WO2022206738A1 - 一种rna质粒递送系统及其应用 - Google Patents

一种rna质粒递送系统及其应用 Download PDF

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WO2022206738A1
WO2022206738A1 PCT/CN2022/083598 CN2022083598W WO2022206738A1 WO 2022206738 A1 WO2022206738 A1 WO 2022206738A1 CN 2022083598 W CN2022083598 W CN 2022083598W WO 2022206738 A1 WO2022206738 A1 WO 2022206738A1
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sirna
sequence
rna
cmv
group
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PCT/CN2022/083598
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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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Priority to JP2023560201A priority Critical patent/JP2024511525A/ja
Priority to EP22778920.3A priority patent/EP4317444A1/en
Publication of WO2022206738A1 publication Critical patent/WO2022206738A1/zh
Priority to US18/374,242 priority patent/US20240141344A1/en

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Definitions

  • the present application relates to the field of biomedical technology, in particular to an RNA plasmid delivery system and its application.
  • 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 and its application to solve the technical defects existing in the prior art.
  • An inventive point of the present application is to provide an RNA plasmid delivery system, the system includes a plasmid, the plasmid carries the RNA fragment to be delivered, the plasmid can be enriched in the organ tissue of the host, and the host Complex structures containing the RNA fragments are formed endogenously and spontaneously in organ tissues, and the complex structures are able to enter and bind to the target tissue to deliver the RNA fragments into the target tissue. After the RNA fragment is delivered to the target tissue, it can inhibit the expression of the matching gene, thereby inhibiting the development of disease in the target tissue.
  • the RNA fragment comprises one, two or more specific RNA sequences with medical significance, and the RNA sequences are siRNA, shRNA or miRNA sequences with medical significance.
  • Figures 39-41 show that the constructed plasmids have the effects of in vivo enrichment, self-assembly and disease treatment when multiple RNA fragments are used alone, in any combination of two or in any combination of three.
  • the plasmid also includes a promoter and a targeting tag
  • the targeting tag can form a targeting structure of the composite structure in the organ tissue of the host, and the composite structure can be found through the targeting structure.
  • the RNA fragments are delivered 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.
  • Figures 42-49 show that 2 different RNA fragments, and the delivery system constructed after any combination of 2 different targeting tags have in vivo enrichment and therapeutic effects. .
  • the plasmid also includes flanking sequences, compensation sequences and loop sequences that enable the circuit to be folded into the correct structure and expressed.
  • flanking sequence includes 5' flanking sequence and 3' flanking sequence;
  • the plasmid includes any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA sequence-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag , 5'-promoter-targeting tag-5'flanking sequence-RNA sequence-loop sequence-compensating sequence-3'flanking sequence.
  • Figure 50 shows the enrichment effect of the promoter-siRNA and promoter-targeted tag-siRNA gene circuits and the detection results of the expression level of EGFR.
  • 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 any 1-5 bases in the RNA fragment deleted, and the purpose of deleting the 1-5 bases of the RNA reverse complementary sequence is to make the sequence not expressed.
  • Figures 51-53 show that the 5' flanking sequence, the loop sequence, the 3' flanking sequence and the homologous sequences of the three are constructed into plasmids, all of which have enrichment and therapeutic effects.
  • 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.
  • sequence 2 is a sequence consisting of 10-50 bases, more preferably, sequence 2 is a sequence consisting of 20-40 bases.
  • adjacent lines are connected by sequence 4 or a sequence with more than 80% homology to sequence 4;
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • Figure 55 shows that the delivery system constructed by sequence 4 and its homologous sequence has both enrichment and therapeutic effects.
  • the plasmid consists of multiple plasmids with different structures, one of which contains a promoter and targeting tag, and the other plasmids that contain a promoter and RNA sequences.
  • the organ tissue is liver
  • the composite structure is exosome
  • the targeting tag is selected from targeting peptide or targeting protein with targeting function, and the targeting structure is located on the surface of the composite structure.
  • 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 desired delivered RNA is 15-25 nucleotides (nt) 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.
  • Figure 54 shows that the delivery systems (plasmids) constructed by RNA sequences of different lengths have in vivo enrichment and therapeutic effects after intravenous injection.
  • the RNA to be delivered is selected from any one or more of the following: siRNA of EGFR gene, siRNA of KRAS gene, siRNA of VEGFR gene, siRNA of mTOR gene, siRNA of TNF- ⁇ gene, integrin- siRNA of ⁇ gene, siRNA of B7 gene, siRNA of TGF- ⁇ 1 gene, siRNA of H2-K gene, siRNA of H2-D gene, siRNA of H2-L gene, siRNA of HLA gene, siRNA of GDF15 gene, miRNA- Antisense strand of 21, antisense strand of miRNA-214, siRNA of TNC gene, siRNA of PTP1B gene, siRNA of mHTT gene, siRNA of Lrrk2 gene, siRNA of ⁇ -synuclein gene, or more than 80 homology with the above sequence % RNA sequence, or nucleic acid molecule encoding the above RNA. It should be noted that the RNA sequences in the "nucleic acid molecules encoding the above RNA.
  • the siRNA of the above-mentioned genes are all RNA sequences that have the function of inhibiting the expression of the gene, and there are many RNA sequences that have the function of inhibiting the expression of each gene. for example.
  • EGFR gene siRNA includes UGUUGCUUCUCUUAAUUCCU, AAAUGAUCUUCAAAAGUGGCC, UCUUUAAGAAGGAAAGAUCAU, AAUAUUCGUAGCAUUUAUGGA, UAAAAAUCCUCACAUAUACUU, other sequences that inhibit EGFR gene expression and sequences with more than 80% homology to the above sequences.
  • the siRNA of KRAS gene includes UGAUUUAGUAUUAUUUAUGGC, AAUUUGUUCUCUAUAAUGGUG, UAAUUUGUUCUCUAUAAUGGU, UUAUGUUUUCGAAUUUCUCGA, UGUAUUUUACAUAAUUACAC AC, other sequences with inhibiting KRAS gene expression and sequences with more than 80% homology to the above sequences.
  • the siRNA of VEGFR gene includes AUUUGAAGAGUUGUAUUAGCC, UAAUAGACUGGUAACUUUCAU, ACAACUAUGUACAUAAUAGAC, UUUAAGACAAGCUUUUCUCCA, AACAAAAGGUUUUUUCAUGGAC, other sequences that inhibit the expression of VEGFR gene and sequences with more than 80% homology to the above sequences.
  • the siRNA of mTOR gene includes AGAUAGUUGGCAAAUCUGCCA, ACUAUUUCAUCCAUAUAAGGU, AAAAUGUUGUCAAAGAAGGGU, AAAAAUGUUGUCAAAGAAGGG, UGAUUUCUUCCAUUUCUUCUC, other sequences that inhibit the expression of mTOR gene and sequences with more than 80% homology to the above sequences.
  • the siRNA of TNF- ⁇ gene includes AAAACAUAAUCAAAAGAAGGC, UAAAAAACAUAAUCAAAAGAA, AAUAAUAAAUAAUCACAAGUG, UUUUCACGGAAAACAUGUCUG, AAACAUAAUCAAAAGAAGGCA, other sequences that inhibit the expression of TNF- ⁇ gene and sequences with more than 80% homology to the above sequences.
  • the siRNA of integrin- ⁇ gene includes AUAAUCAUCUCCAUUAAUGUC, AAACAAUUCCUUUUUUAUCUU, AUUAAAACAGGAAACUUUGAG, AUAAUGAAGGAUAUACAACAG, UUCUUUUAUUCAUAAAAGUCUC, other sequences that inhibit the expression of integrin- ⁇ gene and sequences with more than 80% homology to the above sequences.
  • siRNA of B7 gene UUUUCUUUGGGUAAUCUUCAG, AGAAAAAUUCCACUUUUUCUU, AUUUCAAAGUCAGAUAUACUA, ACAAAAAUUCCAUUUACUGAG, AUUAUUGAGUUAAGUAUUCCU, other sequences that inhibit the expression of B7 gene and sequences with more than 80% homology to the above sequences.
  • 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 siRNA of H2-K gene includes AAAAACAAAUCAAUCAAACAA, UCAAAAAAACAAAUCAAUCAA, UAUGAGAAGACAUUGUCUGUC, AACAAUCAAGGUUACAUUCAA, ACAAAACCUCUAAGCAUUCUC, other sequences that inhibit H2-K gene expression and sequences with more than 80% homology to the above sequences.
  • the siRNA of H2-D gene includes AAUCUCGGAGAGACAUUUCAG, AAUGUUGGUGUAAAGAGAACUG, AACAUCAGACAAUGUUGUGUA, UGUUAACAAUCAAGGUCACUU, AACAAAAAAACCUCUAAGCAU, other sequences that inhibit the expression of H2-D gene, and sequences with more than 80% homology to the above sequences.
  • the siRNA of H2-L gene includes GAUCCGCUCCCAAUACUCCGG, AUCUGCGUGAUCCGCUCCCAA, UCGGAGAGACAUUUCAGAGCU, UCUCGGAGAGACAUUUCAGAG, AAUCUCGGAGAGACAUUUCAG, other sequences that inhibit the expression of H2-L gene and sequences with more than 80% homology to the above sequences.
  • siRNA of HLA gene AUCUGGAUGGUGUGAGAACCG, UGUCACUGCUUGCAGCCUGAG, UCACAAAGGGAAGGGCAGGAA, UUGCAGAAACAAAGUCAGGGU, ACACGAACACACAGACACAUGCA, other sequences that inhibit HLA gene expression, and sequences with more than 80% homology to the above sequences.
  • the siRNA of GDF15 gene includes UAUAAAUACAGCUGUUUGGGC, AGACUUAUAUAAAUACAGCUG, AAUUAAUAAUAAAUAACAGAC, AUCUGAGGCCAUUCACCGUC, UGCAACUCCAGCUGGGGCCGU, other sequences that inhibit the expression of GDF15 gene, and sequences with more than 80% homology to the above sequences.
  • the siRNA of TNC gene includes UAUGAAAUGUAAAAAAAGGGA, AAUAUAUCCUUAAAAUGGAA, UAAUCAUAUCCUUAAAAUGGA, UGAAAAAUCCUUAGUUUUCAU, AGAAGUAAAAAACUAUUGCGA, other sequences with inhibiting TNC gene expression and sequences with more than 80% homology to the above sequences.
  • the siRNA of the PTP1B gene includes UGAUAUAGUCAUUAUCUUCUU, UCCAUUUUUAUCAAACUAGCG, AUUGUUUAAAUAAAUAUGGAG, AAUUUUAAUACAUUAUUGGUU, UUUAUUAUUGUACUUUUUGAU, other sequences that inhibit the expression of the PTP1B gene, and sequences with more than 80% homology to the above sequences.
  • the mHTT gene siRNA includes UAUGUUUUCACAUAUUGUCAG, AUUUAGUAGCCAACUAUAGAA, AUGUUUUUCAAUAAAUGUGCC, UAUGAAUAGCAUUCUUAUCUG, UAUUUGUUCCUCUUAAUACAA, other sequences that inhibit the expression of the mHTT gene, and sequences with more than 80% homology to the above sequences.
  • the siRNA of Lrrk2 gene includes AUUAACAUGAAAAUAUCACUU, UUAACAAUAUCAUAUAAUCUU, AUCUUUAAAAUUUGUUAACGC, UUGAUUUAAGAAAAUAGUCUC, UUUGAUAACAGUAUUUUUCUG, other sequences that inhibit the expression of Lrrk2 gene and sequences with more than 80% homology to the above sequences.
  • the siRNA of ⁇ -synuclein gene includes AUAUAUUAACAAAUUUCACAA, AAGUAUUAUAUAUUAACAA, AUAACUUUUAUAUUUUUGUCCU, UAACUAAAAAAUUAUUUCGAG, UCGAAUAUUAUUUUAUUGUCAG, other sequences that inhibit the expression of ⁇ -synuclein 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 delivery system can be used in mammals including humans.
  • Another inventive point of the present application is to provide an application of the RNA delivery system described in any of the above paragraphs in medicine.
  • the modes of administration of the drug include oral, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection. Intravenous injection is preferred.
  • the drug is for the treatment of cancer, pulmonary fibrosis, colitis, obesity, cardiovascular disease caused by obesity, type 2 diabetes, Huntington's disease, Parkinson's disease, myasthenia gravis, Alzheimer's disease Or drugs for graft-versus-host disease.
  • 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 target tissue, so that the RNA fragments are expressed in the target tissue, and then inhibit the expression of matching genes to achieve the purpose of treating diseases.
  • 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 uses plasmids as carriers and plasmids as mature injectables. 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 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 provided in this application to medicines provides a drug delivery platform through which more RNA-based drugs can be developed, which greatly promotes the development and use of RNA-based 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 the therapeutic effect of mouse EGFR mutant lung cancer tumor provided by an embodiment of the present application.
  • Figure 12 is a mouse HE staining chart, an immunohistochemical staining chart and a coloring statistics chart provided by an embodiment of the present application;
  • FIG. 13 is a comparison diagram of the therapeutic effect of mouse KRAS mutant lung cancer tumor provided by an embodiment of the present application.
  • FIG. 14 is a mouse HE staining chart, an immunohistochemical staining chart, and a coloring situation statistical chart provided by an embodiment of the present application;
  • Figure 15 is a comparison diagram of mouse renal cancer tumor images provided in an embodiment of the present application.
  • FIG. 16 is a comparison diagram of the development of mouse kidney cancer tumor provided by an embodiment of the present application.
  • Figure 17 is a comparison diagram of the development of colitis in mice provided by an embodiment of the present application.
  • Figure 18 is a comparison diagram of mouse colon HE staining provided by an embodiment of the present application.
  • Figure 19 is a comparison diagram of the development of colitis in mice provided by an embodiment of the present application.
  • Fig. 20 is the HE staining comparison diagram of mouse colon provided by an embodiment of the present application.
  • Figure 21 is a comparison diagram of the content of mouse hydroxyproline provided by an embodiment of the application.
  • Figure 22 is a fluorescent staining diagram of mouse lung provided by an embodiment of the present application.
  • Figure 23 is a Masson's trichrome staining diagram of the mouse lung provided by an embodiment of the present application.
  • Fig. 24 is the HE staining diagram of mouse lung provided by an embodiment of the present application.
  • Figure 25 is a comparison diagram of mouse partial protein and mRNA levels provided by an embodiment of the present application.
  • Figure 26 is a comparison diagram of mouse siRNA-related expression provided by an embodiment of the present application.
  • Figure 27 is a comparison diagram of the treatment of glioblastoma in mice provided in an embodiment of the present application.
  • Figure 28 is a comparison diagram of immunohistochemical staining of mouse brain provided in an embodiment of the present application.
  • Figure 29 is a fluorescence microscope image of mouse hypothalamus and liver provided in an embodiment of the present application.
  • Figure 30 is a comparison diagram of the obesity treatment situation in mice provided by an embodiment of the present application.
  • Figure 31 is a comparison diagram of the treatment of obesity fatty liver in mice provided by an embodiment of the present application.
  • Figure 32 is a comparison diagram of the treatment of mouse Huntington's disease provided by an embodiment of the present application.
  • Figure 33 is a comparison diagram of siRNA and protein in mouse liver, cortex, and striatum provided by an example of the present application.
  • Figure 34 is a comparison diagram of the treatment of Huntington's disease in mice provided by an embodiment of the present application.
  • Figure 35 is a comparison diagram of mHTT protein and toxic aggregates in mouse striatum and cortex provided by an embodiment of the present application;
  • Figure 36 is a comparison diagram of the Parkinson's treatment situation of transgenic mice provided in an embodiment of the present application.
  • Figure 37 is a graph of changes in siRNA concentration in cynomolgus monkey whole blood provided by an embodiment of the present application.
  • FIG. 38 is a construction and characterization diagram of a circuit provided by an embodiment of the present application.
  • Figure 39 shows the enrichment effect of plasmids containing 6 different RNAs in plasma, the detection of siRNA in exosomes and the corresponding gene expression levels provided by an example of the present application.
  • A is the enrichment effect of plasmids in plasma and the amount of extracellular siRNA detection results in exosomes
  • B and C are the protein expression and mRNA expression of EGFR
  • D and E are the protein expression and mRNA expression of TNC.
  • Figure 40 is the enrichment effect in plasma, the detection of siRNA in exosomes, and the corresponding gene expression of plasmids containing RNA fragments composed of any 2 RNA sequences out of 6 different RNAs provided by an embodiment of the present application, in the figure, A is the enrichment effect of plasmids in plasma and the detection results of siRNA in exosomes, B is the protein expression of EGFR and TNC, and C is the mRNA expression of EGFR and TNC.
