WO2024065649A1 - Procédé de chargement efficace d'adn dans un exosome - Google Patents

Procédé de chargement efficace d'adn dans un exosome Download PDF

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WO2024065649A1
WO2024065649A1 PCT/CN2022/123200 CN2022123200W WO2024065649A1 WO 2024065649 A1 WO2024065649 A1 WO 2024065649A1 CN 2022123200 W CN2022123200 W CN 2022123200W WO 2024065649 A1 WO2024065649 A1 WO 2024065649A1
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dna
loading
exosomes
protamine
heat shock
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PCT/CN2022/123200
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Chinese (zh)
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王伊
宋海峰
李艳芳
薛苗苗
董亚南
王丹枫
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谛邈生物科技（新加坡）有限公司
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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
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

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  • the present invention relates to the technical field of exosome loading, and specifically to a method for efficiently loading DNA into exosomes, wherein the exosomes are derived from mammalian cells or milk, a polycationic compressor is combined with DNA to form a highly compressed DNA-polycationic compressor complex, and the DNA-polycationic compressor complex is loaded into the exosome membrane by physical means, thereby successfully completing efficient loading of large-fragment DNA molecules and its gene delivery and expression in vivo.
  • DNA-mediated gene therapy is an effective means to treat genetic diseases, malignant tumors and some degenerative diseases.
  • DNA-mediated gene therapy still faces many problems that are difficult to effectively solve: (1) DNA not only needs to enter the cell, but also needs to enter the cell nucleus to be transcribed and expressed; 2) A fully functional DNA sequence needs to include multiple functional elements such as promoter, target fragment, polyA fragment, etc., and its size is usually much larger than the mRNA fragment used for treatment. Therefore, the current mainstream DNA-based gene therapy method is still based on adeno-associated virus AAV. However, AAV vectors themselves also have many serious problems, the most prominent of which is their safety risks in the human body.
  • Exosomes are a type of extracellular vesicles. Nucleic acid delivery is one of the natural functions of exosomes. In addition, exosomes can achieve tissue and cell-specific delivery through engineering modification. As drug transport carriers, they have outstanding advantages such as low immunogenicity, high transport efficiency, good stability, strong targeting, and the ability to cross the blood-brain barrier. If they can be used in the field of DNA delivery, exosomes will become better gene therapy vectors than AAV.
  • RNA mRNA, siRNA, etc.
  • the methods and technologies for loading RNA (mRNA, siRNA, etc.) into exosomes are relatively mature, while the common methods of loading RNA (mRNA, siRNA, etc.) into cell exosomes by incubation or electroporation can only be used to load small nucleic acid fragments such as miRNA, siRNA or ASO, and are powerless for mRNA or even large DNA fragments or DNA plasmids.
  • the present invention provides a method for efficiently loading DNA into mammalian cells or milk-derived exosomes and realizing in vivo gene delivery and expression.
  • This technology not only uses reagents and excipients that have been included in the pharmacopoeias of various countries and are easy to obtain and circulate, but also simplifies the loading and preparation process, reduces costs, facilitates large-scale production, is compatible with existing biopharmaceutical production processes, and significantly improves the loading efficiency of large-fragment DNA molecules, providing a new and efficient tool for gene therapy.
  • the present invention provides a method for efficiently loading DNA into exosomes, wherein the exosomes are derived from mammalian cells or milk, a polycationic compressor is combined with DNA to form a highly compressed DNA-polycationic compressor complex, and the DNA-polycationic compressor complex is loaded into the exosome membrane by physical means, thereby successfully completing efficient DNA in vivo gene delivery and expression.
