WO2022206784A1 - 一种用于治疗肺纤维化的rna递送系统 - Google Patents

一种用于治疗肺纤维化的rna递送系统 Download PDF

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WO2022206784A1
WO2022206784A1 PCT/CN2022/083823 CN2022083823W WO2022206784A1 WO 2022206784 A1 WO2022206784 A1 WO 2022206784A1 CN 2022083823 W CN2022083823 W CN 2022083823W WO 2022206784 A1 WO2022206784 A1 WO 2022206784A1
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sequence
rna
pulmonary fibrosis
delivery system
targeting
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English (en)
French (fr)
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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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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present application relates to the field of biomedical technology, in particular to an RNA delivery system for treating pulmonary fibrosis.
  • Pulmonary fibrosis is the end-stage change of a large class of lung diseases characterized by the proliferation of fibroblasts and the accumulation of a large amount of extracellular matrix accompanied by inflammatory damage and tissue structure destruction. Abnormal (scarring). Pulmonary fibrosis seriously affects the respiratory function of the human body, manifested as dry cough, progressive dyspnea (consciously insufficient air), and with the aggravation of the disease and lung damage, the patient's respiratory function continues to deteriorate. The morbidity and mortality of idiopathic pulmonary fibrosis increase year by year. The average survival time after diagnosis is only 2.8 years, and the mortality rate is higher than that of most tumors. It is called a "tumor-like disease".
  • 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.
  • Virus (Biological virus) is a small individual, simple structure, containing only one nucleic acid (DNA or RNA), must be parasitic in living cells and replicated non-cellular organisms. Viral vectors can bring genetic material into cells. The principle is to use the molecular mechanism of viruses to transmit their genomes into other cells for infection. It can occur in a complete living body (in vivo) or cell culture (in vitro), mainly used in Basic research, gene therapy or vaccines. However, there are few related studies on the use of viruses as vectors to deliver RNA, especially siRNA, using a special self-assembly mechanism.
  • 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 delivery system for the treatment of pulmonary fibrosis, so as to solve the technical defects existing in the prior art.
  • An inventive point of the present application is to provide an RNA delivery system for the treatment of pulmonary fibrosis, the system comprising a viral vector carrying an RNA fragment capable of treating pulmonary fibrosis, and the viral vector can enriched in organ tissue and endogenously spontaneously form in said host organ tissue a composite structure containing said RNA fragment capable of treating pulmonary fibrosis, said composite structure capable of delivering said RNA fragment into the lung, To achieve the treatment of pulmonary fibrosis. After the RNA fragments are delivered to the lungs of the target tissue, they can inhibit the expression of matching genes, thereby inhibiting the development of pulmonary fibrosis.
  • the viral vector is an adenovirus-associated virus.
  • adenovirus-associated virus is adenovirus-associated virus type 5, adenovirus-associated virus type 8 or adenovirus-associated virus type 9.
  • RNA fragment comprises one, two or more specific RNA sequences with medical significance, and the RNA sequences are siRNA, shRNA or miRNA with medical significance.
  • the viral vector comprises 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
  • the targeting structure is located on the surface of the composite structure
  • the The complex structure is capable of finding and binding to the target tissue through the targeting structure, delivering the RNA fragment into the target tissue.
  • the viral vector includes any one of the following circuits or a combination of several circuits: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each of the viral vectors including at least one RNA segment and one targeting tag, the RNA segment and targeting tag are located in the same circuit or are located in different circuits.
  • the viral vector also includes 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 viral vector 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-target To tag, 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 reverse complement of the RNA is to make the sequence unexpressed.
  • 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.
  • organ tissue is liver
  • 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 length of the RNA sequence is 15-25 nucleotides.
  • 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.
  • RNA capable of treating pulmonary fibrosis is selected from any one or more of the following RNAs: antisense strand of miRNA-21, siRNA of TGF- ⁇ 1 gene, or more than 80% homology with the above sequence RNA sequences, or nucleic acid molecules encoding the above RNAs. 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 TGF- ⁇ 1 gene includes ACGGAAAUAACCUAGAUGGGC, UGAACUUGUCAUAGAUUUCGU, UUGAAGAACAUAUAUAUGCUG, UCUAACUACAGUAGUGUUCCC, UCUCAGACUCUGGGGCCUCAG, other sequences that inhibit the expression of TGF- ⁇ 1 gene, and sequences with more than 80% homology to the above sequences.
  • the antisense strand of miRNA-21 is 5'-TCAACATCAGTCTGATAAGCTA-3'.
  • 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 delivery system is a delivery system for use in mammals including humans.
  • Another inventive point of the present application is to provide an application of the above-mentioned RNA delivery system for treating pulmonary fibrosis in medicine.
  • the drug includes the above-mentioned viral vector, specifically, the viral vector here refers to a viral vector carrying RNA fragments, or carrying RNA fragments and targeting tags, and can enter the host body, can be enriched in the liver, and self-assemble. A composite structure exosome is formed, which can deliver RNA fragments to the target tissue, so that the RNA fragments are expressed in the target tissue, thereby inhibiting the expression of matching genes, and achieving the purpose of treating diseases.
  • the viral vector here refers to a viral vector carrying RNA fragments, or carrying RNA fragments and targeting tags, and can enter the host body, can be enriched in the liver, and self-assemble.
  • a composite structure exosome is formed, which can deliver RNA fragments to the target tissue, so that the RNA fragments are expressed in the target tissue, thereby inhibiting the expression of matching genes, and achieving the purpose of treating diseases.
