WO2021004477A1 - Système d'administration de médicament fondé sur des mitochondries et son utilisation - Google Patents

Système d'administration de médicament fondé sur des mitochondries et son utilisation Download PDF

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WO2021004477A1
WO2021004477A1 PCT/CN2020/100825 CN2020100825W WO2021004477A1 WO 2021004477 A1 WO2021004477 A1 WO 2021004477A1 CN 2020100825 W CN2020100825 W CN 2020100825W WO 2021004477 A1 WO2021004477 A1 WO 2021004477A1
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mitochondria
protein
drug
drug delivery
delivery system
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PCT/CN2020/100825
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Chinese (zh)
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唐凌峰
吴忆华
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唐凌峰
吴忆华
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

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  • the present invention relates to a mitochondrial-based drug delivery system, a preparation method of the drug delivery system, the use of the drug delivery system for delivering drugs, and the preparation of the drug delivery system for treating and/or preventing diseases, Use in medicines and/or preparations for cosmetology, weight loss, anti-fatigue, growth promotion, enhancement of body function or anti-aging.
  • a common treatment method is to introduce exogenous biomolecule drugs into target cells to replace, compensate, block or correct defective molecules in the patient's cells, so as to achieve the purpose of preventing and treating diseases.
  • non-viral vector Another common biological macromolecule vector is a non-viral vector.
  • such carriers mainly include cationic polymers, liposomes or other nanoparticles.
  • non-viral vectors still face many challenges, mainly in the aspects of low drug load, low transfection efficiency, and high cytotoxicity. These shortcomings also limit its wide clinical application. Therefore, in the field of drug delivery, there is still a need to develop non-viral biological macromolecule vectors with high transfection efficiency and low toxicity. Summary of the invention
  • Mitochondria are important organelles responsible for energy synthesis, cell differentiation, information transmission and apoptosis of eukaryotic cells. Mitochondria evolved from ancient bacteria 1.5 billion years ago. They still retain some of the characteristics of bacteria and have the "infectious ability" similar to intracellular bacteria. For example, mitochondria are separated from cells and co-cultured with other cells. It can enter these cells quickly and efficiently (Clark, MA and JWShay (1982). "Mitochondrial transformation of mammalian cells.” Nature 295(5850):605-607.).
  • mitochondria can be used as biomacromolecule carriers to achieve safe and effective intracellular delivery of target drugs.
  • the purpose of the present invention is to provide a new, widely applicable and safe mitochondrial loading method, so as to realize the efficient transmembrane delivery of various drugs, especially biological macromolecules such as proteins and nucleic acids.
  • the present invention provides a mitochondrial-based drug delivery system, the drug delivery system comprising:
  • the drug is loaded on the mitochondria through non-free diffusion.
  • the source of mitochondria can be autologous, allogeneic or heterologous and combinations thereof.
  • Drugs can be protein drugs such as peptide drugs, antibody drugs, cytokines, enzymes, protein vaccines, peptide hormones, proteins translated from normal genes, protein derivatives, nucleic acid drugs such as DNA, RNA or nucleic acid analogs , Or any other substances that cannot or are difficult to penetrate the cell membrane, and combinations thereof.
  • the drugs are protein drugs or nucleic acid drugs and combinations thereof.
  • the drug can be reversibly or irreversibly loaded on the outer mitochondrial membrane, inner membrane, interstitial membrane or mitochondrial matrix and combinations thereof in the form of covalent bonds or non-covalent bonds.
  • the drug delivery system according to the present invention can realize the delivery of drugs into cells, where the cells may include the inner surface of the cell membrane, the cytoplasm, the nucleus, and combinations thereof. After the drug is delivered into the cell, it can exist in a state of being bound to or separated from mitochondria.
  • the drug loaded on the mitochondria may be a protein drug.