  • the present invention provides a method for efficiently loading DNA into exosomes, wherein the exosomes are derived from mammalian cells or milk, and the specific steps are:
  • the method may include step (4) of removing unloaded DNA and fragments
  • step (4) heparin is added to dissociate the unloaded DNA-protamine complex, and the method for removing the unloaded free DNA can be selected from: one or more of high-salt nuclease, DNAseI treatment, and Benzonase treatment, and the removal of nucleic acid fragments can be selected from anion exchange chromatography column purification and/or Capto Core 700 chromatography column purification method;
  • the method may include step (5), verifying the loading effect using a qPCR method
  • the exosomes derived from mammalian cells or milk may be unmodified exosomes or engineered exosomes;
  • engineered exosomes are exosomes engineered with syncytin-1;
  • engineered exosomes are exosomes modified with single-chain integrin Integrin ⁇ L ⁇ 2;
  • engineered exosomes are exosomes modified with single-chain integrin Integrin ⁇ D ⁇ 2;
  • step (1) the ratio of DNA:exosomes is 3-20:1;
  • polycationic compressing agent may be selected from one or more of protamine, polylysine, polyarginine, and polyethyleneimine (PEI);
  • polycationic compression agent may be protamine, and the ratio of protamine to DNA is 2-10:1;
  • polycationic compression agent may be protamine, and the ratio of protamine to DNA is 7:1;
  • the resulting precipitate can be separated from the supernatant by centrifugation;
  • the DNA loading method may be an ultrasound method and/or a heat shock/thermal cycle method
  • the number of cycles of the heat shock/thermal cycle loading method is 1-20 times;
  • the number of cycles of the heat shock/thermal cycle loading method is 10-20 times;
  • the heat shock temperature of the heat shock/thermal cycle loading method may be 25-60°C, and the cooling temperature may be 0°C or 4°C;
  • the heat shock temperature for loading milk exosomes was 60°C
  • the cooling temperature was 0°C
  • the number of heat shocks was 20 times
  • the heat shock temperature for loading cell exosomes was 60°C
  • exosomes are pretreated before DNA loading, such as adjusting the pH;
  • auxiliary molecules are added to promote the loading of DNA into the exosome membrane, and the auxiliary factors can be selected from one or two of SM102 and DLin-MC3-DMA;
  • the ratio of DNA to protamine is 1:2;
  • the size of the loaded DNA is 1.9 kbp-9 kbp;
  • the present invention provides an exosome with high efficiency of loading DNA prepared by the above method, wherein the exosome is derived from mammalian cells or milk;
  • the present invention proposes the use of exosomes efficiently loaded with DNA prepared by the above method in the preparation of drugs
  • the drug is a drug used as a non-viral vector for in vivo DNA delivery in gene therapy.
  • the present invention provides a medicine, which comprises exosomes prepared by the preparation method of the present invention.
  • the present invention also provides a gene therapy method, which comprises administering the drug of the present invention.
  • the method for efficiently loading DNA into exosomes realizes the efficient loading of large DNA molecules, and its efficient loading range can be from 1.9 kbp to 9 kbp, which effectively solves the problem of inefficient loading of large DNA molecules in the field, so that when exosomes are used as gene therapy tools, they can carry large pieces of target DNA, expand the range of target gene selection for gene therapy, provide exosomes with a broader drug potential, and provide a powerful tool for gene therapy.
  • the present invention uses one or more of protamine, polylysine, polyarginine, and polyethyleneimine to help DNA molecules fold and thus achieve efficient loading.
  • the above ingredients are all widely used drugs or reagents, thus achieving significant savings in experimental costs and even future treatment costs. It also achieves the highly difficult loading of large DNA molecules in the most conventional loading method, and has significant advantages in efficiency and economy in industrial applications.
  • the present invention further creatively adds one or more auxiliary factors of SM102 and DLin-MC3-DMA to the DNA exosome loading, thereby further improving the loading effect and stability, providing strong support for the application of the exosomes obtained by the above loading method in gene therapy.
  • the exosomes loaded by the present invention may be exosomes modified with syncytin-1 and/or single-chain integrin Integrin ⁇ L ⁇ 2 and/or single-chain integrin Integrin ⁇ D ⁇ 2, which can specifically target inflamed vascular endothelial cells and glomerular podocytes, providing effective tools and directions for the research of corresponding disease models, the development of drugs for clinically corresponding diseases, and the exploration of gene therapy methods.
  • Figure 1 Plasmid map of the mini-circle hRluc (1919 bp) of the present invention
  • Fig. 2 pRP-EGFP (3657 bp) vector map of the present invention
  • Fig. 4 Plasmid map of pRP-Nanoluc (4929 bp) of the present invention
  • FIG5 NanoLuc enzyme activity is detected in the cell supernatant after incubation of the DNA-loaded exosomes of the present invention with cells;
  • Figure 6 Plasmid map of pSGLs-Nanoluc-pp-Fc (8929 bp) of the present invention.
  • FIG7 Nanoluc enzyme activity was detected in the cell supernatant after incubation of the DNA-loaded exosomes of the present invention with cells;
  • FIG8 Nanoluc enzyme activity was detected in the cell supernatant after incubation of the DNA-loaded exosomes of the present invention with cells;
  • FIG. 9 Nanoluc enzyme activity in the cell supernatant of the present invention.
  • FIG10 Immunofluorescence detection of exosome targeting effect of the present invention
  • FIG. 11 Targeted exosome-treated cells of the present invention express nanoluc.
