WO2022206739A1 - 一种基于病毒载体的rna递送系统及其应用 - Google Patents

一种基于病毒载体的rna递送系统及其应用 Download PDF

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WO2022206739A1
WO2022206739A1 PCT/CN2022/083601 CN2022083601W WO2022206739A1 WO 2022206739 A1 WO2022206739 A1 WO 2022206739A1 CN 2022083601 W CN2022083601 W CN 2022083601W WO 2022206739 A1 WO2022206739 A1 WO 2022206739A1
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sequence
sirna
rna
viral vector
gene
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French (fr)
Inventor
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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Nanjing University
Nanjing Tech University
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Nanjing University
Nanjing Tech University
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Priority to JP2023559826A priority Critical patent/JP2024511808A/ja
Priority to EP22778921.1A priority patent/EP4317440A4/en
Publication of WO2022206739A1 publication Critical patent/WO2022206739A1/zh
Priority to US18/374,253 priority patent/US20240141380A1/en
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Definitions

  • the present application relates to the field of biomedical technology, in particular to a viral vector-based RNA 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.
  • Virus (Biological virus) is a small individual, simple structure, containing only one nucleic acid (DNA or RNA), must be parasitic in living cells and replicated non-cellular organisms. Viral vectors can bring genetic material into cells. The principle is to use the molecular mechanism of viruses to transmit their genomes into other cells for infection. It can occur in a complete living body (in vivo) or cell culture (in vitro), mainly used in Basic research, gene therapy or vaccines. However, there are few related studies on the use of viruses as vectors to deliver RNA, especially siRNA, using a special self-assembly mechanism.
  • the Chinese Patent Publication No. CN108624590A discloses a siRNA capable of inhibiting the expression of DDR2 gene; the Chinese Patent Publication No. CN108624591A discloses a siRNA capable of silencing the ARPC4 gene, and the siRNA is modified with ⁇ -phosphorus-selenium;
  • the Chinese Patent Publication No. CN108546702A discloses a siRNA targeting long-chain non-coding RNA DDX11-AS1.
  • the Chinese Patent Publication No. CN106177990A discloses a siRNA precursor that can be used for various tumor treatments. These patents design specific siRNAs to target certain diseases caused by genetic changes.
  • Chinese Patent Publication No. CN108250267A discloses a polypeptide, polypeptide-siRNA induced co-assembly, using polypeptide as a carrier of siRNA.
  • the Chinese Patent Publication No. CN108117585A discloses a polypeptide for promoting apoptosis of breast cancer cells through targeted introduction of siRNA, and the polypeptide is also used as the carrier of siRNA.
  • the Chinese Patent Publication No. CN108096583A discloses a nanoparticle carrier, which can be loaded with siRNA with breast cancer curative effect while containing chemotherapeutic drugs.
  • exosomes can deliver miRNAs to recipient cells, which secrete miRNAs at relatively low concentrations , which can effectively block the expression of target genes.
  • Exosomes are biocompatible with the host immune system and possess the innate ability to protect and transport miRNAs across biological barriers in vivo, thus becoming a potential solution to overcome problems associated with siRNA delivery.
  • the Chinese Patent Publication No. CN110699382A discloses a method for preparing siRNA-delivering exosomes, and discloses the technology of separating exosomes from plasma and encapsulating siRNA into exosomes by electroporation .
  • the embodiments of the present application provide a viral vector-based RNA delivery system and its application, so as to solve the technical defects existing in the prior art.
  • the present application provides an RNA delivery system based on a viral vector.
  • the system includes a viral vector, which carries a desired delivered RNA segment, and the viral vector can be enriched in the organ tissue of the host, and the A complex structure containing the RNA fragment is formed endogenously and spontaneously in the host organ tissue, and the complex structure is able to enter and bind to the target tissue and deliver the RNA fragment 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.
  • Figures 16-17 show that the RNA delivery system constructed by adeno-associated virus vector and lentiviral vector has in vivo enrichment and therapeutic effect.
  • the viral vector is an adeno-associated virus.
  • the adenovirus-associated virus is adeno-associated virus type 5, adenovirus-associated virus type 8 or adenovirus-associated virus type 9.
  • 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.
  • Figures 18-19 show that RNA fragments composed of 2 adenovirus vectors and 6 RNA sequences alone or any 2 or any 3 RNA sequences have in vivo enrichment and therapeutic effects after construction.
  • the viral vector includes a promoter and a targeting tag
  • the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host
  • the targeting structure is located on the surface of the composite structure, so The complex structure can seek and bind to the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
  • the viral vector includes any one of the following circuits or a combination of several circuits: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each of the virus At least one RNA fragment and one targeting tag are included in the vector, and the RNA fragment and targeting tag are located in the same circuit or in different circuits.
  • Figures 20-23 show that the two RNA fragments and the two targeting tags can have enrichment and therapeutic effects in vivo when used alone or in any combination and constructed into a viral vector.
  • the viral vector further comprises a flanking sequence, a compensation sequence and a loop sequence that enable the circuit to be folded into a correct structure and expressed, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence;
  • the viral vector includes any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-target To tag, 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensating sequence-3'flanking sequence.
  • the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence whose homology is greater than 80%;
  • the loop sequence is gttttggccactgactgac or a sequence whose homology is greater than 80%;
  • the 3' flanking sequence is accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence whose homology is greater than 80%;
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the purpose of deleting the 1-5 bases of the RNA reverse complementary sequence is to prevent the sequence from being expressed.
  • Figure 25 shows that the delivery system constructed by the 5' flanking sequence/loop sequence/3' flanking sequence and its homologous sequences all have in vivo 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.
  • adjacent lines are connected by sequence 4 or a sequence with a homology greater than 80% to sequence 4;
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • Figure 26 shows that the viral vector delivery system constructed by sequence 4 and its homologous sequence has both in vivo enrichment and therapeutic effects.
  • the organ tissue is liver
  • the composite structure is exosome
  • the targeting tag is selected from targeting peptides or targeting proteins with targeting function.
  • the targeting peptides include RVG targeting peptides, GE11 targeting peptides, PTP targeting peptides, TCP-1 targeting peptides, and MSP targeting peptides;
  • the targeting proteins include RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein, and MSP-LAMP2B fusion protein.
  • the RNA sequence is 15-25 nucleotides in length.
  • the length of the RNA sequence can be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides.
  • the RNA sequence is 18-22 nucleotides in length.
  • Figure 27 shows that gene circuits constructed by RNA sequences of different lengths have in vivo enrichment and therapeutic effects.
  • the RNA sequence is selected from any one or more of the following RNAs: 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, UGUAUUUUACAUAAUUACACAC, other sequences that inhibit 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.
  • HLA gene siRNA 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 PTP1B gene includes UGAUAUAGUCAUUAUCUUCUU, UCCAUUUUUAUCAAACUAGCG, AUUGUUUAAAUAAAUAUGGAG, AAUUUUAAUACAUUAUUGGUU, UUUAUUAUUGUACUUUUUGAU, other sequences that inhibit the expression of 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.
  • the antisense strand of miRNA-21 is 5'-TCAACATCAGTCTGATAAGCTA-3'.
  • sequences with more than 80% homology may be 85%, 88%, 90%, 95%, 98%, etc. homology.
