WO2023185261A1 - 以外泌体为载体的dna递送系统 - Google Patents

以外泌体为载体的dna递送系统 Download PDF

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WO2023185261A1
WO2023185261A1 PCT/CN2023/075044 CN2023075044W WO2023185261A1 WO 2023185261 A1 WO2023185261 A1 WO 2023185261A1 CN 2023075044 W CN2023075044 W CN 2023075044W WO 2023185261 A1 WO2023185261 A1 WO 2023185261A1
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dna
exosomes
cells
exosome
preparation
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French (fr)
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尹乐
顾雨春
吴理达
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呈诺再生医学科技(珠海横琴新区)有限公司
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Publication of WO2023185261A1 publication Critical patent/WO2023185261A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a DNA delivery system using exosomes as carriers.
  • Exosomes are vesicles with a diameter of 30 to 150 nm secreted by cells. They are intracellular vesicles formed by depressions in the cytoplasmic membrane. They have the same membrane structure as the cell membrane and can carry a large number of components such as RNA and proteins, and interact with cells. There is a close relationship between its biological functions and signal transmission between cells. Due to their special structure, compared with synthetic drug carriers, exosomes have unique advantages as drug carriers for drug transport.
  • exosomes cause extremely low harmful immune responses, and have a good permeability and retention (EPR) effect, thus having a sustained-release effect.
  • EPR permeability and retention
  • the purpose of the present invention is to provide a DNA packaging system that can introduce the DNA sequence of the target gene into exosomes.
  • the invention provides a fusion DNA, the sequence of the fusion DNA includes the DNA binding motif of the DNA binding protein and the DNA sequence shown in SEQ ID NO.1; the DNA binding motif of the DNA binding protein is connected to SEQ ID NO. The 3' end of the DNA sequence shown in 1;
  • the DNA-binding protein includes a TALE protein
  • the DNA binding motif of the DNA protein includes the sequence shown in SEQ ID NO. 2.
  • the present invention provides a vector comprising the aforementioned fusion DNA.
  • the present invention provides a DNA packaging system, which includes the aforementioned carrier exosomes, as well as vectors for expressing exosome proteins and DNA-binding proteins.
  • DNA-binding protein is connected to the C-terminus of the exosome membrane protein.
  • exosome proteins include exosome transmembrane proteins and exosome surface proteins.
  • Exosome proteins that can be used in the present invention may include, but are not limited to: CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, AARDC1, Syntenin-1, Syntenin -2, Lamp2b, TSPAN8, syndecan-1, syndecan-2, syndecan-3, syndecan-4, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2 , CD49d/ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18/ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc receptor, interleukin receptor, immunoglobulin, MHC-I or MHC-II components, CD2, CD3 ⁇ , CD3 ⁇ , CD13,
  • the exosomal protein is CD63.
  • DNA binding protein includes TALE protein.
  • the vector expressing exosome protein and DNA binding protein also expresses Tol2; the DNA packaging system also includes a vector expressing Tol2 transposase.
  • the present invention also provides a DNA delivery system.
  • the DNA delivery system includes the aforementioned DNA packaging system and a targeted delivery vector.
  • the targeted delivery vector expresses exosome proteins and targeting molecules, and the targeted delivery vector expresses exosome proteins and targeting molecules.
  • the molecule recognizes the target tissue or target cells.
  • the targeting molecule is connected to the N-terminus of the exosome membrane protein.
  • exosome proteins include exosome transmembrane proteins and exosome surface proteins.
  • Exosome proteins that can be used in the present invention may include, but are not limited to: CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, AARDC1, Syntenin-1, Syntenin-2, Lamp2b, TSPAN8, syndecan-1, syndecan-2, syndecan-3, syndecan-4, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d/ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18/ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc receptor, interleukin receptor, immunoglobulin, MHC-I or MHC-II components, CD2, CD3 ⁇ , CD3 ⁇ , CD13, CD18,
  • the exosomal protein is Lamp2b.
  • the targeting molecules that can be used in the present invention include targeting peptides that interact with cell membrane surface antigens, antibodies that recognize specific cell surface antigens or antigen-binding fragments thereof, and affibodies.
  • Cell membrane surface antigens include but are not limited to ER, PR, P53, EGFR, IGFR, Her2, CD20, CD25, CD117, CD34, CD138, CD33, VEGFR, BCMA, Mesothelin, CEA, PSCA, MUC1, EpCAM, S100, CD22, CD19 , CD70, CD30, ALK, RANK, GPC2, GPC3, Her3, EGFRvIII, GD2, PD-L1, PD-L2.
  • the targeting molecules include but are not limited to: GE11, T7, RVG, single-chain antibodies that recognize CD19, and single-chain antibodies that recognize BCMA.
  • the DNA delivery system also includes a carrier that promotes the production or secretion of exosomes.
  • the vector that promotes exosome production or secretion expresses STEAP, syndevan-4 and NadB.
  • the present invention also provides an exosome, which includes the aforementioned fusion DNA.
  • the exosomes also include targeting molecules that recognize target tissues or target cells.
  • the targeting molecules include targeting peptides that interact with cell membrane surface antigens and that recognize specific cell surfaces. Antibodies to antigens or antigen-binding fragments and affibodies thereof.
