WO2022206816A1 - Rna plasmid delivery system for treating parkinson's disease - Google Patents

Rna plasmid delivery system for treating parkinson's disease Download PDF

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WO2022206816A1
WO2022206816A1 PCT/CN2022/083979 CN2022083979W WO2022206816A1 WO 2022206816 A1 WO2022206816 A1 WO 2022206816A1 CN 2022083979 W CN2022083979 W CN 2022083979W WO 2022206816 A1 WO2022206816 A1 WO 2022206816A1
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sequence
rna
targeting
plasmid
delivery system
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French (fr)
Chinese (zh)
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张辰宇
陈熹
付正
李菁
张翔
周心妍
张丽
余梦超
郭宏源
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南京大学
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • 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
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    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
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    • 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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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present application relates to the field of biomedical technology, in particular to an RNA plasmid delivery system for treating Parkinson's.
  • Parkinson's disease also known as tremor palsy, is a common neurodegenerative disease of the middle-aged and elderly people. The main lesions are in the substantia nigra and striatum. Parkinson's disease is the fourth most common neurodegenerative disease in older adults.
  • RNA interference (RNAi) therapy has been considered a promising strategy for the treatment of human diseases since its invention, but many problems have been encountered during clinical practice, and the development of this therapy has lagged far behind expectations.
  • RNA cannot exist stably outside the cell for a long time, because RNA will be degraded into fragments by RNases rich in extracellular, so it is necessary to find a method that can make RNA stable outside the cell and can enter specific tissues in a targeted manner. Highlight the effect of RNAi therapy.
  • the Chinese Patent Publication No. CN108624590A discloses a siRNA capable of inhibiting the expression of DDR2 gene; the Chinese Patent Publication No. CN108624591A discloses a siRNA capable of silencing the ARPC4 gene, and the siRNA is modified with ⁇ -phosphorus-selenium;
  • the Chinese Patent Publication No. CN108546702A discloses a siRNA targeting long-chain non-coding RNA DDX11-AS1.
  • the Chinese Patent Publication No. CN106177990A discloses a siRNA precursor that can be used for various tumor treatments. These patents design specific siRNAs to target certain diseases caused by genetic changes.
  • Chinese Patent Publication No. CN108250267A discloses a polypeptide, polypeptide-siRNA induced co-assembly, using polypeptide as a carrier of siRNA.
  • the Chinese Patent Publication No. CN108117585A discloses a polypeptide for promoting apoptosis of breast cancer cells through targeted introduction of siRNA, and the polypeptide is also used as the carrier of siRNA.
  • the Chinese Patent Publication No. CN108096583A discloses a nanoparticle carrier, which can be loaded with siRNA with breast cancer curative effect while containing chemotherapeutic drugs.
  • exosomes can deliver miRNAs to recipient cells, which secrete miRNAs at relatively low concentrations , which can effectively block the expression of target genes.
  • Exosomes are biocompatible with the host immune system and possess the innate ability to protect and transport miRNAs across biological barriers in vivo, thus becoming a potential solution to overcome problems associated with siRNA delivery.
  • the Chinese Patent Publication No. CN110699382A discloses a method for preparing siRNA-delivering exosomes, and discloses the technology of separating exosomes from plasma and encapsulating siRNA into exosomes by electroporation .
  • the embodiments of the present application provide an RNA plasmid delivery system for treating Parkinson's and its application, so as to solve the technical defects existing in the prior art.
  • One of the inventions of the present application is to provide an RNA plasmid delivery system for treating Parkinson's, the system comprising a plasmid carrying an RNA fragment that can be used for treating Parkinson's, and the plasmid can be used in the host's organs and tissues It is enriched in the host organ tissue, and endogenously and spontaneously forms a complex structure containing the RNA fragment in the host organ tissue, and the complex structure can bind to the target tissue, and the RNA fragment is sent into the target tissue to achieve par Treatment of Kinson's disease.
  • the RNA fragment comprises one, two or more specific RNA sequences of medical significance, and the RNA sequences are siRNA, shRNA or miRNA sequences of medical significance that can inhibit or hinder the development of Parkinson's disease .
  • the plasmid also includes a promoter and a targeting tag
  • the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host, and the targeting structure is located on the surface of the composite structure, so The complex structure can seek and bind to the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
  • the plasmid includes any one of the following circuits or a combination of several circuits: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; in each of the plasmids, at least An RNA fragment and a targeting tag are included, the RNA fragment and targeting tag being in the same circuit or in different circuits.
  • the plasmid also includes a flanking sequence, a compensation sequence and a loop sequence that can fold the circuit into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence;
  • the plasmid includes any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag Or 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensating sequence-3'flanking sequence.
  • the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence whose homology is greater than 80%;
  • the loop sequence is gttttggccactgactgac or a sequence whose homology is greater than 80%;
  • the 3' flanking sequence is accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence whose homology is greater than 80%;
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the purpose of deleting bases 1-5 of the RNA reverse complement sequence is to make the sequence unexpressed.
  • siRNA and miRNA precursors due to deletion of the 9th and 10th bases of the reverse complementary strand of the active strand (such as the reverse complementary strand of siRNA), the active strand of siRNA and miRNA will form a bulge. structure, which facilitates more efficient silencing of gene expression. To sum up, it is an accepted conclusion that the silencing efficiency can be improved by deleting the reverse complementary sequence of the 9th and 10th bases.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
  • the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
  • adjacent lines are connected by a sequence composed of sequences 1-3 (sequence 1-sequence 2-sequence 3);
  • sequence 1 is CAGATC
  • sequence 2 is a sequence consisting of 5-80 bases
  • sequence 3 is TGGATC.
  • adjacent lines are connected by sequence 4 or a sequence with more than 80% homology to sequence 4;
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • the organ tissue is liver
  • the composite structure is exosome
  • the targeting tag is selected from targeting peptides or targeting proteins with targeting function.
  • the targeting peptides include RVG targeting peptides, GE11 targeting peptides, PTP targeting peptides, TCP-1 targeting peptides, and MSP targeting peptides;
  • the targeting proteins include RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein, and MSP-LAMP2B fusion protein.
  • the targeting tag is preferably an RVG targeting peptide or an RVG-LAMP2B fusion protein.
  • the RNA sequence is 15-25 nucleotides in length.
  • the length of the RNA sequence can be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides.
  • the RNA sequence is 18-22 nucleotides in length.
  • the RNA capable of treating Parkinson's is selected from: siRNA of the LRRK2 gene, or an RNA sequence with more than 80% homology to the above sequence, or a nucleic acid molecule encoding the above RNA. It should be noted that the RNA sequences in the "nucleic acid molecules encoding the above RNA sequences" here also include RNA sequences with a homology of more than 80% of each RNA.
  • the siRNA of the LRRK2 gene includes AUUAACAUGAAAAUAUCACUU, UUAACAAUAUCAUAUAAUCUU, AUCUUUAAAAUUUGUUAACGC, UUGAUUUAAGAAAAUAGUCUC, UUUGAUAACAGUAUUUUUCUG, other sequences that inhibit the expression of the LRRK2 gene, and sequences with more than 80% homology to the above sequences.
  • sequences with more than 80% homology may be 85%, 88%, 90%, 95%, 98%, etc. homology.
  • the RNA fragment includes an RNA sequence ontology and a modified RNA sequence obtained by modifying the RNA sequence ontology with ribose sugar. That is, the RNA fragment can be composed of only at least one RNA sequence ontology, or only at least one modified RNA sequence, and can also be composed of RNA sequence ontology and modified RNA sequence.
  • the isolated nucleic acid also includes its variants and derivatives.
  • the nucleic acid can be modified by one of ordinary skill in the art using general methods. Modification methods include (but are not limited to): methylation modification, hydrocarbyl modification, glycosylation modification (such as 2-methoxy-glycosyl modification, hydrocarbyl-glycosyl modification, sugar ring modification, etc.), nucleic acid modification, peptide modification Segment modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification) and the like.
  • the modification is an internucleotide linkage, for example selected from: phosphorothioate, 2'-O methoxyethyl (MOE), 2'-fluoro, phosphine Acid alkyl esters, phosphorodithioates, alkyl phosphorothioates, phosphoramidates, carbamates, carbonates, phosphoric triesters, acetamidates, carboxymethyl esters, and combinations thereof.
  • phosphorothioate 2'-O methoxyethyl (MOE), 2'-fluoro
  • phosphine Acid alkyl esters phosphorodithioates, alkyl phosphorothioates, phosphoramidates, carbamates, carbonates, phosphoric triesters, acetamidates, carboxymethyl esters, and combinations thereof.
  • the modification is a modification of nucleotides, such as selected from: peptide nucleic acid (PNA), locked nucleic acid (LNA), arabinose-nucleic acid (FANA), analogs, derivatives objects and their combinations.
  • the modification is a 2' fluoropyrimidine modification.
  • 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on RNA with 2'-F.
  • 2'-F can make RNA not easily recognized by RNase in vivo, thereby increasing the stability of RNA fragment transmission in vivo. sex.
  • the present application also provides an application of the RNA delivery system for treating Parkinson's in medicine.
  • the drug is a drug for the treatment of Parkinson's disease and its related diseases
  • the related diseases here refer to the associated diseases or complications, sequelae, etc. that occur during the formation or development of Parkinson's disease, or have a certain relationship with Parkinson's disease. related other diseases.
  • the administration modes of the drug include oral, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection. Intravenous injection is preferred.
  • the medicine includes the above-mentioned plasmids, specifically, the plasmids here represent plasmids that carry RNA fragments, or carry RNA fragments and targeting tags, and can enter the host body and can be enriched in the liver, self-contained. Assembled to form composite exosomes, the composite structure can deliver RNA fragments to the lungs, so that the RNA fragments are expressed in the brain of the target tissue, and then inhibit the expression of matching genes to achieve the purpose of treating Parkinson's disease.
  • the plasmids here represent plasmids that carry RNA fragments, or carry RNA fragments and targeting tags, and can enter the host body and can be enriched in the liver, self-contained. Assembled to form composite exosomes, the composite structure can deliver RNA fragments to the lungs, so that the RNA fragments are expressed in the brain of the target tissue, and then inhibit the expression of matching genes to achieve the purpose of treating Parkinson's disease.
  • the dosage forms of the drug can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes and the like.
  • RNA delivery system for treating Parkinson's uses plasmid as a carrier and plasmid as a mature injection. Its safety and reliability have been fully verified, and its druggability is very good. The final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of plasmids is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • RNA delivery system for the treatment of Parkinson's provided in this application can be tightly combined with AGO 2 and enriched into a composite structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation, but also maintain its circulation in the circulation. It is stable, and facilitates uptake by recipient cells, intracytoplasmic release and lysosomal escape, and requires a low dose.
  • RNA delivery system for the treatment of Parkinson's disease provided in this application is applied to medicine, that is, it provides a drug delivery platform, which can greatly improve the therapeutic effect of Parkinson's disease, and can also form a research and development basis for more RNA drugs through this platform. , which greatly promotes the development and use of RNA drugs.
  • Fig. 1 is a comparison diagram of plasmid distribution and metabolism in mice provided by an embodiment of the present application
  • Fig. 2 is a comparison diagram of protein expression levels in mice provided by an embodiment of the present application.
  • FIG. 3 is a comparison diagram of related siRNA levels in mice provided by an embodiment of the present application.
  • FIG. 4 is a comparison diagram of absolute siRNA levels in various tissues of mice provided in an embodiment of the present application.
  • Figure 5 is a comparison diagram of the effect of plasmid doses on mouse siRNA levels provided by an embodiment of the present application.
  • Fig. 6 is the metabolic situation comparison diagram of the precursor and the mature body in the mouse liver after injecting the plasmid provided by an embodiment of the present application;
  • FIG. 7 is a comparison diagram of siRNA kinetics and distribution in different tissues of mice provided by an embodiment of the present application.
  • Figure 8 is a comparison diagram of the influence of different promoters on siRNA provided by an embodiment of the present application.
  • FIG. 9 is a comparison diagram of the fluorescence intensity of eGFP in different tissues of mice provided by an embodiment of the present application.
  • Figure 10 is a comparison diagram of mouse alanine aminotransferase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, serum alkaline phosphatase, creatinine content, and thymus gland weight, spleen weight, and peripheral blood cell percentage provided by an embodiment of the present application;
  • Fig. 11 is a comparison diagram of Parkinson's treatment in transgenic mice provided in an embodiment of the present application.
  • FIG. 12 is a graph of in vivo enrichment data when plasmid 1 provided in an example of the present application contains 6 kinds of RNA alone.
  • FIG. 13 is a graph of in vivo enrichment data when plasmid 2 provided by another embodiment of the present application contains 6 kinds of RNAs alone.
  • FIG. 14 is a graph of the in vivo self-assembly data of the exosomes provided in an example of the present application when each of the exosomes individually contains 6 kinds of RNAs.
  • Fig. 15 is a graph showing the therapeutic effect on Parkinson's disease reflected by the expression results of LRRK2 gene when the plasmids provided in an example of the present application contain 6 kinds of RNA alone.
  • FIG. 16 is a graph of in vivo enrichment data when plasmid 1 provided in an example of the present application contains any two RNA sequences respectively.
  • Figure 17 is a graph of the in vivo self-assembly data of exosomes provided in an embodiment of the present application when they contain any two RNA sequences.
  • Fig. 18 is a graph showing the effect of treating Parkinson's disease according to the expression data of LRRK2 mRNA when plasmid 1 provided in an embodiment of the present application contains any two RNA sequences respectively.
  • Fig. 19 is a graph showing the therapeutic effect on Parkinson's disease reflected by the expression result of LRRK2 gene when plasmid 2 provided by another embodiment of the present application contains any two RNA sequences respectively.
  • Fig. 20 is a graph showing the effect of treating Parkinson's disease according to the expression data of LRRK2 mRNA when the plasmid 1 provided in an embodiment of the present application contains any three RNA sequences respectively.
  • Fig. 21 is a diagram showing the therapeutic effect on Parkinson's disease reflected by the expression result of LRRK2 gene when plasmid 2 provided in another embodiment of the present application contains any three RNA sequences respectively.
  • Figure 22 is a graph of the in vivo enrichment data of the plasmid provided in an example of the present application when the sequences in the plasmid contain siRNA and RVG.
  • Figure 23 is a graph showing the effect of treating Parkinson's disease according to the expression data of LRRK2 mRNA when the sequence in the plasmid provided in an example of the present application contains siRNA and RVG.
  • Figure 24 is a data comparison diagram of the therapeutic effect of RVG-LAMP2B fusion protein and other fusion proteins on a plasmid vector for Parkinson's disease provided in an example of the present application, and the data is reflected by the relative level of LRRK2 mRNA.
  • FIG. 25 is a graph of in vivo enrichment data of the gene circuit provided in an example of the present application when three RNA sequences with more than 80% homology to the siRNA sequence of the LRRK2 gene are included.
  • Figure 26 is a data comparison diagram of the therapeutic effect on Parkinson's disease when the gene circuit provided by an embodiment of the present application includes 3 RNA sequences with more than 80% homology to the siRNA sequence of the LRRK2 gene. level to reflect.
  • HE staining Hematoxylin-eosin staining, referred to as HE staining.
  • HE staining is one of the most basic and widely used technical methods in histology and pathology teaching and research.
  • the hematoxylin staining solution is alkaline and can stain the basophilic structure of the tissue (such as ribosome, nucleus and ribonucleic acid in the cytoplasm) into blue-violet; eosin is an acid dye, which can stain the eosinophilic structure of the tissue ( Such as intracellular and intercellular proteins, including Lewy bodies, alcohol bodies, and most of the cytoplasm) stained pink, making the morphology of the entire cell organization clearly visible.
  • the basophilic structure of the tissue such as ribosome, nucleus and ribonucleic acid in the cytoplasm
  • eosin is an acid dye, which can stain the eosinophilic structure of the tissue ( Such as intracellular and intercellular proteins, including Lewy bodies, alcohol bodies, and most of the cytoplasm) stained pink, making the morphology of the entire cell organization clearly visible.
  • HE staining include: sample tissue fixation and sectioning; tissue sample dewaxing; tissue sample hydration; tissue section hematoxylin staining, differentiation and anti-blue; tissue section eosin staining and dehydration; tissue sample section air-drying and sealing; Observe and photograph under the microscope.
  • Masson staining renders collagen fibers blue (stained by aniline blue) or green (stained by bright green) and muscle fibers red (stained by acid fuchsin and Ponceau), which is consistent with the size and organization of the anionic dye molecules of permeability.
  • the fixed tissue is stained sequentially or mixed with a series of anionic water-soluble dyes. It can be found that red blood cells are stained with the smallest molecular anionic dyes, muscle fibers and cytoplasm are stained with medium-sized anionic dyes, and collagen fibers are stained with macromolecular anionic dyes. Dyeing with anionic dyes.
  • red blood cells have the least permeability to anionic dyes, followed by muscle fibers and cytoplasm, and collagen fibers have the largest permeability.
  • Type I and III collagens are green (GBM, TBM, mesangial matrix and renal interstitium are green), and erythropoietin, tubular cytoplasm, and erythrocytes are red.