  • the drug is a drug for the treatment of pulmonary fibrosis 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 the above-mentioned pulmonary fibrosis, or related to pulmonary fibrosis.
  • Other diseases with certain correlation.
  • administration modes of the drug include oral, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection.
  • the dosage forms of the drug can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes and the like.
  • the RNA delivery system for the treatment of pulmonary fibrosis uses virus as a carrier, and the virus carrier is used as a mature injectable substance, 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 delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of viral vectors is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • RNA delivery system 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 and maintain its stability in circulation, but also facilitates Receptor cell uptake, intracytoplasmic release and lysosomal escape require low doses.
  • RNA delivery system for the treatment of pulmonary fibrosis provided in this application is applied to medicines, that is, it provides a drug delivery platform, which can greatly improve the therapeutic effect of pulmonary fibrosis, and can also form the research and development of more RNA drugs through this platform. It will greatly promote the development and use of RNA drugs.
  • Fig. 1 is a comparison diagram of mouse hydroxyproline content and mRNA level provided by an embodiment of the present application
  • Figure 2 is a Masson's trichrome staining diagram of mouse lungs provided in an example of the present application.
  • Fig. 3 is a detection diagram of in vivo enrichment, self-assembly and pulmonary fibrosis treatment effects provided by an embodiment of the present application using adenovirus and lentivirus as viral vectors when RNA fragments are loaded, and the viral vectors are adenovirus/lentivirus , the enrichment results are displayed in terms of siRNA content.
  • A is the enrichment detection map in the lung after the delivery system injection (siRNA-1)
  • B is the enrichment detection map in the lung after the delivery system injection (siRNA-1).
  • C is the enrichment detection chart in blood after delivery system injection (siRNA-1)
  • D is the enrichment detection chart in blood after delivery system injection (siRNA-2).
  • FIG. 4 is a detection diagram of in vivo enrichment, self-assembly and therapeutic effects of pulmonary fibrosis when RNA fragments are loaded using adenovirus and lentivirus as viral vectors provided by another embodiment of the present application, and the viral vectors are adenovirus/lentivirus Virus, the enrichment results are displayed in terms of siRNA content.
  • A is the enrichment detection chart in the lungs after injection of the delivery system without targeting peptide (GE11), and B is the delivery system with targeting peptide (GE11) after injection.
  • the enrichment detection map in the lungs C is the enrichment detection map in the blood (siRNA-1) after injection of the delivery system without targeting peptide (GE11), D is the delivery system with targeting peptide (GE11) After injection, the enrichment profile (siRNA-2) in blood.
  • Fig. 5 is the detection result of in vivo enrichment, self-assembly and therapeutic effect for pulmonary fibrosis when the viral vector system provided by an embodiment of the present application carries a variety of different RNA fragments
  • a in the figure is the mRNA of PTP1B
  • the relative amount detection results, B is the relative amount detection results of PTP1B protein.
  • Fig. 6 shows that when the viral vector delivery system provided by an embodiment of the present application contains multiple RNA fragments and multiple targeting tags (CMV-siRNA-1+2), after intravenous injection, it has in vivo enrichment, self-assembly and lung The detection results of the fibrosis treatment effect.
  • A is the enrichment effect in the lungs (displayed by the siRNA content)
  • B is the enrichment effect in the blood (displayed by the siRNA content).
  • Fig. 7 shows the case where the viral vector delivery system provided by another embodiment of the present application contains multiple RNA fragments and multiple targeting tags (CMV-GE11-siRNA-1+2, CMV-GE11-siRNA-1+CMV- GE11-siRNA-2), after intravenous injection, it has the detection results of in vivo enrichment, self-assembly and treatment effect of pulmonary fibrosis. Enrichment effect in plasma (shown as siRNA content).
  • Fig. 8 shows the case when the viral vector delivery system provided by another embodiment of the present application contains multiple RNA fragments and multiple targeting tags (CMV-GE11-siRNA-1+2, CMV-GE11-siRNA-1+CMV- GE11-siRNA-2), the detection results of pulmonary fibrosis treatment effect after intravenous injection,
  • a and B in the figure are the detection results of the protein content of TGFb1
  • C and D are the detection results of the mRNA content of TGFb1.
  • FIG. 9 is a graph showing the detection results of in vivo enrichment when three 5' flanking sequences/loop sequences/3' flanking sequences with homology greater than 80% are included in the adenoviral vector delivery system provided in an example of the present application (shown as siRNA content in blood).
  • Figure 10 shows the structure constructed when the adenovirus vector provided in an embodiment of the present application carries multiple lines, and adjacent lines are connected by sequence 1-sequence 2-sequence 3, wherein sequence 2 contains multiple bases.
  • the delivery system has a graph of in vivo enrichment assays (shown as siRNA levels in blood).
  • Fig. 11 is a graph showing the detection result of in vivo enrichment of the delivery system constructed by the linker sequence provided in an example of the present application, which is sequence 4 and a sequence with more than 80% homology to sequence 4 (based on the content of siRNA in blood). show).
  • Figure 12 is a graph of the detection results of the delivery system constructed when the lengths of the RNA sequences provided by an embodiment of the present application are respectively 18, 20, and 21, and has the therapeutic effect of pulmonary fibrosis, and A is the PTP1B mRNA of RNA sequences of different lengths in the figure.
  • the relative amount detection results, B is the relative amount detection results of PTP1B protein of different length RNA sequences.
  • FIG. 13 is the result of the detected hydroxyproline content when the gene circuit provided by an embodiment of the present application includes the antisense strand of miRNA-21 and five TGF- ⁇ 1 gene siRNAs.