  • the definition of the targeting molecule is the same as above.
  • the present invention also provides the preparation method of exosomes described above, which preparation method includes any of the following:
  • the invention also provides a cell, which is any one of the following:
  • the present invention also provides a method for preparing the aforementioned cells, which method includes the following steps:
  • the preparation method includes the following steps: introducing the aforementioned DNA packaging system and the aforementioned DNA delivery system into cells that produce exosomes.
  • the cells of the present invention or subsequently mentioned cells capable of secreting exosomes include, but are not limited to, primary cells, cell lines, cells present in multicellular organisms, or essentially any other type of cell source.
  • Cells of the present invention include cells that produce exosomes in vivo.
  • Cells according to the invention may be selected from a wide range of cells and cell lines, such as mesenchymal stem cells or stromal cells (can be selected from, for example, bone marrow, adipose tissue, Wharton's jelly, perinatal tissue, placenta, dental buds, umbilical cord blood, skin tissue, etc.), fibroblasts, amniotic cells and more specifically amniotic epithelial cells optionally expressing various early markers, myelosuppressive cells, M2 polarized macrophages, adipocytes , endothelial cells, fibroblasts, etc.
  • mesenchymal stem cells or stromal cells can be selected from, for example, bone marrow, adipose tissue, Wharton's jelly, perinatal tissue, placenta, dental buds, umbilical cord blood, skin tissue, etc.
  • fibroblasts amniotic cells and more specifically amniotic epithelial cells optionally expressing various early markers, my
  • Cell lines of particular interest include human umbilical cord endothelial cells (HUVEC), human embryonic kidney (HEK) cells, endothelial cell lines such as microvascular endothelial cells or lymphatic endothelial cells, erythrocytes, erythroid progenitor cells, chondrocytes, MSCs from different sources, amniotic membrane cells, amniotic epithelial (AE) cells, any cells obtained by amniocentesis or from the placenta, airway epithelial cells or alveolar epithelial cells, fibroblasts, endothelial cells, etc.
  • HEVEC human umbilical cord endothelial cells
  • HEK human embryonic kidney
  • endothelial cell lines such as microvascular endothelial cells or lymphatic endothelial cells
  • erythrocytes erythroid progenitor cells
  • chondrocytes chondrocytes
  • MSCs from different sources
  • exosomes can be derived from essentially any cell source, whether original or immortalized.
  • Exosome cells can be any embryonic, fetal and adult somatic stem cell type, including induced pluripotent stem cells (iPSCs) and other stem cells derived by any method.
  • iPSCs induced pluripotent stem cells
  • the use of, for example, primary neural cells, astrocytes, oligodendrocytes, microglia and neural progenitor cells may be considered as source cells.
  • the cells can be allogeneic, autologous, or even xenogeneic in nature, that is, the cells can come from the patient himself or from an unrelated, matched or unmatched donor.
  • the present invention also provides a pharmaceutical composition, which includes the aforementioned exosomes and the aforementioned cells.
  • the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not cause significant irritation to the subject and does not eliminate the biological activity and properties of the extracellular vesicles administered in the composition.
  • Pharmaceutically acceptable carriers can enhance or stabilize the composition, or can be used to facilitate preparation of the composition.
  • Pharmaceutically acceptable carriers may include physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the carrier can be selected to minimize adverse side effects in the subject and/or to minimize degradation of the active ingredient or ingredients.
  • Adjuvants may also be included in any of these formulations.
  • pharmaceutically acceptable excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient.
  • Formulations for parenteral administration may, for example, contain excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils or hydrogenated naphthalenes.
  • excipients include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and starches of various types, cellulose derivatives, gelatin, ethylene-vinyl acetate copolymer particles, and surfactants (including, for example, polyethylene glycol). Sorbitate 20).
  • compositions of the present invention may be administered by a variety of methods known in the art.
  • the route and/or mode of administration may vary depending on the desired results.
  • administration is intravitreal, intravenous, intramuscular, intraperitoneal, or subcutaneous.
  • Pharmaceutically acceptable carriers should be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • a pharmaceutical composition comprising at least one exosome and a pharmaceutically acceptable carrier or excipient can be in a form suitable for parenteral administration.
  • pharmaceutical compositions may be in the form of sterile injectable aqueous or suspensions, which may be formulated according to known procedures.
  • the sterile injectable preparation may also be a sterile injectable suspension in a nontoxic parenterally acceptable buffer.
  • the present invention also provides an application, the application includes any of the following:
  • exosomes are the aforementioned exosomes
  • treatment refers to the amelioration, prevention or reversal of a disease or condition or at least one discernible symptom thereof.
  • Treatment means the amelioration, prevention or reversal of at least one measurable physical parameter associated with the disease or condition being treated, which disease or condition is not necessarily discernible in or by the mammal .
  • Treatment means inhibiting or delaying the progression of a disease or condition, either physically (eg, stabilization of discernible symptoms) or physiologically (eg, stabilization of physical parameters), or both.
  • Treatment means delaying the onset of a disease or condition.
  • Prevention refers to the reduction of the risk of acquiring a given disease or condition.