  • Masson staining The specific steps of Masson staining include:
  • Tissues were fixed in Bouin's solution, rinsed with running water overnight, and embedded in conventional dehydration; sections were deparaffinized to water (deparaffinized in xylene for 10 min ⁇ 3 times, and the liquid was blotted dry with absorbent paper; 100% ethanol 5 min ⁇ 2 times, with water absorption Dry the liquid with paper; 95% ethanol for 5min ⁇ 2 times, blot the liquid with absorbent paper; run water for 2min, blot dry with absorbent paper); Weiger's iron hematoxylin staining for 5-10min; ; Rinse with running water for 3min; Stain with Ponceau red acid fuchsin solution for 8min; Rinse slightly with distilled water; Treat with 1% phosphomolybdic acid aqueous solution for about 5min; Do not wash with water, directly counterstain with aniline blue solution or bright green solution for 5min; Treat with 1% glacial acetic acid 1min; dehydrated in 95% ethanol for 5min ⁇ 2 times,
  • Western Blot (Western Blot) is to transfer the protein to the membrane, and then use the antibody for detection.
  • the corresponding antibody can be used as the primary antibody for detection, and the expression product of the new gene can be detected by the fusion part of the antibody. .
  • Western Blot uses polyacrylamide gel electrophoresis, the detected object is protein, the "probe” is an antibody, and the "color development” is a labeled secondary antibody.
  • the protein sample separated by PAGE is transferred to a solid phase carrier (such as nitrocellulose membrane), and the solid phase carrier adsorbs proteins in the form of non-covalent bonds, and can keep the types of polypeptides separated by electrophoresis and their biological activities unchanged.
  • the protein or polypeptide on the solid phase carrier is used as an antigen, which reacts with the corresponding antibody, and then reacts with the enzyme or isotope-labeled secondary antibody to detect the specific target gene separated by electrophoresis through substrate color development or autoradiography.
  • expressed protein components The steps mainly include: protein extraction, protein quantification, gel preparation and electrophoresis, membrane transfer, immunolabeling and development.
  • Immunohistochemistry using antigen-antibody reaction, that is, the principle of specific binding of antigen and antibody, determines the antigen (polypeptide) in tissue cells by developing the color of the chromogenic reagent (fluorescein, enzyme, metal ion, isotope) labeled antibody through chemical reaction. and protein), the localization, qualitative and relative quantitative research, called immunohistochemistry (immunohistochemistry) or immunocytochemistry (immunocytochemistry).
  • chromogenic reagent fluorescein, enzyme, metal ion, isotope
  • the main steps of immunohistochemistry include: section soaking, overnight drying, xylene dewaxing, gradient alcohol dewaxing (100%, 95%, 90%, 80%, 75%, 70%, 50%, 3min each time) , double-distilled water, dropwise addition of 3% hydrogen peroxide solution to remove catalase, water washing, antigen retrieval, dropwise addition of 5% BSA, blocking for 1 h, dilution of primary antibody, washing with PBS buffer, incubation with secondary antibody, washing with PBS buffer , color developing solution, washing with water, hematoxylin staining, dehydration with gradient ethanol, and sealing with neutral gum.
  • the detection of the siRNA level, the protein content and the mRNA content involved in the present invention is to establish the mouse stem cell in vitro model by injecting the RNA delivery system into the mouse.
  • the expression levels of mRNA and siRNA in cells and tissues were detected by qRT-PCR. Absolute quantification of siRNA was determined by plotting a standard curve using the standards.
  • the internal reference gene is U6snRNA (in tissue) or miR-16 (in serum, exosomes)
  • the gene is GAPDH or 18s RNA.
  • Western blotting was used to detect protein expression levels in cells and tissues, and ImageJ software was used for protein quantitative analysis.
  • This embodiment provides an RNA plasmid delivery system for treating Parkinson's, the system comprising a plasmid carrying an RNA fragment that can be used for treating Parkinson's, and the plasmid can be enriched in the organ tissue of a host, And endogenously and spontaneously form a composite structure containing the RNA fragment in the host organ tissue, and the composite structure can send the RNA fragment into the target tissue to achieve the treatment of Parkinson's disease.
  • the plasmid also includes a promoter and a targeting tag.
  • the plasmid includes any one of the following circuits or a combination of several circuits: promoter-RNA sequence, promoter-targeting tag, promoter-RNA sequence-targeting tag, and each of the plasmids includes at least one RNA fragment and a targeting tag, the RNA fragment and targeting tag are located in the same line or in different lines.
  • the plasmid can include only a promoter-RNA sequence-targeting tag, or a combination of a promoter-RNA sequence, a promoter-targeting tag, or a promoter-targeting tag, a promoter- A combination of RNA-seq-targeting tags.
  • the plasmid can also include a flanking sequence, a compensation sequence and a loop sequence that can make the circuit fold into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence; the plasmid includes the following Any one line or combination of several lines: 5'-promoter-5' flanking sequence-RNA sequence-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag, 5'-promoting sub-targeting tag-5' flanking sequence-RNA sequence-loop sequence-compensating sequence-3'flanking sequence.
  • the plasmid vector contains 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensation sequence-3'flanking sequence
  • its RNA delivery system has in vivo enrichment and Parkinson's treatment
  • the effect is shown in Figure 22-23, the targeting tag is RVG, and the RNA fragment is siRNA with therapeutic effect.
  • the Parkinson's treatment effect of the RVG-LAMP2B fusion protein on the plasmid vector is shown in Figure 24, and its treatment effect is reflected by the level of HTT mRNA.
  • the 5' flanking sequence is preferably ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with ggatcctggaggcttgctgaaggctgtatgctgaattc, etc.
  • the loop sequence is preferably gttttggccactgactgac or a sequence with more than 80% homology thereto, including sequences with 85%, 90%, 92%, 95%, 98%, 99% homology with gttttggccactgactgac, and the like.
  • the 3' flanking sequence is preferably accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag, etc.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-5 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-3 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
  • the compensation sequence may be the reverse complementary sequence of the RNA sequence by deleting any 1-3 consecutively arranged bases.
  • the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
  • the compensation sequence may be the reverse complementary sequence of the 9th position and/or the 10th position in the deletion of the RNA sequence. Deleting bases 9 and 10 works best.
  • flanking sequences are not randomly selected, but are determined based on a large number of theoretical studies and experiments. increase the expression rate of RNA fragments.
  • sequence 1 is preferably CAGATC
  • sequence 2 can be composed of 5-80 bases
  • Sequences of composition such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases
  • Any sequence may be used, preferably a sequence consisting of 10-50 bases, more preferably a sequence consisting of 20-40 bases, and sequence 3 is preferably TGGATC.
  • Sequence 2 is specifically shown in Table 1 below.
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • sequence 4 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-1 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-3 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
  • RNA fragments comprise one, two or more specific RNA sequences of medical significance, the RNA sequences can be expressed in the target receptor, and the compensatory sequence cannot be expressed in the target receptor.
  • the RNA sequence can be a siRNA sequence, a shRNA sequence or a miRNA sequence, preferably a siRNA sequence.
  • the length of an RNA sequence is 15-25 nucleotides (nt), preferably 18-22nt, such as 18nt, 19nt, 20nt, 21nt, and 22nt. This range of sequence lengths was not chosen arbitrarily, but was determined through trial and error. A large number of experiments have proved that when the length of the RNA sequence is less than 18nt, especially less than 15nt, the RNA sequence is mostly invalid and will not play a role. The cost of the line is greatly increased, and the effect is not better than the RNA sequence with a length of 18-22nt, and the economic benefit is poor. Therefore, when the length of the RNA sequence is 15-25nt, especially 18-22nt, the cost and the effect can be taken into consideration, and the effect is the best.
  • nt nucleotides
  • the RNA capable of treating Parkinson's disease is selected from: siRNA of LRRK2 gene or nucleic acid molecule encoding the above RNA.
  • the number of required delivery RNA effective sequences is one, two or more.
  • the functional structural region of the plasmid vector can be expressed as: (promoter-siRNA1)-connector sequence-(promoter-siRNA2)-connector sequence- (promoter-targeting tag), or (promoter-targeting tag-siRNA1)-linker-(promoter-targeting tag-siRNA2), or (promoter-siRNA1)-linker-(promoter- Targeting tag-siRNA2) etc.
  • the functional structural region of the plasmid vector can be expressed as: (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-connecting sequence-(5'-promoter - 5' flanking sequence - siRNA2-loop sequence - compensation sequence - 3' flanking sequence) - linking sequence - (5'-promoter-targeting tag), or (5'-promoter-targeting tag-5' flanking sequence-siRNA1-loop sequence-compensation sequence-3' flanking sequence)-linker sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'flanking sequence), or (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-linking sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'
  • the above RNA can also be obtained by ribose modification of the RNA sequence (siRNA, shRNA or miRNA) therein, preferably 2' fluoropyrimidine modification.
  • 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on siRNA, shRNA or miRNA with 2'-F.
  • 2'-F can make it difficult for RNase in the human body to recognize siRNA, shRNA or miRNA, so it can Increases the stability of RNA transport in vivo.
  • the liver will phagocytose exogenous plasmids, and up to 99% of the exogenous plasmids will enter the liver. Therefore, when plasmids are used as vectors, they can be enriched in liver tissue without specific design.
  • the plasmid is opened to release RNA molecules (siRNA, shRNA, or miRNA), and liver tissue spontaneously wraps the above RNA molecules into exosomes, and these exosomes become RNA delivery mechanisms.
  • RNA delivery mechanism in order to make the RNA delivery mechanism (exosome) have the ability of "precision guidance”, we designed a targeting tag in the plasmid injected into the body, and the targeting tag will also be assembled into exosomes by liver tissue , especially when certain specific targeting tags are selected, the targeting tags can be inserted into the surface of exosomes, thereby becoming a targeting structure that can guide exosomes, which greatly improves the RNA delivery mechanism of the present invention.
  • the amount of exogenous plasmids that need to be introduced can be greatly reduced, and on the other hand, the efficiency of potential drug delivery can be greatly improved.
  • the targeting tag is selected from one of the peptides, proteins or antibodies with targeting function.
  • the selection of the targeting tag is a process that requires creative work. On the one hand, it is necessary to select the available targeting tags according to the target tissue. It is ensured that the targeting label can stably appear on the surface of exosomes, so as to achieve the targeting function.
  • Targeting peptides that have been screened so far include, but are not limited to, RVG targeting peptide (nucleotide sequence shown in SEQ ID No: 1), GE11 targeting peptide (nucleotide sequence shown in SEQ ID No: 2), PTP targeting peptide (nucleotide sequence shown in SEQ ID No: 3), TCP-1 targeting peptide (nucleotide sequence shown in SEQ ID No: 4), MSP targeting peptide (nucleotide sequence shown in SEQ ID No: 4) SEQ ID No: 5); targeting proteins include but are not limited to RVG-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6), GE11-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6) : 7), PTP-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 8), TCP-1-LAMP2B fusion protein (nucle
  • RNAs comprise one, two or more specific RNA sequences of medical significance, the RNA sequences can be expressed in the target receptor, and the compensatory sequence cannot be expressed in the target receptor.
  • the plasmid vector can also be composed of multiple plasmids with different structures, one of which contains a promoter promoter and targeting tags, other plasmids contain promoters and RNA fragments. Loading the targeting tag and RNA fragment into different plasmid vectors, and injecting the two plasmid vectors into the body, the targeting effect is no worse than the targeting effect produced by loading the same targeting tag and RNA fragment into one plasmid vector .
  • the plasmid vector containing the RNA sequence can be injected first, and then the plasmid vector containing the targeting tag can be injected after 1-2 hours, so that a better target can be achieved. to the effect.
  • the delivery systems described above can all be used in mammals, including humans.
  • FIG. 1A in order to understand the distribution of plasmids in the body, we carried out a plate test on mice. 720h) sampling, using the plasmid extracted by spectinomycin for transformation, observing the number of clones in liver, plasma, lung, brain, kidney, spleen, the results are shown in Figure 1B, Figure 1C, Figure 1D, it can be seen that the plasmid It is most distributed in the liver of mice, and reaches the peak at about 3 hours after injection, and is basically metabolized at 12 hours after injection.
  • the CMV eGFP siRE circuit co-expressing eGFP protein and EGFR siRNA was injected intravenously into C57BL/6J mice. The results are shown in Figure 2.
  • the eGFP fluorescence in the mouse liver gradually increased over time, reaching a peak at about 12 hours. 48 After hours, it dropped to the background level, and no obvious eGFP signal was seen in other tissues.
  • CMV-scrR The control plasmid
  • CMV-siR E the plasmid expressing EGFR siRNA
  • Figure 3A The related siRNA levels in exosomes, the results are shown in Figure 3A, it can be seen that there is siRNA expression in the exosomes of mouse hepatocytes injected with CMV-siRNA.
  • FIG. 4A After intravenous injection of plasmids into mice, the distribution of mature siRNA in different tissues is shown in Figure 4. It can be seen from Figure 4A that the levels of EGFR-siRNA in plasma, exosomes, and exosome-free plasma show time-dependent changes; from Figure 4B, it can be seen that mouse EGFR-siRNAs in the liver, lung, pancreas, and spleen , The accumulation in the kidney is time-dependent.
  • mice were injected with control plasmid (CMV-scrR), 0.05mg/kg CMV-siR E plasmid, 0.5mg/kg CMV-siR E plasmid, 5mg/kg CMV-siR E plasmid, and detected the liver, Absolute siRNA (EGFR siRNA) levels in spleen, heart, lung, kidney, pancreas, brain, skeletal muscle, CD4 + cells, the results are shown in Figure 5A, it can be seen that there is no siRNA expression in the tissues of mice injected with the control plasmid , in each tissue of mice injected with CMV-siR E plasmid, the level of siRNA expression was positively correlated with the concentration of CMV-siR E plasmid.
  • CMV-scrR control plasmid
  • EGFR siRNA Absolute siRNA
  • fluorescence in situ hybridization assay FISH also confirmed that the level of siRNA expression was positively correlated with the concentration of CMV-siR E plasmid, that is, the tissue distribution of EGFR siRNA was dose-dependent.
  • the plasmid After the plasmid enters the body, it will express the precursor (Precursor) and then process it into the mature body (siRNA), so we tested the metabolism of the precursor (Precursor) and the mature body (siRNA) in the liver after the plasmid was injected into mice. , the results are shown in Figure 6. It can be seen that the expression levels of precursor (Precursor) and mature body (siRNA) in the mouse liver reached a peak at the time point of 6 hours after the injection of the plasmid. Metabolism of the precursor (siRNA) was complete, and the metabolism of the precursor (Precursor) in the mouse liver was complete 48 hours after the injection of the plasmid.
  • siRNA with albumin ALB as the promoter siRNA with CMV as the promoter
  • siRNA without any promoter were injected into mice intravenously.
  • the absolute siRNA levels in the mice were detected at 48 h, and the results are shown in Figure 8. It can be seen that the level of siRNA with CMV as the promoter in mice is the highest, that is, the effect of CMV as the promoter is the best.
  • mice were intravenously injected with PBS or 5 mg/kg CMV-siR G or CMV-RVG-siR G plasmid, and treated for 24 hours After the mice were sacrificed, their eGFP fluorescence levels were detected in cryosections.
  • Figure 9A shows a representative fluorescence microscope image, in which green indicates positive eGFP signal, blue indicates DAPI-stained nuclei, scale bar: 100 ⁇ m, CMV is visible - RVG-siR G plasmid has a more obvious inhibitory effect on mouse eGFP; eGFP transgenic mice were intravenously injected with PBS or CMV-scrR or CMV-siR E plasmid, and the mice were sacrificed after 24 hours of treatment, and they were detected in frozen sections.
  • the fluorescence level of eGFP is a bar graph of the fluorescence intensity (Fluorescence intensity) of the mouse heart, lung, kidney, pancreas, brain, and skeletal muscle injected with PBS, CMV- siRE , and CMV-RVG- siRE . It can be seen that, The contrast of fluorescence intensity in liver, spleen, lung and kidney of mice was more obvious.
  • mice injected with PBS, CMV-scrR, and CMV-siR E their alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), blood urea nitrogen (BUN), serum alkaline phosphatase (ALP), creatinine (CREA) content, thymus weight, spleen weight, and percentage of peripheral blood cells were detected.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • TBIL total bilirubin
  • BUN blood urea nitrogen
  • ALP serum alkaline phosphatase
  • CREA creatinine
  • Figure 10G is a comparison chart of mouse liver, lung, spleen, and kidney tissue
  • Figure 10H -I is a comparison chart of mouse thymus and spleen tissue
  • FIG. 10J is a comparison chart of percentage in peripheral blood cells of mice.
  • mice injected with PBS, CMV- scrR , and CMV-siRE were almost the same.
  • the mice injected with CMV-siRE were compared with those injected with PBS.
  • the liver, lung, spleen, kidney also have no tissue damage.
  • RNA delivery system for treating Parkinson's uses plasmid as a carrier and plasmid as a mature injection, its safety and reliability have been fully verified, and its druggability is very good.