  • 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,
  • 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 delivery system for treating pulmonary fibrosis, the system comprising a viral vector carrying an RNA fragment capable of treating pulmonary fibrosis, the viral vector being capable of enriching in the organ tissue of a host and endogenously and spontaneously form in the host organ tissue a composite structure containing the RNA fragments capable of treating pulmonary fibrosis, the composite structure capable of delivering the RNA fragments into the lungs to achieve pulmonary fibrosis Treatment.
  • Adenovirus and lentivirus are used as viral vectors, and when they load RNA fragments, whether or not they have targeting tags, they have in vivo enrichment, self-assembly and therapeutic effects on pulmonary fibrosis, as shown in Figure 3-4.
  • the aggregation effect is shown by the siRNA content in lung and blood.
  • the viral vector also includes a promoter and a targeting tag.
  • the viral vector 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 viral vectors includes at least one RNA fragments and a targeting tag, either in the same circuit or in different circuits.
  • the viral vector may only include a promoter-RNA sequence-targeting tag, or may include 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.
  • RNA Fragments are siRNA-1, siRNA-2, siRNA-1+siRNA-2.
  • the viral vector may 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 viral vector Including 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, 5' - Promoter - Targeting Tag - 5' Flanking Sequence - RNA Fragment - Loop Sequence - Compensation Sequence - 3' Flanking Sequence.
  • 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.
  • adenovirus vectors containing 3 homologous sequences they also have in vivo enrichment, self-assembly and pulmonary fibrosis therapeutic effects. As shown in Figure 9, the sequences are grouped as follows:
  • sequence 1 is preferably CAGATC
  • sequence 2 can be composed of 5-80 bases
  • Sequence of bases such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases
  • sequence of 10-50 bases is preferable, and the sequence of 20-40 bases is more preferable.
  • Sequence 3 is preferably TGGATC.
  • the adjacent lines are connected by sequence 1-sequence 2-sequence 3, where sequence 2 contains multiple bases, and the constructed delivery system also has in vivo enrichment, self-assembly and lung Fibrosis treatment effect, as shown in Figure 10.
  • Sequence 2 is specifically shown in Table 3 below.
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • sequence 4- 1 is the sequence 4
  • sequences 4-2/4-3/4-4 are the homologous sequences of the sequence 4-1 respectively, and the sequences are specifically shown in Table 4 below.
  • Sequence 4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-4 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 an siRNA sequence, a shRNA sequence or a miRNA sequence, preferably an 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 constructed delivery system also has the therapeutic effects of in vivo enrichment, self-assembly and pulmonary fibrosis, as shown in Figure 12, and the specific sequences are shown in Table 5.
  • RNA capable of treating pulmonary fibrosis is selected from any one or more of the following RNAs: antisense strand of miRNA-21, siRNA of TGF- ⁇ 1 gene, or nucleic acid molecules encoding the above RNAs.
  • the siRNA of TGF- ⁇ 1 gene includes ACGGAAAUAACCUAGAUGGGC, UGAACUUGUCAUAGAUUUCGU, UUGAAGAACAUAUAUAUGCUG, UCUAACUACAGUAGUGUUCCC, UCUCAGACUCUGGGGCCUCAG, other sequences that inhibit the expression of TGF- ⁇ 1 gene, and sequences with more than 80% homology to the above sequences.
  • the antisense strand of miRNA-21 is 5'-TCAACATCAGTCTGATAAGCTA-3'.
  • the gene circuit includes the antisense strand of miRNA-21 and the above-mentioned five TGF- ⁇ 1 gene siRNAs (siRNA-1, siRNA-2, siRNA-3, siRNA-4, siRNA-5), the The in vivo enrichment, self-assembly and treatment effects of pulmonary fibrosis are shown in Figure 13.
  • RNA effective sequences capable of treating pulmonary fibrosis is one, two or more.
  • only the antisense strand of miRNA-21 or the siRNA of the TGF- ⁇ 1 gene can be used on the same viral vector, or the antisense strand of miRNA-21 and the siRNA of the TGF- ⁇ 1 gene can be combined on the same viral vector.
  • the functional structural region of the viral 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 viral vector can be expressed as: (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-connector 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'flanking
  • 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 viruses, and up to 99% of the exogenous viruses will enter the liver. Therefore, when viruses are used as vectors, they can be enriched in liver tissue without specific design. After being opened, RNA molecules (siRNA, shRNA, or miRNA) are released, and liver tissue spontaneously wraps the above RNA molecules into exosomes, and these exosomes become RNA delivery mechanisms.
  • RNA molecules siRNA, shRNA, or miRNA
  • RNA delivery mechanism in order to make the RNA delivery mechanism (exosome) have the ability of "precision guidance”, we design a targeting tag in the viral vector injected into the body, and the targeting tag will also be assembled into exosomes by liver tissue
  • the targeting tags can be inserted into the surface of exosomes to become targeting structures that can guide exosomes, which greatly improves the RNA delivery of the present invention.
  • the accuracy of the mechanism on the one hand, can greatly reduce the amount of viral vector that needs to be introduced, and on the other hand, greatly improves the efficiency of potential drug delivery.
  • 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 tags that have been screened include: targeting peptides, targeting proteins, and antibodies.
  • targeting peptides 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 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) : 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: 7) shown), PTP-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 8), TCP-1-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No:
  • the viral vector can also be composed of multiple viruses with different structures, one of which contains a promoter promoters and targeting tags, other viruses contain promoters and RNA segments. Loading the targeting tag and RNA fragment into different viral vectors, and injecting the two viral 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 viral vector .