  • the RNA fragment includes an RNA sequence ontology and a modified RNA sequence obtained by modifying the RNA sequence ontology with ribose sugar. That is, the RNA fragment can be composed of only at least one RNA sequence ontology, or only at least one modified RNA sequence, and can also be composed of RNA sequence ontology and modified RNA sequence.
  • the isolated nucleic acid also includes its variants and derivatives.
  • the nucleic acid can be modified by one of ordinary skill in the art using general methods. Modification methods include (but are not limited to): methylation modification, hydrocarbyl modification, glycosylation modification (such as 2-methoxy-glycosyl modification, hydrocarbyl-glycosyl modification, sugar ring modification, etc.), nucleic acid modification, peptide modification Segment modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification) and the like.
  • the modification is an internucleotide linkage, for example selected from: phosphorothioate, 2'-O methoxyethyl (MOE), 2'-fluoro, phosphine Acid alkyl esters, phosphorodithioates, alkyl phosphorothioates, phosphoramidates, carbamates, carbonates, phosphoric triesters, acetamidates, carboxymethyl esters, and combinations thereof.
  • phosphorothioate 2'-O methoxyethyl (MOE), 2'-fluoro
  • phosphine Acid alkyl esters phosphorodithioates, alkyl phosphorothioates, phosphoramidates, carbamates, carbonates, phosphoric triesters, acetamidates, carboxymethyl esters, and combinations thereof.
  • the modification is a modification of nucleotides, such as selected from: peptide nucleic acid (PNA), locked nucleic acid (LNA), arabinose-nucleic acid (FANA), analogs, derivatives objects and their combinations.
  • the modification is a 2' fluoropyrimidine modification.
  • 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on RNA with 2'-F.
  • 2'-F can make RNA not easily recognized by RNase in vivo, thereby increasing the stability of RNA fragment transmission in vivo. sex.
  • the delivery system is a delivery system for use in mammals, including humans.
  • the present application also provides the application of the viral vector-based RNA delivery system in medicine as described in any of the above paragraphs.
  • 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 viral vector, specifically, the viral vector here refers to a viral vector carrying an RNA fragment, or carrying an RNA fragment and a targeting tag, and can enter the host and can be enriched in the liver. Assembled, self-assembled to form a composite structure exosome, 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 viral vector here refers to a viral vector carrying an RNA fragment, or carrying an RNA fragment and a targeting tag, and can enter the host and can be enriched in the liver. Assembled, self-assembled to form a composite structure exosome, 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.
  • the viral vector-based RNA delivery system uses the virus as a carrier and the virus as a mature injectable, and its safety and reliability have been fully verified, and it has good druggability.
  • 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 viruses is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • the viral vector-based 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, but also maintain its stability in circulation , and is beneficial to receptor cell absorption, intracytoplasmic release and lysosomal escape, and the required dose is low.
  • the application of the viral vector-based RNA delivery system provided in this application to drugs 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. effect.
  • Fig. 1 is the mouse colitis treatment situation and RNA expression level comparison diagram provided by an embodiment of the present application
  • FIG. 2 is a comparison diagram of mouse cytokine concentration and colon HE staining provided by an embodiment of the present application
  • FIG. 3 is a comparison diagram of the treatment of colitis in mice provided by an embodiment of the present application.
  • FIG. 4 is a comparison diagram of the mouse disease activity index and various siRNA levels provided by an embodiment of the present application.
  • Figure 5 is a comparison diagram of various siRNA and mRNA levels in mice provided by an embodiment of the present application.
  • FIG. 6 is a comparison diagram of the HE staining situation of mouse colon provided by an embodiment of the present application.
  • FIG. 7 is a diagram of the treatment situation of mouse lung cancer based on KRAS siRNA provided in an embodiment of the present application.
  • Figure 8 is a diagram of the treatment situation of mouse lung cancer based on EGFR siRNA provided by an embodiment of the present application.
  • Fig. 9 is a comparison diagram of the content of various enzymes in mice provided by an embodiment of the present application.
  • Figure 10 is a comparison diagram of mouse hydroxyproline content and mRNA level provided by an embodiment of the present application.
  • Figure 11 is a comparison diagram of mouse survival and tumor assessment provided in an example of the present application.
  • Figure 12 is a comparison diagram of the obesity treatment situation in mice provided by an embodiment of the present application.
  • FIG. 13 is a comparison diagram of various obesity indicators in mice provided by an embodiment of the present application.
  • Figure 14 is a comparison diagram of the treatment of mouse Huntington's disease provided by an embodiment of the present application.
  • FIG. 15 is a construction and characterization diagram of a circuit provided by an embodiment of the present application.
  • Figure 16 shows the enrichment effect of adeno-associated virus vectors in liver, lung, plasma, and exosomes and the detection of EGFR gene expression provided by an embodiment of the present application. and the enrichment effect in the lung, B is the enrichment effect of the adeno-associated virus vector carrying the RNA sequence in plasma and exosomes, C and D are the results of the protein expression and mRNA expression of EGFR, respectively.
  • Figure 17 is the enrichment effect of the lentiviral vector provided in an example of the present application in liver, lung, plasma, and exosomes and the detection of EGFR gene expression.
  • the enrichment effect in , B is the enrichment effect of the lentiviral vector carrying the RNA sequence in plasma and exosomes, C and D are the results of protein expression and mRNA expression of EGFR, respectively.
  • Figure 18 is a gene loop containing 6 different RNA sequences constructed with 2 different adenovirus vectors provided in an example of the application, the detection results of the corresponding protein and mRNA after intravenous injection, A is the expression of EGFR protein in the figure B is the expression of TNC protein, C is the expression of EGFR mRNA, and D is the expression of TNC mRNA.
  • Figure 19 shows the protein expression after intravenous injection after the construction of RNA fragments composed of 2 adenovirus vectors and any 2 or any 3 RNA sequences of the 6 RNA sequences provided in an embodiment of the present application, and A in the figure is After the RNA fragments composed of 2 adenovirus vectors and any 2 RNA sequences of the 6 RNA sequences are constructed, the detected protein content of EGFR and TNC, B is the 2 adenovirus vectors and any 2 of the 6 RNA sequences After the construction of RNA fragments composed of RNA sequences, the detected mRNA contents of EGFR and TNC, C is the RNA fragments composed of two adenovirus vectors and any three RNA sequences among the six RNA sequences, the detected EGFR and TNC Protein content, D is the mRNA content of EGFR and TNC detected after the construction of RNA fragments composed of two adenovirus vectors and any four RNA sequences out of six RNA sequences.
  • Figure 20 shows the enrichment effect of EGFR siRNA and TNC siRNA in pancreas and brain obtained after intravenous injection after combining a single RNA fragment and a single targeting tag and constructing a viral vector provided by an example of the present application and the expression levels of EGFR and TNC.
  • AD is the detection result of the enrichment effect of AD2/AD5+PTP/RVG+siR EGFR /siR TNC , respectively
  • EH is the EGFR protein content of AD2/AD5+PTP/RVG+siR EGFR , respectively.
  • IL is the detection results of TNC protein content and mRNA content of AD2/AD5+PTP/RVG+siR TNC , respectively.