  • Figure 41 is the enrichment effect in plasma, the detection of siRNA in exosomes, and the corresponding gene expression of plasmids containing RNA fragments composed of any 3 RNA sequences out of 6 different RNAs provided in an embodiment of the present application, in the figure, A is the enrichment effect of plasmids in plasma and the detection results of siRNA in exosomes, B is the protein expression of EGFR and TNC, and C is the mRNA expression of EGFR and TNC
  • Figure 42 shows the enrichment effects in pancreas, brain, plasma and exosomes when the gene circuit provided by an embodiment of the present application contains two different RNA fragments and two different targeting tags.
  • a and B are the enrichment effects when the RNA fragment is siRNA EGFR and the targeting tag is PTP
  • C and D are the enrichment effects when the RNA fragment is siRNA TNC and the targeting tag is PTP
  • E and F are The enrichment effect when the RNA fragment is siRNA EGFR and the targeting tag is RVG
  • G and H are the enrichment effect when the RNA fragment is siRNA TNC and the targeting tag is RVG.
  • Figure 43 shows the expression levels of EGFR and TNC detected in pancreas and brain when the gene circuit provided in an example of the present application contains two different RNA fragments and two different targeting tags.
  • B is the protein expression and mRNA expression of EGFR when the RNA fragment is siRNA EGFR and the targeting tag is PTP;
  • C and D are the protein expression and mRNA expression of EGFR when the RNA fragment is siRNA EGFR and the targeting tag is RVG
  • E and F are the protein expression and mRNA expression of EGFR when the RNA fragment is siR TNC and the targeting tag is PTP;
  • G and H are the protein expression of EGFR when the RNA fragment is siR TNC and the targeting tag is RVG and mRNA expression.
  • Figure 44 shows the enrichment effect in pancreas, brain, plasma, and exosomes when the gene circuit provided by an embodiment of the present application contains two RNA fragments and two different targeting tags at the same time.
  • a and B are the enrichment effects when the RNA fragment is siRNA EGFR+TNC and the targeting tag is PTP
  • C and D are the enrichment effects when the RNA fragment is siRNA EGFR+TNC and the targeting tag is RVG.
  • Figure 45 shows the expression levels of EGFR and TNC detected in the pancreas and brain when the gene circuit provided in an example of the present application contains two RNA fragments and two different targeting tags at the same time
  • a in the figure and B are the protein expression and mRNA expression of EGFR when the RNA fragment is siRNA EGFR+TNC and the targeting tag is PTP
  • C and D are the protein expression of EGFR when the RNA fragment is siRNA EGFR+TNC and the targeting tag is RVG Expression and mRNA expression
  • E and F are the protein expression and mRNA expression of TNC when the RNA fragment is siRNA EGFR+TNC and the targeting tag is PTP
  • G and H are the RNA fragment is siRNA EGFR+TNC , targeting When the label is RVG, the protein expression and mRNA expression of TNC.
  • Figure 46 shows the enrichment effect in pancreas, brain, plasma, and exosomes when the gene circuit provided by an embodiment of the present application contains two different RNA fragments and two targeting tags at the same time.
  • a and B are the enrichment effects when the RNA fragment is siRNA EGFR and the targeting tag is PTP-RVG
  • C and D are the enrichment effects when the RNA fragment is siRNA TNC and the targeting tag is PTP-RVG.
  • Figure 47 shows the expression levels of EGFR and TNC detected in pancreas and brain when the gene circuit provided in an example of the present application contains two different RNA fragments and two targeting tags at the same time,
  • a in the figure and B are the protein expression and mRNA expression of EGFR when the RNA fragment is siRNA EGFR and the targeting tag is PTP-RVG;
  • C and D are the protein expression of TNC when the RNA fragment is siRNA TNC and the targeting tag is PTP-RVG expression and mRNA expression.
  • Figure 48 shows the enrichment effect in pancreas, brain, plasma, and exosomes when the gene circuit provided by an embodiment of the present application contains two RNA fragments and two targeting tags at the same time
  • a in the figure is the enrichment effect in pancreas and brain when the RNA fragment is siRNA EGFR+TNC and the targeting tag is PTP-RVG
  • B is the enrichment effect in plasma and exocrine when the RNA fragment is siRNA EGFR+TNC and the targeting tag is PTP-RVG enrichment effect in the body.
  • Figure 49 shows the expression levels of EGFR and TNC detected in pancreas and brain when the gene circuit provided in an example of the present application contains two RNA fragments and two targeting tags at the same time
  • a and B in the figure are the protein expression and mRNA expression of EGFR when the RNA fragment is siRNA EGFR+TNC and the targeting tag is PTP-RVG
  • C and D are when the RNA fragment is siRNA EGFR+TNC and the targeting tag is PTP-RVG, TNC protein expression and mRNA expression.
  • Figure 50 shows the enrichment effects of two gene circuits, Albumin-siR EGFR and Albumin-RVG-siR EGFR provided in an example of the present application, in plasma and brain, and the expression levels of EGFR.
  • A is the two genes The enrichment effect of the circuit in plasma
  • B is the enrichment effect of the two gene circuits in the brain
  • C is the protein expression and mRNA expression of EGFR detected by the two gene circuits.
  • Figure 51 shows the lung enrichment effect and therapeutic effect of a delivery system with a 5' flanking sequence homology greater than 80% provided by an embodiment of the present application, in the figure A is two 5' flanking sequences with a homology greater than 80% Lung enrichment effect when RVG is connected and not connected to RVG, B is the protein expression of EGFR when the 5' flanking sequences with more than 80% homology are connected to RVG and not connected to RVG respectively, C is 2 The mRNA expression levels of EGFR when the 5' flanking sequences with more than 80% homology are respectively connected to RVG and not connected to RVG.
  • Figure 52 shows the lung enrichment effect and therapeutic effect of a delivery system with a loop sequence homology greater than 80% provided by an embodiment of the present application.
  • A shows two loop sequences with a homology greater than 80% connected to the RVG respectively and the lung enrichment effect when RVG is not connected
  • B is the protein expression of EGFR when two loop sequences with homology greater than 80% are connected to RVG and not connected to RVG respectively
  • C is two loop sequences with homology greater than 80% The mRNA expression of EGFR when the % loop sequence is connected to RVG and not connected to RVG, respectively.
  • Figure 53 shows the lung enrichment effect and therapeutic effect of a delivery system with a 3' flanking sequence homology greater than 80% provided by an embodiment of the present application, in the figure A is two 3' flanking sequences with a homology greater than 80% Lung enrichment effect when RVG is connected and not connected to RVG, B is the protein expression of EGFR when two 3' flanking sequences with homology greater than 80% are connected to RVG and not connected to RVG, C is 2 The 3' flanking sequences with more than 80% homology were respectively connected to RVG and not connected to RVG, and the mRNA expression of EGFR.
  • Figure 54 is a graph showing the detection results of EGFR expression after intravenous injection of the delivery system containing 3 RNA sequences of different lengths provided in an embodiment of the present application.
  • A is the detection result of the protein expression of EGFR
  • B is the mRNA expression of EGFR Test results.
  • Figure 55 is the EGFR siRNA content in lung tissue 9 hours after intravenous injection of a delivery system containing sequence 4 and two sequences 4-1 and 4-2 with more than 80% homology to sequence 4 provided in an embodiment of the present application ( Enrichment) detection results, in the figure A is the detection result of sequence 4, B is the detection result of sequence 4-1, and C is the detection result of sequence 4-2.
  • 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 (immunocytochem istry).
  • 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, the system comprising a plasmid carrying the RNA fragment to be delivered, the plasmid can be enriched in the organ tissue of the host, and endogenous in the organ tissue of the host A complex structure containing the RNA fragment is spontaneously formed, the complex structure is able to enter and bind the target tissue, and the RNA fragment is transported into the target tissue.
  • 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.
  • Figures 42-49 show the detection results of any combination of two different RNA fragments and two different targeting tags.
  • RNA fragment 1 is siRNA EGFR
  • RNA fragment 2 is siRNA TNC
  • targeting tag 1 is PTP
  • targeting tag 2 is RVG.
  • 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.
  • Albumin is the promoter
  • RVG is the targeting tag
  • siRNA EGFR is the RNA fragment targeting the EGFR protein. Since the gene circuits cannot function without RNA fragments, the enrichment effect of the two gene circuits Albumin-siR EGFR and Albumin-RVG-siR EGFR in plasma and brain and the expression of EGFR were detected.
  • 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 more than 80% homology thereto, including sequences 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.
  • Figures 51-53 respectively show the enrichment effect and therapeutic effect in the lungs of two 5' flanking sequences, loop sequences and 3' flanking sequences with a homology greater than 80% after they are constructed into the delivery system (plasmid).
  • Figure 51 shows the lung enrichment effect and therapeutic effect of plasmids with 5' flanking sequence homology greater than 80%
  • Figure 52 shows the lung enrichment effect and therapeutic effect of plasmids with loop sequence homology greater than 80%
  • Figure 5 53 is the enrichment effect and therapeutic effect of plasmids with 3' flanking sequence homology greater than 80% in lung.
  • 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 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • Figure 55 shows the detection results of EGFR siRNA content in lung tissue 9 hours after intravenous injection of the delivery system constructed by sequence 4 and two sequences 4-1 and 4-2 with more than 80% homology to sequence 4.
  • Sequence 4 is specifically shown in Table 3 below.
  • 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 account, and the effect is the best.
  • nt nucleotides
  • RNA sequences with different lengths are shown in Table 4 below.
  • Figure 54 respectively shows the detection of EGFR expression after intravenous injection of delivery systems (plasmids) constructed with RNA sequences of different lengths, wherein the plasmids with RNA sequence lengths of 18, 20, and 22 respectively correspond to CMV- siRNA (18) , CMV-siR E (20), CMV-siR E (22).
  • plasmids constructed with RNA sequences of different lengths, wherein the plasmids with RNA sequence lengths of 18, 20, and 22 respectively correspond to CMV- siRNA (18) , CMV-siR E (20), CMV-siR E (22).
  • RNA sequence is selected from: siRNA of EGFR gene, siRNA of KRAS gene, siRNA of VEGFR gene, siRNA of mTOR gene, siRNA of TNF- ⁇ gene, siRNA of integrin- ⁇ gene, siRNA of B7 gene, TGF- ⁇ 1 gene siRNA, siRNA of H2-K gene, siRNA of H2-D gene, siRNA of H2-L gene, siRNA of HLA gene, siRNA of GDF15 gene, antisense strand of miRNA-21, antisense strand of miRNA-214, siRNA of TNC gene, siRNA of PTP1B gene, siRNA of mHTT gene, siRNA of Lrrk2 gene, siRNA of ⁇ -synuclein gene, or RNA sequences with more than 80% homology to the above sequences, or nucleic acid molecules encoding the above RNAs.
  • the siRNA of the above-mentioned various genes and the antisense strand of miRNA can inhibit the expression or mutation of the gene or miRNA, thereby achieving the effect of inhibiting disease.
  • diseases include, but are not limited to: cancer, pulmonary fibrosis, colitis, obesity, cardiovascular disease caused by obesity, type 2 diabetes, Huntington's disease, Parkinson's disease, myasthenia gravis, Alzheimer's disease, transplantation Object-versus-host disease and related diseases.
  • the related diseases here refer to the related diseases or complications, sequelae, etc. that occur during the formation or development of any one or more of the above diseases, or other diseases that are related to the above diseases.
  • cancer includes but is not limited to: stomach cancer, kidney cancer, lung cancer, liver cancer, brain cancer, blood cancer, colon cancer, skin cancer, lymphoma, breast cancer, bladder cancer, esophageal cancer, head and neck squamous cell carcinoma, hemangioma, stromal cell tumor, melanoma.
  • the number of RNA sequences in the RNA fragment is one, two or more. For example, if you need to treat glioma, you can use EGFR gene siRNA and TNC gene siRNA on the same plasmid vector in combination; if you need to treat enteritis, you can use TNF- ⁇ gene siRNA and integrin- ⁇ gene siRNA at the same time. siRNA and siRNA of the B7 gene.
  • 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 (self-assembly), 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 design 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 to become targeting structures 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.
  • the targeting tag is selected from one of targeting peptides, targeting proteins or antibodies with targeting function.
  • the selection of targeting tags is a process that requires creative work. On the one hand, it is also necessary to ensure that the targeting tag 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 (nucleot
  • RVG targeting peptide and RVG-LAMP2B fusion protein can precisely target brain tissue; GE11 targeting peptide and GE11-LAMP2B fusion protein can precisely target organs and tissues with high EGFR expression, such as EGFR-mutated lung cancer tissue; PTP targeting Peptides and PTP-LAMP2B fusion proteins can precisely target the pancreas, especially the plectin-1 protein specifically expressed in human and murine pancreatic cancer tissues; TCP-1 targeting peptides and TCP-1-LAMP2B fusion proteins can precisely target To the colon; MSP targeting peptide, MSP-LAMP2B fusion protein can precisely target muscle tissue.
  • RVG targeting peptide, RVG-LAMP2B fusion protein can be used with EGFR gene siRNA, TNC gene siRNA or a combination of the two to treat glioblastoma, and can also be used with PTP1B gene siRNA to treat obesity, and can also be used with mHTT Gene siRNA for Huntington's disease, and LRRK2 siRNA for Parkinson's disease;
  • GE11 targeting peptide and GE11-LAMP2B fusion protein can be combined with EGFR gene siRNA to treat lung cancer and other diseases caused by high EGFR gene expression or mutation;
  • TCP- 1 Targeting peptide or TCP-1-LAMP2B fusion protein can be combined with TNF- ⁇ gene siRNA, integrin- ⁇ gene siRNA, B7 gene siRNA or any combination of the above three to treat colitis or colon cancer.
  • 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.
  • the core circuit consists of a promoter part and an siRNA expression part and is designed to generate and organize siRNA as a payload for exosomes.
  • Other composable components plug-ins
  • plug-ins can be integrated into the framework of the core line for plug-and-play functionality.
  • two types of combinable moieties were combined to optimize the effect of the siRNA: one that modifies the membrane-anchored protein of the exosome for tissue selectivity; the other that co-expresses a second siRNA to inhibit both molecules simultaneously target.
  • EGFR Epidermal growth factor receptor
  • HEK293T Human embryonic kidney 293t cells
  • hep1-6 mouse hepatoma cells
  • siRNA production efficiencies were compared, see Figure 38b.
  • HEK293T cells were transfected with CMV-scrR or CMV-siR E gene, and the exosomes in the cell culture medium were observed.
  • Nanoparticle tracking analysis showed that the number of exosomes secreted in each group was similar, and the size distribution was similar, with a peak between 128-131 nm.
  • Transmission electron microscopy confirmed that the purified exosomes presented typical round vesicle morphology with correct size. Furthermore, enrichment for specific exon markers (CD63, TSG101 and CD9) was only detected in purified exosomes, but not in cell culture medium.
  • a sequence encoding an N-terminally fused targeting tag of the Lamp2b protein was inserted downstream of the CMV promoter, see Figure 38a.
  • This tag is anchored to the surface of exosomes via Lamp2b, thereby guiding the delivery of the composite exosomes to the desired tissue.
  • the central nervous system targeting RVG peptides was selected as a marker to introduce exosomes into the brain (RVG has been shown to facilitate the passage of exosomes across the blood-brain barrier into nerve cells), first assessing promoter initiation Efficiency of RVG-Lamp2b fusion protein expression.
  • the CMV promoter has a certain effect on the production of RVG-Lamp2b mRNA and the marker protein eGFP in HEK293T cells, while the U6 promoter has no effect, which confirms the advantages of the CMV promoter connecting each part of the circuit.
  • Immunoprecipitation was then used to verify the correct expression of the guide targeting tag on the exosome surface. Due to the temporary lack of anti-RVG antibodies in the assay, a Flag tag was used to temporarily replace RVG. Following transfection of HEK293T and Hepa 1-6 cells with the CMV-directed Flag-Lamp2b circuit, intact exosomes were successfully immunoprecipitated with anti-Flag beads, see Fig.
  • TNC tenascin-C
  • HEK293T cells were transfected with a CMV targeting circuit encoding EGFR and TNC siRNAs and an RVG marker (CMV-RVG-siR E ).
  • CMV-RVG-siR E an RVG marker
  • AGO2 is widely expressed in organisms and is a core component of the RNA-induced silencing complex. It has endoribonuclease activity and can inhibit the expression of target genes by promoting the maturation of siRNA and regulating its biosynthesis and function.
  • siRNA processing is dependent on Argonaute 2 (AGO2)
  • AGO2 Argonaute 2
  • RISC RNA-induced silencing complex
  • mice were intravenously injected with a CMV eGFP siRNA E circuit co-expressing eGFP protein and EGFR siRNA, and the results are shown in Figure 2.
  • the eGFP fluorescence in the mouse liver gradually increased over time, about 12 hours It reached the peak value and decreased to the background level after 48 hours, 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 similar to those injected with PBS.
  • the liver, lung, spleen, and kidney also had no tissue damage.