  • the final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of plasmids is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • RNA delivery system for the treatment of Parkinson's disease provided in this example can be tightly combined with AGO 2 and enriched into a complex structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation, but also maintain its circulation in the circulation. It is stable, and is beneficial to receptor cell uptake, intracytoplasmic release and lysosomal escape, and the required dose is low.
  • the medicament comprises a plasmid carrying an RNA fragment that can be used to treat Parkinson's, the plasmid can be enriched in the organ tissue of a host, and endogenously and spontaneously form in the host organ tissue containing the A composite structure of RNA fragments capable of delivering the RNA fragments into target tissues for Parkinson's disease treatment.
  • the RNA fragment comprises one, two or more specific RNA sequences of medical significance, and the RNA sequences are siRNA, shRNA or miRNA sequences of medical significance that can inhibit or hinder the development of Parkinson's disease .
  • a plasmid as a vector, which carries one or more RNA fragments, it has in vivo enrichment, self-assembly and Parkinson's treatment effects.
  • Figure 12-15 shows that the vector carries RNA fragments of 6 separate RNA sequences Data results presented;
  • Figures 16-19 show data results presented by vectors carrying 4 sets of RNA fragments containing any 2 RNA sequences;
  • Figures 20-21 show vectors carrying 3 sets of RNA fragments containing any 3 RNA sequences The therapeutic data results presented by sequenced RNA fragments.
  • CTX stands for cortex and STR stands for Striatum.
  • the plasmid also includes a promoter and a targeting tag
  • the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host, and the targeting structure is located on the surface of the composite structure, so The complex structure can seek and bind to the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
  • the drug can be administered orally, inhaled, subcutaneously injected, intramuscularly injected or intravenously injected into the human body, it can be delivered to the target tissue through the RNA delivery system described in Example 1 to exert a therapeutic effect.
  • the drug can be used in combination with other Parkinson's disease drugs to improve the treatment effect, such as anticholinergics (Antan, etc.), antihistamines (diphenhydramine, amantadine, etc.), levodopa Drugs (Dopa, Sining, Xilaimei, etc.), dopamine receptor agonists (Texida, Xie Liangxing, Crepa, bromocriptine, etc.), monoamine oxidase B inhibitors (Sgining, Jin Siping, etc.) ), catecholamine oxygen methyltransferase inhibitors (Entocapone, etc.).
  • Parkinson's disease drugs such as anticholinergics (Antan, etc.), antihistamines (diphenhydramine, amantadine, etc.), levodopa Drugs (Dopa, Sining, Xilaimei, etc.), dopamine receptor agonists (Texida, Xie Liangxing, Cre
  • the medicine of this embodiment may also include a pharmaceutically acceptable carrier, which includes but is not limited to diluents, buffers, emulsions, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorption Agents, wetting agents, disintegrating agents, absorption enhancers, surfactants, colorants, flavoring agents, adjuvants, desiccants, adsorption carriers, etc.
  • a pharmaceutically acceptable carrier includes but is not limited to diluents, buffers, emulsions, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorption Agents, wetting agents, disintegrating agents, absorption enhancers, surfactants, colorants, flavoring agents, adjuvants, desiccants, adsorption carriers, etc.
  • the dosage forms of the medicine provided in this embodiment can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes, and the like.
  • the medicine provided in this example uses the plasmid as the carrier and the plasmid as the mature injection, and its safety and reliability have been fully verified, and the drugability is very good.
  • the final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the drug can deliver various kinds of small molecule RNAs and has strong versatility. And the preparation of plasmids is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • the drug provided in this application can be closely combined with AGO 2 and enriched into a composite structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation and maintain its stability in circulation, but also benefit the receptor.
  • Cellular uptake, intracytoplasmic release and lysosomal escape require low doses.
  • this embodiment provides an application of an RNA delivery system for treating Parkinson's disease in a drug, and the drug is a drug for treating Parkinson's disease.
  • the application of the RNA delivery system in the treatment of Parkinson's disease is specifically described in conjunction with the following experiments.
  • LRRK2R1441G transgenic mice were selected for the experiment when they were 3 months old, and the experiment set up LPS intervention group and LPS non-intervention group.
  • the LPS intervention group was treated with CMV-scrR/CMV-RVG-siR LRRK2 after 7 days of LPS intervention.
  • Figure 11A is a western bolt image of LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2
  • Figure 11B is a LRRK2R1441G transgenic mouse injected with CMV-scrR/CMV-RVG-siR LRRK2
  • the grayscale analysis of the protein shows that the levels of LRRK2 protein and S935 protein in mice injected with CMV-RVG-si RLRRK2 decreased, indicating that CMV-RVG-siR LRR K2 can pass through the blood brain after the liver releases siRNA and assembles into exosomes
  • the barrier reduces the expression of proteins deep in the brain.
  • FIG. 11C is an immunofluorescence image of TH+ neurons in the substantia nigra of LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2 .
  • the results showed that mice injected with CMV-RVG-siR LRRK2 rescued TH
  • the loss of neurons indicates that CMV-RVG-siR LRRK2 can cross the blood-brain barrier and enter the deep brain to function after the liver releases siRNA and assembles into exosomes.
  • this figure is an immunofluorescence image of the activation level of microglia in LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2 .
  • the results show that injection of CMV-RVG-siR LRRK2 mice can inhibit microglia
  • the activation of CMV-RVG-siR LRRK2 indicates that CMV-RVG-siR LRRK2 can cross the blood-brain barrier and enter the deep brain to function after the liver releases siRNA and assembles into exosomes.

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Abstract

Provided is an RNA delivery system for treating Parkinson's disease, characterized in that the system comprises a plasmid. The plasmid carries an RNA fragment capable of treating Parkinson's disease, and can be enriched in organ tissues of a host. In the organ tissues of the host, the plasmid can endogenously and spontaneously form a composite structure containing the RNA fragment. The composite structure can feed the RNA fragment to target tissues to treat Parkinson's disease. The safety and reliability of the RNA delivery system for treating colitis have been fully verified, and the system has good druggability and high universality.

Description

一种用于治疗帕金森的RNA质粒递送***An RNA plasmid delivery system for the treatment of Parkinson's 技术领域technical field
本申请涉及生物医学技术领域，特别涉及一种用于治疗帕金森的RNA质粒递送***。The present application relates to the field of biomedical technology, in particular to an RNA plasmid delivery system for treating Parkinson's.
背景技术Background technique
帕金森病(PD)又名震颤麻痹，是一种常见的中老年人神经***变性疾病，主要病变在黑质和纹状体，震颤、肌强直及运动减少是本病的主要临床特征。帕金森病是老年人中第四位最常见的神经变性疾病。Parkinson's disease (PD), also known as tremor palsy, is a common neurodegenerative disease of the middle-aged and elderly people. The main lesions are in the substantia nigra and striatum. Parkinson's disease is the fourth most common neurodegenerative disease in older adults.
RNA干扰(RNAi)疗法自从被发明以来，一直被认为是治疗人类疾病的一种很有前途的策略，但在临床实践过程中遇到了许多问题，该疗法的发展进度远远落后于预期。RNA interference (RNAi) therapy has been considered a promising strategy for the treatment of human diseases since its invention, but many problems have been encountered during clinical practice, and the development of this therapy has lagged far behind expectations.
一般认为RNA无法在细胞外长期稳定存在，因为RNA会被细胞外富含的RNase降解成碎片，因此必须找到能够使RNA稳定存在于细胞外，并且能够靶向性地进入特定组织的方法，才能将RNAi疗法的效果凸显出来。It is generally believed that RNA cannot exist stably outside the cell for a long time, because RNA will be degraded into fragments by RNases rich in extracellular, so it is necessary to find a method that can make RNA stable outside the cell and can enter specific tissues in a targeted manner. Highlight the effect of RNAi therapy.
目前与siRNA相关的专利很多，主要聚焦在以下几个方面：1、设计具有医学效果的siRNA。2、对siRNA进行化学修饰，提高siRNA在生物体内的稳定性，提高产率。3、提高设计各种人工载体(如脂质纳米粒子、阳离子聚合物和病毒)，以提高siRNA在体内传递的效率。其中第3方面的专利很多，其根本原因是研究人员们已经意识到目前缺乏合适的siRNA传递***，将siRNA安全地、精确地、高效地输送到目标组织，该问题已经成为制约RNAi疗法的核心问题。At present, there are many patents related to siRNA, mainly focusing on the following aspects: 1. Designing siRNA with medical effects. 2. Chemical modification of siRNA to improve the stability of siRNA in vivo and increase the yield. 3. Improve the design of various artificial carriers (such as lipid nanoparticles, cationic polymers and viruses) to improve the efficiency of siRNA delivery in vivo. Among them, there are many patents in the third aspect. The fundamental reason is that researchers have realized that there is currently a lack of suitable siRNA delivery systems to safely, precisely and efficiently deliver siRNA to target tissues. This problem has become the core restricting RNAi therapy. question.
公开号为CN108624590A的中国专利公开了一种能够抑制DDR2基因表达的siRNA；公开号为CN108624591A的中国专利公开了一种能够沉默ARPC4基因的siRNA，并且对该siRNA进行了α-磷-硒修饰；公开号为CN108546702A的中国专利公开了一种靶向长链非编码RNA DDX11-AS1的siRNA。公开号为CN106177990A的中国专利公开了一种可以用于多种肿瘤治疗的siRNA前体。这些专利均设计了特定的siRNA并且来针对某些由基因变化引起的疾病。The Chinese Patent Publication No. CN108624590A discloses a siRNA capable of inhibiting the expression of DDR2 gene; the Chinese Patent Publication No. CN108624591A discloses a siRNA capable of silencing the ARPC4 gene, and the siRNA is modified with α-phosphorus-selenium; The Chinese Patent Publication No. CN108546702A discloses a siRNA targeting long-chain non-coding RNA DDX11-AS1. The Chinese Patent Publication No. CN106177990A discloses a siRNA precursor that can be used for various tumor treatments. These patents design specific siRNAs to target certain diseases caused by genetic changes.
公开号为CN108250267A的中国专利公开了一种多肽、多肽-siRNA诱导共组装体，使用多肽作为siRNA的载体。公开号为CN108117585A的中国专利公开了一种靶向导入siRNA促进乳腺癌细胞凋亡的多肽，同样使用多肽作为siRNA的载体。公开号为CN108096583A的中国专利公开了一种纳米粒子载体，该载体在包含化疗药物的同时还可以装载具有乳腺癌疗效的siRNA。这些专利均为在siRNA载体方面的发明创造，但是其技术方案具有一个共同特征，那就是载体和siRNA均在体外预先组装，然后再引入宿主体内。事实上，目前绝大部分设计的传递技术均是如此。然而这类传递体系具有共同的问题，那就是这些人工合成的外源性传递体系很容易被宿主的循环***清除，也有可能引起免疫原性反应，甚至可能对特定的细胞类型和组织有毒。Chinese Patent Publication No. CN108250267A discloses a polypeptide, polypeptide-siRNA induced co-assembly, using polypeptide as a carrier of siRNA. The Chinese Patent Publication No. CN108117585A discloses a polypeptide for promoting apoptosis of breast cancer cells through targeted introduction of siRNA, and the polypeptide is also used as the carrier of siRNA. The Chinese Patent Publication No. CN108096583A discloses a nanoparticle carrier, which can be loaded with siRNA with breast cancer curative effect while containing chemotherapeutic drugs. These patents are all inventions and creations in terms of siRNA vectors, but their technical solutions have a common feature, that is, the vectors and siRNA are pre-assembled in vitro and then introduced into the host. In fact, this is the case with most of the delivery technologies currently designed. However, this type of delivery system has a common problem, that is, these synthetic exogenous delivery systems are easily cleared by the host's circulatory system, may also cause immunogenic responses, and may even be toxic to specific cell types and tissues.
本发明的研究团队发现内源性细胞可以选择性地将miRNAs封装到外泌体(exosome)中，外泌体可以将miRNA传递到受体细胞中，其分泌的miRNA在相对较低的浓度下，即可有力阻断靶基因的表达。外泌体与宿主免疫***生物相容，并具有在体内保护和运输miRNA跨越生物屏障的先天能力，因此成为克服与siRNA传递相关的问题的潜在解决方案。例如，公开号为CN110699382A的中国专利就公开了一种递送siRNA的外泌体的制备方法，公开了从血浆中分离外泌体，并将siRNA通过电穿孔的方式封装到外泌体中的技术。The research team of the present invention found that endogenous cells can selectively encapsulate miRNAs into exosomes, and exosomes can deliver miRNAs to recipient cells, which secrete miRNAs at relatively low concentrations , which can effectively block the expression of target genes. Exosomes are biocompatible with the host immune system and possess the innate ability to protect and transport miRNAs across biological barriers in vivo, thus becoming a potential solution to overcome problems associated with siRNA delivery. For example, the Chinese Patent Publication No. CN110699382A discloses a method for preparing siRNA-delivering exosomes, and discloses the technology of separating exosomes from plasma and encapsulating siRNA into exosomes by electroporation .
但是这类在体外分离或制备外泌体的技术，往往需要通过细胞培养获取大量的外泌体，再加上siRNA封装的步骤，这使得大规模应用该产品的临床费用变得非常高，一般患者无法负担；更重要的是，外泌体复杂的生产/纯化过程，使其几乎不可能符合GMP标准。However, such techniques of in vitro isolation or preparation of exosomes often require obtaining a large amount of exosomes through cell culture, coupled with the step of siRNA encapsulation, which makes the clinical cost of large-scale application of this product very high. Patients cannot afford it; more importantly, the complex production/purification process of exosomes makes it almost impossible to comply with GMP standards.
到目前为止，以外泌体为有效成分的药物从未获得CFDA批准，其核心问题就是无法保证外泌体产品的一致性，而这一问题直接导致此类产品无法获得药品生产许可证。如果能解决这一问题，则对推动RNAi疗法治疗帕金森病意义非凡。So far, drugs with exosomes as active ingredients have never been approved by the CFDA. The core problem is that the consistency of exosome products cannot be guaranteed, and this problem directly leads to the inability of such products to obtain drug production licenses. If this problem can be solved, it will be of great significance to promote RNAi therapy for Parkinson's disease.
因此，开发一个安全、精确和高效的siRNA传递***是对提高RNAi治疗效果，推进RNAi疗法至关重要的一环。Therefore, the development of a safe, precise and efficient siRNA delivery system is a crucial part of improving the effect of RNAi therapy and advancing RNAi therapy.
发明内容SUMMARY OF THE INVENTION
有鉴于此，本申请实施例提供了一种用于治疗帕金森的RNA质粒递送***及其应用，以解决现有技术中存在的技术缺陷。In view of this, the embodiments of the present application provide an RNA plasmid delivery system for treating Parkinson's and its application, so as to solve the technical defects existing in the prior art.
本申请的一个发明点为提供一种用于治疗帕金森的RNA质粒递送***，该***包括质粒，所述质粒携带有能够用于治疗帕金森的RNA片段，所述质粒能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述RNA片段的复合结构，所述复合结构能够并结合目标组织，将所述RNA片段送入目标组织，以实现帕金森病的治疗。One of the inventions of the present application is to provide an RNA plasmid delivery system for treating Parkinson's, the system comprising a plasmid carrying an RNA fragment that can be used for treating Parkinson's, and the plasmid can be used in the host's organs and tissues It is enriched in the host organ tissue, and endogenously and spontaneously forms a complex structure containing the RNA fragment in the host organ tissue, and the complex structure can bind to the target tissue, and the RNA fragment is sent into the target tissue to achieve par Treatment of Kinson's disease.
可选地，所述RNA片段包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列是具有医学意义的、能够抑制或阻碍帕金森病发展的siRNA、shRNA或miRNA序列。Optionally, the RNA fragment comprises one, two or more specific RNA sequences of medical significance, and the RNA sequences are siRNA, shRNA or miRNA sequences of medical significance that can inhibit or hinder the development of Parkinson's disease .
可选地，所述质粒还包括启动子和靶向标签，所述靶向标签能够在宿主的器官组织中形成所述复合结构的靶向结构，所述靶向结构位于复合结构的表面，所述复合结构能够通过所述靶向结构寻找并结合目标组织，将所述RNA片段递送进入目标组织。Optionally, the plasmid also includes a promoter and a targeting tag, the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host, and the targeting structure is located on the surface of the composite structure, so The complex structure can seek and bind to the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
可选地，所述质粒包括以下任意一种线路或几种线路的组合：启动子-RNA片段、启动子-靶向标签、启动子-RNA片段-靶向标签；每一个所述质粒中至少包括一个RNA片段和一个靶向标签，所述RNA片段和靶向标签位于相同的线路中或位于不同的线路中。Optionally, the plasmid includes any one of the following circuits or a combination of several circuits: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; in each of the plasmids, at least An RNA fragment and a targeting tag are included, the RNA fragment and targeting tag being in the same circuit or in different circuits.