  • the present invention also provides a method for treating or preventing ALS, which method includes administering the exosomes or cells described above to a subject in need.
  • subject and patient are used interchangeably herein and refer to any human or non-human animal to which treatment (including prophylactic treatment) is provided using the methods and compositions described herein.
  • treatment including prophylactic treatment
  • the term “subject” refers to that particular animal.
  • Non-human animals include all vertebrate animals (eg, mammals and non-mammals), such as any mammal.
  • mammals include humans, mice, rats, rabbits, dogs, monkeys, and pigs.
  • the subject is a human.
  • vector in the present invention refers to any nucleic acid construct capable of directing the expression of a target gene and transferring the gene sequence to a target cell.
  • the term includes cloning and expression vectors, as well as integration vectors.
  • Non-viral vectors include DNA or RNA plasmids, DNA MCs, naked nucleic acids and nucleic acids complexed with delivery vehicles such as liposomes, nanoparticles or poloxamer.
  • Viral vectors include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated virus, vaccinia and herpes simplex virus vectors.
  • Figure 1 shows a schematic diagram of the vector structure of pLenti-EF1 ⁇ -RVG-Lamp2b-Zeo
  • Figure 2 shows a schematic diagram of the vector structure of pLenti-EF1 ⁇ -Tol2transposase
  • Figure 3 shows a schematic diagram of the vector structure of pUC57-Kan-Tol2-CD63-TALE-IRES-Puro;
  • Figure 4 shows a schematic diagram of the vector structure of pAAV-SFFV-pepRNF112-TALE recognition motif
  • Figure 5 shows the fluorescence image of cells under confocal laser microscopy.
  • pRSV-Rev (Plasmid#12253), pMDLg/pRRE (Plasmid#12251), and pMD2.G (Plasmid#12259) are lentivirus packaging auxiliary vectors for lentivirus packaging.
  • a vector for expressing exosome membrane proteins (a schematic diagram of the vector structure is shown in Figure 1).
  • the proteins expressed by this vector include Lamp2b, whose N-terminus (inserted by the BsmBI restriction site) can express, but is not limited to, recognition of neural RVG of metacells.
  • This vector contains the Zeocin selection marker and contains the EF1 ⁇ promoter Start the expression of RVG-Lamp2b fusion protein; the vector also contains the WPRE original element, which can increase the expression of the corresponding protein.
  • RVG-Lamp2b fusion protein The amino acid sequence of RVG-Lamp2b fusion protein is shown in SEQ ID NO.4.
  • a Tol2 transposase vector was constructed (the schematic diagram of the vector structure is shown in Figure 2), which can express Tol2 transposase.
  • CD63-TALE fusion protein (the schematic diagram of the vector structure is shown in Figure 3), which also expresses Rep protein.
  • CD63 protein is an exosome membrane protein, and TALE protein can recognize the specific DNA sequence "TACCACTCACTATA" (SEQ ID NO.2).
  • TALE protein can recognize the specific DNA sequence "TACCACTCACTATA" (SEQ ID NO.2).
  • the vector contains transposable arms.
  • the DNA sequence between the transposable arms of the vector in Figure 3 is integrated into the genome of the target cell, and the Rep protein can continuously replicate the target DNA fragment.
  • CD63-TALE fusion protein The amino acid sequence of CD63-TALE fusion protein is shown in SEQ ID NO.7.
  • pepRNF112 Construct an expression vector containing the TALE recognition motif "TACCACTCACTATA" and the modified RNF112 gene (pepRNF112, the nucleic acid sequence is shown in SEQ ID NO.1) (the schematic diagram of the vector structure is shown in Figure 4).
  • the connection sequence between pepRNF112 and TALE recognition motif is: pepRNF112-TALE.
  • the pepRNF112-TALE nucleic acid sequence was ligated into the pAAV-SFFV vector via the SpeI and EcoRI restriction sites.
  • RVG-lamp2b cell lines Use pLenti-EF1 ⁇ -RVG-Lamp2b-Zeo vector to package lentivirus, infect HEK293T cell line, use Zeocin to screen, and select single clones as engineering cells, named RVG-lamp2b cell lines;
  • the TDP43-EGFP nucleic acid sequence was synthesized and then ligated into the pLenti-EF1a vector through the BsiWI and PflFI restriction sites.
  • the TDP43-EGFP nucleic acid sequence is shown in SEQ ID NO.8.
  • the core plasmid and packaging plasmid take a sterile 1.5ml EP tube, add 400 ⁇ l Opti-MEM culture medium, add 10 ⁇ g core plasmid and 15 ⁇ g virus packaging plasmid (including PMD2G, VSVG, REV), and 75 ⁇ l PEI transfection reagent, fully Mix well and let stand for 15-20 minutes;
  • Collect virus supernatant Aspirate the culture medium after 6-10 hours, and add 10 ml of complete culture medium preheated at 37°C. Continue to incubate the cells in the incubator. The lentivirus-containing supernatant can be collected for about 40 hours. After being filtered through a 45 ⁇ m filter, it can be used for infection or aliquoted and stored at -80°C for later use.