  • Figure 21 shows the enrichment of EGFR siRNA and TNC siRNA obtained in pancreas and brain after intravenous injection after arbitrary combination of two RNA fragments and two targeting tags provided in an example of the present application and constructed into a viral vector
  • AB is the detection result of the enrichment effect of AD2/AD5+PTP/RVG+siR EGFR+TNC
  • CD is the detection result of the enrichment effect of AD2/AD5+(PTP-RVG)+siR EGFR
  • E is AD2 Detection results of enrichment effect of /AD5+(PTP-RVG)+siR EGFR+TNC .
  • Figure 22 shows the expression levels of EGFR and TNC in the pancreas and brain obtained after intravenous injection after arbitrary combination of 2 kinds of RNA fragments and 2 kinds of targeting tags provided in an embodiment of the present application and constructed into a viral vector
  • Figure 22 Middle AD are the detection results of EGFR protein content and mRNA content of AD2/AD5+PTP/RVG+siR EGFR+TNC , respectively
  • EH are the detection results of TNC protein content and mRNA content, respectively.
  • Figure 23 shows the expression levels of EGFR and TNC in the pancreas and brain obtained after intravenous injection after arbitrary combination of 2 kinds of RNA fragments and 2 kinds of targeting tags provided by another embodiment of the application and constructed into a viral vector
  • AB is the detection result of EGFR protein content and mRNA content of AD2/AD5+(PTP-RVG)+siR EGFR
  • CD is the detection result of EGFR protein content and mRNA content of AD2/AD5+(PTP-RVG)+siR EGFR+TNC
  • EF is the detection result of TNC protein content and mRNA content of AD2/AD5+(PTP-RVG)+siR TNC
  • GH is the detection result of TNC protein content and mRNA content of AD2/AD5+(PTP-RVG)+siR EGFR+TNC .
  • Figure 24 shows the enrichment results of siRNA in liver, lung, plasma, and exosomes and the detection results of EGFR protein and mRNA expression after intravenous injection of the delivery system constructed with two routes provided in an embodiment of the present application.
  • A is the enrichment effect of delivery systems AAV- siRE and AAV-GE11- siRE in liver and lung
  • B is the enrichment of delivery systems AAV- siRE and AAV-GE11- siRE in plasma and exosomes Effect
  • C is the detection result of EGFR protein content of delivery system AAV- siRE and AAV-GE11- siRE
  • D is the detection result of EGFR mRNA content of delivery system AAV- siRE and AAV-GE11-siRE.
  • Figure 25 shows the enrichment effect of adenoviral vectors in the lungs provided by an embodiment of the present application containing 2 clear sequences with 5' flanking sequence/loop sequence/3' flanking sequence homology greater than 80%, in the figure A is the enrichment effect of different 5' flanking sequences, B is the enrichment effect of different loop sequences, and C is the enrichment effect of different 3' flanking sequences.
  • Figure 26 shows that sequence 4 and two sequences 4-1 and 4-2 with more than 80% homology to sequence 4 provided in an example of the present application were constructed into an AAV vector, and the sequence enriched in lung tissue 9 hours after intravenous injection Set the effect
  • a in the figure is the detection result of siRNA content of AAV- siR E and AAV-siRT when connected with sequence 4
  • B in the figure is the siRNA content of AAV- siRE and AAV- siRT when connected with sequence 4-1
  • the detection result of siRNA content C in the figure is the detection result of siRNA content of AAV- siR E and AAV-siRT when connected with sequence 4-2.
  • Figure 27 is the detection result of EGFR expression after intravenous injection of gene loops containing sequences of different lengths provided in an embodiment of the present application.
  • A is the detection result of EGFR protein content
  • B is the detection result of EGFR mRNA content.
  • 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.
  • 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 delivery system based on a viral vector, the system comprising a viral vector, the viral vector carrying the RNA fragment to be delivered, the viral vector can be enriched in the organ tissue of the host, and the A complex structure containing the RNA fragment is formed endogenously and spontaneously in the host organ tissue, and the complex structure is able to enter and bind to the target tissue and deliver the RNA fragment into the target tissue.
  • Figures 16-17 show that the RNA delivery system constructed by adeno-associated virus vector and lentiviral vector has in vivo enrichment and therapeutic effects, and Figure 16 shows the enrichment effect of adeno-associated virus vector in liver, lung, plasma, exosome and EGFR gene expression detection, Figure 17 shows the enrichment effect of lentiviral vector in liver, lung, plasma, exosomes and EGFR gene expression detection.
  • the viral vector also includes a promoter and a targeting tag.
  • the viral vector includes any one of the following circuits or a combination of several circuits: promoter-RNA sequence, promoter-targeting tag, promoter-RNA sequence-targeting tag, and each of the viral vectors includes at least one RNA fragments and a targeting tag, either in the same circuit or in different circuits.
  • the viral vector may only include a promoter-RNA sequence-targeting tag, or may include a combination of a promoter-RNA sequence, a promoter-targeting tag, or a promoter-targeting tag, a promoter A combination of RNA-seq-targeting tags.
  • RNA fragment 1 is siRNA EGFR
  • RNA fragment 2 is siRNA TNC
  • targeting tag 1 is PTP
  • targeting tag 2 is RVG
  • two RNA fragments and two targeting tags are used alone or in any combination
  • the viral vector may also include a flanking sequence, a compensation sequence and a loop sequence that can make the circuit fold into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence; the viral vector Including any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag, 5' - Promoter - Targeting Tag - 5' Flanking Sequence - RNA Fragment - Loop Sequence - Compensation Sequence - 3' Flanking Sequence.
  • the 5' flanking sequence is preferably ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with ggatcctggaggcttgctgaaggctgtatgctgaattc, etc.
  • the loop sequence is preferably gttttggccactgactgac or a sequence with more than 80% homology thereto, including sequences with 85%, 90%, 92%, 95%, 98%, 99% homology with gttttggccactgactgac, and the like.
  • the 3' flanking sequence is preferably accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag, etc.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-5 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
  • the compensatory sequence can be the reverse complementary sequence of the RNA sequence in which 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 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.
  • 5'-promoter-5' flanking sequence-RNA sequence-loop sequence-compensation sequence-3' flanking sequence corresponds to the delivery vector name AAV- siRNA
  • 5'-promoter-targeting tag-5' Flanking sequence-RNA sequence-loop sequence-compensation sequence-3' flanking corresponds to the delivery vector name AAV-GE11- siRE
  • Figure 24 shows the enrichment results of siRNA in liver, lung, plasma, exosome species after intravenous injection and EGFR protein and mRNA expression detection results.
  • FIG 25 a set of data on the enrichment effect of adenovirus vectors in the lungs are shown.
  • the sequences in the adenovirus vectors are: 2 sequences with 5' flanking sequence/loop sequence/3' flanking sequence homology greater than 80% Explicit sequence.
  • flanking sequence CTGGAGGCTTGCTGAAGGCTGTATGCTGAATTCG 5' flanking sequence-2 CTGGAGGCTTGCTCGGAGGCTGTATGCTGGCTTCG loop-1 GTTTTGGCCACTGACTGAC loop-2 GTGGGCGGCCACTGACTGAC 3' flanking sequence -1 CACCGGTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC 3' flanking sequence-2 CAGAGGTCAGGACACAAGGCCTACGACTAGCACTCACATGTCTCAAATGGCC
  • sequence 1 is preferably CAGATC
  • sequence 2 can be composed of 5-80 bases
  • Sequence of bases such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases
  • sequence of 10-50 bases is preferable, and the sequence of 20-40 bases is more preferable.