可选地，所述质粒还包括能够使所述线路折叠成正确结构并表达的侧翼序列、补偿序列和loop序列，所述侧翼序列包括5’侧翼序列和3’侧翼序列；Optionally, the plasmid also includes a flanking sequence, a compensation sequence and a loop sequence that can fold the circuit into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence;
所述质粒包括以下任意一种线路或几种线路的组合：5’-启动子-5’侧翼序列-RNA片段-loop序列-补偿序列-3’侧翼序列、5’-启动子-靶向标签或者5’-启动子-靶向标签-5’侧翼序列-RNA片段-loop序列-补偿序列-3’侧翼序列。The plasmid includes any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag Or 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensating sequence-3'flanking sequence.
可选地，所述5’侧翼序列为ggatcctggaggcttgctgaaggctgtatgctgaattc或与其同源性大于80％的序列；Optionally, the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence whose homology is greater than 80%;
所述loop序列为gttttggccactgactgac或与其同源性大于80％的序列；The loop sequence is gttttggccactgactgac or a sequence whose homology is greater than 80%;
所述3’侧翼序列为accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag或与其同源性大于80％的序列；The 3' flanking sequence is accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence whose homology is greater than 80%;
所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-5位碱基。删除RNA反向互补序列的1-5位碱基的目的是使该序列不表达。The compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted. The purpose of deleting bases 1-5 of the RNA reverse complement sequence is to make the sequence unexpressed.
与shRNA不同，在siRNA和miRNA前体中，由于删除作用链的反向互补链(例如siRNA的反向互补链)的第9，10位碱基，导致siRNA和miRNA的作用链会形成凸起结构，以上结构有利于更有效的沉默基因的表达。综上，通过删除第9，10位碱基的反向互补序列来提高沉默效率是目前已公认的结论。Unlike shRNA, in siRNA and miRNA precursors, due to deletion of the 9th and 10th bases of the reverse complementary strand of the active strand (such as the reverse complementary strand of siRNA), the active strand of siRNA and miRNA will form a bulge. structure, which facilitates more efficient silencing of gene expression. To sum up, it is an accepted conclusion that the silencing efficiency can be improved by deleting the reverse complementary sequence of the 9th and 10th bases.
优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-3位碱基。Preferably, the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
更为优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-3位连续排列的碱基。More preferably, the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
最为优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中的第9位和/或第10位碱基。Most preferably, the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
可选地，在质粒中存在至少两种线路的情况下，相邻的线路之间通过序列1-3(序列1-序列2-序列3)组成的序列相连；Optionally, when there are at least two lines in the plasmid, adjacent lines are connected by a sequence composed of sequences 1-3 (sequence 1-sequence 2-sequence 3);
其中，序列1为CAGATC，序列2是由5-80个碱基组成的序列，序列3为TGGATC。Wherein, sequence 1 is CAGATC, sequence 2 is a sequence consisting of 5-80 bases, and sequence 3 is TGGATC.
可选地，在质粒中存在至少两种线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；Optionally, when there are at least two lines in the plasmid, adjacent lines are connected by sequence 4 or a sequence with more than 80% homology to sequence 4;
其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。Wherein, sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
可选地，所述器官组织为肝脏，所述复合结构为外泌体。Optionally, the organ tissue is liver, and the composite structure is exosome.
可选地，所述靶向标签选自具有靶向功能的靶向肽或靶向蛋白。Optionally, the targeting tag is selected from targeting peptides or targeting proteins with targeting function.
可选地，所述靶向肽包括RVG靶向肽、GE11靶向肽、PTP靶向肽、TCP-1靶向肽、MSP靶向肽；Optionally, the targeting peptides include RVG targeting peptides, GE11 targeting peptides, PTP targeting peptides, TCP-1 targeting peptides, and MSP targeting peptides;
所述靶向蛋白包括RVG-LAMP2B融合蛋白、GE11-LAMP2B融合蛋白、PTP-LAMP2B融合蛋白、TCP-1-LAMP2B融合蛋白、MSP-LAMP2B融合蛋白。所述靶向标签优选为RVG靶向肽或RVG-LAMP2B融合蛋白。The targeting proteins include RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein, and MSP-LAMP2B fusion protein. The targeting tag is preferably an RVG targeting peptide or an RVG-LAMP2B fusion protein.
可选地，所述RNA序列的长度为15-25个核苷酸。比如，所述RNA序列的长度可以为16、17、18、19、20、21、22、23、24、25个核苷酸。优选地，所述RNA序列的长度为18-22个核苷酸。Optionally, the RNA sequence is 15-25 nucleotides in length. For example, the length of the RNA sequence can be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides. Preferably, the RNA sequence is 18-22 nucleotides in length.
可选地，所述能够治疗帕金森的RNA选自：LRRK2基因的siRNA，或与上述序列同源性大于80％的RNA序列，或编码上述RNA的核酸分子。需要说明的是，此处“编码上述RNA序列的核酸分子”中的RNA序列也同时包括每种RNA的同源性大于80％的RNA序列。Optionally, the RNA capable of treating Parkinson's is selected from: siRNA of the LRRK2 gene, or an RNA sequence with more than 80% homology to the above sequence, or a nucleic acid molecule encoding the above RNA. It should be noted that the RNA sequences in the "nucleic acid molecules encoding the above RNA sequences" here also include RNA sequences with a homology of more than 80% of each RNA.
可选地，LRRK2基因的siRNA包括AUUAACAUGAAAAUAUCACUU、UUAACAAUAUCAUAUAAUCUU、AUCUUUAAAAUUUGUUAACGC、UUGAUUUAAGAAAAUAGUCUC、UUUGAUAACAGUAUUUUUCUG、其他具有抑制LRRK2基因表达的序列以及与上述序列同源性大于80％的序列。Optionally, the siRNA of the LRRK2 gene includes AUUAACAUGAAAAUAUCACUU, UUAACAAUAUCAUAUAAUCUU, AUCUUUAAAAUUUGUUAACGC, UUGAUUUAAGAAAAUAGUCUC, UUUGAUAACAGUAUUUUUCUG, other sequences that inhibit the expression of the LRRK2 gene, and sequences with more than 80% homology to the above sequences.
需要说明的是，以上所述的“同源性大于80％的序列”可以为同源性为85％、88％、90％、95％、98％等。It should be noted that the above-mentioned "sequences with more than 80% homology" may be 85%, 88%, 90%, 95%, 98%, etc. homology.
可选地，所述RNA片段包括RNA序列本体和对RNA序列本体进行核糖修饰得到的修饰RNA序列。即RNA片段既可以仅由至少一个RNA序列本体组成，也可以仅由至少一个修饰RNA序列组成，还可以由RNA序列本体与修饰RNA序列组成。Optionally, the RNA fragment includes an RNA sequence ontology and a modified RNA sequence obtained by modifying the RNA sequence ontology with ribose sugar. That is, the RNA fragment can be composed of only at least one RNA sequence ontology, or only at least one modified RNA sequence, and can also be composed of RNA sequence ontology and modified RNA sequence.
在本发明中，所述分离的核酸还包括其变体和衍生物。本领域的普通技术人员可以使用通用的方法对所述核酸进行修饰。修饰方式包括(但不限于)：甲基化修饰、烃基修饰、糖基化修饰(如2-甲氧基-糖基修饰、烃基-糖基修饰、糖环修饰等)、核酸化修饰、肽段修饰、脂类修饰、卤素修饰、核酸修饰(如“TT”修饰)等。在本发明的其中一种实施方式中，所述修饰为核苷酸间键合，例如选自：硫代磷酸酯、2'-O甲氧基乙基(MOE)、2'-氟、膦酸烷基酯、二硫代磷酸酯、烷基硫代膦酸酯、氨基磷酸酯、氨基甲酸酯、碳酸酯、磷酸三酯、乙酰胺酯、羧甲基酯及其组合。在本发明的其中一种实施方式中，所述修饰为对核苷酸的修饰，例如选自：肽核酸(PNA)、锁核酸(LNA)、***糖-核酸(FANA)、类似物、衍生物及其组合。优选的，所述修饰为2’氟嘧啶修饰。2’氟嘧啶修饰是将RNA上嘧啶核苷酸的2’-OH用2’-F替代，2’-F能够使RNA不易被体内的RNA酶识别，由此增加RNA片段在体内传输的稳定性。In the present invention, the isolated nucleic acid also includes its variants and derivatives. The nucleic acid can be modified by one of ordinary skill in the art using general methods. Modification methods include (but are not limited to): methylation modification, hydrocarbyl modification, glycosylation modification (such as 2-methoxy-glycosyl modification, hydrocarbyl-glycosyl modification, sugar ring modification, etc.), nucleic acid modification, peptide modification Segment modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification) and the like. In one of the embodiments of the present invention, the modification is an internucleotide linkage, for example selected from: phosphorothioate, 2'-O methoxyethyl (MOE), 2'-fluoro, phosphine Acid alkyl esters, phosphorodithioates, alkyl phosphorothioates, phosphoramidates, carbamates, carbonates, phosphoric triesters, acetamidates, carboxymethyl esters, and combinations thereof. In one of the embodiments of the present invention, the modification is a modification of nucleotides, such as selected from: peptide nucleic acid (PNA), locked nucleic acid (LNA), arabinose-nucleic acid (FANA), analogs, derivatives objects and their combinations. Preferably, the modification is a 2' fluoropyrimidine modification. 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on RNA with 2'-F. 2'-F can make RNA not easily recognized by RNase in vivo, thereby increasing the stability of RNA fragment transmission in vivo. sex.
本申请还提供一种用于治疗帕金森的RNA递送***在药物中的应用。The present application also provides an application of the RNA delivery system for treating Parkinson's in medicine.
可选地，所述药物为治疗帕金森及其相关疾病的药物，这里的相关疾病指的是帕金森的形成或发展过程中出现的关联疾病或并发症、后遗症等，或与帕金森具有一定相关性的其他疾病。Optionally, the drug is a drug for the treatment of Parkinson's disease and its related diseases, and the related diseases here refer to the associated diseases or complications, sequelae, etc. that occur during the formation or development of Parkinson's disease, or have a certain relationship with Parkinson's disease. related other diseases.
所述药物的给药方式包括口服、吸入、皮下注射、肌肉注射、静脉注射。优选静脉注射。The administration modes of the drug include oral, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection. Intravenous injection is preferred.
可选地，所述药物包括上述质粒，具体而言，此处的质粒表示携带有RNA片段、或携带有RNA片段及靶向标签的质粒，并且能够进入宿主体内能够在肝脏部位富集，自组装形成复合结构外泌体，该复合结构能够将RNA片段递送至肺部，使RNA片段在目标组织大脑表达，进而抑制与其匹配的基因的表达，实现治疗帕金森病的目的。Optionally, the medicine includes the above-mentioned plasmids, specifically, the plasmids here represent plasmids that carry RNA fragments, or carry RNA fragments and targeting tags, and can enter the host body and can be enriched in the liver, self-contained. Assembled to form composite exosomes, the composite structure can deliver RNA fragments to the lungs, so that the RNA fragments are expressed in the brain of the target tissue, and then inhibit the expression of matching genes to achieve the purpose of treating Parkinson's disease.
所述药物的剂型可以为片剂、胶囊剂、粉剂、颗粒剂、丸剂、栓剂、软膏剂、溶液剂、混悬剂、洗剂、凝胶剂、糊剂等。The dosage forms of the drug can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes and the like.
本申请的技术效果为：The technical effects of this application are:
本申请提供的用于治疗帕金森的RNA递送***以质粒作为载体，质粒作为成熟的注入物，其安全性和可靠性已被充分验证，成药性非常好。最终发挥效果的RNA序列由内源性外泌体包裹输送，不存在任何免疫反应，无需验证该外泌体的安全性。该递送***可以递送各类小分子RNA，通用性强。并且质粒的制备要比外泌体或是蛋白质、多肽等物质的制备便宜地多，经济性好。本申请提供的用于治疗帕金森的RNA递送***在体内自组装后能够与AGO 2紧密结合并富集为复合结构(外泌体)，不仅能防止其过早降解，维持其在循环中的稳定性，而且有利于受体细胞吸收、胞浆内释放和溶酶体逃逸，所需剂量低。 The RNA delivery system for treating Parkinson's provided by the present application uses plasmid as a carrier and plasmid as a mature injection. Its safety and reliability have been fully verified, and its druggability is very good. The final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes. The delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of plasmids is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances. The RNA delivery system for the treatment of Parkinson's provided in this application can be tightly combined with AGO 2 and enriched into a composite structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation, but also maintain its circulation in the circulation. It is stable, and facilitates uptake by recipient cells, intracytoplasmic release and lysosomal escape, and requires a low dose.
本申请提供的用于治疗帕金森的RNA递送***应用于药物中，即提供了一个药物递送平台，可以大大提高帕金森病的治疗效果，还可以通过该平台形成更多RNA类药物的研发基础，对RNA类药物研发和使用具有极大的推动作用。The RNA delivery system for the treatment of Parkinson's disease provided in this application is applied to medicine, that is, it provides a drug delivery platform, which can greatly improve the therapeutic effect of Parkinson's disease, and can also form a research and development basis for more RNA drugs through this platform. , which greatly promotes the development and use of RNA drugs.
附图说明Description of drawings
图1是本申请一实施例提供的小鼠体内质粒分布与代谢情况对比图；Fig. 1 is a comparison diagram of plasmid distribution and metabolism in mice provided by an embodiment of the present application;
图2是本申请一实施例提供的小鼠体内蛋白表达水平对比图；Fig. 2 is a comparison diagram of protein expression levels in mice provided by an embodiment of the present application;
图3是本申请一实施例提供的小鼠体内相关siRNA水平对比图；FIG. 3 is a comparison diagram of related siRNA levels in mice provided by an embodiment of the present application;
图4是本申请一实施例提供的小鼠各组织中绝对siRNA水平对比图；FIG. 4 is a comparison diagram of absolute siRNA levels in various tissues of mice provided in an embodiment of the present application;
图5是本申请一实施例提供的质粒剂量对小鼠siRNA水平的影响对比图；Figure 5 is a comparison diagram of the effect of plasmid doses on mouse siRNA levels provided by an embodiment of the present application;
图6是本申请一实施例提供的注射质粒后的小鼠肝脏内前体及成熟体的代谢情况对比图；Fig. 6 is the metabolic situation comparison diagram of the precursor and the mature body in the mouse liver after injecting the plasmid provided by an embodiment of the present application;
图7是本申请一实施例提供的小鼠不同组织中siRNA动力学和分布情况对比图；7 is a comparison diagram of siRNA kinetics and distribution in different tissues of mice provided by an embodiment of the present application;
图8是本申请一实施例提供的不同启动子对siRNA的影响对比图；Figure 8 is a comparison diagram of the influence of different promoters on siRNA provided by an embodiment of the present application;
图9是本申请一实施例提供的小鼠不同组织中eGFP荧光强度对比图；FIG. 9 is a comparison diagram of the fluorescence intensity of eGFP in different tissues of mice provided by an embodiment of the present application;
图10是本申请一实施例提供的小鼠谷丙转氨酶、谷草转氨酶、总胆红素、血尿素氮、血清碱性磷酸酶、肌酐含量以及胸腺重量、脾脏重量、外周血细胞百分比对比图；Figure 10 is a comparison diagram of mouse alanine aminotransferase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, serum alkaline phosphatase, creatinine content, and thymus gland weight, spleen weight, and peripheral blood cell percentage provided by an embodiment of the present application;
图11是本申请一实施例提供的转基因小鼠帕金森治疗情况对比图。Fig. 11 is a comparison diagram of Parkinson's treatment in transgenic mice provided in an embodiment of the present application.
图12是本申请一实施例提供的质粒1中分别单独含有6种RNA的情况下，所具有的体内富集数据图。FIG. 12 is a graph of in vivo enrichment data when plasmid 1 provided in an example of the present application contains 6 kinds of RNA alone.
图13是本申请另一实施例提供的质粒2中分别单独含有6种RNA的情况下，所具有的体内富集数据图。FIG. 13 is a graph of in vivo enrichment data when plasmid 2 provided by another embodiment of the present application contains 6 kinds of RNAs alone.
图14是本申请一实施例提供的外泌体分别单独含有6种RNA的情况下，所具有的体内自组装数据图。FIG. 14 is a graph of the in vivo self-assembly data of the exosomes provided in an example of the present application when each of the exosomes individually contains 6 kinds of RNAs.
图15是本申请一实施例提供的质粒中分别单独含有6种RNA的情况下，通过LRRK2基因的表达结果所体现出的针对帕金森的治疗效果图。Fig. 15 is a graph showing the therapeutic effect on Parkinson's disease reflected by the expression results of LRRK2 gene when the plasmids provided in an example of the present application contain 6 kinds of RNA alone.
图16是本申请一实施例提供的质粒1中分别含有任意2种RNA序列的情况下，所具有的体内富集数据图。FIG. 16 is a graph of in vivo enrichment data when plasmid 1 provided in an example of the present application contains any two RNA sequences respectively.
图17是本申请一实施例提供的外泌体分别含有任意2种RNA序列的情况下，所具有的体内自组装数据图。Figure 17 is a graph of the in vivo self-assembly data of exosomes provided in an embodiment of the present application when they contain any two RNA sequences.