  • the RNA delivery system uses a plasmid as a carrier and the plasmid as a mature injectable substance, and 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 provided in this example 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 has It is beneficial to receptor cell absorption, cytoplasmic release and lysosome escape, and the required dose is low.
  • the medicament includes a plasmid carrying the RNA fragment to be delivered, the plasmid can be enriched in the organ tissue of the host, and endogenously and spontaneously form the RNA fragment containing the RNA fragment in the organ tissue of the host.
  • a complex structure capable of entering and binding to a target tissue to deliver the RNA fragment into the target tissue.
  • 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.
  • the six RNAs are: siRNA (target gene is EGFR), siRT (target gene is TNC), shRE (target gene is EGFR), shRT ( target gene is TNC), miR- 7 (the target gene is EGFR), miR-133b (the target gene is EGFR),
  • Figure 39 shows the enrichment effect of the 6 different RNA plasmids in plasma, the detection of siRNA in exosomes and the corresponding gene expression
  • Figure 40 It is the enrichment effect of 4 groups of plasmids in plasma composed of any 2 RNA sequences of the 6 RNA species provided above, the detection of siRNA in exosomes and the corresponding gene expression
  • Figure 41 is any 3 of the 6 RNA species provided above. The enrichment effect of the three groups of plasmids composed of RNA sequences in plasma, the detection of siRNA in exosomes, and the corresponding gene expression.
  • RNA sequences are specifically shown in Table 5 below.
  • siRE precursor sequence ATACCTATTCCGTTACACACTGTTTTTGGCCACTGACTGACAGTTGTGTAGGAATAGGTAT
  • siRT precursor sequence CACACAAGCCATCTACACATGGTTTTGGCCACTGACTGACCATGTGTATGGCTTGTGTG shRE precursor sequence ATACCTATTCCGTTACACACTGTTTTTGGCCACTGACTGACAGTTGTGTAACGGAATAGGTAT shRT precursor sequence CACACAAGCCATCTACACATGGTTTTGGCCACTGACTGACCATGTGTAGATGGCTTGTGTG miR-7 hsa-miR-7 miR-133b hsa-miR-133b
  • 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 may be used to treat cancer, pulmonary fibrosis, colitis, obesity, cardiovascular disease caused by obesity, type 2 diabetes, Huntington's disease, Parkinson's disease, myasthenia gravis, Alzheimer's disease or graft resistance Drugs for host disease.
  • 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 in a medicine, and the medicine is a medicine for treating lung cancer.
  • mice were selected, and mice were injected with mouse lung cancer cells (LLC cells), and then the mice were injected with PBS buffer/CMV-scrR/gefitinib/CMV-siR E every two days.
  • LLC cells mouse lung cancer cells
  • mice were subjected to survival analysis and tumor assessment, respectively, starting on day 30 and ending on day 44.
  • the horizontal axis represents time
  • the vertical axis represents survival rate
  • FIG. 11C the figure shows the 3D modeling of mouse lung tissue based on CT images before and after treatment in mice injected with PBS buffer/CMV-scrR/gefitinib/CMV-siR E .
  • the tumors of mice injected with CMV- siRE were significantly reduced.
  • FIG. 11D is a comparison chart of the tumor volume (mm 3 ) of mice injected with PBS buffer/CMV-scrR/gefitinib/CMV- siRE before and after treatment. It can be seen that, The tumor volume of mice injected with CMV- siRE was significantly reduced. The tumor volume of mice injected with PBS buffer/CMV-scrR/gefitinib not only did not decrease, but also increased to varying degrees.
  • this figure is a comparison chart of western blot of normal mice and mice injected with PBS buffer/CMV-scrR/gefitinib/CMV-si RE . It can be seen that PBS buffer/CMV-scrR/ Gefitinib-treated mice had significantly higher levels of the EGFR gene.
  • FIG. 11F the figure shows the comparison of EGFR miRNA levels in normal mice and mice injected with PBS buffer/CMV-scrR/gefitinib/CMV-siR E. It can be seen that PBS buffer/CMV-scrR was injected Mice treated with gefitinib/gefitinib had relatively higher levels of EGFR miRNAs.
  • CMV-siR E has a significant therapeutic effect on EGFR-mutated lung cancer.
  • HE staining and immunohistochemical staining were performed on mice injected with PBS buffer/CMV-scrR/gefitinib/CMV-siR E , respectively.
  • the results are shown in Figure 12A- Figure 12B. scrR/gefitinib mice had more expression.
  • the staining areas of EGFR and PCNA in mice were counted.
  • the results are shown in Figure 12C- Figure 12D. It can be seen that the staining areas of EGFR and PCNA in mice injected with CMV- siRE are the least, which proves its therapeutic effect on EGFR-mutated lung cancer tumors. most.
  • KRAS G12D p53 -/- mice were selected, and from the 50th day to the 64th day after inhalation of Adv-Cre, the mice were injected with PBS buffer/CMV-scrR/Gefi every two days The mice were treated with tinib/CMV- siRE , and the mice were subjected to survival analysis and tumor assessment, respectively.
  • the horizontal axis represents the time after infection, and the vertical axis represents the survival rate, and it can be seen from this figure that the mice injected with CMV-siR K have a higher survival rate.
  • the figure shows the 3D modeling of mouse lung tissue before and after treatment in mice injected with CMV-scrR/CMV-siR K. It can be seen that the injection of CMV-siR K Can significantly inhibit the growth of lung cancer tumors.
  • the figure is a comparison of the number of tumors (mm 3 ) in mice injected with CMV-scrR/CMV-siR K before and after treatment. It can be seen that the tumors in mice injected with CMV-siR K Volume growth is slow. The tumor volume of mice injected with CMV-scrR increased significantly.
  • FIG. 13G which is a comparison of the related KRAS mRNA levels in mice injected with CMV-scrR/CMV-siR K , it can be seen that the related KRAS mRNA levels in mice injected with CMV-scrR are relatively high.
  • CMV-siR K has a significant therapeutic effect on KRAS-mutated lung cancer tumors.
  • HE staining and immunohistochemical staining were performed on mice injected with CMV-scrR/CMV-siR K respectively.
  • the results are shown in Figure 14A, Figure 14D, and Figure 14E. It can be seen that KRAS and p-AKT in CMV-scrR-injected mice , p-ERK has more expression and higher percentage of staining.
  • Western blot was used to detect the expression levels of related proteins in mice. The results were shown in Figure 14B and Figure 14C, and the related proteins were more expressed in CMV-scrR-injected mice. This also shows that CMV-siR K has a significant inhibitory effect on KRAS-mutated lung cancer tumors.
  • this embodiment provides an application of an RNA delivery system in a medicine, and the medicine is a medicine for treating renal cancer.
  • mice Different mice were injected with PBS buffer/control plasmid/VEGFR siRNA plasmid/mTOR siRNA plasmid/MIX siRNA plasmid (the combination of VEGFR siRNA and mTOR siRNA)/Sunitinib/Everolimus, The development of renal cancer tumors in mice was observed, and the results are shown in Figure 15 and Figure 16 . It can be seen that the development of kidney cancer in mice injected with MIX siRNA plasmid was most significantly inhibited, while the development of kidney cancer in mice injected with PBS buffer/control plasmid was more rapid.
  • VEGFR siRNA and mTOR siRNA has a significant therapeutic effect on renal cancer tumors.
  • this embodiment provides an application of an RNA delivery system in a medicine, and the medicine is a medicine for treating colitis. The effects of treatment are described in detail.
  • the experimental groups were anti-TNF- ⁇ (0.5) group, anti-TNF- ⁇ (5) group, anti-TNF- ⁇ (20 ) group; the control group were mock group, scr-RNA group and IFX group.
  • the anti-TNF- ⁇ (0.5) group, the anti-TNF- ⁇ (5) group, and the anti-TNF- ⁇ (20) group used plasmids to encapsulate the TNF- ⁇ siRNA system (CMV-siR TNF- ⁇ ), respectively, and the tail vein 0.5 ⁇ L, 5 ⁇ L and 20 ⁇ L of CMV-siR TNF- ⁇ solutions were injected into mice.
  • CMV-siR TNF- ⁇ TNF- ⁇ siRNA system
  • the mock group was the negative control group, and the scr-RNA plasmid and IFX (infliximab) were injected into the tail vein of mice in the scr-RNA group and the IFX group, respectively.
  • the in vivo expression of the plasmid system was monitored by small animals, and then the mice were sacrificed to observe the colon.
  • 0.5) group, anti-TNF- ⁇ (5) group and anti-TNF- ⁇ (20) group had relatively longer colon lengths, and the higher the TNF- ⁇ siRNA injection dose, the longer the mice colon lengths were. This indicates that the plasmid-encapsulated TNF- ⁇ siRNA system has different degrees of improvement in the shortening of colon length caused by chronic inflammation.
  • the disease activity index (Disease activity index) of mice was evaluated, and the results are shown in Figure 17C.
  • the disease activity index of mice was higher, while the disease activity index of mice in anti-TNF- ⁇ (20) group and IFX group was lower.
  • TNF- ⁇ mRNA was detected in the colon of mice, and the results were shown in Figure 17D. It can be seen that the CMV-siR TNF- ⁇ system can reduce the expression and secretion of TNF- ⁇ in the colon; TNF- ⁇ was detected in the colon of mice, and the results are shown in the figure As shown in Figure 17E, it can be seen that the AAV system can produce a certain amount of TNF- ⁇ ; the pro-inflammatory factors IL-6, IL-12p70, IL-17A, and IL-23 in the colon were detected, and the results were shown in Figure 17F, It can be seen that the secretion of inflammatory factors in the high-dose group was lower than that in the control group.
  • HE staining was performed on mouse colon sections, and pathological score statistics were performed. The results are shown in Figure 18A and Figure 18B.
  • Anti-TNF- ⁇ (0.5) group, anti-TNF- ⁇ (5) group, anti-TNF- ⁇ group were seen.
  • the experimental groups were anti-TNF- ⁇ group, anti-integrin- ⁇ group, anti-B7 group, anti-mix group, and the control group were respectively Mock group, PBS group, scr-RNA group.
  • the anti-TNF- ⁇ group, anti-integrin- ⁇ group, anti-B7 group and anti-mix group used plasmid-wrapped TNF- ⁇ siRNA system (CMV-siR TNF- ⁇ ), integrin- ⁇ siRNA system (CMV-siR integrin- ⁇ ), B7siRNA system (CMV-siR B7 ), mix siRNA (CMV-siR mix is CMV-siR TNF- ⁇ +integrin- ⁇ +B7 ) system, 20 ⁇ L was injected into mice through tail vein, and the siRNA was monitored in vivo by small animals. The in vivo expression of the system shows that the above system is stably expressed in vivo, especially in the liver.
  • the mock group was the negative control group, and the scr-RNA plasmid and PBS solution (phosphate buffered saline) were injected into the tail vein of mice in the scr-RNA group and the PBS group, respectively.
  • PBS solution phosphate buffered saline
  • the DSS-induced chronic colitis model was constructed, during which daily weighing recordings were performed.
  • the results are shown in Figure 19A. It can be seen that the weight growth of the mice in the anti-mix group was the most stable, that is, the plasmid-encapsulated CMV-siR TNF- ⁇ +integrin
  • the - ⁇ +B7 system can significantly reduce the weight loss of mice with chronic colitis.
  • the mice in the anti-TNF- ⁇ group, anti-integrin- ⁇ group and anti-B7 group also recovered significantly faster during the inflammation remission period. in scr-RNA group and PBS group.
  • the disease activity index of mice was evaluated, and the results are shown in Figure 19C. It can be seen that the disease activity index of mice in the scr-RNA group and PBS group was higher, while the anti-TNF- ⁇ group, anti-integrin- The disease activity index of mice in ⁇ group, anti-B7 group and anti-mix group decreased in turn.
  • TNF- ⁇ mRNA, integrin mRNA and B7 mRNA were detected in mouse plasma, liver and colon.
  • the results are shown in Figure 19D-FIG. 19F. It can be seen that the system generates a certain amount of stably expressed RNA in plasma, liver and colon. , and the system significantly reduced colonic TNF- ⁇ , integrin and B7 mRNA expression.
  • mice in the 4 experimental groups especially the mice in the anti-mix group, have higher colonic mucosal integrity and less infiltration of immune cells. Colonic crypt abscesses and colonic congestion and hemorrhage were also significantly lighter than controls.
  • liver-friendly plasmids to encapsulate the CMV-siR TNF- ⁇ +integrin- ⁇ +B7 circuit can achieve long-term TNF- ⁇ mRNA, B7 mRNA and integrin mRNA expression and silencing of multiple target genes, and significantly relieve colon inflammation. It has great drug potential and clinical research value.
  • this embodiment provides an application of an RNA delivery system in a medicine, and the medicine is a medicine for treating pulmonary fibrosis.
  • 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 experimental groups were Anti-miR-21(1mg/kg) group, Anti-miR-21(5mg/kg) group, Anti-miR-21(10mg/kg) group, TGF- ⁇ 1 siRNA(1mg/kg) group , TGF- ⁇ 1 siRNA (5mg/kg) group, TGF- ⁇ 1 siRNA (10mg/kg) group, Anti-miR-21+TGF- ⁇ 1 siRNA (10mg/kg) group, Pirfenidone (300mg/kg) group, control group They are Normal group, PBS group and scrRNA group, respectively.
  • Anti-miR-21 (1mg/kg) group, Anti-miR-21 (5mg/kg) group and Anti-miR-21 (10mg/kg) group were injected into the tail vein of mice with pulmonary fibrosis respectively 1mg/kg, 5mg/kg, 10mg/kg miR-21 siRNA plasmid, TGF- ⁇ 1 siRNA (1mg/kg) group, TGF- ⁇ 1 siRNA (5mg/kg) group, TGF- ⁇ 1 siRNA (10mg/kg) group
  • Mice with pulmonary fibrosis were injected with 1 mg/kg, 5 mg/kg and 10 mg/kg of TGF- ⁇ 1 siRNA plasmids into the tail vein, and the Anti-miR-21+TGF- ⁇ 1 siRNA (10 mg/kg) group was injected with 1 mg/kg, 5 mg/kg and 10 mg/kg.
  • mice with pulmonary fibrosis were injected with 10 mg/kg Anti-miR-21 and TGF- ⁇ 1 siRNA plasmids in the tail vein, and the Pirfenidone (300 mg/kg) group was injected with 300 mg/kg of pirfenidone in the tail vein of mice with pulmonary fibrosis , Normal group was the normal control group, PBS group and scrRNA group were injected with PBS solution and control plasmid into the tail vein of mice with pulmonary fibrosis, respectively.
  • hydroxyproline is the main component of collagen, and its content reflects the degree of pulmonary fibrosis. It can be seen from Figure 21 that the Anti-miR-21 (5mg/kg) group, Anti-miR-21 (10mg/kg) group , TGF- ⁇ 1 siRNA (10mg/kg) group and Anti-miR-21+TGF- ⁇ 1 siRNA (10mg/kg) group had relatively lower hydroxyproline content, and their pulmonary fibrosis was inhibited.
  • H&E staining was performed on the lungs of mice in each group, and the results are shown in Figure 24. It can be seen that the alveolar space of the mice in the PBS group and the scrRNA group was widened, the inflammatory cells were infiltrated, and the alveolar structure was damaged, while the lung tissue in the experimental group was relatively normal.
  • TGF- ⁇ 1 siRNA (1mg/kg) group TGF- ⁇ 1 siRNA (1mg/kg) group
  • TGF- ⁇ 1 siRNA 5mg/kg
  • TGF- ⁇ 1 siRNA (10mg/kg) group Pirfenidone (300mg/kg) group
  • TGF- ⁇ 1 protein level and TGF- ⁇ 1 mRNA level in mice in TGF- ⁇ 1 siRNA (10 mg/kg) group the results are shown in Figure 25A- Figure 25C, it can be seen that TGF- ⁇ 1 siRNA (10 mg/kg) group mice TGF- ⁇ 1 protein level and TGF- ⁇ 1 mRNA level lowest. This indicated that TGF- ⁇ 1 could be successfully delivered to the lungs to function after the corresponding siRNA expression plasmid was injected into the tail vein.
  • mice in the Normal group, PBS group, scrRNA group, Anti-miR-21 (1mg/kg) group, Anti-miR-21 (5mg/kg) group and Anti-miR-21 (10mg/kg) group were detected respectively.
  • miR-21 level the results are shown in Figure 25D, it can be seen that the anti-miR-21 (10 mg/kg) group has the highest relative miR-21 level. This indicated that the antisense strand of miR-21 could be successfully delivered to the lungs to function after the corresponding antisense strand expression plasmid was injected into the tail vein.
  • liver-affinity plasmids to encapsulate the CMV-siR miR-21 , CMV-siR TGF- ⁇ 1 , CMV-siR miR-21+TGF- ⁇ 1 circuits can significantly alleviate the degree of pulmonary fibrosis, and has great potential for patent medicine potential, and clinical research value.