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Abstract

提供了一种以外泌体为载体的DNA递送系统。该DNA递送系统包括DNA包装系统和靶向递送载体。该DNA包装系统可以把经修饰的RNF112基因的DNA序列导入外泌体中，获得的外泌体施用给受试者可以治疗ALS。

Description

以外泌体为载体的DNA递送系统 技术领域
本发明属于生物医药领域，具体涉及以外泌体为载体的DNA递送系统。
背景技术
外泌体(exosomes)是由细胞分泌的直径为30～150nm的囊泡，细胞质膜凹陷形成的细胞内小泡，具有与细胞膜相同的膜结构，可以携带诸如RNA、蛋白质等大量成分，与细胞的生物学功能及细胞间的信号传递有着密切的关系。因其特殊的结构，相较人工合成的药物载体，外泌体作为药物载体进行药物运输有独特的优势，主要体现在外泌体的膜结构与细胞膜相同，可以提高药物进入细胞的效率，甚至可以透过血脑屏障，同时外泌体引起的有害免疫反应极低，有很好的渗透滞留(EPR)效应从而具有缓释效果等。目前已经尝试用外泌体携带miRNA、siRNA、化学小分子药物等进行基因治疗和肿瘤治疗等研究。但是如何将大分子DNA递送至外泌体从而产生携带DNA的外泌体是本申请需要解决的问题。
发明内容
为了克服现有技术缺陷，本发明的目的在于提供一种DNA包装系统，所述包装系统能够将目的基因DNA序列导入外泌体。
为了实现上述目的，本发明提供了如下技术方案：
本发明提供了一种融合DNA，所述融合DNA的序列包括DNA结合蛋白的DNA结合基序以及SEQ ID NO.1所示的DNA序列；DNA结合蛋白的DNA结合基序连接于SEQ ID NO.1所示的DNA序列的3’端；
优选地，所述DNA结合蛋白包括TALE蛋白；
优选地，所述DNA蛋白的DNA结合基序包括SEQ ID NO.2所示的序列。
本发明提供了一种载体，所述载体包含前面所述的融合DNA。
本发明提供了一种DNA包装系统，所述DNA包装系统包括前面所述的载 体，以及表达外泌体蛋白和DNA结合蛋白的载体。
进一步，DNA结合蛋白连接于外泌体膜蛋白的C端。
进一步，所述外泌体蛋白包括外泌体跨膜蛋白、外泌体表面蛋白。
可用于本发明的外泌体蛋白可以包括但不限于：CD9、CD53、CD63、CD81、CD54、CD50、FLOT1、FLOT2、CD49d、CD71、CD133、CD138、CD235a、ALIX、AARDC1、Syntenin-1、Syntenin-2、Lamp2b、TSPAN8、syndecan-1、syndecan-2、syndecan-3、syndecan-4、TSPAN14、CD37、CD82、CD151、CD231、CD102、NOTCH1、NOTCH2、NOTCH3、NOTCH4、DLL1、DLL4、JAG1、JAG2、CD49d/ITGA4、ITGB5、ITGB6、ITGB7、CD11a、CD11b、CD11c、CD18/ITGB2、CD41、CD49b、CD49c、CD49e、CD51、CD61、CD104、Fc受体、白介素受体、免疫球蛋白、MHC-I或MHC-II成分、CD2、CD3ε、CD3ζ、CD13、CD18、CD19、CD30、CD34、CD36、CD40、CD40L、CD44、CD45、CD45RA、CD47、CD86、CD110、CD111、CD115、CD117、CD125、CD135、CD184、CD200、CD279、CD273、CD274、CD362、COL6A1、AGRN、EGFR、GAPDH、GLUR2、GLUR3、HLA-DM、HSPG2、L1CAM、LAMB1、LAMC1、LFA-1、LGALS3BP、Mac-1α、Mac-1β、MFGE8、SLIT2、STX3、TCRA、TCRB、TCRD、TCRG、VTI1A、VTI1B、其它外泌体多肽及其任何组合组成的组。
在本发明的具体实施方案中，所述外泌体蛋白是CD63。
进一步，所述DNA结合蛋白包括TALE蛋白。
进一步，所述表达外泌体蛋白和DNA结合蛋白的载体还表达Tol2；所述DNA包装系统还包括表达Tol2转座酶的载体。
本发明还提供了一种DNA递送系统，所述DNA递送系统包括前面所述的DNA包装系统和靶向递送载体，所述靶向递送载体表达外泌体蛋白和靶向分子，所述靶向分子识别靶组织或靶细胞。
进一步，所述靶向分子连接于外泌体膜蛋白的N端。
进一步，所述外泌体蛋白包括外泌体跨膜蛋白、外泌体表面蛋白。