图18是本申请一实施例提供的质粒1中分别含有任意2种RNA序列的情况下，通过LRRK2mRNA的表达数据所体现出的针对帕金森治疗的效果图。Fig. 18 is a graph showing the effect of treating Parkinson's disease according to the expression data of LRRK2 mRNA when plasmid 1 provided in an embodiment of the present application contains any two RNA sequences respectively.
图19是本申请另一实施例提供的质粒2中分别含有任意2种RNA序列的情况下，通过LRRK2基因的表达结果体现出的针对帕金森的治疗效果图。Fig. 19 is a graph showing the therapeutic effect on Parkinson's disease reflected by the expression result of LRRK2 gene when plasmid 2 provided by another embodiment of the present application contains any two RNA sequences respectively.
图20是本申请一实施例提供的质粒1中分别含有任意3种RNA序列的情况下，通过LRRK2mRNA的表达数据所体现出的针对帕金森治疗的效果图。Fig. 20 is a graph showing the effect of treating Parkinson's disease according to the expression data of LRRK2 mRNA when the plasmid 1 provided in an embodiment of the present application contains any three RNA sequences respectively.
图21是本申请另一实施例提供的质粒2中分别含有任意3种RNA序列的情况下，通过LRRK2基因的表达结果体现出的针对帕金森的治疗效果图。Fig. 21 is a diagram showing the therapeutic effect on Parkinson's disease reflected by the expression result of LRRK2 gene when plasmid 2 provided in another embodiment of the present application contains any three RNA sequences respectively.
图22是本申请一实施例提供的质粒中的序列包含有siRNA和RVG的情况下，其所具有的体内富集数据图。Figure 22 is a graph of the in vivo enrichment data of the plasmid provided in an example of the present application when the sequences in the plasmid contain siRNA and RVG.
图23是本申请一实施例提供的质粒中的序列包含有siRNA和RVG的情况下，通过LRRK2mRNA的表达数据所体现出的针对帕金森治疗的效果图。Figure 23 is a graph showing the effect of treating Parkinson's disease according to the expression data of LRRK2 mRNA when the sequence in the plasmid provided in an example of the present application contains siRNA and RVG.
图24是本申请一实施例提供的RVG-LAMP2B融合蛋白及其它融合蛋白在质粒载体上针对帕金森治疗效果的数据对比图，数据通过LRRK2mRNA的相对水平来体现。Figure 24 is a data comparison diagram of the therapeutic effect of RVG-LAMP2B fusion protein and other fusion proteins on a plasmid vector for Parkinson's disease provided in an example of the present application, and the data is reflected by the relative level of LRRK2 mRNA.
图25是本申请一实施例提供的基因线路中包括有与LRRK2基因的siRNA序列同源性大于80％的3条RNA序列时，其所具有的体内富集数据图。FIG. 25 is a graph of in vivo enrichment data of the gene circuit provided in an example of the present application when three RNA sequences with more than 80% homology to the siRNA sequence of the LRRK2 gene are included.
图26是本申请一实施例提供的基因线路中包括有与LRRK2基因的siRNA序列同源性大于80％的3条RNA序列时，其针对帕金森治疗效果的数据对比图，数据通过LRRK2mRNA的相对水平来体现。Figure 26 is a data comparison diagram of the therapeutic effect on Parkinson's disease when the gene circuit provided by an embodiment of the present application includes 3 RNA sequences with more than 80% homology to the siRNA sequence of the LRRK2 gene. level to reflect.
具体实施方式Detailed ways
下面结合附图对本申请的具体实施方式进行描述。The specific embodiments of the present application will be described below with reference to the accompanying drawings.
首先，对本发明涉及到的专业名词、试验方法等进行解释说明。First, technical terms, test methods, etc. related to the present invention are explained.
苏木精-伊红染色法(hematoxylin-eosin staining)，简称HE染色。HE染色是组织学、病理学教学与科研中最基础、使用最广泛的技术方法之一。Hematoxylin-eosin staining, referred to as HE staining. HE staining is one of the most basic and widely used technical methods in histology and pathology teaching and research.
苏木精染液为碱性，可以将组织的嗜碱性结构(如核糖体、细胞核及细胞质中的核糖核酸等)染成蓝紫色；伊红为酸性染料，可以将组织的嗜酸性结构(如细胞内及细胞间的蛋白质，包括路易体、酒精小体以及细胞质的大部分)染成粉红色，使整个细胞组织的形态清晰可见。The hematoxylin staining solution is alkaline and can stain the basophilic structure of the tissue (such as ribosome, nucleus and ribonucleic acid in the cytoplasm) into blue-violet; eosin is an acid dye, which can stain the eosinophilic structure of the tissue ( Such as intracellular and intercellular proteins, including Lewy bodies, alcohol bodies, and most of the cytoplasm) stained pink, making the morphology of the entire cell organization clearly visible.
HE染色的具体步骤包括：样本组织固定与切片；组织样本脱蜡；组织样本水化；组织切片苏木素染色、分化与反蓝；组织切片伊红染色与脱水；组织样本切片风干封片；最后在显微镜下观察并拍照。The specific steps of HE staining include: sample tissue fixation and sectioning; tissue sample dewaxing; tissue sample hydration; tissue section hematoxylin staining, differentiation and anti-blue; tissue section eosin staining and dehydration; tissue sample section air-drying and sealing; Observe and photograph under the microscope.
Masson染色使胶原纤维呈蓝色(被苯胺蓝所染)或绿色(被亮绿所染)，肌纤维呈红色(被酸性品红和丽春红所染)，这与阴离子染料分子的大小和组织的渗透性有关。已固定的组织用一系列阴离子水溶性染料先后或混合染色，则可发现红细胞被最小分子的阴离子染料着染，肌纤维与胞质被中等大小的阴离子染料着染，而胶原纤维则被大分子的阴离子染料着染。由此说明了红细胞对阴离子染料的渗透性最小，肌纤维与胞质次之，而胶原纤维具有最大的渗透性。I型、III型胶原呈绿色(GBM、TBM、系膜基质及肾间质呈绿色)，嗜复红蛋白、肾小管胞质、红细胞呈红色。Masson staining renders collagen fibers blue (stained by aniline blue) or green (stained by bright green) and muscle fibers red (stained by acid fuchsin and Ponceau), which is consistent with the size and organization of the anionic dye molecules of permeability. The fixed tissue is stained sequentially or mixed with a series of anionic water-soluble dyes. It can be found that red blood cells are stained with the smallest molecular anionic dyes, muscle fibers and cytoplasm are stained with medium-sized anionic dyes, and collagen fibers are stained with macromolecular anionic dyes. Dyeing with anionic dyes. This shows that red blood cells have the least permeability to anionic dyes, followed by muscle fibers and cytoplasm, and collagen fibers have the largest permeability. Type I and III collagens are green (GBM, TBM, mesangial matrix and renal interstitium are green), and erythropoietin, tubular cytoplasm, and erythrocytes are red.
Masson染色的具体步骤包括：The specific steps of Masson staining include:
组织固定于Bouin氏液，流水冲洗一晚，常规脱水包埋；切片脱蜡至水(二甲苯中脱蜡10min×3次，用吸水纸吸干液体；100％乙醇5min×2次，用吸水纸吸干液体；95％乙醇5min×2次，用吸水纸吸干液体；流水2min，用吸水纸吸干水分)；Weiger氏铁苏木素染5-10min；流水稍洗；0.5％盐酸酒精分化15s；流水冲洗3min；丽春红酸性品红液染8min；蒸馏水稍冲洗；1％磷钼酸水溶液处理约5min；不用水洗，直接用苯胺蓝液或亮绿液复染5min；1％冰醋酸处理1min；95％乙醇脱水5min×2次，用吸水纸吸干液体；100％乙醇5min×2次，用吸水纸吸干液体；二甲苯中透明5min×2次，用吸水纸吸干液体；中性树胶封片。Tissues were fixed in Bouin's solution, rinsed with running water overnight, and embedded in conventional dehydration; sections were deparaffinized to water (deparaffinized in xylene for 10 min × 3 times, and the liquid was blotted dry with absorbent paper; 100% ethanol 5 min × 2 times, with water absorption Dry the liquid with paper; 95% ethanol for 5min × 2 times, blot the liquid with absorbent paper; run water for 2min, blot dry with absorbent paper); Weiger's iron hematoxylin staining for 5-10min; ; Rinse with running water for 3min; Stain with Ponceau red acid fuchsin solution for 8min; Rinse slightly with distilled water; Treat with 1% phosphomolybdic acid aqueous solution for about 5min; Do not wash with water, directly counterstain with aniline blue solution or bright green solution for 5min; Treat with 1% glacial acetic acid 1min; dehydrated in 95% ethanol for 5min×2 times, and blotted dry with absorbent paper; 100% ethanol 5min×2 times, blotted with absorbent paper; transparent in xylene for 5min×2 times, blotted with absorbent paper; Sexual gum seals.
Western免疫印迹(Western Blot)是将蛋白质转移到膜上,然后利用抗体进行检测.对已知表达蛋白,可用相应抗体作为一抗进行检测,对新基因的表达产物,可通过融合部分的抗体检测。Western blot (Western Blot) is to transfer the protein to the membrane, and then use the antibody for detection. For the known expressed protein, the corresponding antibody can be used as the primary antibody for detection, and the expression product of the new gene can be detected by the fusion part of the antibody. .
Western Blot采用的是聚丙烯酰胺凝胶电泳，被检测物是蛋白质，“探针”是抗体，“显色”用标记的二抗。经过PAGE分离的蛋白质样品，转移到固相载体(例如硝酸纤维素薄膜)上，固相载体以非共价键形式吸附蛋白质,且能保持电泳分离的多肽类型及其生物学活性不变，以固相载体上的蛋白质或多肽作为抗原，与对应的抗体起免疫反应，再与酶或同位素标记的第二抗体起反应，经过底物显色或放射自显影以检测电泳分离的特异性目的基因表达的蛋白成分。其步骤主要包括：提取蛋白、蛋白定量、制胶和电泳、转膜、免疫标记及显影。Western Blot uses polyacrylamide gel electrophoresis, the detected object is protein, the "probe" is an antibody, and the "color development" is a labeled secondary antibody. The protein sample separated by PAGE is transferred to a solid phase carrier (such as nitrocellulose membrane), and the solid phase carrier adsorbs proteins in the form of non-covalent bonds, and can keep the types of polypeptides separated by electrophoresis and their biological activities unchanged. The protein or polypeptide on the solid phase carrier is used as an antigen, which reacts with the corresponding antibody, and then reacts with the enzyme or isotope-labeled secondary antibody to detect the specific target gene separated by electrophoresis through substrate color development or autoradiography. expressed protein components. The steps mainly include: protein extraction, protein quantification, gel preparation and electrophoresis, membrane transfer, immunolabeling and development.
免疫组化，应用抗原抗体反应，即抗原与抗体特异性结合的原理，通过化学反应使标记抗体的显色剂(荧光素、酶、金属离子、同位素)显色来确定组织细胞内抗原(多肽和蛋白质)，对其进行定位、定性及相对定量的研究，称为免疫组织化学技术(immunohistochemistry)或免疫细胞化学技术(immunocytochemistry)。Immunohistochemistry, using antigen-antibody reaction, that is, the principle of specific binding of antigen and antibody, determines the antigen (polypeptide) in tissue cells by developing the color of the chromogenic reagent (fluorescein, enzyme, metal ion, isotope) labeled antibody through chemical reaction. and protein), the localization, qualitative and relative quantitative research, called immunohistochemistry (immunohistochemistry) or immunocytochemistry (immunocytochemistry).
免疫组化的主要步骤包括：切片浸泡、过夜晾干、二甲苯脱蜡、梯度酒精脱蜡(100％、95％、90％、80％、75％、70％、50％，每次3min)、双蒸水、滴加3％过氧化氢溶液去除过氧化氢酶、水洗、抗原修复、滴加5％BSA、封闭1h、稀释一抗、PBS缓冲液清洗、孵二抗、PBS缓冲液清洗、显色液显色、水洗、苏木精染色、梯度乙醇脱水、中性树胶封片。The main steps of immunohistochemistry include: section soaking, overnight drying, xylene dewaxing, gradient alcohol dewaxing (100%, 95%, 90%, 80%, 75%, 70%, 50%, 3min each time) , double-distilled water, dropwise addition of 3% hydrogen peroxide solution to remove catalase, water washing, antigen retrieval, dropwise addition of 5% BSA, blocking for 1 h, dilution of primary antibody, washing with PBS buffer, incubation with secondary antibody, washing with PBS buffer , color developing solution, washing with water, hematoxylin staining, dehydration with gradient ethanol, and sealing with neutral gum.
本发明中涉及到的siRNA水平、蛋白含量和mRNA含量的检测，均是通过向小鼠体内注射RNA递送***，建立了小鼠干细胞体外模型。利用qRT-PCR检测细胞、组织中mRNA和siRNA表达水平。对于siRNA的绝对定量利用标准品绘制标准曲线的方式进行确定。每个siRNA或mRNA相对于内参的表达量可以用2-ΔCT表示，其中ΔCT＝C样品-C内参。扩增siRNA时内参基因为U6snRNA(组织中)或miR-16(血清、外泌体中)分子，扩增mRNA时基因为GAPDH或18s RNA。利用Western blotting实验检测细胞、组织中蛋白质的表达水平，用ImageJ软件进行蛋白定量分析。The detection of the siRNA level, the protein content and the mRNA content involved in the present invention is to establish the mouse stem cell in vitro model by injecting the RNA delivery system into the mouse. The expression levels of mRNA and siRNA in cells and tissues were detected by qRT-PCR. Absolute quantification of siRNA was determined by plotting a standard curve using the standards. The expression level of each siRNA or mRNA relative to the internal control can be represented by 2-ΔCT, where ΔCT=C sample-C internal control. When amplifying siRNA, the internal reference gene is U6snRNA (in tissue) or miR-16 (in serum, exosomes), and when amplifying mRNA, the gene is GAPDH or 18s RNA. Western blotting was used to detect protein expression levels in cells and tissues, and ImageJ software was used for protein quantitative analysis.
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的试剂、材料和操作步骤均为相应领域内广泛使用的试剂、材料和常规步骤。In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the reagents, materials and operation steps used herein are the reagents, materials and conventional steps widely used in the corresponding fields.
实施例1Example 1
本实施例提供一种用于治疗帕金森的RNA质粒递送***，该***包括质粒，所述质粒携带有能够用于治疗帕金森的RNA片段，所述质粒能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述RNA片段的复合结构，所述复合结构能够将所述RNA片段送入目标组织，以实现帕金森病的治疗。This embodiment provides an RNA plasmid delivery system for treating Parkinson's, the system comprising a plasmid carrying an RNA fragment that can be used for treating Parkinson's, and the plasmid can be enriched in the organ tissue of a host, And endogenously and spontaneously form a composite structure containing the RNA fragment in the host organ tissue, and the composite structure can send the RNA fragment into the target tissue to achieve the treatment of Parkinson's disease.
在本实施例中，质粒还包括启动子和靶向标签。所述质粒包括以下任意一种线路或几种线路的组合：启动子-RNA序列、启动子-靶向标签、启动子-RNA序列-靶向标签，每一个所述质粒中至少包括一个RNA片段和一个靶向标签，所述RNA片段和靶向标签位于相同的线路中或位于不同的线路中。换而言之，质粒中可以仅包括启动子-RNA序列-靶向标签，也可以包括启动子-RNA序列、启动子-靶向标签的组合，或是启动子-靶向标签、启动子-RNA序列-靶向标签的组合。In this example, the plasmid also includes a promoter and a targeting tag. The plasmid includes any one of the following circuits or a combination of several circuits: promoter-RNA sequence, promoter-targeting tag, promoter-RNA sequence-targeting tag, and each of the plasmids includes at least one RNA fragment and a targeting tag, the RNA fragment and targeting tag are located in the same line or in different lines. In other words, the plasmid can include only a promoter-RNA sequence-targeting tag, or a combination of a promoter-RNA sequence, a promoter-targeting tag, or a promoter-targeting tag, a promoter- A combination of RNA-seq-targeting tags.
进一步地，所述质粒还可以包括能够使所述线路折叠成正确结构并表达的侧翼序列、补偿序列和loop序列，所述侧翼序列包括5’侧翼序列和3’侧翼序列；所述质粒包括以下任意一种线路或几种线路的组合：5’-启动子-5’侧翼序列-RNA序列-loop序列-补偿序列-3’侧翼序列、5’-启动子-靶向标签、5’-启动子-靶向标签-5’侧翼序列-RNA序列-loop序列-补偿序列-3’侧翼序列。Further, the plasmid can also include a flanking sequence, a compensation sequence and a loop sequence that can make the circuit fold into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence; the plasmid includes the following Any one line or combination of several lines: 5'-promoter-5' flanking sequence-RNA sequence-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag, 5'-promoting sub-targeting tag-5' flanking sequence-RNA sequence-loop sequence-compensating sequence-3'flanking sequence.