  • Sequence 3 is preferably TGGATC.
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • Figure 26 shows the results of EGFR siRNA content detection in lung tissue after 9 hours of intravenous injection after constructing sequence 4 and two sequences 4-1 and 4-2 with more than 80% homology to sequence 4 into an AAV vector.
  • 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
  • Figure 27 shows the detection of EGFR expression after intravenous injection of gene loops constructed by RNA sequences of different lengths, wherein the plasmids with RNA sequence lengths of 18, 20, and 22 correspond to AAV-siR E (18) and AAV, respectively.
  • RNA sequences of different lengths are shown in Table 4 below.
  • 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.
  • RNA sequences in the RNA fragment is one, two or more. For example, if you need to treat glioma, you can combine EGFR gene siRNA and TNC gene siRNA on the same viral vector; if you need to treat enteritis, you can use TNF- ⁇ gene siRNA and integrin- ⁇ gene at the same time. siRNA and B7 siRNA.
  • the functional structural region of the viral vector can be expressed as: (promoter-siRNA1)-connector sequence-(promoter-siRNA2)-connector sequence- (promoter-targeting tag), or (promoter-targeting tag-siRNA1)-linker-(promoter-targeting tag-siRNA2), or (promoter-siRNA1)-linker-(promoter- Targeting tag-siRNA2) etc.
  • the functional structural region of the viral vector can be expressed as: (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-connector sequence-(5'-promoter - 5' flanking sequence - siRNA2-loop sequence - compensation sequence - 3' flanking sequence) - linking sequence - (5'-promoter-targeting tag), or (5'-promoter-targeting tag-5' flanking sequence-siRNA1-loop sequence-compensation sequence-3' flanking sequence)-linker sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'flanking sequence), or (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-linking sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'flanking
  • the above RNA can also be obtained by ribose modification of the RNA sequence (siRNA, shRNA or miRNA) therein, preferably 2' fluoropyrimidine modification.
  • 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on siRNA, shRNA or miRNA with 2'-F.
  • 2'-F can make it difficult for RNase in the human body to recognize siRNA, shRNA or miRNA, so it can Increases the stability of RNA transport in vivo.
  • the 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 organ in the host described in this example is preferably the liver.
  • the liver is the most active cell opening and closing tissue of all mammalian tissues and organs.
  • RNA fragments siRNA, shRNA or miRNA
  • the liver tissue can spontaneously wrap the RNA fragments into exosomes, and these exosomes become RNA delivery mechanisms (Composite structure containing the desired delivery RNA and having a targeting structure).
  • targeting tags can be designed in the virus injected into the body, and the targeting tags will also be assembled into the exosomes by the liver tissue, especially when certain specific When targeting the label, the targeting label can be inserted into the surface of the exosome, thereby becoming a targeting structure that can guide the exosome, which can greatly improve the accuracy of the RNA delivery mechanism of the present invention, and greatly improve the potential drug delivery. s efficiency.
  • targeting tags are a process that requires creative work. On the one hand, it is necessary to select the available targeting tags according to the target tissue, and on the other hand, it is necessary to ensure that the targeting tags can stably appear on the surface of exosomes, so as to achieve targeted targeting. Function. Targeting tags that have been screened so far include targeting peptides, targeting proteins, and antibodies.
  • targeting peptides include but are not limited to RVG targeting peptide (nucleotide sequence shown in SEQ ID No: 1), GE11 targeting peptide (nucleotide sequence shown in SEQ ID No: 2), PTP targeting peptide Peptide (nucleotide sequence shown in SEQ ID No: 3), TCP-1 targeting peptide (nucleotide sequence shown in SEQ ID No: 4), MSP targeting peptide (nucleotide sequence shown in SEQ ID No: 4) : 5); targeting proteins include but are not limited to RVG-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6), GE11-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 7) shown), PTP-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 8), TCP-1-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No:
  • 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 viral vectors are preferably adenovirus-associated virus (AAV), more preferably adenovirus-associated virus type 5 (AAV-5), Adeno-associated virus type 8 (AAV-8) or adeno-associated virus type 9 (AAV-9).
  • AAV adenovirus-associated virus
  • AAV-5 adenovirus-associated virus type 5
  • AAV-8 adeno-associated virus type 8
  • AAV-9 adeno-associated virus type 9
  • the viral vector can also be composed of multiple viruses with different structures, one of which contains a promoter promoters and targeting tags, other viruses contain promoters and RNA segments. Loading the targeting tag and RNA fragment into different viral vectors, and injecting the two viral vectors into the body, the targeting effect is no worse than the targeting effect produced by loading the same targeting tag and RNA fragment into one viral vector .
  • the viral vector containing the RNA sequence can be injected first, and then the viral vector containing the targeting tag can be injected after 1-2 hours, so that a better target can be achieved. to the effect.
  • the delivery systems described above can all be used in mammals, including humans.
  • the 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 15b.
  • 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 15a.
  • 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
  • the viral vector-based RNA delivery system uses the virus as the carrier and the viral vector 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 delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of viral vectors is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • the viral vector-based 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, but also maintain its stability in circulation It is also beneficial to receptor cell uptake, intracytoplasmic release and lysosomal escape, and the required dose is low.
  • the medicament includes the system comprising a viral vector carrying the RNA fragment to be delivered, the viral vector being capable of being enriched in the organ tissue of the host, and endogenously forming spontaneously in the organ tissue of the host A complex structure containing the RNA fragments capable of entering and binding to the target tissue, delivering 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 with medical significance.
  • the two adenoviruses are denoted as ADV1 and ADV2, respectively, and the six RNAs are: siR E (target gene is EGFR), siRT (target gene is TNC), shRE (target gene is EGFR), siRT (target gene is TNC), miR-7 (target gene is EGFR), miR-133b (target gene is EGFR).
  • Figure 18 shows the expression levels of the corresponding proteins after intravenous injection of the 12 gene loops constructed by the two adenovirus vectors
  • Figure 19 shows the composition of the two adenovirus vectors and any two or any three RNA sequences among the six RNA sequences. After RNA fragment construction, protein expression after intravenous injection.
  • RNA sequences are specifically shown in Table 5 below.
  • the viral vector includes a promoter and a targeting tag
  • the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host
  • the targeting structure is located on the surface of the composite structure, so The complex structure can seek and bind to the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
  • the drug can be administered orally, inhaled, subcutaneously injected, intramuscularly or intravenously injected into the human body, and then delivered to the target tissue through the viral vector-based 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 virus as the carrier and the virus carrier as the mature injection, and its safety and reliability have been fully verified, and the druggability is very good.
  • the final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the drug can deliver various kinds of small molecule RNAs and has strong versatility. And the preparation of viruses 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.
  • Example 1 or 2 On the basis of Example 1 or 2, this example provides an application of a viral vector-based RNA delivery system in a drug, which is a drug for the treatment of colitis. The efficacy of the vector RNA delivery system in the treatment of colitis is specifically described.