  • this embodiment provides an application of an RNA delivery system in a medicine, and the medicine is a medicine for treating glioblastoma.
  • the application of the RNA delivery system in the treatment of glioblastoma is specifically described in conjunction with the following two experiments.
  • the experimental groups are CMV-siR E group, CMV-siR T group, CMV-RVG-siR E+T group, CMV-siR E+T group, CMV-Flag-siR E+T group, where "E” stands for EGFR , "T” represent TNC, and the control groups are the PBS group, the CMV-scrR group, and the CMV-Flag-scrR group, respectively.
  • E stands for EGFR
  • T represent TNC
  • the control groups are the PBS group, the CMV-scrR group, and the CMV-Flag-scrR group, respectively.
  • the specific experimental process is shown in Figure 26A.
  • the experimental groups were CMV-RVG-siR E group and CMV-RVG-siR E+T group, respectively, and the control group were PBS group and CMV-scrR group, respectively.
  • mice were selected, and glioblastoma cells (U-87MG-Luc cells) were injected into the mice. From the 7th day to the 21st day, the mice were injected every two days. One treatment with PBS buffer/CMV-scrR/CMV-RVG-siR E /CMV-RVG-siR E+T (5 mg/kg), mice were subjected to survival analysis and tumor assessment, respectively. On the 7th, 14th, 28th, and 35th days, the mice were detected by BLI in vivo imaging, respectively.
  • this figure is a comparison chart of BLI in vivo imaging detection of mice on the 7th, 14th, 28th and 35th days. It can be seen that the mice in the CMV-RVG-siR E+T group have glioblastoma The tumor inhibition effect was the most significant.
  • FIG. 27C which is a comparison chart of the survival rate of mice in each group, it can be seen that the mice in the CMV-RVG-siR E+T group have the longest survival time.
  • the graph is a fluorescence comparison graph of each group of mice, which is obtained by luciferase in vivo imaging, and the ordinate reflects the intensity of the lucifer fluorescence signal. Since the gene has been artificially integrated into the implanted tumor, the map reflects tumor progression. It can be seen that the tumors of the mice in the control group developed rapidly, while the tumors of the mice in the experimental group were suppressed to a great extent.
  • this figure is the relative siRNA comparison chart of each group of mice. It can be seen that the level of EGFR siRNA in CMV-RVG-siR E group is higher, and the level of EGFR siRNA in CMV-RVG-siR E+T group is higher. and TNC siRNA levels were higher.
  • the figure is a western blot comparison of mice in each group. It can be seen that the mice in the PBS group, the CMV-scrR group, and the CMV-RVG-siR E group have higher EGFR and TNC gene contents.
  • CMV-RVG-siR E plasmid can inhibit the expression of EGFR and PCNA in the brain
  • CMV-RVG-siR E+T plasmid can inhibit the expression of EGFR, TNC and PCNA in the brain.
  • this embodiment provides an application of an RNA delivery system in a medicine, and the medicine is a medicine for treating obesity.
  • the application of the RNA delivery system in the treatment of obesity is specifically described in conjunction with the following two experiments.
  • the experimental groups were the CMV- siRP group and the CMV-RVG- siRP group, and the control group was the CMV-scrR group, where "P" represented PTP1B.
  • the CMV- siRP group, CMV-RVG- siRP group, and CMV-scrR group were injected with 5 mg/kg of CMV- siRP plasmid, CMV-RVG- siRP plasmid, and CMV-scrR plasmid, respectively, and then obtained each
  • the hypothalamus and liver fluorescence microscope images of the mice in the group were shown in Figure 29. The results showed that PTP1B siRNA could be delivered to the hypothalamus.
  • the experimental groups were CMV- siRP group and CMV-RVG- siRP group respectively, and the control groups were PBS group and CMV-scrR group respectively.
  • Figure 30B is a comparison chart of the body weight of mice in each group, it can be seen that the body weight of mice in the CMV-RVG- siRP group is the most stable.
  • mice The oxygen consumption, respiratory exchange ratio, activity level, and heat production of mice with different treatments were measured for 72 hours in a metabolic cage, and the average values were drawn for statistical analysis.
  • the results are shown in Figure 30D- Figure 30G .
  • the results showed that the CMV-RVG-siR P plasmid can effectively increase the oxygen consumption of mice, which means that this group of mice is in a state of high energy metabolism compared with other groups of mice. Normal mice mainly use glucose as their own energy source.
  • the CMV-RVG-siR P plasmid can reduce the respiratory exchange ratio of mice, which means that this group of mice is more inclined to use protein as their own energy than other groups of mice. source.
  • the activity of mice injected with CMV-RVG- siRP plasmid was significantly increased.
  • the mice in the CMV-RVG- siRP group also had significantly increased thermogenesis.
  • the figure is a comparison chart of the initial body weight curves of mice in each group. It can be seen that the mice in the CMV-RVG- siRP group had the lightest body weight.
  • the figure is a comparison chart of the initial food intake curves of mice in each group. As can be seen, the mice in the CMV-RVG- siRP group had the least food intake.
  • FIG. 30J the figure is a comparison chart of serum leptin content of mice in each group. It can be seen that the serum leptin content of mice in the CMV-RVG- siRP group was the lowest.
  • FIG. 30K the figure is the western blot comparison chart of mice in each group. It can be seen that the CMV-RVG- siRP group mice had the lowest PTP1B protein content.
  • the figure is a comparison chart of blood glucose change curves of mice in each group. It can be seen that the mice in the CMV-RVG- siRP group had the lowest blood glucose levels.
  • the figure is a comparison chart of the basal glucose change curves of mice in each group. It can be seen that the mice in the CMV-RVG- siRP group had the lowest basal glucose content.
  • mice The serum total cholesterol (TC), triglyceride (TG), and low density lipoprotein (LDL) of the mice in each group were measured, and the results are shown in Figure 31A .
  • the liver tissue was collected from the mice in each group after treatment, and compared with the normal control. The results are shown in Figure 31D.
  • the liver tissue pathological sections of the mice in the PBS group and the CMV-scrR group showed obvious pathological characteristics of fatty liver.
  • the fatty liver of the mice in the CMV- siRP group was lighter.
  • this embodiment provides an application of an RNA delivery system in a medicine, and the medicine is a medicine for treating Huntington's disease.
  • This example specifically describes the application of the RNA delivery system in the treatment of Huntington's disease in conjunction with the following five experiments.
  • the experimental groups were the CMV-siR mHTT group and the CMV-RVG- siR mHTT group, respectively, and the control groups were the PBS group and the CMV-scrR group, respectively.
  • the experimental procedure is shown in Figure 32A.
  • the mice in the CMV-siR mHTT group, the CMV-RVG-siR mHTT group, the PBS group, and the CMV-scrR group were injected with the CMV-siR mHTT plasmid and CMV-RVG into the tail vein of mice with Huntington's disease.
  • plasma exosomes were isolated, labeled with PKH26 dye, and co-cultured with cells to observe the absorption of exosomes by cells.
  • FIG 32B which is a comparison chart of siRNA levels in plasma exosomes of mice in each group, it can be seen that the levels of siRNA in plasma exosomes of mice in the two experimental groups are higher.
  • Plasma exosomes extracted from mice in each group after injection of plasmid/solution were labeled with PKH26, co-cultured with cells, and photographed with a confocal microscope. The results are shown in Figure 32C, showing that the siRNA-encapsulated exosomes entered the cells.
  • siR mHTT can reduce the level of HTT protein, indicating that siRNA assembled into exosomes can still exert gene silencing function.
  • this figure is the in situ hybridization of the liver, cortex, and striatum of the mice in each group. It can be seen that the liver tissue sections of the mice in the CMV-siR mHTT group and the CMV-RVG-siR mHTT group have obvious The fluorescence of the mice in the CMV-RVG-siR mHTT group showed obvious fluorescence in the cortex and striatum tissue sections. This indicates that RVG can guide exosomal siRNA to enter and function through the blood-brain barrier.
  • the experimental groups were the CMV-siR GFP group and the CMV-RVG-siR GFP group, and the control groups were the PBS group and the CMV-scrR group, respectively.
  • the figures are the slices of the liver, cortex, and striatum of mice in each group. It can be seen that the GFP transgenic mice injected with CMV-siR GFP /CMV-RVG-siR GFP in the liver The level of GFP fluorescence was decreased, and the level of GFP fluorescence was decreased in cortical striatum injected with CMV-RVG-siR GFP . It shows that RVG can guide exosomal siRNA to enter through the blood-brain barrier and function.
  • the experimental groups were the CMV-siR mHTT group, the CMV-RVG-siR mHT T group, and the control group was the CMV-scrR group.
  • mice 8-week-old N17182Q mice were selected, and they were CMV-siR mHTT group, CMV-RVG-siR mHTT group, and CMV-scrR group.
  • Mice with Huntington's disease were injected with CMV-siR in the tail vein.
  • mHTT plasmid, CMV-RVG-siR mHTT plasmid, CMV-scrR plasmid, spin assays were performed on days 0 and 14, and mice were sacrificed for analysis after 14 days.
  • this figure is a comparison chart of the descending latency of mice in wild-type mice, CMV-scrR group, and CMV-RVG-siR mHTT group. It can be seen that at day 0, CMV-scrR group, CMV-RVG- The descending latency of mice in the siR mHTT group was relatively consistent, and on day 14, the descending latency of the mice in the CMV-scrR group was the shortest.
  • Figure 34C is a western bolt image of the striatum of mice in the CMV-scrR group and the CMV-RVG-siR mHTT group
  • Figure 34D is a small mouse in the CMV-scrR group and the CMV-RVG-siR mHTT group.
  • the comparison of the relative mHTT mRNA levels in the striatum of the mice showed that the N171-mHTT protein content and the relative mHTT mRNA level were also higher in the striatum of the CMV-scrR group mice.
  • the experimental group was the CMV-RVG-siR mHTT group
  • the control group was the CMV-scrR group.
  • mice 3-month-old BACHD mice were selected, and mice in the CMV-RVG-siR mHTT group and the CMV-scrR group with Huntington's disease were injected with the CMV-RVG-siR mHTT plasmid and CMV into the tail vein. -scrR plasmid, mice were sacrificed 14 days later for analysis.
  • Figure 34F is the western bolt image of the cortex and striatum of mice in the CMV-scrR group and the CMV-RVG-siR mHTT group. It can be seen that the cortex and striatum of the CMV-RVG-siR mHTT group mice The contents of mutant HTT (Mutant HTT) and endogenous HTT (Endogenous HTT) were lower.
  • Figure 34G is a comparison chart of the relative mHTT protein levels in the cortex and striatum of mice in the CMV-scrR group and the CMV-RVG-siR mHTT group. Relative mHTT protein levels were lower in mice in the RVG-siR mHTT group.
  • Figure 34H shows the immunofluorescence images of mice in the CMV-scrR group and CMV-RVG-siR mHTT group
  • Figure 35I shows the cortex and The comparison of the relative mHTT mRNA levels in the striatum shows that the relative mHTT mRNA levels of the mice in the CMV-RVG-siR mHTT group were lower in both the mouse cortex and the striatum.
  • the experimental group was the CMV-RVG-siR mHTT group
  • the control group was the CMV-scrR group.
  • mice 6-week-old YAC128 mice were selected, and the mice in the CMV-RVG-siR mHTT group and the CMV-scrR group were injected with the CMV-RVG-siR mHTT plasmid and CMV into the tail vein of the mice with Huntington's disease.
  • - scrR plasmid, rotation test was performed on day 0, week 4, and week 8 of the experiment, and then the mice were sacrificed for analysis.
  • this figure is a comparison chart of the descending latency of wild-type mice, CMV-RVG-siR mHTT group, and CMV-scrR group mice. It can be seen that at day 0, CMV-RVG-siR mHTT group, CMV- The descending latencies of the mice in the scrR group were relatively consistent. At the fourth and eighth weeks, the mice in the CMV-scrR group had the shortest descending latencies.
  • the figure is the western bolt image of the cortex and striatum of mice in the CMV-RVG-siR mHTT group and CMV-scrR group. It can be seen that the mutant HTT in the cortex of the CMV-RVG-siR mHTT group mice and endogenous HTT contents were lower, the striatal mutant had lower HTT content, and striatum endogenous HTT content was higher.
  • this figure is the immunofluorescence image of the cortex and striatum of mice in the CMV-RVG-siR mHTT group and CMV-scrR group. It can be seen that the expression of NeuN and EM48 in the CMV-RVG-siR mHTT group mice were lower than the CMV-scrR group.
  • this embodiment provides an application of an RNA delivery system in a medicine, and the medicine is a medicine 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 36A is a western bolt image of LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2
  • Figure 36B 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- siRLRRK2 decreased, indicating that CMV-RVG-siRLRRK2 can pass through the blood-brain barrier and decrease after the liver releases siRNA and assembles into exosomes. Expression of deep brain proteins.
  • FIG 36C 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 show 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.
  • Figure 37A is a graph of changes in siRNA concentration in cynomolgus monkey whole blood injected with a single injection
  • Figure 37B is a graph of siRNA concentration changes in cynomolgus monkey whole blood injected with multiple injections. It can be seen that a single injection of cynomolgus monkeys The siRNA concentration peaked after 6 hours of intravenous injection and then decreased, the siRNA concentration of multiple injections peaked at 3 hours after intravenous injection, and then decreased, and the decrease rate of siRNA concentration of multiple injections was more slowly .