可用于本发明的外泌体蛋白可以包括但不限于：CD9、CD53、CD63、CD81、 CD54、CD50、FLOT1、FLOT2、CD49d、CD71、CD133、CD138、CD235a、ALIX、AARDC1、Syntenin-1、Syntenin-2、Lamp2b、TSPAN8、syndecan-1、syndecan-2、syndecan-3、syndecan-4、TSPAN14、CD37、CD82、CD151、CD231、CD102、NOTCH1、NOTCH2、NOTCH3、NOTCH4、DLL1、DLL4、JAG1、JAG2、CD49d/ITGA4、ITGB5、ITGB6、ITGB7、CD11a、CD11b、CD11c、CD18/ITGB2、CD41、CD49b、CD49c、CD49e、CD51、CD61、CD104、Fc受体、白介素受体、免疫球蛋白、MHC-I或MHC-II成分、CD2、CD3ε、CD3ζ、CD13、CD18、CD19、CD30、CD34、CD36、CD40、CD40L、CD44、CD45、CD45RA、CD47、CD86、CD110、CD111、CD115、CD117、CD125、CD135、CD184、CD200、CD279、CD273、CD274、CD362、COL6A1、AGRN、EGFR、GAPDH、GLUR2、GLUR3、HLA-DM、HSPG2、L1CAM、LAMB1、LAMC1、LFA-1、LGALS3BP、Mac-1α、Mac-1β、MFGE8、SLIT2、STX3、TCRA、TCRB、TCRD、TCRG、VTI1A、VTI1B、其它外泌体多肽及其任何组合组成的组。
在本发明的具体实施方案中，所述外泌体蛋白是Lamp2b。
可用于本发明的所述靶向分子包括与细胞膜表面抗原相互作用的靶向肽、识别特定细胞表面抗原的抗体或其抗原结合片段、亲合体。
细胞膜表面抗原包括但不限于ER、PR、P53、EGFR、IGFR、Her2、CD20、CD25、CD117、CD34、CD138、CD33、VEGFR、BCMA、Mesothelin、CEA、PSCA、MUC1、EpCAM、S100、CD22、CD19、CD70、CD30、ALK、RANK、GPC2、GPC3、Her3、EGFRvIII、GD2、PD-L1、PD-L2。
作为靶向分子的实施例，所述靶向分子包括但不限于：GE11、T7、RVG，识别CD19的单链抗体、识别BCMA的单链抗体。
更进一步，所述DNA递送系统还包括促进外泌体生产或分泌的载体。
优选地，所述促进外泌体生产或分泌的载体表达STEAP、syndevan-4和NadB。
本发明还提供了一种外泌体，所述外泌体包括前面所述的融合DNA。
优选地，所述外泌体还包括靶向分子，所述靶向分子识别靶组织或靶细胞，优选地，所述靶向分子包括与细胞膜表面抗原相互作用的靶向肽、识别特定细胞表面抗原的抗体或其抗原结合片段、亲合体。
进一步，所述靶向分子的限定同前。
本发明还提供了前面所述的外泌体的制备方法，所述制备方法包括以下任一项：
1)将前面所述的融合DNA导入外泌体中；
2)将前面所述的DNA包装系统或前面所述的DNA递送系统导入产生外泌体的细胞中；培养所述细胞产生外泌体。
本发明还提供了一种细胞，所述细胞为以下任一项：
1)产生并分泌前面所述的外泌体；
2)包含前面所述的融合DNA；
3)导入了前面所述的DNA包装系统或前面所述的DNA递送系统。
本发明还提供了前面所述的细胞的制备方法，所述制备方法包括如下步骤：
1)将前面所述的DNA包装系统引入到产生外泌体的细胞中；
进一步，所述制备方法包括如下步骤：将前面所述的DNA包装系统和前面所述的DNA递送系统引入到产生外泌体的细胞中。
本发明细胞或后续提到的能够分泌外泌体的细胞包括但不限于原代细胞、细胞系、存在于多细胞生物的细胞、或基本上任何其它类型的细胞来源。本发明的细胞包括体内产生外泌体的细胞。根据本发明的细胞可以选自广泛范围的细胞和细胞系，例如间充质干细胞或基质细胞(可从例如骨髓、脂肪组织、脐带胶质(Wharton'sjelly)、围产期组织、胎盘、牙芽、脐带血、皮肤组织等获得)、成纤维细胞、羊膜细胞且更具体地说是任选地表达各种早期标记物的羊膜上皮细胞、骨髓抑制细胞、M2极化巨噬细胞、脂肪细胞、内皮细胞、成纤维细胞等。特别关注的细胞系包括人脐带内皮细胞(HUVEC)、人胚肾(HEK)细胞、内皮细胞系例如微血管内皮细胞或淋巴内皮细胞、红细胞、红系祖细胞、软骨细胞、不同来源的MSC、羊膜细胞、羊膜上皮(AE)细胞、通过羊膜穿刺术或从胎盘获得的任何细胞、气道上皮细胞或肺泡上皮细胞、成纤维细胞、内皮细胞等。此外，免疫细胞例如B细胞、T细胞、NK细胞、巨噬细胞、单核细胞、树突细胞(DC)也在本发明的范围内，并且基本上能够产生细胞外囊泡的任何类型的细胞也包括在本文中。通 常，外泌体基本上可以源自任何细胞来源，无论是原始细胞来源还是永生化细胞系。外泌体细胞可以是任何胚胎的、胎儿的和成年的体干细胞类型，包括诱导多能干细胞(iPSC)和通过任何方法衍生的其它干细胞。当治疗神经系统疾病时，可以考虑利用例如原代神经细胞、星形胶质细胞、少突胶质细胞、小胶质细胞和神经祖细胞作为源细胞。对于待治疗的患者而言，细胞本质上可以是同种异体的、自体的、甚至异种的，即细胞可以来自患者本人或者来自无关的、匹配的或不匹配的供体。