  • the three experimental groups were the AAV-CMV-siR TNF- ⁇ (low) group and the AAV-CMV-siR TNF- ⁇ (medium) group.
  • AAV-CMV-siR TNF- ⁇ (high) group; control group were Normal group and AAV-CMV-scrR group.
  • the experimental process is shown in Figure 1A.
  • the AAV-CMV-siR TNF- ⁇ (low) group, the AAV-CMV-siR TNF- ⁇ (medium) group, and the AAV-CMV-siR TNF- ⁇ (high) group were treated with high hepatic Affinity AAV-5 adeno-associated virus-encapsulated TNF- ⁇ siRNA system (AAV-CMV-siR TNF- ⁇ ), AAV solution with a titer of 10 12 Vg/ml, 25 ⁇ L, 50 ⁇ L, 100 ⁇ L was injected into mice through the tail vein in vivo.
  • the in vivo expression of the AAV system was monitored by small animals. The results are shown in Figure 1B. After 3 weeks, it can be seen that the AAV system is stably expressed in vivo, especially in the liver.
  • the AAV-CMV-siR TNF- ⁇ (high) group has an average The Average Radiance reached 8.42*105 (p/sec/cm 2 /sr), and the expression site was in the liver, which indicated that the expression of the AAV system had a dose-dependent effect.
  • the disease index of the mice in each group was scored and counted, and the results are shown in Figure 1E. It can be seen that the disease index of the mice in the AAV-CMV-siR TNF- ⁇ (high) group was lower than that of the AAV-CMV-siR TNF- ⁇ (low) group, AAV-CMV-siR TNF- ⁇ (medium) group and AAV-CMV-scrR group.
  • TNF- ⁇ siRNA The levels of TNF- ⁇ siRNA in the mice in each group were detected respectively, and the results are shown in Figure 1F. It can be seen that the levels of TNF- ⁇ siRNA in the three experimental groups were higher, while the AAV-CMV-scrR group in the control group had higher levels of TNF- ⁇ siRNA. There is almost no expression of TNF- ⁇ siRNA, which indicates that the above-mentioned AAV system can produce a certain amount of TNF- ⁇ siRNA.
  • TNF- ⁇ mRNA in the mice in each group were detected respectively, and the results are shown in Figure 1G. It can be seen that the levels of TNF- ⁇ mRNA in the mice in the Normal group and the three experimental groups were relatively low, while the AAV-CMV-scrR group was lower. The level of TNF- ⁇ mRNA in mice was higher, which indicated that AAV system could reduce the expression and secretion of colonic TNF- ⁇ .
  • the pro-inflammatory cytokines IL-6, IL-12, and IL -23 in the colon of mice were detected, and the results were shown in Figure 2A.
  • the secretion of inflammatory cytokines was the least, and the secretion of pro-inflammatory cytokines was the most in the AAV-CMV-scrR group.
  • HE staining and pathological score statistics were performed on the mouse colon sections. The results are shown in Figure 2B and Figure 2C. It can be seen that the experimental group, especially the AAV-CMV-siR TNF- ⁇ (high) group, had better colonic mucosa integrity. The degree of infiltration of immune cells was more shallow, and the colonic crypt abscess and the congestion and hemorrhage of the colon were also significantly lighter than those in the AAV-CMV-scrR group.
  • liver-friendly AAV to encapsulate the CMV-siR TNF- ⁇ circuit can achieve long-term TNF- ⁇ siRNA expression and long-term TNF- ⁇ silencing, and can relieve colitis to a certain extent. potential and clinical research value.
  • the experimental groups were AAV-CMV-siR T+B+I (low) group, AAV-CMV-siR T+B+I (medium) group, AAV-CMV-siR T+B+I (high) group ;
  • the control groups were Normal group and AAV-CMV-scrR group.
  • AAV-CMV-siR T+B+I (low) group, AAV-CMV-siR T+B+I (medium) group, and AAV-CMV-siR T+B+I (high) group used liver high affinity
  • the AAV-5 adeno-associated virus-encapsulated TNF- ⁇ siRNA, B7-siRNA and Integrin ⁇ 4 siRNA element tandem drug delivery system (AAV-CMV-siR T+B+I ) was injected through the tail vein with a titer of 10 12 Vg/ml AAV solution 25 ⁇ L, 50 ⁇ L, 100 ⁇ L into mice.
  • the in vivo expression of the AAV system was monitored by small animals. The results are shown in Figure 3A. After 3 weeks, it can be seen that the AAV system is stably expressed in vivo, especially in the liver, and the expression of the AAV system has a dose-dependent effect.
  • the disease index of mice in each group was scored and counted. The results are shown in Figure 4A. It can be seen that the disease index of mice in the AAV-CMV-siR T+B+I (high) group was lower than that of AAV-CMV-siR T +B+I (low) group, AAV-CMV-siR T+B+I (medium) group and AAV-CMV-scrR group.
  • TNF- ⁇ siRNA, B7 siRNA and integrin ⁇ 4 siRNA in mouse plasma were detected.
  • the results are shown in Figure 4B, Figure 4C, and Figure 4D. It can be seen that the AAV-encapsulated CMV-siR T+B+I system was generated in mouse plasma. A certain amount of stably expressed siRNA showed a dose-dependent effect.
  • TNF- ⁇ mRNA, B7mRNA and integri ⁇ 4 mRNA in the mouse colon were detected.
  • the results are shown in Figure 5D, Figure 5E and Figure 5F. It can be seen that the AAV-encapsulated CMV-siR T+B+I system significantly reduced TNF- ⁇ in the mouse colon. - ⁇ , B7 and integrin ⁇ 4 mRNA expression.
  • liver-friendly AAV to encapsulate the CMV-siR T+B+I circuit can achieve long-term TNF- ⁇ siRNA, B7 siRNA and integrin ⁇ 4 siRNA expression and multiple target gene silencing, and significantly alleviate the degree of colon inflammation.
  • great drug potential and clinical research value great drug potential and clinical research value.
  • this embodiment provides an application of a viral vector-based RNA delivery system in a drug, which is a drug for the treatment of lung cancer.
  • a drug which is a drug for the treatment of lung cancer.
  • the effect of the RNA delivery system in the treatment of lung cancer is specifically described.
  • liver high-affinity AAV-5 adeno-associated virus-encapsulated EGFR siRNA system AAV-CMV-EGFR siRNA
  • KRAS siRNA system AAV-CMV-KRAS siRNA
  • mice mouse lung cancer cells
  • CT scanning technology was used to observe the progress of mouse model construction.
  • the mice in the successfully constructed mice were administered once every two days, that is, the mice in the PBS group/AAV-CMV-scrR group/AAV-CMV-KRAS siRNA group were injected with PBS buffer/AAV-CMV once every two days -scrR/AAV-CMV-KRAS siRNA treatment, survival analysis and tumor assessment were performed in mice, respectively, and the treatment was stopped after 7 doses.
  • FIG. 7B “PBS pre” indicates the PBS group before administration, “PBS post” indicates the PBS group after administration; “AAV-CMV-scrR pre” indicates the AAV-CMV-scrR group before administration, “ “AAV-CMV-scrR post” indicates the AAV-CMV-scrR group after administration; “AAV-CMV-KRAS-siRNA pre” indicates the AAV-CMV-KRAS siRNA group before administration, “AAV-CMV-KRAS-siRNA pre” post” indicates the AAV-CMV-KRAS siRNA group after administration.