  • Example 1 Loading small molecular weight plasmid mini-circle hRluc DNA (1919 bp) into milk exosomes, and comparing the effects of different polycations such as poly-lysine, poly-arginine, protamine and polyethyleneimine (PEI) on DNA loading.
  • polycations such as poly-lysine, poly-arginine, protamine and polyethyleneimine (PEI) on DNA loading.
  • protamine or poly-lysine, poly-arginine, polyethyleneimine
  • the anion exchange chromatography column-low salt elution fraction contained the exosome peak after loading, and about 16-63 DNA plasmid copies were loaded per 100 exosome particles.
  • the protamine group had the best loading effect, and no mini-circle hRluc DNA loading was detected in the experimental group without polycations.
  • Example 2 Loading small molecular weight plasmid mini-circle hRuc DNA (1919bp) into milk exosomes to verify whether protamine is the key reagent for DNA loading.
  • high salt nuclease at a final concentration of 5U/mL
  • the nucleic acid fragments were purified and removed using Capto Core700 chromatography column and anion exchange chromatography column (equilibrium solution 20mM Tris pH 8.0, 100mM NaCl, elution solution equilibration solution 20mM Tris pH 8.0, 1M NaCl, gradient elution method).
  • Example 3 Compare the effects of centrifugation or non-centrifugation during the loading process on the efficiency of exosome loading with mini-circle hRluc DNA.
  • Protamine is positively charged and can bind to negatively charged DNA through electrostatic interaction, thereby compressing the DNA.
  • the loading efficiency of exosomes for DNA can be changed by controlling the loading method. Since obvious precipitate will be generated during the loading process, heparin is added to dissolve the precipitate with or without centrifugation, and nuclease is added to digest the free nucleic acid. After removing the free protamine, heparin, and nucleic acid fragments in the system through Capto Core700, the effect of centrifugation on the DNA loading effect can be compared.
  • DNA: exosome 20:1 (copy number: particle number).
  • the OMEM-DNA-protamine mixture was added to the milk exosomes, mixed while adding, and then the sample was placed in a metal bath at 37°C, 120 rpm, for 30 min, and then immediately placed in an ice-water mixture (4°C) and allowed to stand for 10 min. This was one cycle; the heat shock was repeated 10 times. After the sample was heat-shocked, it was divided into two equal parts: one part was directly added with heparin, mixed evenly, and then DNaseI enzyme was added to digest at 37°C overnight; the other part was centrifuged at 12000rpm for 20min, and the precipitate and supernatant were collected respectively.
  • Embodiment 4 is a diagrammatic representation of Embodiment 4:
  • DNA loading The DNA plasmid loaded was mini-circle hRluc DNA (plasmid vector, target fragment was hRluc, plasmid size was 1919 bp), and the DNA: exosome ratio was 20:1 (copy number: particle number).
  • OMEM + protamine 200 ⁇ L was mixed and allowed to stand for 1 min, and 200 ⁇ L of OMEM + 10 ⁇ g of DNA was mixed and allowed to stand for 1 min; then OMEM-DNA was added to OMEM + protamine (protamine: DNA mass ratio was 2:1, 5:1, 7:1, 10:1), then vortexed for 1 min, and allowed to stand at 25°C for 10 min.
  • Example 5 The medium molecular weight plasmid pRP-EGFP (3657 bp) was loaded into cells and exosomes, the ratio of protamine to DNA was fixed, and the difference in loading efficiency between cell exosomes and milk exosomes was verified.
  • sample name pRP-EGFP DNA ( ⁇ g) Protamine ( ⁇ g) Exosomes Heparin ( ⁇ g) DNaseI Exosomes 152.6 1068.2 2.27E+12 3205 - Exosomes+DNaseI 152.6 1068.2 2.27E+12 3205 0.1mg/mL Milk exosomes 30 210 1.05E+12 630 - Milk exosomes + DNaseI 30 210 1.05E+12 630 0.1mg/mL
  • Example 6 Loading the medium molecular weight plasmid pRP-Nanoluc (4929 bp) into cell or milk exosomes, and comparing the effects of different heat shock methods on the loading efficiency of exosomes from different sources during the loading process.
  • OMEM + protamine 2 mL was mixed and allowed to stand for 1 min
  • 2 mL of OMEM + 3 ⁇ g of DNA was mixed and allowed to stand at 25°C for 1 min
  • the OMEM-DNA-protamine mixture was added to the cell exosomes or milk exosomes, and mixed while adding.