本发明还提供了一种药物组合物，所述药物组合物包括前面所述的外泌体、前面所述的细胞。
进一步，所述药物组合物还包括药学上可接受的载体或赋形剂。
术语“药学上可接受的载体”是指不会对受试者造成明显刺激并且不会消除组合物中所施用的细胞外囊泡的生物学活性和性质的载体或稀释剂。药学上可接受的载体可以增强或稳定组合物，或者可以用于促进组合物的制备。药学上可接受的载体可以包括生理上相容的溶剂、分散介质、包衣、抗细菌和抗真菌剂、等渗剂和吸收延迟剂等。可以选择载体以最小化受试者的不良副作用和/或最小化一种或多种活性成分的降解。这些配制品中的任何一种中也可以包含佐剂。
术语“药学上可接受的赋形剂”是指添加到药物组合物中以进一步促进活性成分的施用的惰性物质。肠胃外施用的配制品可以例如含有赋形剂如无菌水或盐水，聚亚烷基二醇如聚乙二醇，植物油或氢化萘。其他示例性赋形剂包括但不限于碳酸氢钙、磷酸钙、各种糖和各种类型的淀粉、纤维素衍生物、明胶、乙烯-乙酸乙烯酯共聚物颗粒和表面活性剂(包括例如聚山梨酯20)。
本发明的药物组合物可以通过本领域已知的多种方法施用。给予的途径和/或方式可以根据所期望的结果而变化。在一些实施例中，施用是玻璃体内、静脉内、肌内、腹膜内或皮下施用。药学上可接受的载体应适合于玻璃体内、静脉内、肌内、皮下、肠胃外、脊髓或表皮施用(例如，通过注射或输注)。在一些实施例中，包含至少一种外泌体和药学上可接受的载体或赋形剂的药物组合物可以处于适于肠胃外施用的形式。在一些实施例中，药物组合物可以是无菌可注射的水性或悬浮液的形式，其可以根据已知程序进行配制。无菌可注射制剂也可以是在无毒的肠胃外可接受的缓冲液中的无菌可注射悬浮液。
本发明还提供了一种应用，所述应用包括以下任一项：
1)前面所述的DNA包装系统在制备装载外源DNA的外泌体中的应用；优选地，所述外泌体是前面所述的外泌体；
2)前面所述的DNA递送系统在在制备装载外源DNA的外泌体中的应用；优选地，所述外泌体是前面所述的外泌体；
3)前面所述的细胞在制备装载外源DNA的外泌体中的应用；优选地，所述外泌体是前面所述的外泌体；
4)前面所述的外泌体在制备治疗ALS的药物中的应用；
5)前面所述的细胞在制备治疗ALS的药物中的应用；
6)前面所述的DNA包装系统在制备治疗ALS的药物中的应用；
7)前面所述的DNA递送系统在制备治疗ALS的药物中的应用；
8)前面所述的融合DNA在制备治疗ALS的药物中的应用；
9)前面所述的融合DNA在制备装载外源DNA的外泌体中的应用；优选地，所述外泌体是前面所述的外泌体；
术语"治疗"是指疾病或病症或它的至少一种可辨别症状的改善、预防或逆转。"治疗"是指至少一种与所治疗的疾病或病症有关的可测量的物理参数的改善、预防或逆转，所述疾病或病症不一定是哺乳动物中可辨别的或可被哺乳动物辨别的。"治疗"是指物理上(例如可辨别症状的稳定)或生理上(例如物理参数的稳定)或既在物理上又在生理上抑制或延缓疾病或病症的进展。"治疗"是指延迟疾病或病症的发病。
本文所用的"预防"是指获得给定疾病或病症的风险的降低。
本发明还提供了一种治疗或预防ALS的方法，所述方法包括将前面所述的外泌体或细胞给有需要的受试者施用。
术语“受试者”和“患者”在本文可互换使用，是指用本文描述的方法和组合物向其提供治疗(包括预防性治疗)的任何人或非人动物。对于治疗对特定动物如人受试者具有特异性的病状或疾病状态，术语“受试者”是指该特定动物。非人动物包括所有脊椎动物(例如，哺乳动物和非哺乳动物)，例如任何哺乳动物。 哺乳动物的非限制性实例包括人、小鼠、大鼠、兔、狗、猴和猪。在一些实施例中，受试者是人。
本发明的术语“载体”是指能够指导目的基因表达并可以转移基因序列到靶细胞的任何核酸构建体。该术语包括克隆和表达载体，以及整合载体。
可用于本发明的载体包括非病毒载体和病毒载体。非病毒载体包括DNA或RNA质粒，DNA MC，裸核酸以及与诸如脂质体，纳米颗粒或泊洛沙姆的递送运载体复合的核酸。病毒载体包括但不限于逆转录病毒，慢病毒，腺病毒，腺相关病毒，牛痘和单纯疱疹病毒载体。
附图说明
图1显示pLenti-EF1α-RVG-Lamp2b-Zeo的载体结构示意图；
图2显示pLenti-EF1α-Tol2transposase的载体结构示意图；
图3显示pUC57-Kan-Tol2-CD63-TALE-IRES-Puro的载体结构示意图；
图4显示pAAV-SFFV-pepRNF112-TALE识别基序的载体结构示意图；
图5显示激光共聚焦显微镜的细胞荧光图。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实验例1构建相关载体
