in biological-grade ultrapure water, and the diluents of different concentrations were neutralized with pseudoviruses according to the method in Example 2, and the neutralization efficiency was calculated. Then, the freshly prepared NCs solution and the optimized NCs/HA/sucrose lyophilized powder were stored in a 4°C refrigerator for 1 month and then reconstituted. The pseudovirus neutralization experiment was performed under the same conditions. The related results are shown in Figure 5.
  • Figure 5a is a graph showing the neutralization effect of different lyoprotectants and NCs mixed solutions on pseudoviruses.
  • the pseudovirus neutralization efficiency is the most important indicator. The higher the pseudovirus neutralization efficiency, the better the lyophilization protection effect. It can be seen from the figure that the NCs/HA/sucrose group, NCs/HA/lactose and sucrose, NCs/ There was no significant difference between the HA/sucrose and trehalose, NCs/HA/trehalose and mannitol groups and the newly prepared nanovesicles, and they all maintained a high level;
  • Figure 5b shows the NCs solution and the optimized NCs/HA/sucrose jelly The neutralization effect of dry powder on pseudovirus after being stored at 4°C for one month. The titer of the reconstituted NCs retained approximately 90%. The results indicated that the NCs/HA/sucrose lyophilized powder better retained the virus neutralization ability of NCs.
  • mice Male immunodeficient NSG mice were anesthetized, and hACE2-encoding replication-deficient adenovirus (AdV-hACE2) (1 ⁇ 1010 PFU, 50 ⁇ L) was administered through the bronchi to express hACE2 in mouse lung tissue to establish a hACE2-expressing mouse model. After 5 days, the mouse lung tissue was removed, and part of it was used to prepare single cell suspension for hACE2 flow antibody staining, and the other part was used to prepare cell lysate to detect the expression of hACE2 by Western blotting. The results are shown in Figure 6.
  • AdV-hACE2 replication-deficient adenovirus
  • Figure 6 shows the expression of hACE2 in mouse lung tissue, wherein AdV-Empty represents a replication-deficient adenovirus that does not carry genetic information.
  • mice expressing hACE2 were divided into three groups, inhaled 50 ⁇ L of phosphate buffered saline (PBS), NCs/sucrose, and NCs/HA/sucrose, respectively, and inhaled twice after 4 h and 8 h of SARS-CoV-2 encoding LUCI containing SARS-CoV-2 Pseudovirus with viral S protein coat.
  • PBS phosphate buffered saline
  • NCs/sucrose NCs/HA/sucrose
  • Figure 7a In the flow cytometry results, the percentages of LUCI-positive cells in the PBS control group, NCs/sucrose and NCs/HA/sucrose groups were 6.5%, 2.1% and 0%, respectively; Figure 7b Western blot analysis of LUCI expression in the PBS group Highest. The above results suggest that inhalation of hACE2-containing NCs and HA exhibited potent pseudovirus inhibition in a hACE2-expressing mouse model by prolonging pulmonary retention.
  • NCs/HA/sucrose 200 ⁇ g of NCs membrane protein
  • mouse serum and whole blood samples were collected on days 1 and 7 for serum biochemistry and whole blood analysis.
  • concentrations of inflammatory cytokines (tumor necrosis factor alpha, interleukin 6, interleukin 12) in mouse serum were determined by ELISA kit.
  • the biosafety results of NCs/HA/sucrose in vivo are shown in Figure 8.
  • Figure 8a shows that all blood markers were not significantly different from the PBS treated group;
  • Figure 8b serum inflammatory cytokine concentrations were at baseline levels. All these results indicate that the NCs/HA/sucrose complex has excellent biocompatibility.

Abstract

La présente invention concerne un agent de nano-piégeage qui inhibe le SARS-CoV-2, comprenant des nanovésicules contenant hACE2, et un ou deux parmi un agent protecteur de lyophilisation et une matière auxiliaire mucoadhésive, les nanovésicules contenant hACE2 étant préparées et obtenues par les étapes suivantes : transfection de cellules 293T à l'aide d'un lentivirus qui code pour hACE2, pour construire des cellules 293T qui expriment de manière stable hACE2; et extraction des membranes cellulaires des cellules obtenues pour préparer des nanovésicules contenant hACE2. L'agent protecteur de lyophilisation est le saccharose, le saccharose et le lactose, le saccharose et le tréhalose, ou le tréhalose et le mannitol; et la matière auxiliaire mucoadhésive comprend de l'acide hyaluronique ou de l'alcool polyvinylique.
PCT/CN2021/141286 2021-04-15 2021-12-24 Agent de nano-piégeage inhibant le sars-cov -2 WO2022217966A1 (fr)

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CN113509546A (zh) * 2021-04-15 2021-10-19 苏州大学 一种抑制SARS-CoV-2的纳米捕集剂
CN114557971B (zh) * 2022-04-25 2023-05-23 康希诺生物股份公司 一种核酸-脂质纳米颗粒的冷冻干燥保护剂及其制备方法和应用
CN115058344B (zh) * 2022-08-05 2022-11-18 深圳湾实验室 去除废水中的SARS-CoV-2及其变异株的诱饵微机器人、其制备方法及应用

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