质粒载体中包含有5'-启动子--靶向标签-5'侧翼序列-RNA片段-loop序列-补偿序列-3'侧翼序列时，其RNA递送***所具有的体内富集和帕金森治疗效果如图22-23所示，靶向标签为RVG，RNA片段为具有治疗作用的siRNA。When the plasmid vector contains 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensation sequence-3'flanking sequence, its RNA delivery system has in vivo enrichment and Parkinson's treatment The effect is shown in Figure 22-23, the targeting tag is RVG, and the RNA fragment is siRNA with therapeutic effect.
RVG-LAMP2B融合蛋白在质粒载体上所具有的帕金森治疗效果如图24所示，其治疗效果通过HTT mRNA的水平来体现。The Parkinson's treatment effect of the RVG-LAMP2B fusion protein on the plasmid vector is shown in Figure 24, and its treatment effect is reflected by the level of HTT mRNA.
其中，所述5’侧翼序列优选为ggatcctggaggcttgctgaaggctgtatgctgaattc或与其同源性大于80％的序列，包括与ggatcctggaggcttgctgaaggctgtatgctgaattc同源性为85％、90％、92％、95％、98％、99％的序列等。Wherein, the 5' flanking sequence is preferably ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with ggatcctggaggcttgctgaaggctgtatgctgaattc, etc.
所述loop序列优选为gttttggccactgactgac或与其同源性大于80％的序列，包括与gttttggccactgactgac同源性为85％、90％、92％、95％、98％、99％的序列等。The loop sequence is preferably gttttggccactgactgac or a sequence with more than 80% homology thereto, including sequences with 85%, 90%, 92%, 95%, 98%, 99% homology with gttttggccactgactgac, and the like.
所述3’侧翼序列优选为accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag或与其同源性大于80％的序列，包括与accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag同源性为85％、90％、92％、95％、98％、99％的序列等。The 3' flanking sequence is preferably accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag, etc.
所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-5位碱基。在RNA片段中仅包含一个RNA序列时，所述补偿序列可以为该RNA序列的删除其中任意1-5位碱基的反向互补序列。The compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted. When the RNA fragment contains only one RNA sequence, the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-5 bases therein.
优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-3位碱基。在RNA片段中仅包含一个RNA序列时，所述补偿序列可以为该RNA序列的删除其中任意1-3位碱基的反向互补序列。Preferably, the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted. When the RNA fragment contains only one RNA sequence, the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-3 bases therein.
更为优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-3位连续排列的碱基。在RNA片段中仅包含一个RNA序列时，所述补偿序列可以为该RNA序列的删除其中任意1-3位连续排列的碱基的反向互补序列。More preferably, the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted. When the RNA fragment contains only one RNA sequence, the compensation sequence may be the reverse complementary sequence of the RNA sequence by deleting any 1-3 consecutively arranged bases.
最为优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中的第9位和/或第10位碱基。在RNA片段中仅包含一个RNA序列时，所述补偿序列可以为该RNA序列的删除其中第9位和/或第10位的反向互补序列。删除第9位和第10位碱基效果最优。Most preferably, the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted. When the RNA fragment contains only one RNA sequence, the compensation sequence may be the reverse complementary sequence of the 9th position and/or the 10th position in the deletion of the RNA sequence. Deleting bases 9 and 10 works best.
需要说明的是，上述侧翼序列、补偿序列、loop序列均不是随意选择的，而是基于大量的理论研究和试验确定的，在上述特定侧翼序列、补偿序列、loop序列的配合下，能够最大程度的提高RNA片段的表 达率。It should be noted that the above-mentioned flanking sequences, compensation sequences and loop sequences are not randomly selected, but are determined based on a large number of theoretical studies and experiments. increase the expression rate of RNA fragments.
在质粒携带两个或多个线路的情况下，相邻的线路之间可以通过序列1-序列2-序列3相连；其中，序列1优选为CAGATC，序列2可以为由5-80个碱基组成的序列，比如10个、15个、20个、25个、30个、35个、40个、45个、50个、55个、60个、65个、70个、75个碱基组成的序列均可，优选为10-50个碱基组成的序列，更优选为20-40个碱基组成的序列，序列3优选为TGGATC。In the case that the plasmid carries two or more lines, the adjacent lines can be connected by sequence 1-sequence 2-sequence 3; wherein, sequence 1 is preferably CAGATC, and sequence 2 can be composed of 5-80 bases Sequences of composition, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases Any sequence may be used, preferably a sequence consisting of 10-50 bases, more preferably a sequence consisting of 20-40 bases, and sequence 3 is preferably TGGATC.
序列2具体如下表1所示。 Sequence 2 is specifically shown in Table 1 below.
Figure PCTCN2022083979-appb-000001
Figure PCTCN2022083979-appb-000001
更为优选地，在质粒携带两个或多个线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。More preferably, when the plasmid carries two or more lines, adjacent lines are connected by sequence 4 or a sequence with a homology greater than 80% to sequence 4; wherein, sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
序列具体如下表2所示。The specific sequence is shown in Table 2 below.
序列4sequence 4 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATCCAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-1Sequence 4-1 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATCCAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-2Sequence 4-2 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATCCAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-3Sequence 4-3 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATCCAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
以上所述的RNA片段包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列能够在目标受体中被表达，所述补偿序列在目标受体中不能被表达。RNA序列可以为siRNA序列、shRNA序列 或miRNA序列，优选为siRNA序列。The above-mentioned RNA fragments comprise one, two or more specific RNA sequences of medical significance, the RNA sequences can be expressed in the target receptor, and the compensatory sequence cannot be expressed in the target receptor. The RNA sequence can be a siRNA sequence, a shRNA sequence or a miRNA sequence, preferably a siRNA sequence.
一个RNA序列的长度为15-25个核苷酸(nt)，优选为18-22nt，比如18nt、19nt、20nt、21nt、22nt均可。此序列长度的范围并不是随意选择的，而是经过反复的试验后确定的。大量试验证明，在RNA序列的长度小于18nt，特别是小于15nt的情况下，该RNA序列大多无效，不会发挥作用，而在RNA序列的长度大于22nt，特别是大于25nt的情况下，则不仅线路的成本大大提高，而且效果也并未优于长度为18-22nt的RNA序列，经济效益差。因此，在RNA序列的长度为15-25nt，特别是18-22nt时，能够兼顾成本与作用的发挥，效果最好。The length of an RNA sequence is 15-25 nucleotides (nt), preferably 18-22nt, such as 18nt, 19nt, 20nt, 21nt, and 22nt. This range of sequence lengths was not chosen arbitrarily, but was determined through trial and error. A large number of experiments have proved that when the length of the RNA sequence is less than 18nt, especially less than 15nt, the RNA sequence is mostly invalid and will not play a role. The cost of the line is greatly increased, and the effect is not better than the RNA sequence with a length of 18-22nt, and the economic benefit is poor. Therefore, when the length of the RNA sequence is 15-25nt, especially 18-22nt, the cost and the effect can be taken into consideration, and the effect is the best.
所述能够治疗帕金森的RNA选自：LRRK2基因的siRNA或编码上述RNA的核酸分子。The RNA capable of treating Parkinson's disease is selected from: siRNA of LRRK2 gene or nucleic acid molecule encoding the above RNA.
以LRRK2基因的siRNA为例，设计的3条同源性分别为85％、90％、98％的序列，均有良好的基因沉默作用，分别负载有siRNA三个同源序列的递送***，其所具有的体内富集和帕金森治疗效果如图25-26所示。Taking the siRNA of the LRRK2 gene as an example, the designed three sequences with homology of 85%, 90% and 98% have good gene silencing effect. The resulting in vivo enrichment and Parkinson's treatment effects are shown in Figures 25-26.
所需递送RNA有效序列的数量为1条、2条或多条。The number of required delivery RNA effective sequences is one, two or more.
以在同一个质粒载体上联合使用“siRNA1”和“siRNA2”为例，该质粒载体的功能结构区可以表示为：(启动子-siRNA1)-连接序列-(启动子-siRNA2)-连接序列-(启动子-靶向标签)，或(启动子-靶向标签-siRNA1)-连接序列-(启动子-靶向标签-siRNA2)，或(启动子-siRNA1)-连接序列-(启动子-靶向标签-siRNA2)等。Taking the combined use of "siRNA1" and "siRNA2" on the same plasmid vector as an example, the functional structural region of the plasmid vector can be expressed as: (promoter-siRNA1)-connector sequence-(promoter-siRNA2)-connector sequence- (promoter-targeting tag), or (promoter-targeting tag-siRNA1)-linker-(promoter-targeting tag-siRNA2), or (promoter-siRNA1)-linker-(promoter- Targeting tag-siRNA2) etc.
更加具体地，该质粒载体的功能结构区可以表示为：(5’-启动子-5’侧翼序列-siRNA1-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-5’侧翼序列-siRNA2-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-靶向标签)，或(5’-启动子-靶向标签-5’侧翼序列-siRNA1-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-靶向标签-5’侧翼序列-siRNA2-loop序列-补偿序列-3’侧翼序列)，或(5’-启动子-5’侧翼序列-siRNA1-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-靶向标签-5’侧翼序列-siRNA2-loop序列-补偿序列-3’侧翼序列)、(5’-启动子-靶向标签-5’侧翼序列-siRNA1-siRNA2-loop序列-补偿序列-3’侧翼序列)等。其他情况均可以此类推，在此不再赘述。以上连接序列可以为“序列1-序列2-序列3”或“序列4”，一个括号表示一个完整的线路(circuit)。More specifically, the functional structural region of the plasmid vector can be expressed as: (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-connecting sequence-(5'-promoter - 5' flanking sequence - siRNA2-loop sequence - compensation sequence - 3' flanking sequence) - linking sequence - (5'-promoter-targeting tag), or (5'-promoter-targeting tag-5' flanking sequence-siRNA1-loop sequence-compensation sequence-3' flanking sequence)-linker sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'flanking sequence), or (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-linking sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence- Compensation sequence-3'flanking sequence), (5'-promoter-targeting tag-5'flanking sequence-siRNA1-siRNA2-loop sequence-compensating sequence-3'flanking sequence), etc. Other situations can be deduced by analogy, and details are not repeated here. The above connecting sequence can be "sequence 1-sequence 2-sequence 3" or "sequence 4", and a bracket indicates a complete circuit.
优选地，上述RNA还可以通过对其中的RNA序列(siRNA、shRNA或miRNA)进行核糖修饰得到，优选2’氟嘧啶修饰。2’氟嘧啶修饰是将siRNA、shRNA或miRNA上嘧啶核苷酸的2’-OH用2’-F替代，2’-F能够使人体内的RNA酶不易识别siRNA、shRNA或miRNA，如此能够增加RNA在体内传输的稳定性。Preferably, the above RNA can also be obtained by ribose modification of the RNA sequence (siRNA, shRNA or miRNA) therein, preferably 2' fluoropyrimidine modification. 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on siRNA, shRNA or miRNA with 2'-F. 2'-F can make it difficult for RNase in the human body to recognize siRNA, shRNA or miRNA, so it can Increases the stability of RNA transport in vivo.
具体地，肝脏会吞噬外源性的质粒，高达99％的外源性质粒会进入肝脏，因此当以质粒作为载体时并不需要做特异性设计即可在肝脏组织中富集，随后外源性质粒被打开，释放出RNA分子(siRNA、shRNA或miRNA)，肝脏组织自发地将上述RNA分子包裹进外泌体内部，这些外泌体就变成了RNA输送机构。Specifically, the liver will phagocytose exogenous plasmids, and up to 99% of the exogenous plasmids will enter the liver. Therefore, when plasmids are used as vectors, they can be enriched in liver tissue without specific design. The plasmid is opened to release RNA molecules (siRNA, shRNA, or miRNA), and liver tissue spontaneously wraps the above RNA molecules into exosomes, and these exosomes become RNA delivery mechanisms.
优选地，为了使该RNA输送机构(外泌体)具有“精确制导”的能力，在注入体内的质粒中我们设计了靶向标签，该靶向标签也会被肝脏组织组装到外泌体中，尤其是当选择某些特定的靶向标签时，靶向标签能够被***外泌体表面，从而成为能够引导外泌体的靶向结构，这就大大提高了本发明所述的RNA输送机构的精准性，一方面可以使所需引入的外源性质粒的用量大大减少，另一方面还大大提高了潜在药物递送的效率。Preferably, in order to make the RNA delivery mechanism (exosome) have the ability of "precision guidance", we designed a targeting tag in the plasmid injected into the body, and the targeting tag will also be assembled into exosomes by liver tissue , especially when certain specific targeting tags are selected, the targeting tags can be inserted into the surface of exosomes, thereby becoming a targeting structure that can guide exosomes, which greatly improves the RNA delivery mechanism of the present invention. On the one hand, the amount of exogenous plasmids that need to be introduced can be greatly reduced, and on the other hand, the efficiency of potential drug delivery can be greatly improved.
靶向标签选自具有靶向功能的肽、蛋白质或抗体中的一种，靶向标签的选择是需要创造性劳动的过程，一方面需要根据目标组织选取可用的靶向标签，另一方面还需要保证该靶向标签能够在稳定地出现在外泌体的表面，从而达到靶向功能。目前已经筛选出的靶向肽包括但不限于RVG靶向肽(核苷酸序列如SEQ  ID No：1所示)、GE11靶向肽(核苷酸序列如SEQ ID No：2所示)、PTP靶向肽(核苷酸序列如SEQ ID No：3所示)、TCP-1靶向肽(核苷酸序列如SEQ ID No：4所示)、MSP靶向肽(核苷酸序列如SEQ ID No：5所示)；靶向蛋白包括但不限于RVG-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：6所示)、GE11-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：7所示)、PTP-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：8所示)、TCP-1-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：9所示)、MSP-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：10所示)。优选采用GE11靶向肽、GE11-LAMP2B融合蛋白。The targeting tag is selected from one of the peptides, proteins or antibodies with targeting function. The selection of the targeting tag is a process that requires creative work. On the one hand, it is necessary to select the available targeting tags according to the target tissue. It is ensured that the targeting label can stably appear on the surface of exosomes, so as to achieve the targeting function. Targeting peptides that have been screened so far include, but are not limited to, RVG targeting peptide (nucleotide sequence shown in SEQ ID No: 1), GE11 targeting peptide (nucleotide sequence shown in SEQ ID No: 2), PTP targeting peptide (nucleotide sequence shown in SEQ ID No: 3), TCP-1 targeting peptide (nucleotide sequence shown in SEQ ID No: 4), MSP targeting peptide (nucleotide sequence shown in SEQ ID No: 4) SEQ ID No: 5); targeting proteins include but are not limited to RVG-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6), GE11-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6) : 7), PTP-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 8), TCP-1-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 9), MSP- LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No: 10). Preferably, GE11 targeting peptide, GE11-LAMP2B fusion protein is used.
以上所述的RNA包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列能够在目标受体中被表达，所述补偿序列在目标受体中不能被表达。The above-mentioned RNAs comprise one, two or more specific RNA sequences of medical significance, the RNA sequences can be expressed in the target receptor, and the compensatory sequence cannot be expressed in the target receptor.
此外，为了达到精准递送的目的，我们实验了多种质粒载体搭载的方案，得出另一优化的方案：所述质粒载体还可以由具有不同结构的多种质粒构成，其中一种质粒包含启动子和靶向标签，其他质粒包含启动子和RNA片段。即将靶向标签与RNA片段装载到不同的质粒载体中，将两种质粒载体注入体内，其靶向效果不差于将相同的靶向标签与RNA片段装载在一个质粒载体中产生的靶向效果。In addition, in order to achieve the purpose of precise delivery, we experimented with a variety of plasmid vector loading schemes, and came up with another optimized scheme: the plasmid vector can also be composed of multiple plasmids with different structures, one of which contains a promoter promoter and targeting tags, other plasmids contain promoters and RNA fragments. Loading the targeting tag and RNA fragment into different plasmid vectors, and injecting the two plasmid vectors into the body, the targeting effect is no worse than the targeting effect produced by loading the same targeting tag and RNA fragment into one plasmid vector .
更优选地，两种不同的质粒载体注入宿主体内时，可以先将装有RNA序列的质粒载体注入，在1-2小时后再注入含有靶向标签的质粒载体，如此能够达到更优的靶向效果。More preferably, when two different plasmid vectors are injected into the host, the plasmid vector containing the RNA sequence can be injected first, and then the plasmid vector containing the targeting tag can be injected after 1-2 hours, so that a better target can be achieved. to the effect.
以上所述的递送***均可用于包括人在内的哺乳动物。The delivery systems described above can all be used in mammals, including humans.
如图1A所示，为了了解质粒在体内的分布情况，我们对小鼠进行了平板试验，对小鼠注射质粒后按时间点(1h、3h、6h、9h、12h、24h、72h、168h、720h)取样，采用大观霉素提取的质粒进行转化，观测肝脏、血浆、肺、大脑、肾脏、脾脏中克隆体的数量，结果如图1B、图1C、图1D所示，可以看出，质粒在小鼠肝脏中分布最多，并且在注射后3h左右达到峰值，注射后12h已基本代谢。As shown in Figure 1A, in order to understand the distribution of plasmids in the body, we carried out a plate test on mice. 720h) sampling, using the plasmid extracted by spectinomycin for transformation, observing the number of clones in liver, plasma, lung, brain, kidney, spleen, the results are shown in Figure 1B, Figure 1C, Figure 1D, it can be seen that the plasmid It is most distributed in the liver of mice, and reaches the peak at about 3 hours after injection, and is basically metabolized at 12 hours after injection.