  • Two heat shock methods samples treated at 60°C were shaken at 120rpm for 1h in a 60°C shaker, then placed in an ice bath (0°C) for 5min until the samples were completely cooled. This was one cycle and repeated 3 times. Samples treated at 42°C were first placed in an ice-water bath (0°C) and allowed to stand for 30min, then heat-shocked at 42°C for 90s, then placed in an ice-water bath (0°C) for 3min. This was one cycle and repeated 10 times. After heat shock, all samples were added with heparin and vortexed at 25°C for 1min.
  • every 100 exosome particles can be loaded with up to 50 plasmid copies.
  • the loading capacity of cell exosomes for pRP-Nanoluc plasmid is relatively high
  • the loading capacity of milk exosomes for pRP-Nanoluc plasmid is relatively high.
  • Example 7 The 42°C heat shock method was used to determine whether milk exosomes could be successfully loaded with the high molecular weight plasmid pSGLs-Nanoluc-pp-Fc (8929 bp).
  • DNA loading The DNA plasmid was pSGLs-Nanoluc-pp-Fc DNA (expression vector, target fragment was Nanoluc, plasmid size was 8929 bp), and the DNA: exosomes were loaded at a ratio of 10:1 (copy number: particle number).
  • Example 8 Loading high molecular weight plasmid pSGLs-Nanoluc-pp-Fc into cell- and milk-derived exosomes, and comparing the effects of different heat shock methods on the efficiency of DNA loading in exosomes from different sources during the loading process.
  • the OMEM-DNA-protamine mixture was added to 2.0E+11 cell exosomes or 2.0E+11 milk exosomes.
  • the control group samples included all other samples except exosomes.
  • Two heat shock methods For samples treated at 60°C, oscillate at 120 rpm for 1 hour in a 60°C shaker, then place in an ice bath (0°C) for 5 minutes until the sample is completely cooled. This is one cycle and is repeated three times. For samples treated at 42°C, first place the sample in an ice-water mixed bath (0°C), let it stand for 30 minutes, then heat shock at 42°C for 90 seconds, then place in an ice-water mixed bath (0°C) for 3 minutes. This is one cycle and is repeated 10 times.
  • Exosomes are not digested at 60°C 6.46E+10 10.9 ⁇ 1.45 38 ⁇ 7 Digestion of exosomes at 60°C 9.50E+10 10.98 ⁇ 1.15 28 ⁇ 3 Exosomes 60°CBlank 1.35E+11 22.57 ⁇ 0.21 0 ⁇ 2 Exosomes are not digested at 42°C 6.05E+10 17.65 ⁇ 0.87 3 ⁇ 6 Digestion of exosomes at 42°C 8.91E+10 19.9 ⁇ 0.30 0 ⁇ 5 Exosomes 42°CB1ank 5.70E+10 23.23 ⁇ 0.70 0 ⁇ 7 Milk exosomes undigested at 60°C 4.79E+10 16.57 ⁇ 0.42 7 ⁇ 3 Digestion of milk exosomes at 60°C 3.98E+10 16.77 ⁇ 0.70 2 ⁇ 6 Milk Exosomes 60°CBlank 4.68E+10 21.80 ⁇ 0.2
  • Example 9 In order to improve the efficiency of loading the high molecular weight plasmid pSGLs-Nanoluc-pp-Fc into exosomes and to express Nanoluc with enzymatic activity after entry into the cells, different heat shock temperatures and times during the loading process were compared.
  • samples that were heat-shocked once at different temperatures were placed on ice for 10 minutes, heated at the corresponding temperature for 9 minutes, and then placed on a preheated shaker at the corresponding temperature for 175 minutes at 120 rpm, and then placed on ice for 5 minutes; for samples that were heat-shocked 5 times at different temperatures, the samples were placed on ice for 10 minutes, heated at the corresponding temperature for 9 minutes, and then placed on a preheated shaker at the corresponding temperature for 31 minutes at 120 rpm, and then placed on ice for 5 minutes, and the heat shock was repeated 5 times; for samples that were heat-shocked 10 times at different temperatures, the samples were placed on ice for 10 minutes, heated at the corresponding temperature for 9 minutes, and then placed on a preheated shaker at the corresponding temperature for 13 minutes at 120 rpm, and then placed on ice for 5 minutes, and the heat shock was repeated 10 times; for samples that were heat-shocked 20 times at different temperatures, the samples were placed on
  • heparin was added to all samples and mixed by inversion. For all samples, 1 mM MgCl2 and a final concentration of 5 U/mL Benzonase were added and digested at 37°C overnight. The digested samples were treated with Capto Core700, and the flow-through was collected to remove free protamine-heparin-Benzonase.