1、实验材料
常规载体pRSV-Rev(Plasmid#12253)、pMDLg/pRRE(Plasmid#12251)、pMD2.G(Plasmid#12259)为慢病毒包装辅助载体，用于慢病毒包装。
2、实验方法
1)pLenti-EF1α-Lamp2b-RVG-Zeo载体构建
构建一种表达外泌体膜蛋白的载体(载体结构示意图如图1所示)，该载体表达的蛋白包括Lamp2b，其N端(由BsmBI酶切位点插入)可表达包括但不限于识别神经元细胞的RVG。该载体含有Zeocin筛选标记，含有EF1α启动子 启动表达RVG-Lamp2b融合蛋白；该载体还包含有WPRE原件，可增加相应蛋白表达。
pLenti-EF1α-RVG-Lamp2b-Zeo载体全序列如SEQ ID NO.3所示。
RVG-Lamp2b融合蛋白氨基酸序列如SEQ ID NO.4所示。
2)pLenti-EF1α-Tol2 transposase载体构建
构建一种Tol2转座酶载体(载体结构示意图如图2所示)，可表达Tol2转座酶。
pLenti-EF1α-Tol2 transposase载体全序列如SEQ ID NO.5所示。
3)pUC57-Kan-Tol2-CD63-TALE-IRES-Puro载体构建
构建一种表达CD63-TALE融合蛋白的载体(载体结构示意图如图3所示)，同时也表达Rep蛋白，CD63蛋白为外泌体膜蛋白，TALE蛋白可识别特定DNA序列“TACCACTCACTATA”(SEQ ID NO.2)，同时载体上含有转座臂，可通过与图2载体共转染细胞，使图3载体转座臂间DNA序列整合进目的细胞基因组中，Rep蛋白可持续复制目的DNA片段。
构建方法：合成SEQ ID NO.6所示的核酸序列经NotI、SphI酶切位点连入pUC57载体。
CD63-TALE融合蛋白氨基酸序列如SEQ ID NO.7所示。
4)pAAV-SFFV-pepRNF112-TALE识别基序载体构建
构建一种表达含有TALE识别基序“TACCACTCACTATA”及经修饰的RNF112基因(pepRNF112，核酸序列如SEQ ID NO.1所示)的载体(载体结构示意图如图4所示)。pepRNF112和TALE识别基序的连接序列为：pepRNF112-TALE。将pepRNF112-TALE核酸序列经SpeI、EcoRI酶切位点连入pAAV-SFFV载体。
实施例2构建产生外泌体的工程细胞株以及分离外泌体
1、利用pLenti-EF1α-RVG-Lamp2b-Zeo载体包装慢病毒，感染HEK293T细胞株，并使用Zeocin筛选，并挑取单克隆作为工程细胞，命名为RVG-lamp2b 细胞株；
2、以pUC57-Kan-Tol2-CD63-TALE-IRES-Puro载体与pLenti-EF1α-Tol2transposase载体瞬时转染步骤1制备好的RVG-lamp2b细胞株，并使用puromycin筛选，构建RVG-lamp2b-CD63-TALE-CX43细胞株；
3、以pAAV-SFFV-pepRNF112-TALE识别基序载体瞬时转染rAAV侵染步骤2中获得的细胞，制备形成分泌外泌体工程细胞株，该细胞株命名为293T-RVG-lamp2b-CD63-TALE-PepRNF112。扩大培养，收集上清获得外泌体。
4、293T-RVG-lamp2b-CD63-TALE-PepRNF112细胞在T175培养瓶中扩增培养，收集上清，0.45μm滤膜过滤后，加入外泌体分离试剂4℃过夜，隔天10000g，1h离心，离心后弃去上清，用400μl DPBS重悬后置于-80℃储藏。
实施例3外泌体侵染293T-TDP43-EGFP细胞模型
1、构建293T-TDP43-EGFP细胞模型
1)包装TDP43-EGFP慢病毒
合成TDP43-EGFP核酸序列后经BsiWI、PflFI酶切位点连入pLenti-EF1a载体。TDP43-EGFP核酸序列如SEQ ID NO.8所示。
慢病毒包装流程：
通过胰酶消化收集293T细胞，用适当的完全培养基平铺细胞于10cm培养皿上(根据实验需要选择培养皿，使细胞贴壁后所占面积达到培养皿总面积的80％以上)。将细胞置于含5％CO2的37℃温箱中孵育8-24h，当细胞贴壁完全后即可开始转染；
混匀核心质粒和包装质粒：取无菌1.5ml EP管，加入400μl Opti-MEM培养基，加入10μg核心质粒和15μg病毒包装质粒(包含PMD2G、VSVG、REV)，及75μl PEI转染试剂，充分混匀，静置15-20min；
孵育：将这400μl的混合液逐滴加入上述单层细胞的细胞培养基中，轻轻摇动平皿混匀后置于含5％CO2的37℃温箱孵育；