向C57BL/6J小鼠静脉注射共表达eGFP蛋白和EGFR siRNA的CMV eGFP siRE circuit，结果如图2所示，随着时间的推移，小鼠肝脏内eGFP荧光逐渐增强，约12小时达到峰值，48小时降至背景水平，其他组织未见明显的eGFP信号。The CMV eGFP siRE circuit co-expressing eGFP protein and EGFR siRNA was injected intravenously into C57BL/6J mice. The results are shown in Figure 2. The eGFP fluorescence in the mouse liver gradually increased over time, reaching a peak at about 12 hours. 48 After hours, it dropped to the background level, and no obvious eGFP signal was seen in other tissues.
分别向小鼠注射对照质粒(CMV-scrR)、表达EGFR siRNA的质粒(CMV-siR E)，并建立小鼠肝细胞体外模型，分别检测注射CMV-scrR和CMV-siR E的小鼠肝细胞外泌体中相关siRNA水平，结果如图3A所示，可见在注射CMV-siR E的小鼠肝细胞外泌体中存在siRNA的表达。 The control plasmid (CMV-scrR) and the plasmid expressing EGFR siRNA (CMV-siR E ) were injected into mice respectively, and the mouse liver cell model was established in vitro, and the mouse liver cells injected with CMV-scrR and CMV-siR E were detected respectively. The related siRNA levels in exosomes, the results are shown in Figure 3A, it can be seen that there is siRNA expression in the exosomes of mouse hepatocytes injected with CMV-siRNA.
我们通常认为与Ago2蛋白结合是siRNA发挥功能的必要条件，即外泌体中的siRNA可以与Ago2蛋白结合，因此我们进行Ago2免疫沉淀实验，结果如图3B、图3C所示。其中，Input代表不经过免疫沉淀，直接将外泌体裂解并进行检测的样品，代表阳性对照。We generally believe that binding to Ago2 protein is a necessary condition for siRNA to function, that is, siRNA in exosomes can bind to Ago2 protein, so we performed Ago2 immunoprecipitation experiments, and the results are shown in Figure 3B and Figure 3C. Among them, Input represents the sample in which the exosomes were directly lysed and detected without immunoprecipitation, representing the positive control.
对小鼠静脉注射质粒后，成熟siRNA在不同组织中的分布如图4所示。从图4A可以看出，血浆、外泌体、无外泌体的血浆中EGFR-siRNA水平呈时间依赖性变化；从图4B可以看出，小鼠EGFR-siRNA在肝、肺、胰腺、脾脏、肾脏中的积累具有时间依赖性。After intravenous injection of plasmids into mice, the distribution of mature siRNA in different tissues is shown in Figure 4. It can be seen from Figure 4A that the levels of EGFR-siRNA in plasma, exosomes, and exosome-free plasma show time-dependent changes; from Figure 4B, it can be seen that mouse EGFR-siRNAs in the liver, lung, pancreas, and spleen , The accumulation in the kidney is time-dependent.
分别对小鼠注射对照质粒(CMV-scrR)、0.05mg/kg的CMV-siR E质粒、0.5mg/kg的CMV-siR E质粒、5mg/kg的CMV-siR E质粒，检测小鼠肝脏、脾脏、心脏、肺、肾脏、胰腺、脑、骨骼肌、CD4 +细胞中绝对siRNA(EGFR siRNA)水平，结果如图5A所示，可以看出，注射对照质粒的小鼠组织中无siRNA的表达，注射CMV-siR E质粒的小鼠各组织中，siRNA表达的水平与CMV-siR E质粒浓度呈正相关。如图5B所示，荧光原位杂交试验(FISH)同样证实了siRNA表达的水平与CMV-siR E质粒浓度呈正相关，即EGFR siRNA的组织分布具有剂量依赖性。 The mice were injected with control plasmid (CMV-scrR), 0.05mg/kg CMV-siR E plasmid, 0.5mg/kg CMV-siR E plasmid, 5mg/kg CMV-siR E plasmid, and detected the liver, Absolute siRNA (EGFR siRNA) levels in spleen, heart, lung, kidney, pancreas, brain, skeletal muscle, CD4 + cells, the results are shown in Figure 5A, it can be seen that there is no siRNA expression in the tissues of mice injected with the control plasmid , in each tissue of mice injected with CMV-siR E plasmid, the level of siRNA expression was positively correlated with the concentration of CMV-siR E plasmid. As shown in Figure 5B, fluorescence in situ hybridization assay (FISH) also confirmed that the level of siRNA expression was positively correlated with the concentration of CMV-siR E plasmid, that is, the tissue distribution of EGFR siRNA was dose-dependent.
由于质粒进入体内后，会表达前体(Precursor)，再加工成成熟体(siRNA)，故我们对小鼠注射质粒之 后肝脏中前体(Precursor)和成熟体(siRNA)的代谢情况进行了检测，结果如图6所示。可以看出，在注射质粒后6个小时的时间节点，小鼠肝脏中前体(Precursor)和成熟体(siRNA)的表达水平达到峰值，在注射质粒后36个小时，小鼠肝脏中的成熟体(siRNA)代谢完成，在注射质粒后48个小时，小鼠肝脏中前体(Precursor)代谢完成。After the plasmid enters the body, it will express the precursor (Precursor) and then process it into the mature body (siRNA), so we tested the metabolism of the precursor (Precursor) and the mature body (siRNA) in the liver after the plasmid was injected into mice. , the results are shown in Figure 6. It can be seen that the expression levels of precursor (Precursor) and mature body (siRNA) in the mouse liver reached a peak at the time point of 6 hours after the injection of the plasmid. Metabolism of the precursor (siRNA) was complete, and the metabolism of the precursor (Precursor) in the mouse liver was complete 48 hours after the injection of the plasmid.
对小鼠进行胆总管注射外源性siRNA后分别检测小鼠无外泌体血浆(exosome-free)、外泌体(exosome)、血浆中绝对siRNA水平，结果如图7A所示。对小鼠进行胆总管注射外源性siRNA后分别检测小鼠脾脏、心脏、肺、肾脏、胰腺、脑、骨骼肌、CD4 +细胞中siRNA的水平，结果如图7B所示。这两张图反映出siRNA在不同组织中动力学几乎相同，在不同组织中siRNA的分布有显著差别。 After injection of exogenous siRNA into the common bile duct of mice, the absolute levels of siRNA in exosome-free plasma (exosome-free), exosome (exosome) and plasma were detected respectively, and the results are shown in Figure 7A. After injection of exogenous siRNA into the common bile duct of mice, the levels of siRNA in the spleen, heart, lung, kidney, pancreas, brain, skeletal muscle, and CD4 + cells of the mice were detected, and the results are shown in Figure 7B. These two figures reflect that the kinetics of siRNA in different tissues are almost the same, and the distribution of siRNA in different tissues is significantly different.
分别向小鼠体内静脉注射以白蛋白ALB为启动子的siRNA、以CMV为启动子的siRNA、不含任何启动子的siRNA，在注射后0h、3h、6h、9h、12h、24h、36h、48h分别检测小鼠体内的绝对siRNA水平，结果如图8所示。可见，小鼠体内以CMV为启动子的siRNA的水平最高，即以CMV作为启动子效果最优。siRNA with albumin ALB as the promoter, siRNA with CMV as the promoter, and siRNA without any promoter were injected into mice intravenously. The absolute siRNA levels in the mice were detected at 48 h, and the results are shown in Figure 8. It can be seen that the level of siRNA with CMV as the promoter in mice is the highest, that is, the effect of CMV as the promoter is the best.
我们通过荧光试验观察自组装的eGFP siRNA对小鼠体内eGFP水平的抑制，过程如下：对eGFP转基因小鼠静脉注射PBS或5mg/kg CMV-siR G或CMV-RVG-siR G质粒，治疗24小时后处死小鼠，在冷冻切片中检测其eGFP荧光水平，图9A所示为具有代表性的荧光显微镜图像，其中绿色表示阳性eGFP信号，蓝色显表示DAPI染色的细胞核，比例尺：100μm，可见CMV-RVG-siR G质粒对小鼠eGFP的抑制效果更为明显；对eGFP转基因小鼠静脉注射PBS或CMV-scrR或CMV-siR E质粒，治疗24小时后处死小鼠，在冷冻切片中检测其eGFP荧光水平，图9B为注射PBS、CMV-siR E、CMV-RVG-siR E的小鼠心脏、肺、肾脏、胰腺、脑、骨骼肌的荧光强度(Fluorescence intensity)柱形对比图，可见，在肝脏、脾脏、肺、肾脏部位小鼠荧光强度对比更为明显。 We observed the inhibition of eGFP levels in mice by self-assembled eGFP siRNA by fluorescence assay. The process is as follows: eGFP transgenic mice were intravenously injected with PBS or 5 mg/kg CMV-siR G or CMV-RVG-siR G plasmid, and treated for 24 hours After the mice were sacrificed, their eGFP fluorescence levels were detected in cryosections. Figure 9A shows a representative fluorescence microscope image, in which green indicates positive eGFP signal, blue indicates DAPI-stained nuclei, scale bar: 100 μm, CMV is visible - RVG-siR G plasmid has a more obvious inhibitory effect on mouse eGFP; eGFP transgenic mice were intravenously injected with PBS or CMV-scrR or CMV-siR E plasmid, and the mice were sacrificed after 24 hours of treatment, and they were detected in frozen sections. The fluorescence level of eGFP, Figure 9B is a bar graph of the fluorescence intensity (Fluorescence intensity) of the mouse heart, lung, kidney, pancreas, brain, and skeletal muscle injected with PBS, CMV- siRE , and CMV-RVG- siRE . It can be seen that, The contrast of fluorescence intensity in liver, spleen, lung and kidney of mice was more obvious.
分别对于注射PBS、CMV-scrR、CMV-siR E的小鼠其谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TBIL)、血尿素氮(BUN)、血清碱性磷酸酶(ALP)、肌酐(CREA)含量以及胸腺重量、脾脏重量、外周血细胞百分比(percentage peripheral blood cells)进行检测，结果如图10所示，图10A-F为分别注射PBS、小鼠CMV-scrR、CMV-siR E的谷丙转氨酶、谷草转氨酶、总胆红素、血尿素氮、血清碱性磷酸酶、肌酐含量对比图，图10G为小鼠肝脏、肺、脾脏、肾脏组织对比图，图10H-I为小鼠胸腺、脾脏组织对比图，图10J为小鼠外周血细胞百分比(percentage in peripheral blood cells)对比图。 For the mice injected with PBS, CMV-scrR, and CMV-siR E , their alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), blood urea nitrogen (BUN), serum alkaline phosphatase (ALP), creatinine (CREA) content, thymus weight, spleen weight, and percentage of peripheral blood cells were detected. Comparison of the contents of alanine aminotransferase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, serum alkaline phosphatase, and creatinine in CMV-siR E. Figure 10G is a comparison chart of mouse liver, lung, spleen, and kidney tissue, and Figure 10H -I is a comparison chart of mouse thymus and spleen tissue, and FIG. 10J is a comparison chart of percentage in peripheral blood cells of mice.
结果显示注射PBS、CMV-scrR、CMV-siR E的小鼠ALT、AST等含量以及胸腺重量、脾脏重量、外周血细胞百分比均相差无几，注射CMV-siRE的小鼠与注射PBS的小鼠相比，其肝脏、肺、脾脏、肾脏也无组织损伤。 The results showed that the contents of ALT, AST, thymus, spleen, and peripheral blood cells in the mice injected with PBS, CMV- scrR , and CMV-siRE were almost the same. The mice injected with CMV-siRE were compared with those injected with PBS. , the liver, lung, spleen, kidney also have no tissue damage.
上述试验可以证明，本实施例提供的用于治疗帕金森的RNA递送***以质粒作为载体，质粒作为成熟的注入物，其安全性和可靠性已被充分验证，成药性非常好。最终发挥效果的RNA序列由内源性外泌体包裹输送，不存在任何免疫反应，无需验证该外泌体的安全性。该递送***可以递送各类小分子RNA，通用性强。并且质粒的制备要比外泌体或是蛋白质、多肽等物质的制备便宜地多，经济性好。本实施例提供的用于治疗帕金森的RNA递送***在体内自组装后能够与AGO 2紧密结合并富集为复合结构(外泌体)，不仅能防止其过早降解，维持其在循环中的稳定性，而且有利于受体细胞吸收、胞浆内释放和溶酶体逃逸，所需剂量低。 The above test can prove that the RNA delivery system for treating Parkinson's provided in this example uses plasmid as a carrier and plasmid as a mature injection, its safety and reliability have been fully verified, and its druggability is very good. The final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes. The delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of plasmids is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances. The RNA delivery system for the treatment of Parkinson's disease provided in this example can be tightly combined with AGO 2 and enriched into a complex structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation, but also maintain its circulation in the circulation. It is stable, and is beneficial to receptor cell uptake, intracytoplasmic release and lysosomal escape, and the required dose is low.
实施例2Example 2
在实施例1的基础上，本实施例提供一种药物。该药物包括质粒，所述质粒携带有能够用于治疗帕金 森的RNA片段，所述质粒能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述RNA片段的复合结构，所述复合结构能够将所述RNA片段送入目标组织，以实现帕金森病的治疗。On the basis of Embodiment 1, this embodiment provides a medicine. The medicament comprises a plasmid carrying an RNA fragment that can be used to treat Parkinson's, the plasmid can be enriched in the organ tissue of a host, and endogenously and spontaneously form in the host organ tissue containing the A composite structure of RNA fragments capable of delivering the RNA fragments into target tissues for Parkinson's disease treatment.
可选地，所述RNA片段包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列是具有医学意义的、能够抑制或阻碍帕金森病发展的siRNA、shRNA或miRNA序列。Optionally, the RNA fragment comprises one, two or more specific RNA sequences of medical significance, and the RNA sequences are siRNA, shRNA or miRNA sequences of medical significance that can inhibit or hinder the development of Parkinson's disease .
以质粒为载体，其在携带有1个或多个RNA片段的情况下，具有体内富集、自组装和帕金森治疗效果，图12-15显示了载体携带有6种单独RNA序列的RNA片段所呈现的数据结果；图16-19显示了载体携带有4组包含有任意2种RNA序列的RNA片段所呈现的数据结果；图20-21显示了载体携带有3组包含有任意3种RNA序列的RNA片段所呈现的治疗数据结果。Using a plasmid as a vector, which carries one or more RNA fragments, it has in vivo enrichment, self-assembly and Parkinson's treatment effects. Figure 12-15 shows that the vector carries RNA fragments of 6 separate RNA sequences Data results presented; Figures 16-19 show data results presented by vectors carrying 4 sets of RNA fragments containing any 2 RNA sequences; Figures 20-21 show vectors carrying 3 sets of RNA fragments containing any 3 RNA sequences The therapeutic data results presented by sequenced RNA fragments.
具体序列如下表3所示。The specific sequence is shown in Table 3 below.
Figure PCTCN2022083979-appb-000002
Figure PCTCN2022083979-appb-000002
CTX代表皮层(cortex)，STR代表纹状体(Striatum)。CTX stands for cortex and STR stands for Striatum.
可选地，所述质粒还包括启动子和靶向标签，所述靶向标签能够在宿主的器官组织中形成所述复合结构的靶向结构，所述靶向结构位于复合结构的表面，所述复合结构能够通过所述靶向结构寻找并结合目标组织，将所述RNA片段递送进入目标组织。Optionally, the plasmid also includes a promoter and a targeting tag, the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host, and the targeting structure is located on the surface of the composite structure, so The complex structure can seek and bind to the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
关于本实施例中上述质粒、RNA片段、靶向标签等的解释说明均可以参考实施例1，在此不再赘述。For explanations about the above-mentioned plasmids, RNA fragments, targeting tags, etc. in this embodiment, reference may be made to Embodiment 1, which will not be repeated here.
该药物可以通过口服、吸入、皮下注射、肌肉注射或静脉注射的方式进入人体后，通过实施例1所述的RNA递送***递送至目标组织，发挥治疗作用。After the drug can be administered orally, inhaled, subcutaneously injected, intramuscularly injected or intravenously injected into the human body, it can be delivered to the target tissue through the RNA delivery system described in Example 1 to exert a therapeutic effect.
该药物可以与其他治疗帕金森病的药物联合使用，以提高治疗效果，比如抗胆碱能药(安坦等)、抗组织胺药(苯海拉明、金刚烷胺等)、左旋多巴类药(多芭、息宁、西莱美等)、多巴胺受体激动剂(泰舒达、协良行、克瑞帕、溴隐亭等)、单胺氧化酶B抑制剂(司吉宁、金思平等)、儿茶酚胺氧位甲基转移酶抑制剂(恩托卡朋等)。The drug can be used in combination with other Parkinson's disease drugs to improve the treatment effect, such as anticholinergics (Antan, etc.), antihistamines (diphenhydramine, amantadine, etc.), levodopa Drugs (Dopa, Sining, Xilaimei, etc.), dopamine receptor agonists (Texida, Xie Liangxing, Crepa, bromocriptine, etc.), monoamine oxidase B inhibitors (Sgining, Jin Siping, etc.) ), catecholamine oxygen methyltransferase inhibitors (Entocapone, etc.).