  • the viral vector containing the RNA sequence can be injected first, and then the viral 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.
  • the RNA delivery system provided in this example uses the virus as the carrier and the virus carrier as the mature injectable material. 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 delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of viral vectors is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • RNA delivery system 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 and maintain its stability in circulation, but also facilitates Receptor cell uptake, intracytoplasmic release and lysosomal escape require low doses.
  • the medicament comprises a viral vector carrying an RNA fragment capable of treating pulmonary fibrosis, the viral vector being capable of being enriched in the organ tissue of a host, and endogenously forming spontaneously in the organ tissue of the host containing the pulmonary fibrosis
  • the RNA fragment comprises one, two or more specific RNA sequences with medical significance, and the RNA sequences are siRNA, shRNA or miRNA with medical significance.
  • RNA fragments When the viral vector system carries a variety of different RNA fragments, it has in vivo enrichment, self-assembly and therapeutic effects on pulmonary fibrosis, as shown in Figure 5, where the RNA fragments are grouped as follows:
  • RNA-1 alone siRNA-2 alone, shRNA-1 alone, shRNA-2 alone, miRNA-1 alone, miRNA-2 alone;
  • RNA-1+siRNA-2 siRNA-1+siRNA-2, shRNA-1+shRNA-2, miRNA-1+miRNA-2;
  • RNA-1+siRNA-2+shRNA-1 siRNA-1+siRNA-2+shRNA-2
  • siRNA-1+siRNA-2+miRNA- 1 siRNA-1+siRNA-2+miRNA-2.
  • RNA sequences are specifically shown in Table 1 below.
  • the viral vector 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
  • 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 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 drug can also be used in combination with other drugs for the treatment of pulmonary fibrosis to improve the therapeutic effect, such as: glucocorticoids, immunosuppressants, anticoagulants, etc.
  • the medicine provided in this example uses the virus as the carrier and the virus carrier as the mature injection, and its safety and reliability have been fully verified, and the 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 drug can deliver various kinds of small molecule RNAs and has strong versatility. And the preparation of viral vectors 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 the application of the RNA delivery system for treating pulmonary fibrosis in medicine, and the medicine is a medicine for treating pulmonary fibrosis.
  • This example specifically illustrates the application of the RNA delivery system in the treatment of pulmonary fibrosis with the following experiments.
  • the anti-miR-21 (miR-21 antisense strand)/TGF- ⁇ 1 siRNA/anti-miR-21+TGF- ⁇ 1 siRNA system was encapsulated by the liver high-affinity AAV-5 adeno-associated virus.
  • AAV-anti-miR21/AAV-TGF- ⁇ 1 siRNA/AAV-MIX were obtained respectively, and 100 ⁇ L of AAV solution with a titer of 10 12 Vg/ml was injected into mice through tail vein.
  • the in vivo expression of the AAV system was monitored by small animals. After 3 weeks, it was found that the AAV system was stably expressed in vivo, especially in the liver.
  • mice were then selected for modeling. After successful modeling, mice were injected with PBS buffer/AAV-scrR/AAV-anti-miR21/AAV-TGF- ⁇ 1 siRNA/AAV-MIX (10 mg/kg) to form PBS group/AAV-scrR group/AAV-anti-miR21 group/AAV-TGF- ⁇ 1siRNA group/AAV-MIX group.
  • Hydroxyproline is the main component of collagen, and its content reflects the degree of pulmonary fibrosis.
  • the hydroxyproline content of mice in each group was detected, and the results are shown in Figure 1A. It can be seen that the hydroxyproline content in the PBS group and AAV-scrR group was the highest, and the AAV-anti-miR21 group and AAV-TGF- ⁇ 1 siRNA The hydroxyproline content of mice in the AAV-anti-miR21 group, AAV-TGF- ⁇ 1 siRNA group and AAV-MIX group was inhibited.