收取病毒上清：6-10h后吸去培养基，加入10ml 37℃预热的完全培养基， 继续将细胞放置温箱孵育，40h左右可以收集含慢病毒的上清，经45μm滤膜过滤后，可用于感染或者分装后置于－80℃储存待用。
2)使用1)中慢病毒感染293T细胞，并使用puromycin进行筛选；
3)2)中细胞挑取单克隆培养作为细胞模型
2、使用实施例2收集到的外泌体侵染293T-TDP43-EGFP细胞，连续侵染数次；
3、使用激光共聚焦显微镜观察293T-TDP43-EGFP细胞胞质荧光变化。
4、结果
1)293T-TDP43-EGFP细胞胞质荧光检测结果如表1和图5所示。
表1荧光检测值
上述结果表明，使用携带表达pepRNF112基因DNA片段的外泌体侵染293T-TDP43-EGFP细胞，细胞胞质中TDP43-EGFP积聚蛋白被降解。

Claims (10)

  1. 一种融合DNA，所述融合DNA的序列包括DNA结合蛋白的DNA结合基序以及SEQ ID NO.1所示的DNA序列；DNA结合蛋白的DNA结合基序连接于SEQ ID NO.1所示的DNA序列的3’端；
    优选地，所述DNA结合蛋白包括TALE蛋白；
    优选地，所述DNA蛋白的DNA结合基序包括SEQ ID NO.2所示的序列。
  2. 一种载体，所述载体包含权利要求1所述的融合DNA。
  3. 一种DNA包装系统，所述DNA包装系统包括权利要求2所述的载体，以及表达外泌体蛋白和DNA结合蛋白的载体；所述DNA结合蛋白连接于所述外泌体蛋白的C端；
    优选地，所述外泌体蛋白包括CD63、Lamp2b、CD9；优选地，所述外泌体蛋白是CD63；
    优选地，所述表达外泌体蛋白和DNA结合蛋白的载体还表达Tol2；所述DNA包装系统还包括表达Tol2转座酶的载体。
  4. 一种DNA递送系统，所述DNA递送系统包括权利要求3所述的DNA包装系统和靶向递送载体，所述靶向递送载体表达外泌体蛋白和靶向分子，所述靶向分子识别靶组织或靶细胞；
    优选地，所述外泌体蛋白包括CD63、Lamp2b、CD9；优选地，所述外泌体膜蛋白是Lamp2b；
    优选地，所述靶向分子包括与细胞膜表面抗原相互作用的靶向肽、识别特定细胞表面抗原的抗体或其抗原结合片段、亲合体；
    优选地，所述DNA递送系统还包括促进外泌体生产或分泌的载体；优选地，所述载体表达STEAP、syndevan-4和NadB。
  5. 一种外泌体，所述外泌体包括权利要求1所述的融合DNA；优选地，所述外泌体还包括靶向分子，所述靶向分子识别靶组织或靶细胞；优选地，所述靶 向分子包括与细胞膜表面抗原相互作用的靶向肽、识别特定细胞表面抗原的抗体或其抗原结合片段、亲合体。
  6. 权利要求5所述的外泌体的制备方法，其特征在于，所述制备方法包括以下任一项：
    1)将权利要求1所述的融合DNA导入外泌体中；
    2)将权利要求3所述的DNA包装系统或权利要求4所述的DNA递送系统导入产生外泌体的细胞中；培养所述细胞产生外泌体。
  7. 一种细胞，所述细胞为以下任一项：
    1)产生并分泌权利要求5所述的外泌体；
    2)包含权利要求1所述的融合DNA；
    3)导入了权利要求3所述的DNA包装系统或权利要求4所述的DNA递送系统。
  8. 权利要求7所述的细胞的制备方法，其特征在于，所述制备方法包括如下步骤：将权利要求3所述的DNA包装系统或权利要求4所述的DNA递送系统引入到产生外泌体的细胞中。
  9. 一种药物组合物，所述药物组合物包括权利要求5所述的外泌体、权利要求7所述的细胞；优选地，所述药物组合物还包括药学上可接受的载体或赋形剂。
  10. 一种应用，其特征在于，所述应用包括以下任一项：
    1)权利要求1所述的融合DNA在制备装载外源DNA的外泌体中的应用；优选地，所述外泌体是权利要求5所述的外泌体；
    2)权利要求3所述的DNA包装系统在制备装载外源DNA的外泌体中的应用；优选地，所述外泌体是权利要求5所述的外泌体；
    3)权利要求4所述的DNA递送系统在制备装载外源DNA的外泌体中的应用；优选地，所述外泌体是权利要求5所述的外泌体；
    4)权利要求7所述的细胞在制备装载外源DNA的外泌体中的应用；优选地，所述外泌体是权利要求5所述的外泌体；
    5)权利要求5所述的外泌体在制备治疗ALS的药物中的应用；
    6)权利要求7所述的细胞在制备治疗ALS的药物中的应用；
    7)权利要求1所述的融合DNA在制备治疗ALS的药物中的应用；
    8)权利要求1所述的DNA包装系统在制备治疗ALS的药物中的应用；
    9)权利要求4所述的DNA递送系统在制备治疗ALS的药物中的应用。
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