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WO2022262050A1 - Vecteur non viral, sa méthode de préparation et son utilisation - Google Patents

Vecteur non viral, sa méthode de préparation et son utilisation Download PDF

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WO2022262050A1
WO2022262050A1 PCT/CN2021/106991 CN2021106991W WO2022262050A1 WO 2022262050 A1 WO2022262050 A1 WO 2022262050A1 CN 2021106991 W CN2021106991 W CN 2021106991W WO 2022262050 A1 WO2022262050 A1 WO 2022262050A1
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protamine
plasmid
viral vector
lip
distearoylphosphatidylethanolamine
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Chinese (zh)
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张学农
王钰
黄归
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Suzhou University
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Suzhou University
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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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • 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
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • 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

Definitions

  • the invention belongs to the technical field of gene editing, and in particular relates to a non-viral vector and its preparation method and application.
  • CRISPR/Cas9 As the third-generation gene editing technology, CRISPR/Cas9 has powerful gene editing capabilities, and can realize gene silencing or correction at the DNA level. Once edited successfully, the effect will be permanent. It can be used to treat blood diseases, hereditary retinal diseases and tumors Some progress has been made in clinical trials of the disease, and it has broad application prospects in tumor-targeted gene therapy.
  • CRISPR/Cas9 system There are three implementation forms of CRISPR/Cas9 system, such as delivery of plasmids encoding both Cas9 and sgRNA, delivery of Cas9 mRNA and sgRNA, or delivery of Cas9 protein and sgRNA complex, that is, ribonucleoprotein complex.
  • Gene editing occurs in the nucleus, and the ability of the vector to target the nucleus is critical when delivering a plasmid and ribonucleoprotein complex.
  • CRISPR/Cas9 system delivery methods include physical methods, viral vectors and non-viral vectors.
  • Physical methods include microinjection, electroporation, nuclear infection, and membrane deformation, which are mainly used for gene editing of cells and tissues in vitro or in vitro, which have poor biocompatibility in vivo; viral vectors, such as: lentivirus, adenovirus, Adenovirus and other related viruses have been widely used in the in vivo and in vitro drug delivery of the CRISPR/Cas9 system, but there are safety issues such as carcinogenicity, insertion mutation, and immunogenicity, and the loading capacity is limited, which seriously affects the clinical application of viral vectors. Transformation: Non-viral vectors have the advantages of strong loading capacity, low immunogenicity, and easy assembly, and are ideal delivery methods.
  • Non-viral vectors load CRISPR/Cas9 systems through electrostatic interactions, van der Waals forces, hydrogen bonds and covalent bonds, etc., mainly liposomes (lipsomes), polymers, polypeptides or proteins, vesicles, DNA nanocoils, inorganic nano Granules etc.
  • liposomes liposomes
  • non-viral vectors need to efficiently deliver the CRISPR/Cas9 system to the cytoplasm of target cells.
  • delivering it to the nucleus is more conducive to promoting the expression of the plasmid Cas9 protein and sgRNA to improve editing efficiency.
  • the existing non-viral vectors still have problems of low targeting and stability during the delivery process.
  • the present invention provides a non-viral vector for gene editing.
  • the non-viral vector of the present invention relies on passive targeting and active targeting effects, can efficiently accumulate in tumor tissues, increase the uptake of tumor cells and promote Nucleic acid drugs enter the nucleus to accelerate the subsequent transcription and translation processes, providing a new candidate system for the delivery of gene editing plasmids.