  • the tumor volume of the mice in the AAV-CMV-KRAS siRNA group decreased significantly after administration, while the tumor volume of the mice in the PBS group and AAV-CMV-scrR group not only did not decrease after administration, but also showed increased to varying degrees.
  • 1 experimental group and 2 control groups were set, wherein the experimental group was the AAV-CMV-EGFR siRNA group, and the control groups were the PBS group and the AAV-CMV-scrR group.
  • mice were administered to the successfully constructed mice, once every two days, that is, to the PBS group/AAV-CMV-scrR group/
  • the mice in the AAV-CMV-EGFR siRNA group were injected with PBS buffer/AAV-CMV-scrR/AAV-CMV-EGFR siRNA once for treatment, and the mice were subjected to survival analysis and tumor assessment respectively, and the treatment was stopped after 7 administrations.
  • CT scans were performed on the mice in each group before and after administration.
  • the CT images are shown in Figure 8E.
  • 3D modeling was performed on the lung tissue of the mice, and the tumor volume was calculated.
  • the results are shown in Figure 8B. .
  • PBS pre indicates the PBS group before administration
  • PBS post indicates the PBS group after administration
  • AAV-CMV-scrR pre indicates the AAV-CMV-scrR group before administration
  • AAV-CMV-scrR post indicates the AAV-CMV-scrR group after administration
  • AAV-CMV-EGFR siRNA pre indicates the AAV-CMV-EGFRsiRNA group before administration
  • AAV-CMV-EGFR siRNA post indicates AAV-CMV-EGFR siRNA group after administration.
  • the tumor volume of the mice in the AAV-CMV-EGFR siRNA group decreased significantly after administration, while the tumor volume of the mice in the PBS group and AAV-CMV-scrR group not only did not decrease after administration, but also showed increased to varying degrees.
  • the experimental group was AAV-CMV-KRAS siRNA group, AAV-CMV-EGFR siRNA group, and the control group was PBS group and AAV-CMV-scrR Group.
  • mice were administered to the successfully constructed mice, once every two days, that is, to the PBS group/AAV-CMV-scrR group/
  • the mice in the AAV-CMV-EGFR siRNA group/AAV-CMV-KRAS siRNA group were injected once with PBS buffer/AAV-CMV-scrR/AAV-CMV-EGFR siRNA/AAV-CMV-KRAS siRNA for treatment.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • TBIL total bilirubin
  • ALP serum alkaline phosphatase
  • CREA creatinine
  • BUN blood urea nitrogen
  • this embodiment provides the application of a viral vector-based RNA delivery system in a drug, which is a drug for the treatment of pulmonary fibrosis.
  • a drug which is a drug for the treatment of pulmonary fibrosis.
  • the efficacy of the RNA delivery system in the treatment of pulmonary fibrosis is specifically described.
  • the anti-miR-21/TGF- ⁇ 1 siRNA/anti-miR-21+TGF- ⁇ 1 siRNA system was encapsulated by AAV-5 adeno-associated virus with high affinity in the liver to obtain AAV-anti-miR21/
  • AAV-TGF- ⁇ 1 siRNA/AAV-MIX 100 ⁇ L of AAV solution with a titer of 10 12 Vg/ml was injected into mice via tail vein.
  • the in vivo expression of the AAV system was monitored by small animals. After 3 weeks, it was found that the AAV system was stably expressed in vivo, especially in the liver.
  • mice were then selected for modeling. After successful modeling, mice were injected with PBS buffer/AAV-scrR/AAV-anti-miR21/A AV-TGF- ⁇ 1 siRNA/AAV-MIX (10 mg/kg), respectively. PBS group/AAV-scrR group/AAV-anti-miR21 group/AAV-TGF- ⁇ 1 siRNA group/AAV-MIX group were formed.
  • mice The relative TGF- ⁇ 1 mRNA levels of normal mice, PBS group mice, AAV-scrR group mice, and AAV-TGF- ⁇ 1 siRNA group mice were detected respectively. The results are shown in Figure 10B. It can be seen that AAV-TGF- ⁇ 1 siRNA The relative TGF- ⁇ 1 mRNA level of mice in the group was relatively low.
  • Hydroxyproline is the main component of collagen, and its content reflects the degree of pulmonary fibrosis.
  • the hydroxyproline content of mice in each group was detected respectively, and the results are shown in Figure 10A. It can be seen that the hydroxyproline content in the PBS group and AAV-scrR group was the highest, and the AAV-anti-miR21 group and AAV-TGF- ⁇ 1 siRNA The hydroxyproline content of mice in the AAV-anti-miR21 group, AAV-TGF- ⁇ 1 siRNA group and AAV-MIX group was inhibited.
  • this embodiment provides an application of a viral vector-based RNA delivery system in a drug, which is a drug for the treatment of glioblastoma.
  • a drug which is a drug for the treatment of glioblastoma.
  • the effect of viral vector RNA delivery system in glioblastoma treatment is described in detail.
  • the EGFR siRNA system (AAV-CMV-RVG-siRE) and the EGFR siRNA and TNC siRNA system (AAV-CMV-RVG-siRE+T) were encapsulated by the liver high-affinity AAV-5 adeno-associated virus.
  • the in vivo expression of the AAV system was monitored by small animals. After 3 weeks, it was found that the AAV system was stably expressed in vivo, especially in the liver.
  • mice were randomly selected and injected with glioblastoma cells (U-87 MG-Luc cells) into the mice. From the 7th day to the 21st day, the mice were injected with PBS buffer/AAV- CMV-scrR/AAV-CMV-RVG-siR E /AAV-CMV-RVG-siR E+T (5 mg/kg) was treated to form PBS group/AAV-scrR group/AAV-CMV-RVG-siR E group /AAV-CMV-RVG-siR E+T group.
  • PBS buffer/AAV- CMV-scrR/AAV-CMV-RVG-siR E /AAV-CMV-RVG-siR E+T 5 mg/kg
  • this embodiment provides the application of a viral vector-based RNA delivery system in medicine, which is a medicine for treating obesity.
  • RNA delivery system in obesity treatment is specified.
  • the PTP1B siRNA system (AAV-CMV-siR P /AAV-CMV-RVG- siRP ) was encapsulated by the high-affinity AAV-5 adeno-associated virus in the liver, and 100 ⁇ L of AAV solution with a titer of 10 12 Vg/ml was injected into the tail vein. into mice.
  • the in vivo expression of the AAV system was monitored by small animals. After 3 weeks, it was found that the AAV system was stably expressed in vivo, especially in the liver.
  • mice C57BL/6 mice were selected and injected with PBS buffer/AAV-CMV-scrR/AAV-CMV- siRP /AAV-CMV-RVG- siRP after 12 weeks to form PBS group/AAV-CMV-scrR group/AAV -CMV- siRP group/AAV-CMV-RVG- siRP group and injected every two days for 24 days.
  • Changes in body weight, weight of covered fat pads, initial food intake, serum leptin content, blood glucose content, basal glucose content, serum total cholesterol (TC), triglyceride (TG), low-density lipid The detection and statistics of protein (LDL), body length and food intake, the results are as follows.