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Abstract

提供一种RNA质粒递送系统及其应用。该RNA质粒递送系统包括携带有所需递送的RNA片段的质粒，该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片段的外泌体，进而能够进入并结合目标组织，将RNA片段送入目标组织。该RNA递送系统安全、可靠、成药性好、通用性强。

Description

一种RNA质粒递送系统及其应用 技术领域
本申请涉及生物医学技术领域，特别涉及一种RNA质粒递送系统及其应用。
背景技术
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、阳离子聚合物和质粒)，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。
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R NAi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RNA质粒递送系统及其应用，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RNA质粒递送系统，该系统包括质粒，所述质粒携带有所需递送的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，所述复合结构能够进入并结合目标组织，以将所述RNA片段送入目标组织。RNA片段送入目标组织后，能够抑制与其相匹配的基因的表达，进而抑制目标组织中疾病的发展。
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序列。
通过图39-41，显示出多种RNA片段单独、任意2种组合、任意3种组合时，所构建的质粒均具有体内富集、自组装及疾病治疗的效果。
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。
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。
通过图42-49表明了2种不同的RNA片段，2种不同的靶向标签任意组合后构建的递送系统均具有体内富集和治疗效果。。
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。其中，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。
通过图50表明了启动子-siRNA及启动子-靶向标签-siRNA基因线路的富集效果以及EGFR的表达量检测结果。
可选地，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片段的删除其中任意1-5位碱基的反向互补序列，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。
通过图51-53，显示将5’侧翼序列、loop序列、3’侧翼序列及三者的同源序列构建质粒后，均具有富集和治疗效果。
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。
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。优选地，序列2是由10-50个碱基组成的序列，更为优选地，序列2是由20-40个碱基组成的序列。
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；
其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
通过图55表明了序列4及其同源序列构建的递送系统均具有富集和治疗效果。可选地，所述质粒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序列。
可选地，所述器官组织为肝脏，所述复合结构为外泌体。
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，所述靶向结构位于复合结构的表面。
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；
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。
可选地，所述所需递送的RNA长度为15-25个核苷酸(nt)。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。
通过图54表明了不同长度的RNA序列所构建的递送系统(质粒)静脉注射后均具有体内富集及治疗效果。
可选地，所述所需递送的RNA选自以下任意一种或几种：EGFR基因的siRNA，KRAS基因的siRNA，VEGFR基因的siRNA，mTOR基因的siRNA，TNF-α基因的siRNA，integrin-α基因的siRNA，B7基因的siRNA，TGF-β1基因的siRNA，H2-K基因的siRNA，H2-D基因的siRNA，H2-L基因的siRNA，HLA基因的siRNA，GDF15基因的siRNA，miRNA-21的反义链，miRNA-214的反义链，TNC基因的siRNA，PTP1B基因的siRNA，mHTT基因的siRNA，Lrrk2基因的siRNA，α-synuclein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。
其中，上述各基因的siRNA均为具有抑制该基因表达的功能的RNA序列，具有抑制各基因表达的功能的RNA序列数量繁多，在此无法一一列举，仅以下述部分效果较优的序列进行举例说明。
EGFR基因的siRNA包括UGUUGCUUCUCUUAAUUCCU、AAAUGAUCUUCAAAAGUGCCC、UCUUUAAGAAGGAAAGAUCAU、AAUAUUCGUAGCAUUUAUGGA、UAAAAAUCCUCACAUAUACUU、其他具有抑制EGFR基因表达的序列以及与上述序列同源性大于80％的序列。
KRAS基因的siRNA包括UGAUUUAGUAUUAUUUAUGGC、AAUUUGUUCUCUAUAAUGGUG、UAAUUUGUUCUCUAUAAUGGU、UUAUGUUUUCGAAUUUCUCGA、UGUAUUUACAUAAUUACAC AC、其他具有抑制KRAS基因表达的序列以及与上述序列同源性大于80％的序列。
VEGFR基因的siRNA包括AUUUGAAGAGUUGUAUUAGCC、UAAUAGACUGGUAACUUUCAU、ACAACUAUGUACAUAAUAGAC、UUUAAGACAAGCUUUUCUCCA、AACAAAAGGUUUUUCAUGGAC、其他具有抑制VEGFR基因表达的序列以及与上述序列同源性大于80％的序列。
mTOR基因的siRNA包括AGAUAGUUGGCAAAUCUGCCA、ACUAUUUCAUCCAUAUAAGGU、AAAAUGUUGUCAAAGAAGGGU、AAAAAUGUUGUCAAAGAAGGG、UGAUUUCUUCCAUUUCUUCUC、其他具有抑制mTOR基因表达的序列以及与上述序列同源性大于80％的序列。
TNF-α基因的siRNA包括AAAACAUAAUCAAAAGAAGGC、UAAAAAACAUAAUCAAAAGAA、AAUAAUAAAUAAUCACAAGUG、UUUUCACGGAAAACAUGUCUG、AAACAUAAUCAAAAGAAGGCA、其他具有抑制TNF-α基因表达的序列以及与上述序列同源性大于80％的序列。
integrin-α基因的siRNA包括AUAAUCAUCUCCAUUAAUGUC、AAACAAUUCCUUUUUUAUCUU、AUUAAAACAGGAAACUUUGAG、AUAAUGAAGGAUAUACAACAG、UUCUUUAUUCAUAAAAGUCUC、其他具有抑制integrin-α基因表达的序列以及与上述序列同源性大于80％的序列。
B7基因的siRNA、UUUUCUUUGGGUAAUCUUCAG、AGAAAAAUUCCACUUUUUCUU、AUUUCAAAGUCAGAUAUACUA、ACAAAAAUUCCAUUUACUGAG、AUUAUUGAGUUAAGUAUUCCU、其他具有抑制B7基因表达的序列以及与上述序列同源性大于80％的序列。
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。
H2-K基因的siRNA包括AAAAACAAAUCAAUCAAACAA、UCAAAAAAACAAAUCAAUCAA、UAUGAGAAGACAUUGUCUGUC、AACAAUCAAGGUUACAUUCAA、ACAAAACCUCUAAGCAUUCUC、其他具有抑制H2-K基因表达的序列以及与上述序列同源性大于80％的序列。
H2-D基因的siRNA包括AAUCUCGGAGAGACAUUUCAG、AAUGUUGUGUAAAGAGAACUG、AACAUCAGACAAUGUUGUGUA、UGUUAACAAUCAAGGUCACUU、AACAAAAAAACCUCUAAGCAU、其他具有抑制H2-D基因表达的序列以及与上述序列同源性大于80％的序列。
H2-L基因的siRNA包括GAUCCGCUCCCAAUACUCCGG、AUCUGCGUGAUCCGCUCCCAA、UCGGAGAGACAUUUCAGAGCU、UCUCGGAGAGACAUUUCAGAG、AAUCUCGGAGAGACAUUUCAG、其他具有抑制H2-L基因表达的序列以及与上述序列同源性大于80％的序列。
HLA基因的siRNA、AUCUGGAUGGUGUGAGAACCG、UGUCACUGCUUGCAGCCUGAG、UCACAAAGGGAAGGGCAGGAA、UUGCAGAAACAAAGUCAGGGU、ACACGAACACAGACACAUGCA、其他具有抑制HLA基因表达的序列以及与上述序列同源性大于80％的序列。
GDF15基因的siRNA包括UAUAAAUACAGCUGUUUGGGC、AGACUUAUAUAAAUACAGCUG、AAUUAAUAAUAAAUAACAGAC、AUCUGAGAGCCAUUCACCGUC、UGCAACUCCAGCUGGGGCCGU、其他具有抑制GDF15基因表达的序列以及与上述序列同源性大于80％的序列。
TNC基因的siRNA包括UAUGAAAUGUAAAAAAAGGGA、AAUCAUAUCCUUAAAAUGGAA、UAAUCAUAUCCUUAAAAUGGA、UGAAAAAUCCUUAGUUUUCAU、AGAAGUAAAAAACUAUUGCGA、其他具有抑制TNC基因表达的序列以及与上述序列同源性大于80％的序列。
PTP1B基因的siRNA包括UGAUAUAGUCAUUAUCUUCUU、UCCAUUUUUAUCAAACUAGCG、 AUUGUUUAAAUAAAUAUGGAG、AAUUUUAAUACAUUAUUGGUU、UUUAUUAUUGUACUUUUUGAU、其他具有抑制PTP1B基因表达的序列以及与上述序列同源性大于80％的序列。
mHTT基因的siRNA包括UAUGUUUUCACAUAUUGUCAG、AUUUAGUAGCCAACUAUAGAA、AUGUUUUUCAAUAAAUGUGCC、UAUGAAUAGCAUUCUUAUCUG、UAUUUGUUCCUCUUAAUACAA、其他具有抑制mHTT基因表达的序列以及与上述序列同源性大于80％的序列。
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。
α-synuclein基因的siRNA包括AUAUAUUAACAAAUUUCACAA、AAGUAUUAUAUAUAUUAACAA、AUAACUUUAUAUUUUUGUCCU、UAACUAAAAAAUUAUUUCGAG、UCGAAUAUUAUUUAUUGUCAG、其他具有抑制α-synuclein基因表达的序列以及与上述序列同源性大于80％的序列。
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。
可选地，该递送系统可用于包括人在内的哺乳动物。
本申请的另一个发明点为提供一种如上任意一段所述的RNA递送系统在药物中的应用。
可选地，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。优选静脉注射。
可选地，所述药物为治疗癌症、肺纤维化、结肠炎、肥胖症、由肥胖症引起的心血管疾病、二型糖尿病、亨廷顿病、帕金森病、重症肌无力、阿尔兹海默病或移植物抗宿主病的药物。
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。
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RNA递送系统以质粒作为载体，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。该递送系统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体内质粒分布与代谢情况对比图；
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；
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；
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；
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；
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；
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；
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；
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；
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；
图11是本申请一实施例提供的小鼠EGFR突变肺癌肿瘤治疗效果对比图；
图12是本申请一实施例提供的小鼠HE染色图、免疫组织染色图以及着色情况统计图；
图13是本申请一实施例提供的小鼠KRAS突变肺癌肿瘤治疗效果对比图；
图14是本申请一实施例提供的小鼠HE染色图、免疫组织染色图以及着色情况统计图；
图15是本申请一实施例提供的小鼠肾癌肿瘤影像对比图；
图16是本申请一实施例提供的小鼠肾癌肿瘤发展情况对比图；
图17是本申请一实施例提供的小鼠结肠炎发展情况对比图；
图18是本申请一实施例提供的小鼠结肠HE染色情况对比图；
图19是本申请一实施例提供的小鼠结肠炎发展情况对比图；
图20是本申请一实施例提供的小鼠结肠HE染色对比图；
图21是本申请一实施例提供的小鼠羟脯氨酸含量对比图；
图22是本申请一实施例提供的小鼠肺部荧光染色图；
图23是本申请一实施例提供的小鼠肺部Masson三色染色图；
图24是本申请一实施例提供的小鼠肺部HE染色图；
图25是本申请一实施例提供的小鼠部分蛋白、mRNA水平对比图；
图26是本申请一实施例提供的小鼠siRNA相关表达对比图；
图27是本申请一实施例提供的小鼠胶质母细胞瘤治疗情况对比图；
图28是本申请一实施例提供的小鼠脑部免疫组织染色对比图；
图29是本申请一实施例提供的小鼠下丘脑、肝脏的荧光显微镜图像；
图30是本申请一实施例提供的小鼠肥胖症治疗情况对比图；
图31是本申请一实施例提供的小鼠肥胖症脂肪肝治疗情况对比图；
图32是本申请一实施例提供的小鼠亨廷顿病治疗情况对比图；
图33是本申请一实施例提供的小鼠肝脏、皮质、纹状体中siRNA、蛋白对比图；
图34是本申请一实施例提供的小鼠亨廷顿病治疗情况对比图；
图35是本申请一实施例提供的小鼠纹状体和皮层mHTT蛋白和毒性聚集体情况对比图；
图36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；
图37是本申请一实施例提供的食蟹猕猴全血siRNA浓度变化图；
图38是本申请一实施例提供的线路的构建与表征图。
图39是本申请一实施例提供的含有6种不同RNA的质粒在血浆中的富集效果、外泌体内siRNA检测以及相应基因表达量，图中，A为质粒在血浆中富集效果和外泌体内siRNA检测结果，B和C为EGFR的蛋白表达量和mRNA表达量，D和E为TNC的蛋白表达量和mRNA表达量。
图40是本申请一实施例提供的含有6种不同RNA中的任意2种RNA序列组成的RNA片段的质粒在血浆中的富集效果、外泌体内siRNA检测以及相应基因表达量，图中，A为质粒在血浆中富集效果和外泌体内siRNA检测结果，B为EGFR和TNC的蛋白表达量，C为EGFR和TNC的mRNA表达量。
图41是本申请一实施例提供的含有6种不同RNA中的任意3种RNA序列组成的RNA片段的质粒在血浆中的富集效果、外泌体内siRNA检测以及相应基因表达量，图中，A为质粒在血浆中富集效果和外泌体内siRNA检测结果，B为EGFR和TNC的蛋白表达量，C为EGFR和TNC的mRNA表达量
图42是本申请一实施例提供的基因线路中含有不同的2种RNA片段和不同的2种靶向标签的情况下，在胰腺、脑、血浆和外泌体中的富集效果，图中A和B为RNA片段为siR EGFR、靶向标签为PTP时所呈现的富集效果，C和D为RNA片段为siR TNC、靶向标签为PTP时所呈现的富集效果，E和F为RNA片段为siR EGFR、靶向标签为RVG时所呈现的富集效果，G和H为RNA片段为siR TNC、靶向标签为RVG时所呈现的富集效果。
图43是本申请一实施例提供的基因线路中含有不同的2种RNA片段和不同的2种靶向标签的情况下，在胰腺、脑中检测的EGFR和TNC的表达量，图中A和B为RNA片段为siR EGFR、靶向标签为PTP时，EGFR的蛋白表达量和mRNA表达量；C和D为RNA片段为siR EGFR、靶向标签为RVG时，EGFR的蛋白表达量和mRNA表达量；E和F为RNA片段为siR TNC、靶向标签为PTP时，EGFR的蛋白表达量和mRNA表达量；G和H为RNA片段为siR TNC、靶向标签为RVG时，EGFR的蛋白表达量和mRNA表达量。
图44是本申请一实施例提供的基因线路中同时含有2种RNA片段、且含有不同的2种靶向标签的情况下，在胰腺、脑、血浆、外泌体中的富集效果，图中A和B为RNA片段为siR EGFR+TNC、靶向标签为PTP时的富集效果，C和D为RNA片段为siR EGFR+TNC、靶向标签为RVG时的富集效果。
图45是本申请一实施例提供的基因线路中同时含有2种RNA片段、且含有不同的2种靶向标签的情况下，在胰腺、脑中检测的EGFR和TNC的表达量，图中A和B为RNA片段为siR EGFR+TNC、靶向标签为PTP时，EGFR的蛋白表达量和mRNA表达量；C和D为RNA片段为siR EGFR+TNC、靶向标签为RVG时，EGFR的蛋白表达量和mRNA表达量；E和F为RNA片段为siR EGFR+TNC、靶向标签为PTP时，TNC的蛋白表达量和mRNA表达量；G和H为RNA片段为siR EGFR+TNC、靶向标签为RVG时，TNC的蛋白表达量和mRNA表达量。
图46是本申请一实施例提供的基因线路中含有不同的2种RNA片段、且同时含有2种靶向标签的情况下，在胰腺、脑、血浆、外泌体中的富集效果，图中A和B为RNA片段为siR EGFR、靶向标签为PTP-RVG时的富集效果，C和D为RNA片段为siR TNC、靶向标签为PTP-RVG时的富集效果。
图47是本申请一实施例提供的基因线路中含有不同的2种RNA片段、且同时含有2种靶向标签的情况下，在胰腺、脑中检测的EGFR和TNC的表达量，图中A和B为RNA片段为siR EGFR、靶向标签为PTP-RVG时，EGFR的蛋白表达量和mRNA表达量；C和D为RNA片段为siR TNC、靶向标签为PTP-RVG时，TNC的蛋白表达量和mRNA表达量。