本实施例的药物还可以包括药学上可以接受的载体，该载体包括但不限于稀释剂、缓冲剂、乳剂、包囊剂、赋形剂、填充剂、粘合剂、喷雾剂、透皮吸收剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、着 色剂、矫味剂、佐剂、干燥剂、吸附载体等。The medicine of this embodiment may also include a pharmaceutically acceptable carrier, which includes but is not limited to diluents, buffers, emulsions, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorption Agents, wetting agents, disintegrating agents, absorption enhancers, surfactants, colorants, flavoring agents, adjuvants, desiccants, adsorption carriers, etc.
本实施例提供的药物的剂型可以为片剂、胶囊剂、粉剂、颗粒剂、丸剂、栓剂、软膏剂、溶液剂、混悬剂、洗剂、凝胶剂、糊剂等。The dosage forms of the medicine provided in this embodiment can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes, and the like.
本实施例提供的药物以质粒作为载体，质粒作为成熟的注入物，其安全性和可靠性已被充分验证，成药性非常好。最终发挥效果的RNA序列由内源性外泌体包裹输送，不存在任何免疫反应，无需验证该外泌体的安全性。该药物可以递送各类小分子RNA，通用性强。并且质粒的制备要比外泌体或是蛋白质、多肽等物质的制备便宜地多，经济性好。本申请提供的药物在体内自组装后能够与AGO 2紧密结合并富集为复合结构(外泌体)，不仅能防止其过早降解，维持其在循环中的稳定性，而且有利于受体细胞吸收、胞浆内释放和溶酶体逃逸，所需剂量低。 The medicine provided in this example uses the plasmid as the carrier and the plasmid as the mature injection, and its safety and reliability have been fully verified, and the drugability is very good. The final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes. The drug can deliver various kinds of small molecule RNAs and has strong versatility. And the preparation of plasmids is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances. The drug provided in this application can be closely combined with AGO 2 and enriched into a composite structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation and maintain its stability in circulation, but also benefit the receptor. Cellular uptake, intracytoplasmic release and lysosomal escape require low doses.
实施例3Example 3
在实施例1或2的基础上，本实施例提供一种用于治疗帕金森的RNA递送***在药物中的应用，该药物为治疗帕金森的药物。本实施例结合以下试验对RNA递送***在帕金森治疗方面的应用进行具体说明。On the basis of Embodiment 1 or 2, this embodiment provides an application of an RNA delivery system for treating Parkinson's disease in a drug, and the drug is a drug for treating Parkinson's disease. In this example, the application of the RNA delivery system in the treatment of Parkinson's disease is specifically described in conjunction with the following experiments.
在此试验中，选择LRRK2R1441G转基因小鼠3月龄时进行试验，试验设置LPS干预组和LPS非干预组。LPS干预组LPS干预7天后进行CMV-scrR/CMV-RVG-siR LRRK2的治疗。 In this experiment, LRRK2R1441G transgenic mice were selected for the experiment when they were 3 months old, and the experiment set up LPS intervention group and LPS non-intervention group. The LPS intervention group was treated with CMV-scrR/CMV-RVG-siR LRRK2 after 7 days of LPS intervention.
如图11A和图11B所示，图11A为注射CMV-scrR/CMV-RVG-siR LRRK2的LRRK2R1441G转基因小鼠western bolt图，图11B注射CMV-scrR/CMV-RVG-siR LRRK2的LRRK2R1441G转基因小鼠蛋白灰度分析图，可以看出注射CMV-RVG-si RLRRK2的小鼠LRRK2蛋白和S935蛋白水平降低，说明CMV-RVG-siR LRR K2在肝脏释放siRNA组装进入外泌体后可以穿过血脑屏障降低脑深部蛋白的表达。 As shown in Figure 11A and Figure 11B, Figure 11A is a western bolt image of LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2 , and Figure 11B is a LRRK2R1441G transgenic mouse injected with CMV-scrR/CMV-RVG-siR LRRK2 The grayscale analysis of the protein shows that the levels of LRRK2 protein and S935 protein in mice injected with CMV-RVG-si RLRRK2 decreased, indicating that CMV-RVG-siR LRR K2 can pass through the blood brain after the liver releases siRNA and assembles into exosomes The barrier reduces the expression of proteins deep in the brain.
如图11C所示，该图为注射CMV-scrR/CMV-RVG-siR LRRK2的LRRK2R1441G转基因小鼠黑质区TH+神经元免疫荧光图，结果表明注射CMV-RVG-siR LRRK2的小鼠挽救了TH神经元的丢失，说明CMV-RVG-siR LRRK2在肝脏释放siRNA组装进入外泌体后可以穿过血脑屏障进入脑深部发挥功能。 As shown in Figure 11C, which is an immunofluorescence image of TH+ neurons in the substantia nigra of LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2 , the results showed that mice injected with CMV-RVG-siR LRRK2 rescued TH The loss of neurons indicates that CMV-RVG-siR LRRK2 can cross the blood-brain barrier and enter the deep brain to function after the liver releases siRNA and assembles into exosomes.
如图11D所示，该图为注射CMV-scrR/CMV-RVG-siR LRRK2的LRRK2R1441G转基因小鼠小胶质细胞激活水平免疫荧光图，结果表明注射CMV-RVG-siR LRRK2小鼠能够抑制小胶质的激活，说明CMV-RVG-siR LRRK2在肝脏释放siRNA组装进入外泌体后可以穿过血脑屏障进入脑深部发挥功能。 As shown in Figure 11D, this figure is an immunofluorescence image of the activation level of microglia in LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2 . The results show that injection of CMV-RVG-siR LRRK2 mice can inhibit microglia The activation of CMV-RVG-siR LRRK2 indicates that CMV-RVG-siR LRRK2 can cross the blood-brain barrier and enter the deep brain to function after the liver releases siRNA and assembles into exosomes.
以上试验可以说明，静脉注射CMV-RVG-siR LRRK2质粒有助于抑制多巴胺能神经元中的LRRK2，从而减轻帕金森PD小鼠的神经病理发展。 The above experiments can illustrate that intravenous injection of CMV-RVG-siR LRRK2 plasmid helps to inhibit LRRK2 in dopaminergic neurons, thereby reducing the neuropathological development of Parkinson's PD mice.
在本文中，“上”、“下”、“前”、“后”、“左”、“右”等仅用于表示相关部分之间的相对位置关系，而非限定这些相关部分的绝对位置。In this document, "upper", "lower", "front", "rear", "left", "right", etc. are only used to indicate the relative positional relationship between related parts, rather than limit the absolute positions of these related parts .
在本文中，“第一”、“第二”等仅用于彼此的区分，而非表示重要程度及顺序、以及互为存在的前提等。In this document, "first", "second", etc. are only used to distinguish each other, but do not indicate the degree of importance and order, and the premise of mutual existence.
在本文中，“相等”、“相同”等并非严格的数学和/或几何学意义上的限制，还包含本领域技术人员可以理解的且制造或使用等允许的误差。In this paper, "equal", "same" and the like are not limitations in strict mathematical and/or geometric senses, and also include errors that can be understood by those skilled in the art and allowed by manufacturing or use.
除非另有说明，本文中的数值范围不仅包括其两个端点内的整个范围，也包括含于其中的若干子范围。Unless otherwise indicated, numerical ranges herein include not only the entire range between its two endpoints, but also several subranges subsumed therein.
上面结合附图对本申请优选的具体实施方式和实施例作了详细说明，但是本申请并不限于上述实施方式和实施例，在本领域技术人员所具备的知识范围内，还可以在不脱离本申请构思的前提下做出各种变化。The preferred specific embodiments and embodiments of the present application have been described in detail above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned embodiments and embodiments. Various changes are made under the premise of the application concept.

Claims (16)

  1. 一种用于治疗帕金森的RNA质粒递送***，其特征在于，该***包括质粒，所述质粒携带有能够用于治疗帕金森的RNA片段，所述质粒能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述RNA片段的复合结构，所述复合结构能够将所述RNA片段送入目标组织，以实现帕金森病的治疗。An RNA plasmid delivery system for treating Parkinson's, characterized in that the system comprises a plasmid, the plasmid carries an RNA fragment that can be used for treating Parkinson's, and the plasmid can be enriched in the organ tissue of a host, And endogenously and spontaneously form a composite structure containing the RNA fragment in the host organ tissue, and the composite structure can send the RNA fragment into the target tissue to achieve the treatment of Parkinson's disease.
  2. 如权利要求1所述的用于治疗帕金森的RNA质粒递送***，其特征在于，所述RNA片段包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列是具有医学意义的、能够抑制或阻碍帕金森病发展的siRNA、shRNA或miRNA序列。The RNA plasmid delivery system for treating Parkinson's according to claim 1, wherein the RNA fragment comprises one, two or more specific RNA sequences with medical significance, and the RNA sequences are medically significant. A meaningful siRNA, shRNA or miRNA sequence capable of inhibiting or hindering the progression of Parkinson's disease.
  3. 如权利要求2所述的用于治疗帕金森的RNA质粒递送***，其特征在于，所述质粒还包括启动子和靶向标签，所述靶向标签能够在宿主的器官组织中形成所述复合结构的靶向结构，所述靶向结构位于复合结构的表面，所述复合结构能够通过所述靶向结构寻找并结合目标组织，将所述RNA片段递送进入目标组织。The RNA plasmid delivery system for treating Parkinson's according to claim 2, wherein the plasmid further comprises a promoter and a targeting tag, and the targeting tag can form the complex in the organ tissue of the host The targeting structure of the structure, the targeting structure is located on the surface of the composite structure, and the composite structure can find and bind the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
  4. 如权利要求3所述的用于治疗帕金森的RNA质粒递送***，其特征在于，所述质粒包括以下任意一种线路或几种线路的组合：启动子-RNA片段、启动子-靶向标签、启动子-RNA片段-靶向标签；每一个所述质粒中至少包括一个RNA片段和一个靶向标签，所述RNA片段和靶向标签位于相同的线路中或位于不同的线路中。The RNA plasmid delivery system for treating Parkinson's according to claim 3, wherein the plasmid comprises any one of the following lines or a combination of several lines: promoter-RNA fragment, promoter-targeting tag , promoter-RNA fragment-targeting tag; each of the plasmids includes at least one RNA fragment and one targeting tag, and the RNA fragment and the targeting tag are located in the same circuit or in different circuits.
  5. 如权利要求4所述的用于治疗帕金森的RNA质粒递送***，其特征在于，所述质粒还包括能够使所述线路折叠成正确结构并表达的侧翼序列、补偿序列和loop序列，所述侧翼序列包括5’侧翼序列和3’侧翼序列；The RNA plasmid delivery system for treating Parkinson's according to claim 4, wherein the plasmid further comprises a flanking sequence, a compensation sequence and a loop sequence that can make the circuit fold into a correct structure and express, the Flanking sequences include 5' flanking sequences and 3' flanking sequences;
    所述质粒包括以下任意一种线路或几种线路的组合：5’-启动子-5’侧翼序列 -RNA片段-loop序列-补偿序列-3’侧翼序列、5’-启动子-靶向标签或者5’-启动子-靶向标签-5’侧翼序列-RNA片段-loop序列-补偿序列-3’侧翼序列。The plasmid includes any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag Or 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensating sequence-3'flanking sequence.
  6. 如权利要求5所述的用于治疗帕金森的RNA质粒递送***，其特征在于，所述5’侧翼序列为ggatcctggaggcttgctgaaggctgtatgctgaattc或与其同源性大于80％的序列；The RNA plasmid delivery system for treating Parkinson's according to claim 5, wherein the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with more than 80% homology to it;
    所述loop序列为gttttggccactgactgac或与其同源性大于80％的序列；The loop sequence is gttttggccactgactgac or a sequence whose homology is greater than 80%;
    所述3’侧翼序列为accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag或与其同源性大于80％的序列；The 3' flanking sequence is accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence whose homology is greater than 80%;
    所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-5位碱基。The compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  7. 如权利要求4所述的用于治疗帕金森的RNA质粒递送***，其特征在于，在质粒中存在至少两种线路的情况下，相邻的线路之间通过序列1-3组成的序列相连；The RNA plasmid delivery system for treating Parkinson's according to claim 4, characterized in that, when there are at least two lines in the plasmid, adjacent lines are connected by a sequence consisting of sequences 1-3;
    其中，序列1为CAGATC，序列2是由5-80个碱基组成的序列，序列3为TGGATC。Wherein, sequence 1 is CAGATC, sequence 2 is a sequence consisting of 5-80 bases, and sequence 3 is TGGATC.
  8. 如权利要求7所述的用于治疗帕金森的RNA质粒递送***，其特征在于，在质粒中存在至少两种线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；The RNA plasmid delivery system for the treatment of Parkinson's as claimed in claim 7, characterized in that, when there are at least two lines in the plasmid, adjacent lines pass through sequence 4 or homology with sequence 4 More than 80% of the sequences are connected;
    其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。Wherein, sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  9. 如权利要求1所述的用于治疗帕金森的RNA质粒递送***，其特征在于，所述器官组织为肝脏，所述复合结构为外泌体。The RNA plasmid delivery system for treating Parkinson's according to claim 1, wherein the organ tissue is liver, and the composite structure is exosome.
  10. 如权利要求3所述的用于治疗帕金森的RNA质粒递送***，其特征在于， 所述靶向标签选自具有靶向功能的靶向肽或靶向蛋白。The RNA plasmid delivery system for treating Parkinson's according to claim 3, wherein the targeting tag is selected from targeting peptides or targeting proteins with targeting function.
  11. 如权利要求10所述的用于治疗帕金森的RNA质粒递送***，其特征在于，所述靶向肽包括RVG靶向肽、GE11靶向肽、PTP靶向肽、TCP-1靶向肽、MSP靶向肽；The RNA plasmid delivery system for treating Parkinson's according to claim 10, wherein the targeting peptide comprises RVG targeting peptide, GE11 targeting peptide, PTP targeting peptide, TCP-1 targeting peptide, MSP targeting peptide;
    所述靶向蛋白包括RVG-LAMP2B融合蛋白、GE11-LAMP2B融合蛋白、PTP-LAMP2B融合蛋白、TCP-1-LAMP2B融合蛋白、MSP-LAMP2B融合蛋白。The targeting proteins include RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein, and MSP-LAMP2B fusion protein.
  12. 如权利要求3所述的用于治疗帕金森的RNA质粒递送***，其特征在于，所述RNA序列的长度为15-25个核苷酸。The RNA plasmid delivery system for treating Parkinson's according to claim 3, wherein the length of the RNA sequence is 15-25 nucleotides.
  13. 如权利要求12所述的用于治疗帕金森的RNA质粒递送***，其特征在于，所述能够治疗帕金森的RNA选自：LRRK2基因的siRNA，或与上述序列同源性大于80％的RNA序列，或编码上述RNA的核酸分子。The RNA plasmid delivery system for treating Parkinson's according to claim 12, wherein the RNA capable of treating Parkinson's is selected from the group consisting of: siRNA of LRRK2 gene, or RNA with more than 80% homology to the above sequence sequence, or nucleic acid molecule encoding the above RNA.
  14. 如权利要求13所述的用于治疗帕金森的RNA质粒递送***，其特征在于，LRRK2基因的siRNA包括AUUAACAUGAAAAUAUCACUU、UUAACAAUAUCAUAUAAUCUU、AUCUUUAAAAUUUGUUAACGC、UUGAUUUAAGAAAAUAGUCUC、UUUGAUAACAGUAUUUUUCUG、其他具有抑制LRRK2基因表达的序列以及与上述序列同源性大于80％的序列。The RNA plasmid delivery system for treating Parkinson's according to claim 13, wherein the siRNA of LRRK2 gene comprises AUUAACAUGAAAAUAUCACUU, UUAACAAUAUCAUAUAAUCUU, AUCUUUAAAAUUUGUUAACGC, UUGAUUUAAGAAAAUAGUCUC, UUUGAUAACAGUAUUUUUCUG, other sequences that inhibit the expression of LRRK2 gene, and the same sequence as the above sequence. Sequences with an origin greater than 80%.
  15. 一种权利要求1-14任意一项所述的用于治疗帕金森的RNA递送***在药物中的应用。A use of the RNA delivery system for treating Parkinson's according to any one of claims 1-14 in medicine.
  16. 如权利要求15所述的应用，其特征在于，所述药物为治疗帕金森及其相关疾病的药物，所述药物的给药方式包括口服、吸入、皮下注射、肌肉注射、静脉注射。The application according to claim 15, wherein the medicine is a medicine for the treatment of Parkinson's and related diseases, and the administration mode of the medicine comprises oral administration, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection.
PCT/CN2022/083979 2021-03-30 2022-03-30 Rna plasmid delivery system for treating parkinson's disease WO2022206816A1 (en)

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