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Abstract

提供了一种用于治疗肺纤维化的RNA递送系统。该系统包括病毒载体，该病毒载体携带有能够治疗肺纤维化的RNA片段，该病毒载体能够在宿主的器官组织中富集，并在该宿主器官组织中内源性地自发形成含有该RNA片段的复合结构，该复合结构能够将该RNA片段送入肺部，实现肺纤维化的治疗。

Description

一种用于治疗肺纤维化的RNA递送系统 技术领域
本申请涉及生物医学技术领域，特别涉及一种用于治疗肺纤维化的RNA递送系统。
背景技术
肺纤维化是以成纤维细胞增殖及大量细胞外基质聚集并伴炎症损伤、组织结构破坏为特征的一大类肺疾病的终末期改变，也就是正常的肺泡组织被损坏后经过异常修复导致结构异常(疤痕形成)。肺纤维化严重影响人体呼吸功能，表现为干咳、进行性呼吸困难(自觉气不够用)，且随着病情和肺部损伤的加重，患者呼吸功能不断恶化。特发性肺纤维化发病率和死亡率逐年增加，诊断后的平均生存期仅2.8年，死亡率高于大多数肿瘤，被称为一种“类肿瘤疾病”。
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。
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。
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传递系统，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。
病毒(Biological virus)是一种个体微小，结构简单，只含一种核酸(DNA或RNA)，必须在活细胞内寄生并以复制方式增殖的非细胞型生物。病毒载体可将遗传物质带入细胞，原理是利用病毒具有传送其基因组进入其他细胞，进行感染的分子机制，可发生于完整活体(in vivo)或是细胞培养(in vitro)中，主要应用于基础研究、基因疗法或疫苗。但是目前很少有针对将病毒作为载体利用特殊的自组装机制递送RNA，特别是siRNA的相关研究。
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。
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系统清除，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。
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。外泌体与宿主免疫系统生物相容，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。
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。
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，则对推动肺纤维化的RNAi疗法意义非凡。
因此，开发一个安全、精确和高效的siRNA传递系统是对提高肺纤维化的RNAi治疗效果，推进RNAi疗法至关重要的一环。
发明内容
有鉴于此，本申请实施例提供了一种用于治疗肺纤维化的RNA递送系统，以解决现有技术中存在的技术缺陷。
本申请的一个发明点为提供一种用于治疗肺纤维化的RNA递送系统，该系统包括病毒载体，所述病毒载体携带有能够治疗肺纤维化的RNA片段，所述病毒载体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能够治疗肺纤维化的所述RNA片段的复合结构，所述复合结构能够将所述RNA片段送入肺部，实现肺纤维化的治疗。RNA片段送入目标组织肺部后，能够抑制与其相匹配的基因的表达，进而抑制肺纤维化的发展。
进一步地，所述病毒载体为腺病毒相关病毒。
进一步地，所述腺病毒相关病毒为腺病毒相关病毒5型、腺病毒相关病毒8型或腺病毒相关病毒9型。
进一步地，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siRNA、shRNA或miRNA。
进一步地，所述病毒载体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。
进一步地，所述病毒载体中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；每一个所述病毒载体中至少包括一个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。
进一步地，所述病毒载体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；
所述病毒载体中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、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。
进一步地，所述5’侧翼序列为ggatcctggaggcttgctgaaggctgtatgctgaattc或与其同源性大于80％的序列；
所述loop序列为gttttggccactgactgac或与其同源性大于80％的序列；
所述3’侧翼序列为accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag或与其同源性大于80％的序列；
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。
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。
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。
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。
进一步地，在病毒载体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(序列1-序列2-序列3)相连；
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。
进一步地，在病毒载体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；
其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
进一步地，所述器官组织为肝脏，所述复合结构为外泌体。
进一步地，所述靶向标签选自具有靶向功能的靶向肽或靶向蛋白。
进一步地，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；
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。
进一步地，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。
进一步地，所述能够治疗肺纤维化的RNA选自以下RNA中的任意一种或几种：miRNA-21的反义链、TGF-β1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。
TGF-β1基因的siRNA包括ACGGAAAUAACCUAGAUGGGC、UGAACUUGUCAUAGAUUUCGU、UUGAAGAACAUAUAUAUGCUG、UCUAACUACAGUAGUGUUCCC、UCUCAGACUCUGGGGCCUCAG、其他具有抑制TGF-β1基因表达的序列以及与上述序列同源性大于80％的序列。
miRNA-21的反义链为5’-TCAACATCAGTCTGATAAGCTA-3’。
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。
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。
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)、阿拉伯糖-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。
进一步地，所述递送系统为用于包括人在内的哺乳动物中的递送系统。
本申请的另一个发明点为提供一种如上所述的用于治疗肺纤维化的RNA递送系统在药物中的应用。
所述药物包括上述病毒载体，具体而言，此处的病毒载体表示携带有RNA片段、或携带有RNA片段及靶向标签的病毒载体，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片段递送至目标组织，使RNA片段在目标组织中表达，进而抑制与其匹配的基因的表达，实现治疗疾病的目的。