  • Protein drugs can be bound to mitochondria by any method, including but not limited to: (1) Protein drugs and mitochondrial localization sequences, such as mitochondrial outer membrane localization sequences such as FIS1.MTS, and mitochondrial matrix localization sequences such as COX8.MTS to form fusion proteins , Bind to mitochondria through the positioning sequence; (2) Protein drugs and other proteins that can specifically bind to mitochondria, such as antibodies, antibody-binding variable regions of antigens, nanobodies, or other non-antibodies that can bind to mitochondria The amino acid sequence is fused to form a fusion protein to bind to mitochondria; (3) Through chemical bonds such as covalent bonds, ionic bonds, secondary bonds such as van der Waals forces, hydrogen bonds, aromatic ring stacking, hydrophobic forces and halogen bonds, and Combines to mitochondria.
  • the protein drug loaded on the mitochondria can be a single protein or a combination of multiple proteins.
  • the protein drug loaded on the mitochondria can be a complete protein, such as GFP, or each part of a protein divided into several parts, such as GFP1-11 and GFP12 of Split-GFP.
  • the protein drugs loaded on the mitochondria can be labeled with fluorescent protein or other protein tags (such as His-Tag, etc.) or not.
  • the drug loaded on the mitochondria may be a nucleic acid drug.
  • Nucleic acid drugs can bind to mitochondria through nucleic acid binding proteins or nucleic acid binding domains of nucleic acid binding proteins, or through chemical bonds such as covalent bonds, ionic bonds, secondary bonds such as van der Waals forces, hydrogen bonds, aromatic ring stacking, and hydrophobic forces. And the halogen bond, and its combination, bind to the mitochondria.
  • DNA is negatively charged. If protein X is positively charged, X-MOM (Mitochondrial outer membrane, mitochondrial outer membrane positioning sequence) is anchored to the outer membrane of mitochondria, then DNA can bind to the protein X, and then load the outer membrane of the mitochondria. ⁇ The membrane.
  • Nucleic acid drugs loaded on mitochondria can be one gene or multiple genes.
  • Nucleic acid drugs can be DNA, RNA, DNA and RNA analogs, and combinations thereof.
  • DNA can be circular or linear, single-stranded or double-stranded, including but not limited to plasmids, PCR products, genomic DNA, etc.;
  • RNA includes but not limited to mRNA, miRNA, shRNA, siRNA or aptomer.
  • DNA or RNA analogs refer to substances that can be transcribed, replicated, or bind to DNA or RNA through similar base complementation, such as morpholino and oligos.
  • the present invention also provides the use of the above-mentioned drug delivery system for delivering drugs, such as protein drugs, nucleic acid drugs or other macromolecular drugs.
  • the present invention also provides the above-mentioned drug delivery system in the preparation of preparations and/or compositions for treating and/or preventing diseases, beauty, weight loss, anti-fatigue, promoting growth and development, enhancing body function or delaying aging use.
  • the preparations or compositions prepared by the drug delivery system of the present invention can be used to treat and/or prevent diseases including nervous system diseases, immune system diseases, respiratory system diseases, circulatory system diseases, urinary system diseases, reproductive system diseases, and sports system diseases , Endocrine system disease, digestive system disease, single gene genetic disease, polygenic genetic disease, infectious disease, tumor, diabetes, neurodegenerative disease or cardiovascular and cerebrovascular disease and combinations thereof.
  • Fig. 1 shows that the target protein NeoR in Example 1 according to the present invention is loaded on the outer membrane of mitochondria through the mitochondrial outer membrane targeting sequence, and then is introduced into the cell.
  • A CellMask Deep-red marks the cell membrane.
  • B The red fluorescent protein mScarlet-i shows the target protein NeoR.
  • C The cell membrane signal overlaps with the target protein signal, showing that the target protein NeoR is delivered into the cell.
  • Fig. 2 shows that the target protein NeoR in Example 2 of the present invention is loaded on the outer membrane of mitochondria through an optogenetic protein pair and then introduced into the cell.
  • A CellMask Deep-red marks the cell membrane.
  • B The red fluorescent protein mScarlet-i shows the target protein NeoR.
  • C The cell membrane signal overlaps with the target protein signal, showing that the target protein NeoR is delivered into the cell.
  • FIG. 3 shows that in Example 3 according to the present invention, mitochondria simultaneously deliver two target proteins into the cell.
  • A CellMask Deep-red marks the cell membrane.
  • B The red fluorescent protein mScarlet-i shows the target protein 1 (Catalase).
  • C Green fluorescent protein GFP shows target protein 2 (IL10).
  • D The cell membrane signal overlaps with the target protein signal, showing that the target protein Catalase and IL10 are delivered into the cell.