  • Figure 12A 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 AAV-CMV-RVG- siRP group is the most stable.
  • FIG. 12B which is a comparison chart of epididymal fat pad weights of mice in each group, it can be seen that the epididymal fat pad of mice in the AAAV-CMV-RVG- siRP group has the lightest 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 AAV-CMV-RVG- siRP group had the least food intake.
  • FIG 12D 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 AAV-CMV-RVG- siRP group was the lowest.
  • FIG 12E the figure is a comparison chart of blood glucose change curves of mice in each group. It can be seen that the blood glucose content of mice in the AAV-CMV-RVG- siRP group was the lowest.
  • 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 AAV-CMV-RVG- siRP group had the lowest basal glucose content.
  • the three graphs are the comparison graphs of serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL) of mice in each group. It can be seen that AAV-CMV -RVG- siRP group had the lowest TC, TG and LDL.
  • FIG. 13D which is a comparison chart of the body lengths of the mice in each group, it can be seen that the body lengths of the four groups of mice are almost the same.
  • FIG. 13E which is a comparison chart of the HFD food intake of the mice in each group, it can be seen that the HFD food intake of the four groups of mice is also similar.
  • this embodiment provides the application of a viral vector-based RNA delivery system in medicine, which is a medicine for the treatment of Huntington's disease.
  • RNA delivery systems in the treatment of Huntington's disease are specifically described.
  • the HTT siRNA system (AAV-CMV-siR mHTT /AA V-CMV-RVG-siR mHTT ) was encapsulated by the liver high-affinity AAV-5 adeno-associated virus, and 100 ⁇ L was injected into the tail vein at a titer of 10 12 Vg/ml of AAV solution into mice.
  • the in vivo expression of the AAV system was monitored by small animals. After 3 weeks, it was found that the AAV system was stably expressed in vivo, especially in the liver.
  • mice were then selected for modeling. After modeling, mice were injected with PBS buffer/AAV-CMV-scrR/AAV-CMV-siR mHTT /AAV-CMV-RVG-siR mHTT to form a PBS group/AAV-CMV- scrR group/AAV-CMV-siR mHTT group/AAV-CMV-RVG-siR mHTT group.
  • plasma exosomes were isolated, labeled with PKH26 dye, and co-cultured with cells to observe the absorption of exosomes by cells. The results are as follows.
  • FIG 14A the figure shows the comparison of siRNA levels in plasma exosomes of mice in each group. It can be seen that the plasma exosomes of AAV-CMV-siR mHTT group and AAV-CMV-RVG-siR mHTT group siRNA levels are higher in vivo.
  • this figure is a comparison of the relative mHTT mRNA levels of mice in each group after co-culture of mouse plasma exosomes with cells. It can be seen that the AAV-CMV-siR mHTT group and AAV-CMV-RVG The relative mHTT mRNA level of mice in the -siR mHTT group was lower, which indicated that AAV-CMV-siRmHTT and AAV-CMV-RVG-siRmHTT could reduce the level of HTT mRNA, that is, siRNA assembled into exosomes could still exert gene silencing function.
  • FIG. 14C which is a comparison of absolute levels of siRNA in mouse liver, it can be seen that the absolute levels of siRNA in mice in the AAV-CMV-siR mHTT group and the AAV-CMV-RVG-siR mHTT group are higher.
  • FIG. 14D which is a comparison of absolute levels of siRNA in mouse plasma, it can be seen that the absolute levels of siRNA in mice in the AAV-CMV-siR mHTT group and the AAV-CMV-RVG-siR mHTT group are higher.
  • the figure is a comparison chart of the descending latency of mice in wild-type mice (WT), AAV-CMV-scrR group, and AAV-CMV-RVG-siR mHTT group. It can be seen that at 0 weeks, three The descending latencies of the mice in the groups were relatively consistent. At the 4th and 8th weeks, the mice in the CMV-scrR group had the shortest descending latencies.
  • the figure is a comparison of the relative mHTT mRNA levels in the cortex and striatum of mice in the AAV-CMV-scrR group and the AAV-CMV-RVG-siR mHTT group.
  • the mHTT mRNA levels of mice in the AAV-CMV-RVG-siR mHTT group were lower than those in the AAV-CMV-scrR group.

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Abstract

提供一种基于病毒载体的RNA递送系统及其应用。该RNA递送系统包括病毒载体，所述病毒载体携带有所需递送的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述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。该病毒载体具有靶向标签。该复合结构为外泌体。该病毒载体的RNA递送系统安全可靠，成药性好，通用性强。

Description