图48是本申请一实施例提供的基因线路中同时含有2种RNA片段且同时含有2种靶向标签的情况下，在胰腺、脑、血浆、外泌体中的富集效果，图中A为RNA片段为siR EGFR+TNC、靶向标签为PTP-RVG时，胰腺和脑中的富集效果，B为RNA片段为siR EGFR+TNC、靶向标签为PTP-RVG时，血浆和外泌体中的富集效果。
图49是本申请一实施例提供的基因线路中同时含有2种RNA片段且同时含有2种靶向标签的情况下，在胰腺、脑中检测的EGFR和TNC的表达量，图中A和B为RNA片段为siR EGFR+TNC、靶向标签为PTP-RVG时，EGFR的蛋白表达量和mRNA表达量；C和D为RNA片段为siR EGFR+TNC、靶向标签为PTP-RVG时，TNC的蛋白表达量和mRNA表达量。
图50是本申请一实施例提供的提供的Albumin-siR EGFR和Albumin-RVG-siR EGFR两种基因环路在血浆和脑中的富集效果以及EGFR的表达量，图中A为两种基因环路在血浆中的富集效果，B为两种基因环路在脑中的富集效果，C为两种基因环路检测得到的EGFR的蛋白表达量和mRNA表达量。
图51是本申请一实施例提供的5’侧翼序列同源性大于80％的递送系统在肺部富集效果以及治疗效果，图中A为2条同源性大于80％的5’侧翼序列在分别连接RVG和不连接RVG时的肺部富集效果，B为2条同源性大于80％的5’侧翼序列在分别连接RVG和不连接RVG时，EGFR的蛋白表达量，C为2条同源性大于80％的5’侧翼序列在分别连接RVG和不连接RVG时，EGFR的mRNA表达量。
图52是本申请一实施例提供的loop序列同源性大于80％的递送系统在肺部富集效果以及治疗效果，图中A为2条同源性大于80％的loop序列在分别连接RVG和不连接RVG时的肺部富集效果，B为2条同源性大于80％的loop序列在分别连接RVG和不连接RVG时，EGFR的蛋白表达量，C为2条同源性大于80％的loop序列在分别连接RVG和不连接RVG时，EGFR的mRNA表达量。
图53是本申请一实施例提供的3’侧翼序列同源性大于80％的递送系统在肺部富集效果以及治疗效果，图中A为2条同源性大于80％的3’侧翼序列在分别连接RVG和不连接RVG时的肺部富集效果，B为2条同源性大于80％的3’侧翼序列在分别连接RVG和不连接RVG时，EGFR的蛋白表达量，C为2条同源性大于80％的3’侧翼序列在分别连接RVG和不连接RVG时，EGFR的mRNA表达量。
图54是本申请一实施例提供的含有3种不同长度RNA序列的递送系统在静脉注射后EGFR表达量检测结果图，图中A为EGFR的蛋白表达量检测结果，B为EGFR的mRNA表达量检测结果。
图55是本申请一实施例提供的含有序列4以及2条与序列4同源性大于80％的序列4-1、4-2的递送系统静脉注射9小时后肺部组织的EGFR siRNA含量(富集)检测结果，图中A为序列4的检测结果，B为序列4-1的检测结果，C为序列4-2的检测结果。
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。
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。
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。
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。
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。
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。
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。
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(immunocytochem istry)。
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。
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RNA质粒递送系统，该系统包含质粒，所述质粒携带所需递送的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，所述复合结构能够进入并结合目标组织，将所述RNA片段送入目标组织。
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。
图42-49显示了2种不同的RNA片段、2种不同的靶向标签任意组合后的检测结果，具体的，RNA片段1为siR EGFR，RNA片段2为siR TNC，靶向标签1为PTP，靶向标签2为RVG，将RNA片段和靶向标签任意组合后，检测了EGFR siRNA和TNC siRNA在胰腺、脑、血浆和外泌体中的富集效果以及EGFR和TNC的表达量。
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。
图50中，Albumin是启动子，RVG是靶向标签，siR EGFR是靶向EGFR蛋白的RNA片段。由于无RNA片段时，基因线路无法发挥作用，因此检测了Albumin-siR EGFR和Albumin-RVG-siR EGFR两种基因线路在血浆和脑中的富集效果以及EGFR的表达量。
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。
图51-53分别显示了2种同源性大于80％的5’侧翼序列、loop序列以及3’侧翼序列构建进递送系统(质粒)后，在肺部的富集效果以及治疗效果，具体的，图51为5’侧翼序列同源性大于80％的质粒在肺部富集效果以及治疗效果，图52为loop序列同源性大于80％的质粒在肺部富集效果以及治疗效果，图53为3’侧翼序列同源性大于80％的质粒在肺部富集效果以及治疗效果。
上述各序列具体如下表1所示。
名称 序列
5flank1 ggataatggaggcttgctgcaggctgtatgctgaattc
5flank2 ggatactggacgcttgcttaaggctgtatggtgaattc
Loop1 gacttggccactgactgac
Loop2 gttttggccactggctgtc
3flank1 agccgtcaggacatgaggcctgttactagcactcacgtggctcaaatggcagagatctggctacactccag
3flank2 actggtcacgacacaaggcctattactagcagtcacattgaacaaatggccaagatctcgccgcactgtag
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。
序列2具体如下表2所示。
Figure PCTCN2022083598-appb-000001
Figure PCTCN2022083598-appb-000002
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。
图55显示了序列4以及2条与序列4同源性大于80％的序列4-1、4-2构建的递送系统，静脉注射9小时后肺部组织的EGFR siRNA含量检测结果。
序列4具体如下表3所示。
名称 序列
序列4 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-1 CAGATGATTCCGCACTCGAGGTCATGAGTCGACCAGTGGATC
序列4-2 CAGATCTAAGGTCACTCGAGGTAGTGCTACGACCAGTGGATC
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。
长度不同的RNA序列具体如下表4所示。
名称 序列
siRE(18) ACCTATTCCGTTACACACT
siRE(20) ATACCTATTCCGTTACACAC
siRE(22) ATACCTATTCCGTTACACACTT
图54分别显示了不同长度的RNA序列所构建的递送系统(质粒)静脉注射后的EGFR表达量检测， 其中，RNA序列长度分别为18、20、22的质粒分别对应CMV-siR E(18)、CMV-siR E(20)、CMV-siR E(22)。
所述RNA序列选自：EGFR基因的siRNA，KRAS基因的siRNA，VEGFR基因的siRNA，mTOR基因的siRNA，TNF-α基因的siRNA，integrin-α基因的siRNA，B7基因的siRNA，TGF-β1基因的siRNA，H2-K基因的siRNA，H2-D基因的siRNA，H2-L基因的siRNA，HLA基因的siRNA，GDF15基因的siRNA，miRNA-21的反义链，miRNA-214的反义链，TNC基因的siRNA，PTP1B基因的siRNA，mHTT基因的siRNA，Lrrk2基因的siRNA，α-synuclein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。
上述各种基因的siRNA以及miRNA的反义链均可以通过抑制该基因、miRNA的表达或突变，从而达到抑制疾病的效果。这些疾病包括但不限于：癌症、肺纤维化、结肠炎、肥胖症、由肥胖症引起的心血管疾病、二型糖尿病、亨廷顿病、帕金森病、重症肌无力、阿尔兹海默病、移植物抗宿主病及其相关疾病。这里的相关疾病指的是上述任意一种或几种疾病的形成或发展过程中出现的关联疾病或并发症、后遗症等，或与上述疾病具有一定相关性的其他疾病。其中，癌症包括但不限于：胃癌、肾癌、肺癌、肝癌、脑癌、血癌、肠癌、皮肤癌、淋巴癌、乳腺癌、膀胱癌、食管癌、头颈部鳞癌、血管瘤、胶质细胞瘤、黑色素瘤。
RNA片段中RNA序列的数量为1条、2条或多条。比如若需治疗脑胶质瘤，则可以在同一个质粒载体上联合使用EGFR基因的siRNA和TNC基因的siRNA；若需治疗肠炎，则可以同时使用TNF-α基因的siRNA、integrin-α基因的siRNA和B7基因的siRNA。
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。
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)。
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(自组装)，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。
靶向标签选自具有靶向功能的靶向肽、靶向蛋白或抗体中的一种，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)。
其中，RVG靶向肽、RVG-LAMP2B融合蛋白可以精准靶向脑组织；GE11靶向肽、GE11-LAMP2B融合蛋白可以精准靶向EGFR高表达的器官组织，比如EGFR突变的肺癌组织；PTP靶向肽、PTP-LAMP2B融合蛋白可以精准靶向胰腺，特别是人源及鼠源胰腺癌组织中特异性表达的plectin-1蛋白；TCP-1靶向肽、TCP-1-LAMP2B融合蛋白可以精准靶向结肠；MSP靶向肽、MSP-LAMP2B融合蛋白可以精准靶向肌肉组织。
在实际应用中，靶向标签可以灵活搭配各种不同的RNA片段，不同的靶向标签搭配不同的RNA片段可以起到不同的作用。比如：RVG靶向肽、RVG-LAMP2B融合蛋白可以搭配EGFR基因的siRNA、TNC基因的siRNA或二者的组合治疗胶质母细胞瘤，还可以搭配PTP1B基因的siRNA治疗肥胖症，也可以搭配mHTT基因的siRNA治疗亨廷顿舞蹈症，以及搭配LRRK2基因的siRNA治疗帕金森；GE11靶向肽、GE11-LAMP2B融合蛋白可以搭配EGFR基因的siRNA治疗由EGFR基因高表达或突变诱导的肺癌等疾病；TCP-1靶向肽或TCP-1-LAMP2B融合蛋白可以搭配TNF-α基因的siRNA、integrin-α基因的siRNA、B7基因的siRNA或上述三者的任意组合治疗结肠炎或结肠癌等。
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。
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。
以上所述的递送系统均可用于包括人在内的哺乳动物。
为了验证递送系统的可行性，我们进行了如下试验：
首先，参见图38a，我们合理地设计了核心线路(genetic circuit)的基础结构，允许不同功能模块的自由组合。核心线路由启动子部分和siRNA表达部分组成，旨在产生和组织siRNA，作为外泌体(exosomes)的有效载荷。其他可组合部件(插件)可以集成到核心线路的框架中，以实现即插即用功能。比如合了两种类型的可组合部分以优化siRNA的作用：一种修饰外显体的膜锚定蛋白以实现组织选择性；另一种能够共同表达第二个siRNA，以同时抑制两个分子靶标。
对于核心线路构建体，我们设计了在启动子部分的控制下编码优化的siRNA表达骨架部分的方案，以最大化引导链(guide strand)表达，同时最小化不希望的过客链(passenger strand)表达，参见图38a。表皮生长因子受体(Epidermal growth factor receptor，EGFR)是一种在多种人类肿瘤(如肺癌和胶质母细胞瘤)中频繁突变和高表达的癌基因，将其选为siRNA靶点。并选择人胚肾293t细胞(HEK293T)和小鼠肝癌细胞(hep1-6)作为siRNA体外组装的底盘细胞(cell chassis)。为了优化siRNA产生效率，我们比较了两种设计方案：一种是使用CMV启动子表达miRNA前体(pre-miRNA)并用siRNA替换miRNA序列，另一种是使用U6启动子表达短发夹RNA(shRNA)，我们首先研究了一系列miRNA前体结构，并选择pre-miR-155作为产生siRNA的最佳骨架。然后，比较了编码EGFR siRNA(CMV-siR E)和U6定向EGFR shRNA(U6-siR E)的CMV定向(CMV-directed)的pre-miRNA的siRNA产生效率，参见图38b。两种方案在驱动EGFR siRNA导向链转录方面具有相似的效率；但是，与shRNA方法相比，pre-miRNA方法产生的乘客链更少或没有(在成熟的引导链的生物发生过程中，乘客链似乎被降解)，参见图38b。因此，决定选择CMV驱动的pre-miRNA设计来避免脱靶效应。
接下来，我们检查核心线路是否能够引导siRNA自主加载到外泌体。采用CMV-scrR或CMV-siR E基因转染HEK293T细胞，观察细胞培养液中的外泌体。纳米粒追踪分析(NTA)显示各组分泌的外泌体数量相似，大小分布相似，峰值在128-131nm之间。透射电子显微镜(TEM)证实纯化的外泌体呈现典型的圆形囊泡形态，大小正确。此外，特定外显子标记物(CD63、TSG101和CD9)的富集仅在纯化的外泌体中检测到，而在细胞培养基中未检测到。这些结果表明，线路转染并不影响HEK293T细胞产生的外泌体的大小、结构或数量。最后，在CMV-siR E线路转染的HEK293T和Hepa 1–6细胞衍生的外泌体中检测到大量的EGFR siRNA，参见图38c。当这些外泌体与小鼠Lewis肺癌(LLC)细胞一起孵育时，EGFR表达的剂量依赖性降低，参见图38d、图38e，表明外泌体siRNA具有生物学功能。
为了设计线路的靶向标签，将编码Lamp2b蛋白(一种典型的外体膜蛋白)的N末端融合的靶向标签的序列插入CMV启动子的下游，参见图38a。这个标签通过Lamp2b锚定在外泌体表面，从而引导复合结构外泌体运送到所需的组织。具体而言，选择以RVG肽为靶点的中枢神经系统作为标记，将外泌体导入大脑(RVG已被证明有助于外泌体穿过血脑屏障进入神经细胞)，首先评估启动子启动RVG-Lamp2b融合蛋白表达的效率。经过试验证明，CMV启动子在HEK293T细胞中产生RVG-Lamp2b mRNA和标记蛋白eGFP方面有一定的效果，而U6启动子则没有效果，这证实了CMV启动子连接线路各部分的优势。然后使用免疫沉淀法验证引导靶向标签在外泌体表面正确表达。由于试验暂时缺乏抗RVG抗体，因此采用Flag标签暂时代替RVG。在用CMV引导的Flag-Lamp2b电路转染HEK293T和Hepa 1-6细胞后，用抗Flag珠成功地免疫沉淀完整的外泌体，参见图38f，证明了靶向标签的精确定位。为了设计额外的siRNA表达部分，使用与许多癌症特别是胶质母细胞瘤相关的关键癌基因tenascin-C(TNC)作为第二个siRNA靶点。TNC-siRNA也被嵌入前miR-155骨架中，并插入EGFR-siRNA的下游，参见图38。无论单个(CMV siR E或CMV siR T)或串联(CMV siR E)转录，均检测到了相差无几的EGFR和TNC siRNA，参见图38g。总之，这些结果表明，不论是siRNA还是靶向标签，其都可以在线路中发挥其各自的作用。
接下来，我们研究了线路是否能够自组装成外泌体。采用编码EGFR和TNC siRNAs的CMV导向电路以及RVG标记(CMV-RVG-siR E)转染HEK293T细胞。结果显示来自细胞培养基的外泌体显示出典型的形态和大小分布，表明用复合核心线路修饰不会改变外泌体的物理性质。此外，我们构建了一个完整的线路，包括EGFR和TNC-siRNAs和一个靶向标签(CMV-Flag-siR E)，用其转染的HEK293T和hep1-6细胞产生的外泌体成功地进行了免疫沉淀，EGFR和TNC siRNA都大量存在于免疫沉淀的外泌体中，参见图38h。
此外，AGO2在生物体内广泛表达，是RNA诱导沉默复合体的核心成分，其具有核糖核酸内切酶活性，可通过促进siRNA的成熟并调节其生物合成及功能，进而抑制靶基因的表达。
由于siRNA的加工依赖于Argonaute 2(AGO2)，并且将siRNA适当地装载到AGO2中有望增强siRNA的靶向作用，因此我们进行了另一项免疫沉淀实验以评估AGO2与外泌体中siRNA的关联。试验证明在用AGO2抗体(anti-AGO2)沉淀的外泌体中很容易检测到EGFR和TNC siRNA，这表明我们的设计可确保将siRNA加载到RNA诱导的沉默复合物(RISC)中，并促进AGO2结合的siRNA高效转运到外泌体。最后，为了研究体外组装的siRNA是否具有功能，将用CMV-RVG-siR E+T线路转染的HEK293T细胞衍生的外泌体与U87MG胶质母细胞瘤细胞一起孵育。在U87MG细胞中实现了EGFR和TNC表达的剂量依赖性下调，参见图38i、图38j。此外，外泌体表面的RVG标签不影响EGFR和TNC siRNA对靶标的沉默效果。这些结果将线路确立为多个可组合部分的有机组合，正是这些部分的巧妙配合使得RNA能够自组装和释放。
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，如图1A所示，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。
向C57BL/6J小鼠静脉注射共表达eGFP蛋白和EGFR siRNA的CMV eGFP siR E线路(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。
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。
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。
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。
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。
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。
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。
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。
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。
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。