  • the first object of the present invention is to provide a kind of non-viral vector, and described non-viral vector comprises the protamine that compresses plasmid drug, the cationic liposome coated on the surface of protamine that has compressed nucleic acid drug, and modification Distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid (DSPE-PEG-HA) polymer on the surface of cationic liposomes.
  • DSPE-PEG-HA Distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid
  • the cationic liposome is DOPE/DOTAP/Chol.
  • the plasmid drug is a gene editing plasmid.
  • the gene editing plasmid is one or more of pCas9/sgMTH1, pCas9/sgKRAS, pCas9/sgPLK1, pCas9/sgMETTL3.
  • the second object of the present invention is to provide the preparation method of said non-viral vector, comprising the following steps:
  • step S1 hyaluronic acid (HA) reacts with distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG-NH 2 ) after the carboxyl group is activated by an activator.
  • step S3 the protamine solution and the plasmid drug solution form a complex through electrostatic interaction.
  • step S4 cationic liposomes are coated on the surface of the complex through electrostatic interaction.
  • the distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer (DSPE-PEG-HA) is inserted into the liposome through hydrophobic interaction and modified on the surface of the non-viral carrier.
  • step S3 incubation is carried out at 20-30° C. for 4-6 minutes, and the mass ratio of protamine to plasmid drug is 1.8-2.2:1.
  • step S4 the incubation is carried out at 20-30° C. for 15-25 min, and the mass ratio of the protamine/plasmid drug complex to the cationic liposome is 3:11-11.5.
  • step S5 the incubation is at 50-60°C for 15-25min, in the aqueous solution of distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer, distearoylphosphatidylethanolamine-polyethylene glycol
  • concentration of ethylene glycol-hyaluronic acid polymer was 19-21 mg ⁇ mL -1 .
  • step S1 hyaluronic acid is activated by an activator and then reacted with distearoylphosphatidylethanolamine-polyethylene glycol, and the activator is 1-(3-dimethylaminopropyl)-3- One or more of ethylcarbodiimide hydrochloride (EDC ⁇ HCl), N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC).
  • EDC ⁇ HCl ethylcarbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • DCC dicyclohexylcarbodiimide
  • the third object of the present invention is to provide the application of the non-viral vector in delivering CRISPR/Cas9 system.
  • the protamine used in the present invention has the functions of compressing plasmids and targeting the nucleus, but it cannot protect the stable circulation of nucleic acid drugs in the blood and deliver the drugs to tumor tissues when used alone.
  • the present invention compresses protamine After the nucleic acid drug forms a negatively charged complex, the cationic liposome is coated on the outer layer, and the surface is modified with DSPE-PEG-HA, thereby protecting the nucleic acid drug from nuclease degradation in the blood circulation, reducing the interaction between the carrier and blood components, and at the same time The carrier is endowed with active targeting.
  • the invented carrier can increase the accumulation of the carrier in the tumor site and mediate the carrier into the cell. After the carrier is taken up by the cell, it depends on the phase transition of the liposome components to escape from the endosome and release the protamine/nucleic acid drug complex into the cytoplasm.
  • the nuclear localization signal (NLS) sequence in protamine can mediate the complex Drugs enter the nucleus through nuclear pores, thereby promoting the function of nucleic acid drugs.
  • the carrier constructed in this way can realize the nuclear delivery of some nucleic acid drugs, and provide a new type of carrier for plasmid drugs such as CRISPR/Cas9 plasmids.
  • the present invention has at least the following advantages:
  • the non-viral vector of the present invention can efficiently accumulate in tumor tissue, increase the uptake of tumor cells and promote the entry of nucleic acid drugs into the nucleus, accelerate the subsequent transcription and translation processes, and provide a new way for the delivery of gene editing plasmids.
  • a new candidate system was developed.
  • Fig. 1 is the 1 H-NMR spectrum of HA (a), DSPE-PEG (b) and DSPE-PEG-HA (c) in Example 1 of the present invention
  • Fig. 2 is the infrared spectrum of HA (a), DSPE-PEG (b) and DSPE-PEG-HA (c) in Example 1 of the present invention
  • Figure 3 is the ability of the vector to coat the plasmid in Example 1 of the present invention, wherein, a: Marker; b: naked pMTH1; c: PS/pMTH1; d: PS@HA-Lip/pMTH1;
  • Fig. 4 is the particle size of the carrier detected by the dynamic light scattering particle size analyzer DLS in Example 1 of the present invention, wherein, A: the particle size distribution of different carriers; B: the zeta potential of different carriers;
  • Fig. 5 is an electron micrograph of PS, PS@Lip and PS@HA-Lip in Example 1 of the present invention
  • Figure 6 is the serum stability of the carrier in Example 1 of the present invention.
  • Figure 7 is the in vitro safety of the CCK-8 experiment in Example 1 of the present invention to investigate the non-pharmaceutical carrier A: HUVEC cells, B: A549 cells;
  • Fig. 8 is the laser confocal microscopy investigation of the cellular uptake of the carrier in Example 1 of the present invention.
  • Figure 9 is the quantitative investigation of the cellular uptake of the carrier by flow cytometry in Example 1 of the present invention
  • Figure 10 is a laser confocal microscope in Example 1 of the present invention to investigate the carrier delivery plasmid into the nucleus;
  • Figure 11 is a schematic diagram of a non-viral vector of the present invention.
  • the preparation of non-viral vector comprises the following steps:
  • HA hyaluronic acid
  • DOTAP, DOPE and cholesterol (Chol) were precisely weighed, dissolved in a mixed organic solvent of chloroform and methanol (2:1, v/v) to prepare a mother solution (20 mg.mL -1 ).
  • a mother solution (20 mg.mL -1 ).
  • DOTAP mother solution 200 ⁇ L of DOPE mother solution, and 100 ⁇ L of cholesterol mother solution, and add them to a vial, then add 2 mL of mixed organic reagent and 10 mL of deionized water, ultrasonically (200W, 5min), transfer to an eggplant-shaped bottle, and remove by rotary evaporation at 45°C.
  • Organic reagents were filtered through a 0.45 ⁇ m filter membrane to obtain liposome Lip (1 mg.mL -1 ).
  • step (3) Mix the complex obtained in step (3) with the liposome according to the ratio determined in the experiment, vortex for 30 s, and incubate at room temperature for 20 min to obtain the liposome-coated protamine/plasmid complex.
  • step (4) Mix the liposome-coated protamine/plasmid complex obtained in step (4) with the aqueous solution of DSPE-PEG-HA obtained in step (1), and incubate at 55°C for 20min to obtain PS@ HA-Lip/pMTH1.
  • the 1 H-NMR spectrum is shown in the figure Shown in 1: the peaks at 5.91ppm and 4.50ppm are the characteristic peaks of methine hydrogen on the HA sugar ring, the peak at 1.24ppm is the characteristic peak of methylene hydrogen on the distearoyl carbon chain, and the peak at 3.50ppm is The characteristic peaks of methylene hydrogen on PEG, the characteristic peaks of HA and DSPE-PEG appeared in the hydrogen spectrum of DSPE-PEG-HA, indicating that DSPE-PEG-HA has been successfully synthesized.
  • the infrared spectrum of DSPE-PEG-HA is shown in Figure 2: 2916cm -1 and 2868cm -1 are the stretching vibration peaks of PEG methylene; due to the reaction to generate new amide bonds, in the infrared spectrum of DSPE-PEG-HA, 3329cm
  • the intensity of the stretching vibration peak of the primary amide at -1 increased; the carbonyl stretching vibration peak of distearyl ester appeared at 1733cm -1 , the stretching vibration peak of the carbonyl group of the amide bond at 1637cm -1 and 1573cm -1 strengthened, and the stretching vibration peak of the NH at 843cm -1
  • PS, PS@Lip and PS@HA-Lip are round and spherical; after coating liposomes, the particle size of PS@Lip and PS@HA-Lip becomes larger than that of PS, and the surface of PS can be seen A layer of shell is uniformly distributed, which proves that the carrier is successfully prepared and has a suitable particle size.