进一步地，所述药物为治疗肺纤维化及其相关疾病的药物，这里的相关疾病指的是上述肺纤维化的形成或发展过程中出现的关联疾病或并发症、后遗症等，或与肺纤维化具有一定相关性的其他疾病。
进一步地，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。
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。
本申请的技术效果为：
本申请提供的用于治疗肺纤维化的RNA递送系统以病毒作为载体，病毒载体作为成熟的注入物，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。该递送系统可以递送各类小分子RNA，通用性强。并且病毒载体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RNA递送系统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。
本申请提供的用于治疗肺纤维化的RNA递送系统应用于药物中，即提供了一个药物递送平台，可以大大提高肺纤维化的治疗效果，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。
附图说明
图1是本申请一实施例提供的小鼠羟脯氨酸含量、mRNA水平对比图；
图2是本申请一实施例提供的小鼠肺部Masson三色染色图。
图3是本申请一实施例提供的以腺病毒和慢病毒做为病毒载体，负载RNA片段时具有体内富集、自组装及肺纤维化治疗效果的检测图，病毒载体为腺病毒/慢病毒，富集结果以siRNA含量显示，图中A为递送系统注射后，在肺部的富集检测图(siRNA-1)，B为递送系统注射后，在肺部的富集检测图(siRN A-2)，C为递送系统注射后，在血液中的富集检测图(siRNA-1)，D为递送系统注射后，在血液中的富集检测图(siRNA-2)。
图4是本申请另一实施例提供的以腺病毒和慢病毒做为病毒载体，负载RNA片段时具有体内富集、自组装及肺纤维化治疗效果的检测图，病毒载体为腺病毒/慢病毒，富集结果以siRNA含量显示，图中A为无靶向肽(GE11)的递送系统注射后，在肺部的富集检测图，B为有靶向肽(GE11)的递送系统注射后，在肺部的富集检测图，C为无靶向肽(GE11)的递送系统注射后，在血液中的富集检测图(siRNA-1)，D为有靶向肽(GE11)递送系统注射后，在血液中的富集检测图(siRNA-2)。
图5是本申请一实施例提供的病毒载体系统中携带有多种不同RNA片段的情况下，具有体内富集、自组装及针对肺纤维化治疗效果的检测结果，图中A为PTP1B的mRNA相对量检测结果，B为PTP1B的蛋白相对量检测结果。
图6是本申请一实施例提供的病毒载体递送系统中包含有多个RNA片段和多个靶向标签时(CMV-siRNA-1+2)，静脉注射后具有体内富集、自组装及肺纤维化治疗效果的检测结果，图中A为肺部的富集效果(以siRNA含量显示)，B为血液中的富集效果(以siRNA含量显示)。
图7是本申请另一实施例提供的病毒载体递送系统中包含有多个RNA片段和多个靶向标签时(CMV-GE11-siRNA-1+2、CMV-GE11-siRNA-1+CMV-GE11-siRNA-2)，静脉注射后具有体内富集、自组装及肺纤维化治疗效果的检测结果，图中A和B为肺部的富集效果(以siRNA含量显示)，C和D为血浆中的富集效果(以siRNA含量显示)。
图8是本申请再一实施例提供的病毒载体递送系统中包含有多个RNA片段和多个靶向标签时(CMV-GE11-siRNA-1+2、CMV-GE11-siRNA-1+CMV-GE11-siRNA-2)，静脉注射后的肺纤维化治疗效果检测结果，图中A和B为TGFb1的蛋白含量检测结果，C和D为TGFb1的mRNA含量检测结果。
图9是本申请一实施例提供的腺病毒载体递送系统中，包含有同源性大于80％的3条5’侧翼序列/loop序列/3’侧翼序列时，具有体内富集的检测结果图(以血液中的siRNA含量显示)。
图10是本申请一实施例提供的腺病毒载体携带多个线路时，相邻线路之间以序列1-序列2-序列3相连，其中序列2含有多个碱基的情况下，所构建的递送系统具有体内富集的检测结果图(以血液中的siRNA含量显示)。
图11是本申请一实施例提供的连接序列为序列4以及与序列4同源性大于80％的序列时，其构建的递送系统也具有体内富集的检测结果图(以血液中的siRNA含量显示)。
图12是本申请一实施例提供的RNA序列长度分别为18、20、21时，所构建的递送系统，具有肺纤维化治疗效果的检测结果图，图中A为不同长度RNA序列的PTP1B mRNA相对量检测结果，B为不同长度RNA序列的PTP1B蛋白相对量检测结果。
图13是本申请一实施例提供的基因线路在包括有miRNA-21的反义链及5条TGF-β1基因siRNA的情况下，所检测到的羟脯氨酸含量结果。
具体实施方式
下面结合附图对本申请的具体实施方式进行描述。
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染色的具体步骤包括：
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。
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递送系统，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。
在本发明所提供的图示中，“*”表示P<0.05，“**”表示P<0.01,“***”表示P<0.005。
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。
实施例1
本实施例提供一种用于治疗肺纤维化的RNA递送系统，该系统包括病毒载体，所述病毒载体携带有能够治疗肺纤维化的RNA片段，所述病毒载体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能够治疗肺纤维化的所述RNA片段的复合结构，所述复合结构能够将所述RNA片段送入肺部，实现肺纤维化的治疗。
将腺病毒和慢病毒做为病毒载体，其负载RNA片段时，无论是否带有靶向标签，其均具有体内富集、自组装及肺纤维化治疗效果，如图3-4所示，富集效果以肺、血液中的siRNA含量显示。
在本实施例中，病毒载体还包括启动子和靶向标签。所述病毒载体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，每一个所述病毒载体中至少包括一个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，病毒载体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。
病毒载体递送系统中包含有多个RNA片段和多个靶向标签时，其具有体内富集、自组装及肺纤维化治疗效果，如图6-8所示，其中靶向标签为GE11，RNA片段为siRNA-1、siRNA-2、siRNA-1+siRNA-2。
进一步地，所述病毒载体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；所述病毒载体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、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。
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。
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。
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。
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。
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。
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。
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。
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。
腺病毒载体中，含有3种同源序列的情况下，也具有体内富集、自组装及肺纤维化治疗效果，如图9所示，序列分组如下：