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-siR E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。
因此，本实施例提供的RNA递送系统以质粒作为载体，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。该递送系统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。该药物包括质粒，所述质粒携带有所需递送的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，所述复合结构能够进入并结合目标组织，以将所述RNA片段送入目标组织。
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。
图39-41中，6种RNA分别为：siR E(靶基因为EGFR)、siR T(靶基因为TNC)、shR E(靶基因为 EGFR)、shR T(靶基因为TNC)、miR-7(靶基因为EGFR)、miR-133b(靶基因为EGFR)，图39是提供的6种不同RNA的质粒在血浆中的富集效果、外泌体内siRNA检测以及相应基因表达量；图40是以上提供的6种RNA种任意2种RNA序列组成的4组质粒在血浆中的富集效果、外泌体内siRNA检测以及相应基因表达量；图41是以上提供的6种RNA种任意3种RNA序列组成的3组质粒在血浆中的富集效果、外泌体内siRNA检测以及相应基因表达量。
RNA序列具体如下表5所示。
名称 序列
siRE前体序列 ATACCTATTCCGTTACACACTGTTTTGGCCACTGACTGACAGTGTGTAGGAATAGGTAT
siRT前体序列 CACACAAGCCATCTACACATGGTTTTGGCCACTGACTGACCATGTGTATGGCTTGTGTG
shRE前体序列 ATACCTATTCCGTTACACACTGTTTTGGCCACTGACTGACAGTGTGTAACGGAATAGGTAT
shRT前体序列 CACACAAGCCATCTACACATGGTTTTGGCCACTGACTGACCATGTGTAGATGGCTTGTGTG
miR-7 hsa-miR-7
miR-133b hsa-miR-133b
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。
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。
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。
该药物可以为治疗癌症、肺纤维化、结肠炎、肥胖症、由肥胖症引起的心血管疾病、二型糖尿病、亨廷顿病、帕金森病、重症肌无力、阿尔兹海默病或移植物抗宿主病的药物。
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。
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。
实施例3
在实施例1或2的基础上，本实施例提供一种RNA递送系统在药物中的应用，该药物为治疗肺癌的药物。
在此通过以下试验进行具体说明。
如图11A所示，选取小鼠，向小鼠体内注射小鼠肺癌细胞(LLC细胞)，而后每两日向小鼠注射一次PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR E进行治疗，分别对小鼠进行生存分析和肿瘤评估，第30天 治疗开始，第44天治疗结束。
如图11B所示，横轴表示时间，纵轴表示生存率，从该图中可以看出，注射CMV-siR E的小鼠生存率最高。
如图11C所示，该图为注射PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR E的小鼠在治疗之前和治疗之后根据CT影像图对小鼠肺组织进行3D建模，可以看出，注射CMV-siR E的小鼠肿瘤显著减小。
如图11D所示，该图为注射PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR E的小鼠在治疗之前和治疗之后的肿瘤体积(mm 3)对比图，可以看出，注射CMV-siR E的小鼠肿瘤体积显著减小。而注射PBS缓冲液/CMV-scrR/吉非替尼的小鼠肿瘤体积不仅没有减小，还呈现不同程度的增加。
如图11E所示，该图为正常小鼠、注射PBS缓冲液/CMV-scrR/吉非替尼/CMV-si RE的小鼠的western blot对比图，可见注射PBS缓冲液/CMV-scrR/吉非替尼的小鼠其EGFR基因含量明显较高。
如图11F所示，该图为正常小鼠、注射PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR E的小鼠相关EGFR miRNA水平对比图，可见注射PBS缓冲液/CMV-scrR/吉非替尼的小鼠其相关EGFRmiRNA水平相对较高。
综上，CMV-siR E对EGFR突变的肺癌肿瘤具有显著的治疗效果。
分别对注射PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR E的小鼠进行HE染色和免疫组织染色，结果如图12A-图12B所示，EGFR在注射PBS缓冲液/CMV-scrR/吉非替尼的小鼠中具有更多的表达。统计小鼠中EGFR和PCNA的着色面积，结果如图12C-图12D所示，可见注射CMV-siR E的小鼠EGFR和PCNA的着色面积均最少，证明其对EGFR突变的肺癌肿瘤的治疗效果最好。
如图13A所示，选取KRAS G12D p53 -/-小鼠，使小鼠吸入Adv-Cre后的第50天至第64天，每两日向小鼠注射一次PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR E进行治疗，分别对小鼠进行生存分析和肿瘤评估。
如图13B所示，横轴表示感染后的时间，纵轴表示生存率，从该图中可以看出，注射CMV-siR K的小鼠生存率更高。
如图13C所示，该图为注射CMV-scrR/CMV-siR K的小鼠在治疗之前和治疗之后根据CT影像图对小鼠肺组织进行3D建模，可以看出，注射CMV-siR K能够显著抑制肺癌肿瘤的增长。
如图13D所示，该图为注射CMV-scrR/CMV-siR K的小鼠在治疗之前和治疗之后的肿瘤数量对比图，可以看出，注射CMV-siR K的小鼠肿瘤数量增长显著更少。
如图13E所示，该图为注射CMV-scrR/CMV-siR K的小鼠在治疗之前和治疗之后的肿瘤数量(mm 3)对比图，可以看出，注射CMV-siR K的小鼠肿瘤体积增长缓慢。而注射CMV-scrR的小鼠肿瘤体积增长显著。
如图13F所示，该图为注射CMV-scrR/CMV-siR K的小鼠的western blot对比图，可见注射CMV-scrR的小鼠其KRAS基因含量明显较高。
如图13G所示，该图为注射CMV-scrR/CMV-siR K的小鼠相关KRAS mRNA水平对比图，可见注射CMV-scrR的小鼠其相关KRAS mRNA水平相对较高。
综上，CMV-siR K对KRAS突变的肺癌肿瘤具有显著的治疗效果。
分别对注射CMV-scrR/CMV-siR K的小鼠进行HE染色和免疫组织染色，结果如图14A、图14D、图14E所示，可见在注射CMV-scrR的小鼠中KRAS、p-AKT、p-ERK具有更多的表达，着色百分比更高。采 用western blot免疫印迹法检测小鼠体内相关蛋白的表达水平，结果如图14B、图14C所示，在注射CMV-scrR的小鼠中相关蛋白具有更多的表达。这也说明CMV-siR K对KRAS突变的肺癌肿瘤具有显著的抑制作用。
实施例4
在实施例1或2的基础上，本实施例提供一种RNA递送系统在药物中的应用，该药物为治疗肾癌的药物。
在此通过以下试验进行具体说明。
分别对不同小鼠注射PBS缓冲液/对照质粒/VEGFR siRNA质粒/mTOR siRNA质粒/MIX siRNA质粒(VEGFR siRNA和mTOR siRNA联合使用)/舒尼替尼(Sunitinib)/依维莫司(Everolimus)，观察小鼠肾癌肿瘤的发展情况，结果如图15、图16所示。可见，注射MIX siRNA质粒的小鼠其肾癌的发展得到了最为显著的抑制，而注射PBS缓冲液/对照质粒的小鼠肾癌发展则较为迅速。
综上，VEGFR siRNA和mTOR siRNA联合使用对肾癌肿瘤具有显著的治疗效果。
实施例5
在实施例1或2的基础上，本实施例提供一种RNA递送系统在药物中的应用，该药物为治疗结肠炎的药物，本实施例将通过以下两个试验对RNA递送系统在结肠炎治疗方面的效果进行具体说明。
在第一个试验中，我们设置3组试验组和3组对照组，试验组分别为anti-TNF-α(0.5)组、anti-TNF-α(5)组、anti-TNF-α(20)组；对照组分别为mock组、scr-RNA组、IFX组。
其中，anti-TNF-α(0.5)组、anti-TNF-α(5)组、anti-TNF-α(20)组分别利用质粒包裹TNF-αsiRNA系统(CMV-siR TNF-α)，尾静脉注射0.5μL、5μL、20μL的CMV-siR TNF-α的溶液至小鼠体内。
mock组为阴性对照组，scr-RNA组、IFX组分别向小鼠尾静脉注射scr-RNA质粒和IFX(英夫利西单抗)。
随即开始构建DSS诱导的慢性结肠炎模型，其间每天进行称重记录，结果如图17A所示，可见scr-RNA组的小鼠体重下降最快，anti-TNF-α(0.5)组、anti-TNF-α(5)组、anti-TNF-α(20)组的小鼠体重下降较缓，并且TNF-αsiRNA溶液的剂量越高，小鼠体重下降越缓，这说明质粒包裹的TNF-αsiRNA系统能够减轻结肠炎小鼠的体重下降情况。
模型构建结束，通过小动物活体监测质粒系统的体内表达情况，而后处死小鼠进行结肠的观察，结果如图17B所示，可见scr-RNA组的小鼠结肠长度最短，anti-TNF-α(0.5)组、anti-TNF-α(5)组、anti-TNF-α(20)组的小鼠结肠长度相对较长，并且TNF-αsiRNA注射剂量越高，小鼠结肠长度相对越长。这说明质粒包裹的TNF-αsiRNA系统对慢性炎症导致的结肠长度缩短有着不同程度的改善。
对小鼠疾病活动指数(Disease activity index)进行评估，结果如图17C所示，可见，注射scr-RNA组、anti-TNF-α(0.5)组、anti-TNF-α(5)组的小鼠疾病活动指数较高，而anti-TNF-α(20)组、IFX组的小鼠疾病活动指数则较低。
对小鼠结肠进行TNF-αmRNA检测，结果如图17D所示，可见该CMV-siR TNF-α系统能够降低结肠TNF-α的表达及分泌；对小鼠结肠进行TNF-α检测，结果如图17E所示，可见，该AAV系统能够产生一定量的TNF-α；对结肠内的促炎因子IL-6、IL-12p70、IL-17A、IL-23进行检测，结果如图17F所示，可见高剂量组的炎症因子分泌整体较低于对照组。
对小鼠结肠切片进行HE染色，以及病理评分统计，结果如图18A和图18B所示，可见anti-TNF-α (0.5)组、anti-TNF-α(5)组、anti-TNF-α(20)组的小鼠，尤其是anti-TNF-α(20)组的小鼠结肠黏膜完整性更高，且免疫细胞的浸润程度更浅，结肠隐窝脓肿以及结肠的充血和出血情况也显著轻于对照组。
以上试验可证明，在改善结肠炎的表现方面，使用质粒包裹TNF-αsiRNA系统治疗比IFX更有效或至少与IFX一样有效。
在第二个试验中，我们设置4组试验组和3组对照组，试验组分别为anti-TNF-α组、anti-integrin-α组、anti-B7组、anti-mix组，对照组分别为mock组、PBS组、scr-RNA组。
anti-TNF-α组、anti-integrin-α组、anti-B7组、anti-mix组分别利用质粒包裹TNF-αsiRNA系统(CMV-siR TNF-α)、integrin-αsiRNA系统(CMV-siR integrin-α)、B7siRNA系统(CMV-siR B7)、mix siRNA(CMV-siR mix即CMV-siR TNF-α+integrin-α+B7)系统，尾静脉注射20μL至小鼠体内，通过小动物活体监测该系统的体内表达情况，可见上述系统在体内尤其是肝脏稳定表达。
mock组为阴性对照组，scr-RNA组、PBS组分别向小鼠尾静脉注射scr-RNA质粒和PBS溶液(磷酸缓冲盐溶液)。
随即开始构建DSS诱导的慢性结肠炎模型，其间每天进行称重记录，结果如图19A所示，可见anti-mix组小鼠的体重增长最为平稳，即质粒包裹的CMV-siR TNF-α+integrin-α+B7系统能够显著减轻慢性结肠炎小鼠的体重下降情况，anti-TNF-α组、anti-integrin-α组、anti-B7组的小鼠在炎症缓解期体重回复的速度也显著快于scr-RNA组和PBS组。
模型构建结束，通过小动物活体监测质粒系统的体内表达情况，而后处死小鼠进行结肠的观察，结果如图19B所示，可见4个试验组小鼠结肠的红肿情况有不同程度的减轻，慢性炎症导致的结肠长度缩短也有不同程度的改善。
对小鼠疾病活动指数(Disease activity index)进行评估，结果如图19C所示，可见，scr-RNA组和PBS组小鼠疾病活动指数较高，而anti-TNF-α组、anti-integrin-α组、anti-B7组、anti-mix组的小鼠疾病活动指数则依次降低。
对小鼠血浆、肝脏以及结肠进行TNF-αmRNA、integrin mRNA以及B7mRNA检测，结果如图19D-图19F所示，可见，该系统在血浆、肝脏以及结肠内，生成了一定量的稳定表达的RNA，同时该系统显著降低了结肠TNF-α，integrin以及B7mRNA表达。
对小鼠结肠切片进行HE染色，结果如图20所示，可见4个试验组的小鼠，尤其是anti-mix组的小鼠结肠黏膜完整性更高，且免疫细胞的浸润程度更浅，结肠隐窝脓肿以及结肠的充血和出血情况也显著轻于对照组。
以上试验说明，利用亲和肝脏的质粒包裹CMV-siR TNF-α+integrin-α+B7线路，能够实现长期的TNF-αmRNA、B7mRNA以及integrin mRNA表达以及多个靶基因沉默，并且显著缓解结肠炎症程度，具有极大的成药潜力以及临床研究价值。
实施例6
在实施例1或2的基础上，本实施例提供一种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。
本实施例设置8组试验组和3组对照组。试验组分别为Anti-miR-21(1mg/kg)组、Anti-miR-21(5mg/kg)组、Anti-miR-21(10mg/kg)组、TGF-β1 siRNA(1mg/kg)组、TGF-β1 siRNA(5mg/kg)组、TGF-β1 siRNA(10mg/kg)组、Anti-miR-21+TGF-β1 siRNA(10mg/kg)组、Pirfenidone(300mg/kg)组，对照组分别为Normal组、PBS组、scrRNA组。
其中，Anti-miR-21(1mg/kg)组、Anti-miR-21(5mg/kg)组、Anti-miR-21(10mg/kg)组分别向患有肺纤维化的小鼠尾静脉注射1mg/kg、5mg/kg、10mg/kg的miR-21 siRNA质粒，TGF-β1 siRNA(1mg/kg)组、TGF-β1 siRNA(5mg/kg)组、TGF-β1 siRNA(10mg/kg)组分别向患有肺纤维化的小鼠尾静脉注射1mg/kg、5mg/kg、10mg/kg的TGF-β1 siRNA质粒，Anti-miR-21+TGF-β1 siRNA(10mg/kg)组向患有肺纤维化的小鼠尾静脉注射10mg/kgAnti-miR-21和TGF-β1 siRNA质粒，Pirfenidone(300mg/kg)组向患有肺纤维化的小鼠尾静脉注射300mg/kg的吡非尼酮，Normal组为正常对照组，PBS组、scrRNA组分别向患有肺纤维化的小鼠尾静脉注射PBS溶液和对照质粒。
分别检测各组小鼠的羟脯氨酸含量，结果如图21所示。羟脯氨酸是胶原的主要成分，其含量反映了肺纤维化程度，从图21中可以看出，Anti-miR-21(5mg/kg)组、Anti-miR-21(10mg/kg)组、TGF-β1 siRNA(10mg/kg)组、Anti-miR-21+TGF-β1 siRNA(10mg/kg)组小鼠的羟脯氨酸含量相对较低，其肺纤维化得到抑制。
分别对各组小鼠肺部进行荧光染色，结果如图22所示，图中绿色部分表示I型胶原(Collagen I)，红色部分表示α-SMA，蓝色部分表示DAPI。可以看出，PBS组、scrRNA组小鼠I型胶原、α-SMA含量较多，而试验组小鼠的I型胶原、α-SMA含量均相对较少，尤其是Anti-miR-21(5mg/kg)组、Anti-miR-21(10mg/kg)组、Anti-miR-21+TGF-β1 siRNA(10mg/kg)组几乎无I型胶原、α-SMA的表达。
分别对各组小鼠肺部进行Masson三色染色，结果如图23所示。可以看出PBS组和scrRNA组小鼠肺泡间隙被严重破坏，造成肺间质胶原，而试验组则显著减轻了这些现象。
分别对各组小鼠肺部进行H&E染色，结果如图24所示。可以看出PBS组和scrRNA组小鼠肺泡间隙增宽、炎性细胞被浸润、肺泡结构被损害，而试验组肺组织则较为正常。
通过western blot分别检测Normal组、PBS组、scrRNA组、TGF-β1 siRNA(1mg/kg)组、TGF-β1 siRNA(5mg/kg)组、TGF-β1 siRNA(10mg/kg)组、Pirfenidone(300mg/kg)组小鼠TGF-β1蛋白水平、TGF-β1mRNA水平，结果如图25A-图25C所示，可见TGF-β1 siRNA(10mg/kg)组小鼠TGF-β1蛋白水平以及TGF-β1mRNA水平最低。这说明了尾静脉注射相应siRNA表达质粒后，TGF-β1能够成功递送至肺部发挥功能。
分别检测Normal组、PBS组、scrRNA组、Anti-miR-21(1mg/kg)组、Anti-miR-21(5mg/kg)组、Anti-miR-21(10mg/kg)组小鼠的相对miR-21水平，结果如图25D所示，可见Anti-miR-21(10mg/kg)组小鼠的相对miR-21水平最高。这说明了尾静脉注射相应反义链表达质粒后，miR-21的反义链能够成功递送至肺部发挥功能。
以上试验说明，利用亲和肝脏的质粒包裹CMV-siR miR-21、CMV-siR TGF-β1、CMV-siR miR-21+TGF-β1线路，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。
实施例7
在实施例1或2的基础上，本实施例提供一种RNA递送系统在药物中的应用，该药物为治疗胶质母细胞瘤的药物。本实施例结合以下两个试验对RNA递送系统在胶质母细胞瘤治疗方面的应用进行具体说明。
在第一个试验中，我们设置5个试验组和3个对照组。试验组分别为CMV-siR E组、CMV-siR T组、CMV-RVG-siR E+T组、CMV-siR E+T组、CMV-Flag-siR E+T组，其中“E”表示EGFR、“T”表示TNC，对照组分别为PBS组、CMV-scrR组、CMV-Flag-scrR组，具体试验过程参见图26A。
分别检测不同组别小鼠的CD63蛋白表达含量、siRNA表达水平，结果如图26B-图26D所示，这表 明静脉注射CMV-RVG-siR E+T线路可将siRNA传递至大脑。
在第二个试验中，我们设置2个试验组和2个对照组。试验组分别为CMV-RVG-siR E组、CMV-RVG-siR E+T组，对照组分别为PBS组、CMV-scrR组。
具体试验过程如图27A所示，选取小鼠，向小鼠体内注射胶质母细胞瘤细胞(U-87MG-Luc细胞)，自第7天开始至第21天，期间每两日向小鼠注射一次PBS缓冲液/CMV-scrR/CMV-RVG-siR E/CMV-RVG-siR E+T(5mg/kg)进行治疗，分别对小鼠进行生存分析和肿瘤评估。在第7天、14天、28天、35天分别对小鼠进行BLI活体成像检测。