  • Example 10 SGLs-Nanoluc-pp-Fc plasmid was loaded into cell exosomes to verify whether the addition of different auxiliary molecules (including ionizable lipid molecules or CaCl2) during the loading process could promote the loading of DNA by exosomes, and the effect of different ratios of protamine to DNA on the loading efficiency of exosomes under the action of the addition of auxiliary molecules.
  • auxiliary molecules including ionizable lipid molecules or CaCl2
  • the sample group includes OMEM culture medium, protamine, SM102/DLin-MC3-DMA, DNA and cell exosomes, and the control group includes all other samples except exosomes.
  • the above mixture was mixed with 1E+11 cell exosomes and then added with a final concentration of 20 mM CaCl 2 .
  • the sample group includes OMEM culture medium, protamine, CaCl 2 , DNA and cell exosomes, and the control group includes all other samples except exosomes.
  • NanoFCM detects the number of sample particles after loading.
  • Example 11 SGLs-Nanoluc-pp-Fc plasmid DNA is loaded into cell exosomes, and after entry into the cell, Nanoluc with enzymatic activity can be expressed; verify whether the cell entry ability of exosomes engineered to express syncytin-1 is improved.
  • Detection of Nanoluc expression by microplate reader Detect Nanoluc enzyme activity in the cell supernatant after 72 h of culture.
  • Example 12 Engineered cell exosomes expressing single-chain integrin Integrin ⁇ L ⁇ 2 (or single-chain Integrin ⁇ D ⁇ 2) on the membrane surface can be loaded with pSGLs-Nanoluc-pp-Fc plasmid, specifically targeting inflamed vascular endothelial cells and glomerular podocytes, respectively.
  • DNA loading The DNA plasmid was pSGLs-Nanoluc-pp-Fc DNA (expression vector, target fragment was Nanoluc, plasmid size was 8929 bp), and the DNA: exosome ratio was 10:1 (copy number: particle number).
  • OMEM medium 2 mL of OMEM medium was mixed with protamine, and 2 mL of OMEM medium was mixed with 3 ⁇ g of DNA.
  • the OMEM-DNA-protamine mixture was added to 1E+11 engineered cell exosomes.
  • ICAM-1 positive cells and glomerular podocytes were inoculated at 2E5 cells per well in a 24-well plate with a slide placed in advance. After 24 hours, the slide was removed and gently washed 3 times with PBS. Fix with 500 ⁇ L paraformaldehyde for 10 minutes and wash 3 times with PBS for 5 minutes each time. Add 1 mL 5% BSA/PBS buffer to block at room temperature for 2 hours, wash 3 times with PBS for 5 minutes each time, and add unlabeled wild-type EVs at a cell to EV ratio of 1:30000 to continue blocking for 2 hours.
  • ICAM-1 positive cells and glomerular podocytes were seeded into 24-well plates at a density of 2E5 cells per well. After 24 h, wild-type EVs were added at a cell to EV ratio of 1:30,000 and blocked at room temperature for 2 h. 5E9 engineered EVs loaded with single-chain integrin Integrin ⁇ L ⁇ 2 (or single-chain Integrin ⁇ D ⁇ 2) and wild-type EVs (Blank and loaded with DNA) were added to the cells and incubated at 37°C for 72 h. The cells were then harvested for nanoluc enzyme activity detection.

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Abstract

L'invention concerne un procédé de chargement efficace d'ADN dans un exosome, l'exosome étant dérivé de cellules de mammifère ou de lait. Un complexe d'agent de compression polycationique et d'ADN hautement comprimé est formé par la combinaison d'un agent de compression polycationique avec un ADN, et le complexe d'agent de compression polycationique et d'ADN est chargé dans la membrane de l'exosome au moyen d'un moyen physique, l'agent de compression cationique pouvant être choisi parmi un ou plusieurs éléments parmi la protamine, la polylysine, la polyarginine et la polyéthylèneimine ; par conséquent, une administration de gène in vivo efficace et l'expression d'ADN à grands fragments sont réalisées en douceur en utilisant la fonction de transport de l'exosome. L'exosome obtenu par le procédé est censé devenir un outil efficace pour le chargement, le transport et l'expression d'ADN à grands fragments dans le domaine de la thérapie génique.
PCT/CN2022/123200 2022-09-30 2022-09-30 Procédé de chargement efficace d'adn dans un exosome WO2024065649A1 (fr)

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