1、3条同源性大于80％的5’侧翼序列；
2、3条同源性大于80％的loop序列；
3、3条同源性大于80％的3’侧翼序列。
序列具体如下表2所示。
Figure PCTCN2022083823-appb-000001
Figure PCTCN2022083823-appb-000002
在病毒载体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。
腺病毒载体携带多个线路时，相邻线路之间以序列1-序列2-序列3相连，其中序列2含有多个碱基，所构建的递送系统也同样具有体内富集、自组装和肺纤维化治疗效果，如图10所示。
序列2具体如下表3所示。
Figure PCTCN2022083823-appb-000003
更为优选地，在病毒载体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。
连接序列为序列4以及与序列4同源性大于80％的序列时，构建的递送系统也具有体内富集、自组装和肺纤维化治疗效果，如图11所示，图11中序列4-1即为所述序列4，序列4-2/4-3/4-4分别为序列4-1的同源序列，序列具体如下表4所示。
序列4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
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。
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。
RNA序列长度分别为18、20、21时，所构建的递送系统，也具有体内富集、自组装和肺纤维化的治疗效果，如图12所示，具体序列如表5所示。
21nt序列 TATCTTTGCTGTCACAAGAGC
19nt序列 TAAAGTCAATGTACAGCTG
18nt序列 TTCATGTCATGGATGGTG
所述能够治疗肺纤维化的RNA选自以下RNA中的任意一种或几种：miRNA-21的反义链、TGF-β1基因的siRNA或编码上述RNA的核酸分子。
TGF-β1基因的siRNA包括ACGGAAAUAACCUAGAUGGGC、UGAACUUGUCAUAGAUUUCGU、UUGAAGAACAUAUAUAUGCUG、UCUAACUACAGUAGUGUUCCC、UCUCAGACUCUGGGGCCUCAG、其他具有抑制TGF-β1基因表达的序列以及与上述序列同源性大于80％的序列。
miRNA-21的反义链为5’-TCAACATCAGTCTGATAAGCTA-3’。
基因线路在包括有miRNA-21的反义链及上述5条TGF-β1基因siRNA(siRNA-1、siRNA-2、siRN A-3、siRNA-4、siRNA-5)的情况下，所具有的体内富集、自组装及肺纤维化治疗效果，如图13所示。
能够治疗肺纤维化的RNA有效序列的数量为1条、2条或多条。比如可以在同一个病毒载体上仅使用miRNA-21的反义链或TGF-β1基因的siRNA，还可以在同一个病毒载体上联合使用miRNA-21的反义链和TGF-β1基因的siRNA。
以在同一个病毒载体上联合使用“siRNA1”和“siRNA2”为例，该病毒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。
更加具体地，该病毒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)。
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。
具体地，肝脏会吞噬外源性的病毒，高达99％的外源性病毒会进入肝脏，因此当以病毒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，随后病毒载体被打开，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。
优选地，为了使该RNA输送机构(外泌体)具有“精确制导”的能力，在注入体内的病毒载体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，靶向标签能够被插入外泌体表面，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病毒载体的用量大大减少，另一方面还大大提高了潜在药物递送的效率。
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标签包括：靶向肽、靶向蛋白、抗体。其中，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)。
此外，为了达到精准递送的目的，我们实验了多种病毒载体搭载的方案，得出另一优化的方案：所述 病毒载体还可以由具有不同结构的多种病毒构成，其中一种病毒包含启动子和靶向标签，其他病毒包含启动子和RNA片段。即将靶向标签与RNA片段装载到不同的病毒载体中，将两种病毒载体注入体内，其靶向效果不差于将相同的靶向标签与RNA片段装载在一个病毒载体中产生的靶向效果。
更优选地，两种不同的病毒载体注入宿主体内时，可以先将装有RNA序列的病毒载体注入，在1-2小时后再注入含有靶向标签的病毒载体，如此能够达到更优的靶向效果。
以上所述的递送系统均可用于包括人在内的哺乳动物。
本实施例提供的RNA递送系统以病毒作为载体，病毒载体作为成熟的注入物，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。该递送系统可以递送各类小分子RNA，通用性强。并且病毒载体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RNA递送系统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。
实施例2
在实施例1的基础上，本实施例提供一种药物。该药物包括病毒载体，所述病毒载体携带有能够治疗肺纤维化的RNA片段，所述病毒载体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能够治疗肺纤维化的所述RNA片段的复合结构，所述复合结构能够将所述RNA片段送入肺部，实现肺纤维化的治疗。
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siRNA、shRNA或miRNA。
病毒载体系统中携带有多种不同RNA片段的情况下，具有体内富集、自组装及针对肺纤维化治疗效果，如图5所示，其中RNA片段分组如下所示：
1、6种RNA单独使用：siRNA-1单独、siRNA-2单独、shRNA-1单独、shRNA-2单独、miRNA-1单独、miRNA-2单独；
2、6种RNA序列中的任意2种组合成RNA片段：siRNA-1+siRNA-2、shRNA-1+shRNA-2、miRNA-1+miRNA-2；
3、6种RNA序列中的任意3种组合成RNA片段：siRNA-1+siRNA-2+shRNA-1、siRNA-1+siRNA-2+shRNA-2、siRNA-1+siRNA-2+miRNA-1、siRNA-1+siRNA-2+miRNA-2。
RNA序列具体如下表1所示。
Figure PCTCN2022083823-appb-000004
Figure PCTCN2022083823-appb-000005
可选地，所述病毒载体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。
关于本实施例中上述病毒载体、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。
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递送系统递送至目标组织，发挥治疗作用。
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。
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。
该药物还可以与其他治疗肺纤维化的药物联合使用，以提高治疗效果，比如：糖皮质激素、免疫抑制剂、抗凝剂等。
本实施例提供的药物以病毒作为载体，病毒载体作为成熟的注入物，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。并且病毒载体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。
实施例3
在实施例1或2的基础上，本实施例提供用于治疗肺纤维化的RNA递送系统在药物中的应用，该药物为治疗肺纤维化的药物。本实施例结合以下试验对RNA递送系统在肺纤维化治疗方面的应用进行具体说明。