  • Fig. 4 shows the introduction of a target protein into a cell by an antibody bound to the outer mitochondrial membrane protein in Example 4 according to the present invention.
  • A CellMask Deep-red staining, marking the cell membrane.
  • B GFP labels the outer mitochondrial membrane and mimics the outer mitochondrial membrane protein.
  • C The target protein NeoR linked to the anti-GFP Nanobody.
  • D After overlapping, it shows that the target protein NeoR has been introduced into the cell.
  • Fig. 6 shows that in Example 6 of the present invention, mitochondria is used as a vector to introduce mRNA into a cell and successfully express it.
  • A CellMask Deep-red staining, marking the cell membrane.
  • B The red fluorescent protein labeled with mitochondria indicates that Mito-mScarlet-i has been introduced into the cell and translated.
  • C Overlap (A) and (B), showing that the translated protein is located in the cell, indicating that the mRNA is successfully delivered into the cell and expressed.
  • the drug can be fused with FIS1.MTS protein and then loaded on the outer membrane of mitochondria, where FIS1.MTS protein is a targeting polypeptide (MTS, mitochondrial targeting sequence) of the outer mitochondrial membrane protein FIS1 . After the drug is fused with FIS1.MTS protein, it contains the targeting part of FIS1, so it can be loaded on the outer membrane of mitochondria.
  • FIS1.MTS protein is a targeting polypeptide (MTS, mitochondrial targeting sequence) of the outer mitochondrial membrane protein FIS1 .
  • the drug can be fused with the fluorescent protein and FIS1.MTS protein, and then loaded on the surface of the mitochondria.
  • fluorescent protein the role of fluorescent protein is to mark drugs, only for tracking and monitoring drug delivery, not a necessary means to achieve drug delivery.
  • Fluorescent proteins include but are not limited to green fluorescent protein GFP, red fluorescent protein RFP, enhanced green fluorescent protein EGFR, red fluorescent protein mScarlet-I and so on.
  • the drug is loaded onto the outer membrane of the mitochondria by optogenetic methods, such as pMag and nMag, Cry2-CIB or any other optogenetic protein pair.
  • optogenetic methods such as pMag and nMag, Cry2-CIB or any other optogenetic protein pair.
  • the drug forms a fusion protein with pMag
  • nMag forms a fusion protein with the mitochondrial outer membrane positioning sequence such as FIS1.MTS protein
  • the pair of pMag and nMag proteins are paired and bound under the action of blue light, so that The drug is loaded on the outer membrane of the mitochondria.
  • the drug delivery system according to the present invention can also use proteins that bind under the action of chemical substances, such as FKBP12 and FRB regulated by rapamycin, or any other reversibility. Or irreversibly bound protein pair.
  • a pair of protein pairs can be used between the drug and the mitochondrial outer membrane localization sequence, or multiple pairs of proteins can be used, one type of protein pair can be used, or multiple types of protein pairs can be used. Protein pair.
  • the drug can be fused with the fluorescent protein and the aforementioned protein pair, and then loaded on the outer membrane of the mitochondria.
  • the fluorescent protein plays the role of labeling drugs, only for tracking and monitoring the delivery of drugs, and is not necessary to achieve drug delivery.
  • Fluorescent proteins include but are not limited to green fluorescent protein GFP, red fluorescent protein RFP, enhanced green fluorescent protein EGFR, red fluorescent protein mScarlet-I and so on.
  • the drug delivery system according to the present invention is prepared by a method including the following steps:
  • X-Y-FIS1.MTS protein where the protein includes three parts: X is any target drug; Y is a fluorescent protein used to label the target drug; FIS1.MTS is a targeting polypeptide of the mitochondrial outer membrane protein FIS1;
  • the drug delivery system according to the present invention is prepared by a method including the following steps:
  • X is any target drug
  • Y1 and Y2 are fluorescent proteins for labeling
  • pMag and nMagHigh1 are a pair of optogenetic proteins
  • FIS1 is the targeting peptide of the outer mitochondrial membrane protein FIS1.
  • the fluorescent protein can be GFP, EGFP, mScarlet-i, YFP, CFP or other fluorescent proteins.
  • the transfected cells can be HEK 293T, CHO-K1, COS-7, HeLa, NIH-3T3, PC-3, A549, MCF-7, HuH-7, U-2OS, HepG2 or any other transfectable cells.
  • the transfection reagent can be Lipofectamine, X-tremeGENE 9, Escort TM III, Escort TM IV, NeuroPorter TM DOTAP methosulfate, Calcium Phosphate or any other transfection reagent.