如图27B所示，该图为第7天、14天、28天、35天小鼠BLI活体成像检测对比图，可以看出，CMV-RVG-siR E+T组的小鼠其胶质母细胞瘤抑制效果最为显著。
如图27C所示，该图为各组小鼠生存率对比图，可见，CMV-RVG-siR E+T组的小鼠其生存时间最长。
如图27D所示，该图为各组小鼠的荧光对比图，该图通过luciferase生物活体成像得到，纵坐标反应lucifer荧光信号强弱。由于种植的肿瘤中已经人工整合了该基因，因此，该图能够反应肿瘤的进展情况。可以看出对照组小鼠肿瘤发展均比较迅速，而试验组小鼠的肿瘤则得到了很大程度的抑制。
如图27E所示，该图为各组小鼠的相对siRNA对比图，可见CMV-RVG-siR E组的小鼠EGFR siRNA水平较高，CMV-RVG-siR E+T组的小鼠EGFR siRNA和TNC siRNA水平均较高。
如图27F所示，该图为各组小鼠的western blot对比图，可见PBS组、CMV-scrR组、CMV-RVG-siR E组的小鼠其EGFR、TNC基因含量较高。
以上试验数据说明了静脉注射CMV-RVG-siR E+T质粒能够将siRNA传递到大脑并抑制胶质母细胞瘤的生长。
分别对各组小鼠脑部进行免疫组织染色处理，并统计每视野中EGFR、TNC、PCNA着色比例，结果如图28所示。可以看出，CMV-RVG-siR E+T组的小鼠脑部EGFR、TNC、PCNA含量最低，CMV-RVG-siR E组的小鼠脑部EGFR、PCNA含量较低。可见注射CMV-RVG-siR E质粒能够抑制脑部EGFR、PCNA的表达，注射CMV-RVG-siR E+T质粒能够抑制脑部EGFR、TNC、PCNA的表达。
实施例8
在实施例1或2的基础上，本实施例提供一种RNA递送系统在药物中的应用，该药物为治疗肥胖症的药物。本实施例结合以下两个试验对RNA递送系统在肥胖症治疗方面的应用进行具体说明。
在第一个试验中，设置2个试验组和1个对照组。试验组分别为CMV-siR P组、CMV-RVG-siR P组，对照组为CMV-scrR组，其中“P”表示PTP1B。
CMV-siR P组、CMV-RVG-siR P组、CMV-scrR组分别对小鼠注射5mg/kg的CMV-siR P质粒、CMV-RVG-siR P质粒、CMV-scrR质粒，而后分别获取各组小鼠的下丘脑、肝脏荧光显微镜图像，结果如图29所示，结果显示PTP1B siRNA能够向下丘脑传递。
在第二个试验中，设置2个试验组和2个对照组，试验组分别为CMV-siR P组、CMV-RVG-siR P组，对照组分别为PBS组、CMV-scrR组。
具体试验过程如图30A所示，选取C57BL/6小鼠，12周后注射PBS缓冲液/CMV-scrR/CMV-siR P/CMV-RVG-siR P，在24天内每两天注射一次，最后统计小鼠的肥胖和能源支出、瘦素敏感性、胰岛素敏感度。
如图30B所示，该图为各组小鼠体重对比图，可以看出，CMV-RVG-siR P组的小鼠体重最为稳定。
如图30C所示，该图为各组小鼠附睾脂肪垫重量对比图，可以看出，CMV-RVG-siR P组的小鼠附睾脂 肪垫重量最轻。
利用代谢笼对不同处理小鼠的氧气消耗量、呼吸交换比、活动量、产热量进行连续72小时的检测，再取平均值绘图进行统计分析，结果如图30D-图30G所示。结果表明CMV-RVG-siR P质粒可以有效提高小鼠的氧气消耗量，这意味着这组小鼠相比于其他组小鼠处于高能量代谢状态。正常小鼠主要以葡萄糖作为自己的能量来源，CMV-RVG-siR P质粒可以降低小鼠的呼吸交换比，这意味着这组小鼠相对于其他组小鼠更倾向于利用蛋白质作为自己的能量来源。注射CMV-RVG-siR P质粒的小鼠活动量明显增加。而且，CMV-RVG-siR P组的小鼠产热也明显提高。
如图30H所示，该图为各组小鼠初始体重曲线对比图。可以看出，CMV-RVG-siR P组的小鼠体重最轻。
如图30I所示，该图为各组小鼠初始食物摄入量曲线对比图。可以看出，CMV-RVG-siR P组的小鼠食物摄入量最少。
如图30J所示，该图为各组小鼠血清瘦素含量对比图。可以看出，CMV-RVG-siR P组的小鼠血清瘦素含量最低。
如图30K所示，该图为各组小鼠的western blot对比图。可以看出，CMV-RVG-siR P组小鼠的PTP1B蛋白含量最低。
如图30L所示，该图为各组小鼠血糖变化曲线对比图。可以看出，CMV-RVG-siR P组的小鼠血糖含量最低。
如图30M所示，该图为各组小鼠基础葡萄糖变化曲线对比图。可以看出，CMV-RVG-siR P组的小鼠基础葡萄糖含量最低。
以上试验可以得出，静脉注射CMV-RVG-siR P质粒能够降低肥胖小鼠模型的肥胖。
对各组小鼠的血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)进行测定，结果如图31A所示，可以看出，CMV-RVG-siR P组的小鼠TC、TG、LDL最低。
对各组小鼠的体长进行测定，结果如图31B所示，可以看出，四组小鼠体长相差无几。
统计各组小鼠的HFD食物摄入量，结果如图31C所示，可以看出，四组小鼠HFD食物摄入量相差无几。
对各组小鼠分别进行治疗后取肝组织，与正常对照进行对比，结果如图31D所示，PBS组、CMV-scrR组小鼠的肝组织病理切片中可见有明显的脂肪肝病理特征，CMV-siR P组小鼠的脂肪肝较轻。
以上试验说明静脉注射CMV-RVG-siR P质粒能够减轻肥胖小鼠的脂肪肝。
实施例9
在实施例1或2的基础上，本实施例提供一种RNA递送系统在药物中的应用，该药物为治疗亨廷顿病的药物。本实施例结合以下5个试验对RNA递送系统在亨廷顿病治疗方面的应用进行具体说明。
在第一个试验中，设置2个试验组和2个对照组。试验组分别为CMV-siR mHTT组、CMV-RVG-siR mHT T组，对照组分别为PBS组、CMV-scrR组。
试验流程如图32A所示，分别对CMV-siR mHTT组、CMV-RVG-siR mHTT组、PBS组、CMV-scrR组患有亨廷顿病的小鼠尾静脉注射CMV-siR mHTT质粒、CMV-RVG-siR mHTT质粒、PBS溶液、CMV-scrR质粒后，分离血浆外泌体，用PKH26染料标记后与细胞进行共培，观察细胞对外泌体的吸收情况。
如图32B所示，该图为各组小鼠血浆外泌体中siRNA水平对比图，可以看出，2个试验组小鼠血浆 的外泌体中siRNA水平较高。
对各组小鼠注射质粒/溶液后提取的血浆外泌体，采用PKH26标记、与细胞共培养并用共聚焦显微镜进行拍照。结果如图32C所示，显示包裹siRNA的外泌体进入细胞。
提取各组小鼠血浆外泌体与细胞共培后，检测各组小鼠的HTT蛋白水平以及mRNA水平的变化，结果如图32D-图32F所示，表明CMV-siR mHTT与CMV-RVG-siR mHTT可以降低HTT蛋白水平，说明组装进入外泌体的siRNA仍然可以发挥基因沉默功能。
将提取的小鼠血浆外泌体与细胞共培后，观察并统计各组小鼠HTT蛋白的聚集情况，结果如图32G-图32H所示，表明CMV-siR mHTT与CMV-RVG-siR mHTT可以降低亨廷顿HTT聚集细胞模型中病理HTT蛋白的聚集，说明组装进入外泌体的siRNA仍然可以发挥基因沉默功能，并可以有效降低突变蛋白聚集。
分别检测统计各组小鼠肝脏、血浆、皮质、纹状体中绝对siRNA的表达水平。如图33A所示，该图为各组小鼠肝脏siRNA绝对水平对比图，可以看出，CMV-siR mHTT组、CMV-RVG-siR mHTT组的小鼠绝对siRNA水平较高；如图33B所示，该图为各组小鼠血浆中绝对siRNA水平对比图，可以看出，CMV-siR m HTT组、CMV-RVG-siR mHTT组的小鼠绝对siRNA水平较高；如图33C所示，该图为各组小鼠皮层和纹状体绝对siRNA水平对比图，可以看出，注射CMV-RVG-siRmHTT的小鼠绝对siRNA水平较高。
如图33D所示，该图为各组小鼠肝、皮层、纹状体组织原位杂交图，可以看出，CMV-siR mHTT组、CMV-RVG-siR mHTT组小鼠肝脏组织切片有明显的荧光，CMV-RVG-siR mHTT组小鼠皮层、纹状体组织切片有明显的荧光。这说明RVG可以引导外泌体siRNA进入通过血脑屏障并发挥功能。
在第二个试验中，设置2个试验组和2个对照组。试验组分别为CMV-siR GFP组、CMV-RVG-siR GFP组，对照组分别为PBS组、CMV-scrR组。分别对CMV-siR GFP组、CMV-RVG-siR GFP组、PBS组、CMV-scrR组GFP转基因小鼠的小鼠尾静脉注射CMV-siR GFP质粒、CMV-RVG-siR GFP质粒、PBS溶液、CMV-scrR质粒。
如图33E、图33F所示，该图为各组小鼠肝、皮层、纹状体组织切片图，可以看出，肝脏中注射CMV-siR GFP/CMV-RVG-siR GFP的GFP转基因小鼠GFP荧光水平降低，皮层纹状体中注射CMV-RVG-siR GFP的GFP荧光水平降低。说明RVG可以引导外泌体siRNA进入通过血脑屏障并发挥功能。
在第三个试验中，设置两个试验组和一个对照组，试验组分别为CMV-siR mHTT组、CMV-RVG-siR mHT T组，对照组为CMV-scrR组。
试验过程如图34A所示，选取8周龄的N17182Q小鼠，分别为CMV-siR mHTT组、CMV-RVG-siR mHTT组、CMV-scrR组患有亨廷顿病的小鼠尾静脉注射CMV-siR mHTT质粒、CMV-RVG-siR mHTT质粒、CMV-scrR质粒，在第0天和第14天进行旋转试验，14天后处死小鼠进行分析。
如图34B所示，该图为野生型小鼠、CMV-scrR组、CMV-RVG-siR mHTT组的小鼠下降潜伏期对比图，可以看出在0天时，CMV-scrR组、CMV-RVG-siR mHTT组的小鼠的下降潜伏期比较一致，在第14天时，CMV-scrR组的小鼠下降潜伏期最短。
如图34C和图34D所示，图34C为CMV-scrR组、CMV-RVG-siR mHTT组小鼠纹状体的western bolt图，图34D为CMV-scrR组、CMV-RVG-siR mHTT组小鼠纹状体的相对mHTT mRNA水平对比图，可以看出，CMV-scrR组小鼠纹状体中N171-mHTT蛋白含量较高、相对mHTT mRNA水平同样较高。
在第四个试验中，设置一个试验组和一个对照组，试验组为CMV-RVG-siR mHTT组，对照组为CMV-scrR组。
试验过程如图34E所示，选取3月龄的BACHD小鼠，分别为CMV-RVG-siR mHTT组、CMV-scrR组 患有亨廷顿病的小鼠尾静脉注射CMV-RVG-siR mHTT质粒、CMV-scrR质粒，14天后处死小鼠进行分析。
如图34F所示，图34F为CMV-scrR组、CMV-RVG-siR mHTT组小鼠皮质和纹状体的western bolt图，可以看出CMV-RVG-siR mHTT组小鼠皮质和纹状体的突变体HTT(Mutant HTT)、内源HTT(Endogenous HTT)含量均较少。
如图34G所示，图34G为CMV-scrR组、CMV-RVG-siR mHTT组小鼠皮质和纹状体的相对mHTT蛋白水平对比图，可见，不论是小鼠皮质还是纹状体，CMV-RVG-siR mHTT组小鼠的相对mHTT蛋白水平均较低。
如图34H和图35I所示，图34H为CMV-scrR组、CMV-RVG-siR mHTT组小鼠的免疫荧光图，图35I为CMV-scrR组、CMV-RVG-siR mHTT组小鼠皮质和纹状体的相对mHTT mRNA水平对比图，可见，不论是小鼠皮质还是纹状体，CMV-RVG-siR mHTT组小鼠的相对mHTT mRNA水平均较低。
以上试验证明了静脉注射MV-RVG-siR mHTT质粒有助于抑制纹状体和皮质的mHTT，从而改善HD小鼠的运动能力和减轻神经病理学。
在第五个试验中，设置一个试验组和一个对照组，试验组为CMV-RVG-siR mHTT组，对照组为CMV-scrR组。
试验过程如图35A所示，选取6周龄的YAC128小鼠，分别为CMV-RVG-siR mHTT组、CMV-scrR组患有亨廷顿病的小鼠尾静脉注射CMV-RVG-siR mHTT质粒、CMV-scrR质粒，在试验开始第0天、第4周、第8周时分别进行旋转试验，而后处死小鼠进行分析。
如图35B所示，该图为野生型小鼠、CMV-RVG-siR mHTT组、CMV-scrR组小鼠下降潜伏期对比图，可以看出在0天时，CMV-RVG-siR mHTT组、CMV-scrR组的小鼠的下降潜伏期比较一致，在第四周和第八周时，CMV-scrR组的小鼠下降潜伏期最短。
如图35C所示，该图为CMV-RVG-siR mHTT组、CMV-scrR组小鼠皮质和纹状体的western bolt图，可以看出，CMV-RVG-siR mHTT组小鼠皮质突变体HTT和内源HTT含量均较低，其纹状体突变体HTT含量较低，纹状体内源HTT含量较高。
如图35D、图35E所示，这两张图分别为CMV-RVG-siR mHTT组、CMV-scrR组小鼠皮质和纹状体的相对mHTT mRNA水平对比图、相对mHTT蛋白水平对比图，可以看出，不论是皮质还是纹状体，CMV-RVG-siR mHTT组小鼠的相对mHTT mRNA水平、相对mHTT蛋白水平均相对更低。
如图35F所示，该图是CMV-RVG-siR mHTT组、CMV-scrR组小鼠皮质和纹状体的免疫荧光图，可以看出CMV-RVG-siR mHTT组小鼠的NeuN、EM48表达均低于CMV-scrR组。
以上试验可以说明，静脉注射MV-RVG-siR mHTT质粒有助于纹状体和皮层mHTT蛋白和毒性聚集体减少，从而改善行为缺陷和纹状体和皮层神经病理学。
实施例10
在实施例1或2的基础上，本实施例提供一种RNA递送系统在药物中的应用，该药物为治疗帕金森的药物。本实施例结合以下试验对RNA递送系统在帕金森治疗方面的应用进行具体说明。
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。
如图36A和图36B所示，图36A为注射CMV-scrR/CMV-RVG-siR LRRK2的LRRK2R1441G转基因小鼠western bolt图，图36B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-siRLRRK2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。
如图36C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。
如图36D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。
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。
实施例11
为了进一步研究这一关键问题并阐明体内自组装siRNA的药代动力学和药效学，我们对食蟹猕猴(Macaca fascicularis)进行了更详细的研究，食蟹猕猴是一种用于安全性评估研究的著名非人灵长类动物模型。经伦理学批准，我们用4只成年猕猴静脉注射5mg/kg CMV-siR E质粒，在注射前或注射后不同时间点采集血样。一个月后，这些猕猴每天静脉注射5mg/kg CMV-siR E质粒，共注射5次，并在注射前或最后一次注射后的不同时间点采集血样。
如图37所示，图37A为单次注射的食蟹猕猴全血siRNA浓度变化图，图37B位多次注射的食蟹猕猴全血siRNA浓度变化图，可以看出单次注射的食蟹猕猴siRNA浓度在静脉注射6小时后达到峰值，随即降低，多次注射的食蟹猕猴siRNA浓度在静脉注射3小时后达到峰值，随即降低，多次注射的食蟹猕猴siRNA浓度的降低速度更为缓慢。
以上实验可以说明，CMV-siR E质粒能够对食蟹猕猴等灵长类动物安全且有效，应用前景十分广泛。
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。

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  1. 一种RNA质粒递送系统，其特征在于，该系统包括质粒，所述质粒携带有所需递送的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，所述复合结构能够进入并结合目标组织，以将所述RNA片段送入目标组织。
  2. 如权利要求1所述的RNA质粒递送系统，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序列。
  3. 如权利要求1所述的RNA质粒递送系统，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。
  4. 如权利要求3所述的RNA质粒递送系统，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。
  5. 如权利要求4所述的RNA质粒递送系统，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；
    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、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。
  6. 如权利要求5所述的RNA质粒递送系统，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。
  7. 如权利要求4所述的RNA质粒递送系统，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；
    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。
  8. 如权利要求7所述的RNA质粒递送系统，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；
    其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
  9. 如权利要求1所述的RNA质粒递送系统，其特征在于，所述器官组织为肝脏，所述复合结构为外泌体。
  10. 如权利要求3所述的RNA质粒递送系统，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。
  11. 如权利要求10所述的RNA质粒递送系统，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。
  12. 如权利要求2所述的RNA质粒递送系统，其特征在于，所述RNA序列的长度为15-25个核苷酸。
  13. 如权利要求12中任一所述的RNA质粒递送系统，其特征在于，所述RNA序列选自以下任意一种或几种：EGFR基因的siRNA，KRAS基因的siRNA，VEGFR基因的siRNA，mTOR基因的siRNA，TNF-α基因的siRNA，integrin-α基因的siRNA，B7基因的siRNA，TGF-β1基因的siRNA，H2-K基因的siRNA，H2-D基因的siRNA，H2-L基因的siRNA，HLA基因的siRNA，GDF15基因的siRNA，miRNA-21的反义链，miRNA-214的反义链，TNC基因的siRNA，PTP1B基因的siRNA，mHTT基因的siRNA，Lrrk2基因的siRNA，α-synuclein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。
  14. 如权利要求1所述的RNA质粒递送系统，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。
  15. 一种权利要求1-14任意一项所述的RNA质粒递送系统在药物中的应用。
  16. 如权利要求15所述的应用，其特征在于，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。
  17. 如权利要求15所述的应用，其特征在于，所述药物为治疗癌症、肺纤维化、结肠炎、肥胖症、由肥胖症引起的心血管疾病、二型糖尿病、亨廷顿病、帕金森病、重症肌无力、阿尔兹海默病或移植物抗宿主病的药物。
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