在此试验中，利用肝脏高亲和的AAV-5型腺相关病毒包裹抗-miR-21(miR-21反义链)/TGF-β1 siRNA/抗-miR-21+TGF-β1 siRNA系统，分别得到AAV-anti-miR21/AAV-TGF-β1 siRNA/AAV-MIX，尾静脉注射100μL滴度为10 12V.g/ml的AAV溶液至小鼠体内。通过小动物活体监测AAV系统的体内表达情况，3周后可见AAV系统在体内尤其是肝脏，稳定表达。
随即选取小鼠进行造模，造模成功后，分别向小鼠注射注射PBS缓冲液/AAV-scrR/AAV-anti-miR21/AAV-TGF-β1 siRNA/AAV-MIX(10mg/kg)，形成PBS组/AAV-scrR组/AAV-anti-miR21组/AAV-TGF-β1siRNA组/AAV-MIX组。
分别检测正常小鼠、PBS组小鼠、AAV-scrR组小鼠、AAV-TGF-β1 siRNA组小鼠相对TGF-β1 mRNA水平，结果如图1B所示，可见，AAV-TGF-β1 siRNA组小鼠相对TGF-β1 mRNA水平相对较低。
分别检测正常小鼠、PBS组小鼠、AAV-scrR组小鼠、AAV-anti-miR21组小鼠相对miR21 mRNA水平，结果如图1C所示，可见，AAV-anti-miR21组小鼠相对miR21 mRNA水平相对较低。
羟脯氨酸是胶原的主要成分，其含量反映了肺纤维化的程度。分别检测各组小鼠羟脯氨酸含量，结果如图1A所示，可见PBS组、AAV-scrR组小鼠体内羟脯氨酸含量最高，AAV-anti-miR21组、AAV-TGF-β1 siRNA组、AAV-MIX组小鼠体内羟脯氨酸含量均较低，说明AAV-anti-miR21组、AAV-TGF-β1 siRNA组、AAV-MIX组小鼠的肺纤维化得到抑制。
分别对各组小鼠肺部进行Masson三色染色，结果如图2所示，可以看出PBS组和AAV-scrR组小鼠肺泡间隙被严重破坏，造成肺间质胶原，而AAV-anti-miR21组、AAV-TGF-β1 siRNA组、AAV-MIX组这些试验组则显著减轻了这些现象。
以上试验说明，利用亲和肝脏的病毒包裹CMV-siR miR-21、CMV-siR TGF-β1、CMV-siR miR-21+TGF-β1回路，能够显著缓解肺纤维化程度，具有极大的成药潜力，以及临床研究价值。
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。
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。
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。
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。
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。

Claims (19)

  1. 一种用于治疗肺纤维化的RNA递送系统，其特征在于，该系统包括病毒载体，所述病毒载体携带有能够治疗肺纤维化的RNA片段，所述病毒载体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能够治疗肺纤维化的所述RNA片段的复合结构，所述复合结构能够将所述RNA片段送入肺部，实现肺纤维化的治疗。
  2. 如权利要求1所述的用于治疗肺纤维化的RNA递送系统，其特征在于，所述病毒载体为腺病毒相关病毒。
  3. 如权利要求2所述的用于治疗肺纤维化的RNA递送系统，其特征在于，所述腺病毒相关病毒为腺病毒相关病毒5型、腺病毒相关病毒8型或腺病毒相关病毒9型。
  4. 如权利要求1所述的用于治疗肺纤维化的RNA递送系统，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siRNA、shRNA或miRNA。
  5. 如权利要求1所述的用于治疗肺纤维化的RNA递送系统，其特征在于，所述病毒载体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。
  6. 如权利要求5所述的用于治疗肺纤维化的RNA递送系统，其特征在于，所述病毒载体中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；每一个所述病毒载体中至少包括一个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。
  7. 如权利要求6所述的用于治疗肺纤维化的RNA递送系统，其特征在于，所述病毒载体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；
    所述病毒载体中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、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。
  8. 如权利要求7所述的用于治疗肺纤维化的RNA递送系统，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；
    所述loop序列为gttttggccactgactgac或与其同源性大于80％的序列；
    所述3’侧翼序列为accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag或与其同源性大于80％的序列；
    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。
  9. 如权利要求6所述的用于治疗肺纤维化的RNA递送系统，其特征在于，在病毒载体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；
    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；
  10. 如权利要求9所述的用于治疗肺纤维化的RNA递送系统，其特征在于，在病毒载体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；
    其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
  11. 如权利要求1所述的用于治疗肺纤维化的RNA递送系统，其特征在于，所述器官组织为肝脏，所述复合结构为外泌体。
  12. 如权利要求5所述的用于治疗肺纤维化的RNA递送系统，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。
  13. 如权利要求12所述的用于治疗肺纤维化的RNA递送系统，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；
    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。
  14. 如权利要求4所述的基于病毒载体的RNA递送系统，其特征在于，所述RNA序列的长度为15-25个核苷酸。
  15. 如权利要求14所述的用于治疗肺纤维化的RNA递送系统，其特征在于，所述能够治疗肺纤维化的RNA选自以下RNA中的任意一种或几种：miRNA-21的反义链、TGF-β1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。
  16. 如权利要求15所述的用于治疗肺纤维化的RNA递送系统，其特征在于，TGF-β1基因的siRNA包括ACGGAAAUAACCUAGAUGGGC、UGAACUUGUCAUAGAUUUCGU、UUGAAGAACAUAUAUAUGCUG、UCUAACUACAGUAGUGUUCCC、UCUCAGACUCUGGGGCCUCAG、其他具有抑制TGF-β 1基因表达的序列以及与上述序列同源性大于80％的序列。
  17. 如权利要求1所述的基于病毒载体的RNA递送系统，其特征在于，所述递送系统为用于包括人在内的哺乳动物中的递送系统。
  18. 一种权利要求1-17任意一项所述的用于治疗肺纤维化的RNA递送系统在药物中的应用。
  19. 如权利要求18所述的应用，其特征在于，所述药物为治疗肺纤维化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。
PCT/CN2022/083823 2021-03-30 2022-03-29 一种用于治疗肺纤维化的rna递送系统 WO2022206784A1 (zh)

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