  • the transfection method can be liposome transfection method, calcium phosphate transfection method, electroporation transfection method or any other transfection method.
  • the co-cultivation time of mitochondria and cells can be 0.5-120 hours, preferably 6-24 hours.
  • NeoR-mScarlet-i-pMag and nMagHigh1-GFP-FIS1.MTS design proteins that NeoR-mScarlet-i-pMag and nMagHigh1-GFP-FIS1.MTS.
  • NeoR is the target drug
  • mScarlet-i is the red fluorescent protein used to label the target drug.
  • the nMagHigh1-GFP-FIS1.MTS fusion protein can bind to the outer mitochondrial membrane through the mitochondrial outer membrane targeting sequence FIS1.MTS.
  • pMag and nMagHigh1 are a pair of optogenetic proteins that can bind to each other under blue light irradiation.
  • pcDNA3.1-NeoR-mScarlet-i-pMag and pcDNA3.1-nMagHigh1-GFP-FIS1.MTS were co-transfected into 293T cells. After 24 hours, the cells were irradiated with strong light for 10 minutes to make NeoR-mScarlet-i-pMag fusion protein Combine with nMagHigh1-GFP-FIS1.MTS fusion protein to load the target protein NeoR onto the outer membrane of mitochondria. Then extract the mitochondria loaded with NeoR protein (mScarlet-i label), and dilute the extracted mitochondria with DMEM complete medium, and then add them to the cultured unlabeled 293A cells.
  • NeoR protein mScarlet-i label
  • NeoR is the target drug
  • mScarlet-i is a red fluorescent protein used to label the target drug
  • NB.GFP is a nanobody that can specifically bind to GFP.
  • the role of GFP is to mark mitochondria and mimic mitochondrial outer membrane protein.
  • FIS1.MTS is the target sequence of mitochondrial outer membrane protein FIS1. Two 293T cells in a 10cm culture dish were transfected with the two plasmids.
  • NeoR-mScarlet-i-NB.GFP After 24 hours, the cells expressing NeoR-mScarlet-i-NB.GFP were harvested. The cells were swelled with distilled water for 5 minutes, and then 1/9 volume of 10X PBS was added. The cells were broken by a glass homogenizer, centrifuged at 10,000g for 10 minutes, and the supernatant containing NeoR-mScarlet-i-NB.GFP was taken for later use.
  • the cells expressing GFP-FIS1.MTS were harvested, mitochondria were extracted, and resuspended in the supernatant containing NeoR-mScarlet-i-NB.GFP, placed at room temperature for 10 minutes, and then co-cultured with unlabeled 293A cells, 37°C medium After 6-8 hours of incubation, the cell membrane was stained with the cell membrane dye CellMask Deep-red, and then imaged with a laser confocal microscope. It was found that some GFP-labeled mitochondria had successfully transferred mScarlet-i labeled NeoR into the cells. The test results are shown in Figure 3.
  • the pcDNA3.1 vector was used to construct the COX8.MTS-hCatalase-mScarlet-i plasmid (see the sequence list, SEQ ID NO: 6) and the IL10-mNeonGreen-FIS1.MTS plasmid (see the sequence list, SEQ ID NO: 7) 293T cells were co-transfected with two plasmids, 24 hours later, mitochondria loaded with hCatalase (mScarlet-i label) and/or IL10 (mNeonGreen label) were extracted, and the extracted mitochondria were diluted with DMEM complete medium and added to the culture In the unlabeled 293A cells, after incubating in 37°C medium for 6-8 hours, stain the cell membrane with the cell membrane dye CellMask Deep-red, and then use laser confocal microscope imaging to detect some mNeonGreen-labeled hCatalase and mScarlet-i labeled IL10 has entered the cell.
  • the test results are shown in
  • telomere Construct the plasmid pcDNA3.1-hTFAM1-CRT-mScarlet-i-FIS1.MTS (see Sequence Listing, SEQ ID NO: 8), where hTFAM1 is the DNA binding domain of human TFAM1, CRT is the DNA binding domain of TopII alpha, both Can bind to DNA.
  • mScarlet-i is a red fluorescent protein used to label mitochondria carrying the fusion protein; FIS1.MTS is a signal peptide targeting the outer mitochondrial membrane.
  • the plasmid pcDNA3.1-UP1-FIS1.MTS (see Sequence Listing, SEQ ID NO: 10) was constructed, where UP1 is the non-specific RNA binding domain of human hnRNP A1 protein, and FIS1.MTS is a signal peptide targeting the outer mitochondrial membrane.
  • 293T cells were transfected with pcDNA3.1-UP1-FIS1.MTS, mitochondrial 107 cell extracts taken after 24 hours, resuspended in mitochondria 100 ⁇ LDMEM complete medium, plus the mRNA 200ng Mito-mScarlet-i (manufactured by plasmid pcDNA3.1-Mito -mScarlet-I (see sequence listing, SEQ ID NO: 11) was transcribed in vitro), incubated at room temperature for 0.5 hours, then co-cultured mitochondria with unlabeled 293A cells, and stained the cell membrane with the cell membrane dye CellMask Deep-red after 24 hours , Laser confocal microscope imaging shows that some cells express red signals that mark mitochondria, indicating that mRNA has been successfully transferred into cells and translated. The test results are shown in Figure 6.