  • the carrier was co-incubated with fetal bovine serum, and the relative turbidity of Lip, P-Lip, HA-Lip, and PS@HA-Lip at predetermined time points was recorded to evaluate the serum stability of the carrier.
  • Lip has a high positive charge, it is easy to interact with negatively charged proteins in serum to produce precipitation, resulting in an increase in relative turbidity; PEG modification is beneficial to improve the serum stability of Lip, significantly reducing The relative turbidity of the mixed system; although there is a difference in charge between HA-Lip and PS@HA-Lip, the relative turbidity is not much different, indicating that both can exist stably in serum, and the modification of HA can further reduce the carrier and serum protein interaction, improve the stability of the carrier, and facilitate long-term circulation in the body.
  • A549 cells and HUVEC cells with logarithmic growth were taken and cultured in 96-well plates (3000 cells/well). After 12 hours, they were co-cultured with media containing different concentrations of carriers for 24 hours. 20 ⁇ L of CCK-8 solution was added and incubated for 4 hours. The absorbance value OD 450 was measured using a multifunctional microplate reader. The cell viability was calculated according to the following formula, and the safety of the vector to normal cells and tumor cells was evaluated. Lip, P-Lip, HA-Lip, PS@HA-Lip loaded with non-drug-effective negative control plasmid p-null at different concentrations were administered.
  • OD 450(treated) Absorbance of wells with cells, loading body and CCK-8 solution
  • OD 0 Absorbance of wells without cells, medium and CCK-8 solution
  • OD 450(nontreated) cells, plus CCK-8 8. Absorbance of unloaded volumetric wells.
  • the cell survival rate of HA-Lip and PS@HA-Lip is also higher than 95% at higher concentrations, which can be regarded as the presence of the two on the cells. There is no impact on various life activities, and it has high safety.
  • pMTH1 itself does not have a fluorescent label
  • FAM-labeled siRNA Take A549 cells with logarithmic growth, inoculate an appropriate amount into a confocal small dish, culture for 12 hours, and mix with P-Lip/siRNA FAM , HA-Lip/siRNA FAM , PS@HA-Lip/siRNA FAM , HA+PS@HA-Lip/ siRNA FAM (siRNA FAM concentration: 50nM) co-incubation.
  • A549 cells with logarithmic growth were inoculated into 6-well plates (about 300,000 per well), cultured for 12 hours, and mixed with P-Lip/siRNA FAM , HA-Lip/siRNA FAM , PS@HA-Lip/siRNA FAM , HA +PS@HA-Lip/siRNA FAM (siRNA FAM concentration: 50nM) co-incubation. After 2 hours, the cells were washed 3 times with PBS, digested with trypsin, washed 3 times with PBS, and finally the cells were resuspended with 0.3 mL of PBS, and the uptake was analyzed by flow cytometry. The uptake experiment was carried out with MDA-MB-231 cells and HepG2 cells to investigate the effect of the expression level of CD44 receptor on the uptake of the vector cells, and the results are shown in FIG. 9 .
  • a proper amount of A549 cells with logarithmic growth was inoculated into confocal small dishes (150,000 per dish), cultured for 12 hours, and co-incubated with HA-Lip/siRNA FAM and PS@HA-Lip/siRNA FAM .

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Abstract

L'invention concerne un vecteur non viral, ainsi que sa méthode de préparation et son utilisation. Le vecteur non viral comprend de la protamine avec un médicament plasmidique comprimé, un liposome cationique enrobant la surface de la protamine avec le médicament d'acide nucléique comprimé, et un polymère de distéaroylphosphatidyléthanolamine-polyéthylène glycol-acide hyaluronique (DSPE-PEG-HA) modifié sur la surface du liposome cationique. Le vecteur non viral fournit un système candidat pour l'administration de plasmides d'édition génétique.
PCT/CN2021/106991 2021-06-17 2021-07-19 Vecteur non viral, sa méthode de préparation et son utilisation Ceased WO2022262050A1 (fr)

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