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Abstract

Système d'administration de médicament fondé sur des mitochondries. Ce système d'administration de médicament comprend des mitochondries et un médicament chargé sur les mitochondries, le médicament étant chargé sur les mitochondries par diffusion non libre. Le système d'administration de médicament peut permettre d'administrer efficacement une protéine, un acide nucléique ou d'autres médicaments dans des cellules, peut être utilisé pour traiter des maladies monogéniques, des maladies polygéniques ou d'autres maladies, et peut également être utilisé chez des sujets sains afin de retarder l'apparition de la sénilité, d'améliorer les fonctions corporelles ou de prévenir des maladies.
PCT/CN2020/100825 2019-07-09 2020-07-08 Système d'administration de médicament fondé sur des mitochondries et son utilisation WO2021004477A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112999357A (zh) * 2021-03-03 2021-06-22 中国药科大学 一种外源线粒体载体、复合物及其制备方法和应用
WO2022228223A1 (fr) * 2021-04-26 2022-11-03 重庆理工大学 Mitochondries modifiées et leur procédé de préparation
WO2023237788A1 (fr) * 2022-06-10 2023-12-14 Cellvie Ag Mitochondries en tant que plateforme d'administration ciblée

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WO2017192102A1 (fr) * 2016-05-06 2017-11-09 National University Of Singapore Distribution mitochondriale d'acides nucléiques recombinants
WO2017201313A1 (fr) * 2016-05-18 2017-11-23 Shengkan Jin Nouveaux découplants mitochondriaux pour le traitement de maladies métaboliques et du cancer
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CN109952379A (zh) * 2016-11-14 2019-06-28 白雁生物技术公司 将外源性线粒体递送到细胞中的方法

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AU2008216018B2 (en) * 2007-02-16 2012-10-25 John Guy Mitochondrial nucleic acid delivery systems
EP2992892A1 (fr) * 2014-09-05 2016-03-09 Universität zu Köln Protéine de fusion pour une utilisation dans le traitement de maladies mitochondriales

Patent Citations (6)

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Publication number Priority date Publication date Assignee Title
WO2017124037A1 (fr) * 2016-01-15 2017-07-20 The Children's Medical Center Corporation Utilisation thérapeutique de mitochondries et d'agents mitochondriaux combinés
WO2017192102A1 (fr) * 2016-05-06 2017-11-09 National University Of Singapore Distribution mitochondriale d'acides nucléiques recombinants
WO2017201313A1 (fr) * 2016-05-18 2017-11-23 Shengkan Jin Nouveaux découplants mitochondriaux pour le traitement de maladies métaboliques et du cancer
WO2018088875A2 (fr) * 2016-11-14 2018-05-17 주식회사 파이안에스테틱스 Cellule tueuse naturelle contenant une mitochondrie exogène et composition pharmaceutique la comprenant
CN109952379A (zh) * 2016-11-14 2019-06-28 白雁生物技术公司 将外源性线粒体递送到细胞中的方法
WO2018101708A1 (fr) * 2016-11-30 2018-06-07 차의과학대학교 산학협력단 Composition pharmaceutique contenant des mitochondries

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112999357A (zh) * 2021-03-03 2021-06-22 中国药科大学 一种外源线粒体载体、复合物及其制备方法和应用
WO2022228223A1 (fr) * 2021-04-26 2022-11-03 重庆理工大学 Mitochondries modifiées et leur procédé de préparation
WO2023237788A1 (fr) * 2022-06-10 2023-12-14 Cellvie Ag Mitochondries en tant que plateforme d'administration ciblée

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