一种基于病毒载体的RNA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，特别涉及一种基于病毒载体的RNA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、阳离子聚合物和病毒)，以提高siRNA在体内传递的效率。其中第3方面的专利很多，其根本原因是研究人员们已经意识到目前缺乏合适的siRNA传递系统，将siRNA安全地、精确地、高效地输送到目标组织，该问题已经成为制约RNAi疗法的核心问题。
病毒(Biological virus)是一种个体微小，结构简单，只含一种核酸(DNA或RNA)，必须在活细胞内寄生并以复制方式增殖的非细胞型生物。病毒载体可将遗传物质带入细胞，原理是利用病毒具有传送其基因组进入其他细胞，进行感染的分子机制，可发生于完整活体(in vivo)或是细胞培养(in vitro)中，主要应用于基础研究、基因疗法或疫苗。但是目前很少有针对将病毒作为载体利用特殊的自组装机制递送RNA，特别是siRNA的相关研究。
公开号为CN108624590A的中国专利公开了一种能够抑制DDR2基因表达的siRNA；公开号为CN108624591A的中国专利公开了一种能够沉默ARPC4基因的siRNA，并且对该siRNA进行了α-磷-硒修饰；公开号为CN108546702A的中国专利公开了一种靶向长链非编码RNA DDX11-AS1的siRNA。公开号为CN106177990A的中国专利公开了一种可以用于多种肿瘤治疗的siRNA前体。这些专利均设计了特定的siRNA并且来针对某些由基因变化引起的疾病。
公开号为CN108250267A的中国专利公开了一种多肽、多肽-siRNA诱导共组装体，使用多肽作为siRNA的载体。公开号为CN108117585A的中国专利公开了一种靶向导入siRNA促进乳腺癌细胞凋亡的多肽，同样使用多肽作为siRNA的载体。公开号为CN108096583A的中国专利公开了一种纳米粒子载体，该载体在包含化疗药物的同时还可以装载具有乳腺癌疗效的siRNA。这些专利均为在siRNA载体方面的发明创造，但是其技术方案具有一个共同特征，那就是载体和siRNA均在体外预先组装，然后再引入宿主体内。事实上，目前绝大部分设计的传递技术均是如此。然而这类传递体系具有共同的问题，那就是这些人工合成的外源性传递体系很容易被宿主的循环系统清除，也有可能引起免疫原性反应，甚至可能对特定的细胞类型和组织有毒。
本发明的研究团队发现内源性细胞可以选择性地将miRNAs封装到外泌体(exosome)中，外泌体可以将miRNA传递到受体细胞中，其分泌的miRNA在相对较低的浓度下，即可有力阻断靶基因的表达。外泌体与宿主免疫系统生物相容，并具有在体内保护和运输miRNA跨越生物屏障的先天能力，因此成为克服与siRNA传递相关的问题的潜在解决方案。例如，公开号为CN110699382A的中国专利就公开了一种递送siRNA的外泌体的制备方法，公开了从血浆中分离外泌体，并将siRNA通过电穿孔的方式封装到外泌体中的技术。
但是这类在体外分离或制备外泌体的技术，往往需要通过细胞培养获取大量的外泌体，再加上siRNA封装的步骤，这使得大规模应用该产品的临床费用变得非常高，一般患者无法负担；更重要的是，外泌体复杂的生产/纯化过程，使其几乎不可能符合GMP标准。
到目前为止，以外泌体为有效成分的药物从未获得CFDA批准，其核心问题就是无法保证外泌体产品的一致性，而这一问题直接导致此类产品无法获得药品生产许可证。如果能解决这一问题，则对推动RNAi疗法意义非凡。
因此，开发一个安全、精确和高效的siRNA传递系统是对提高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基于病毒载体的RNA递送系统及其应用，以解决现有技术中存在的技术缺陷。
本申请提供一种基于病毒载体的RNA递送系统，该系统包括病毒载体，所述病毒载体携带有所需递送的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述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。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。
通过图16-17表明了腺相关病毒载体和慢病毒载体构建的RNA递送系统具有体内富集和治疗效果。
可选地，所述病毒载体为腺病毒相关病毒。
可选地，所述腺病毒相关病毒为腺病毒相关病毒5型、腺病毒相关病毒8型或腺病毒相关病毒9型。
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。
通过图18-19，表明了2种腺病毒载体与6种RNA序列单独或其中任意2种或其中任意3种RNA序列组成的RNA片段构建后均具有体内富集和治疗效果。
可选地，所述病毒载体包括启动子和靶向标签，所述靶向标签能够在宿主的器官组织中形成所述复合结构的靶向结构，所述靶向结构位于复合结构的表面，所述复合结构能够通过所述靶向结构寻找并结合目标组织，将所述RNA片段递送进入目标组织。
可选地，所述病毒载体中包括以下任意一种线路或几种线路的组合：启动子-RNA片段、启动子-靶向标签、启动子-RNA片段-靶向标签；每一个所述病毒载体中至少包括一个RNA片段和一个靶向标签，所述RNA片段和靶向标签位于相同的线路中或位于不同的线路中。
通过图20-23表明了2种RNA片段和2种靶向标签单独使用或任意组合并构建进病毒载体均具有体内富集及治疗效果。
可选地，所述病毒载体还包括能够使所述线路折叠成正确结构并表达的侧翼序列、补偿序列和loop序列，所述侧翼序列包括5’侧翼序列和3’侧翼序列；
所述病毒载体中包括以下任意一种线路或几种线路的组合：5'-启动子-5'侧翼序列-RNA片段-loop序列-补偿序列-3'侧翼序列、5'-启动子-靶向标签、5'-启动子-靶向标签-5'侧翼序列-RNA片段-loop序列-补偿序列-3'侧翼序列。
通过图24表明了5’-启动子-5’侧翼序列-RNA序列-loop序列-补偿序列-3’侧翼序列和5’-启动子-靶向 标签-5’侧翼序列-RNA序列-loop序列-补偿序列-3’侧翼构建的递送载体均具有体内富集及治疗效果。
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片段的反向互补序列，并删除其中任意1-5位碱基，删除RNA反向互补序列的1-5位碱基的目的是使该序列不表达。
图25表明了5’侧翼序列/loop序列/3’侧翼序列及其同源序列构建的递送系统均具有体内富集及治疗效果。
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，在病毒载体中存在至少两种线路的情况下，相邻的线路之间通过序列1-3组成的序列(序列1-序列2-序列3)相连；
其中，序列1为CAGATC，序列2是由5-80个碱基组成的序列，序列3为TGGATC。
可选地，在病毒载体中存在至少两种线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；
其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
通过图26表明了序列4及其同源序列构建的病毒载体递送系统，均具有体内富集及治疗效果。
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。
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RNA序列的长度为15-25个核苷酸。比如，所述RNA序列的长度可以为16、17、18、19、20、21、22、23、24、25个核苷酸。优选地，所述RNA序列的长度为18-22个核苷酸。
通过图27表明了不同长度的RNA序列构建的基因线路均具有体内富集及治疗效果。
可选地，所述RNA序列选自以下RNA中的任意一种或几种：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、UGUAUUUACAUAAUUACACAC、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。
miRNA-21的反义链为5’-TCAACATCAGTCTGATAAGCTA-3’。
需要说明的是，以上所述的“同源性大于80％的序列”可以为同源性为85％、88％、90％、95％、98％等。
可选地，所述RNA片段包括RNA序列本体和对RNA序列本体进行核糖修饰得到的修饰RNA序列。即RNA片段既可以仅由至少一个RNA序列本体组成，也可以仅由至少一个修饰RNA序列组成，还可以由RNA序列本体与修饰RNA序列组成。
在本发明中，所述分离的核酸还包括其变体和衍生物。本领域的普通技术人员可以使用通用的方法对所述核酸进行修饰。修饰方式包括(但不限于)：甲基化修饰、烃基修饰、糖基化修饰(如2-甲氧基-糖基修饰、烃基-糖基修饰、糖环修饰等)、核酸化修饰、肽段修饰、脂类修饰、卤素修饰、核酸修饰(如“TT”修饰)等。在本发明的其中一种实施方式中，所述修饰为核苷酸间键合，例如选自：硫代磷酸酯、2'-O甲氧基乙基(MOE)、2'-氟、膦酸烷基酯、二硫代磷酸酯、烷基硫代膦酸酯、氨基磷酸酯、氨基甲酸酯、碳酸酯、磷酸三酯、乙酰胺酯、羧甲基酯及其组合。在本发明的其中一种实施方式中，所述修饰为对核苷酸的修饰，例如选自：肽核酸(PNA)、锁核酸(LNA)、阿拉伯糖-核酸(FANA)、类似物、衍生物及其组合。优选的，所述修饰为2’氟嘧啶修饰。2’氟嘧啶修饰是将RNA上嘧啶核苷酸的2’-OH用2’-F替代，2’-F能够使RNA不易被体内的RNA酶识别，由此增加RNA片段在体内传输的稳定性。
可选地，所述递送系统为用于包括人在内的哺乳动物中的递送系统。
本申请还提供一种如上任意一段所述的基于病毒载体的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，所述药物包括上述病毒载体，具体而言，此处的病毒载体表示携带有RNA片段、或携带有RNA片段及靶向标签的病毒载体，并且能够进入宿主体内能够在肝脏部位富集，自组装形成复合结构外泌体，该复合结构能够将RNA片段递送至目标组织，使RNA片段在目标组织中表达，进而抑制与其匹配的基因的表达，实现治疗疾病的目的。
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：
本申请提供的基于病毒载体的RNA递送系统以病毒作为载体，病毒作为成熟的注入物，其安全性和可靠性已被充分验证，具有良好的成药性。最终发挥效果的RNA序列由内源性外泌体包裹输送，不存在任何免疫反应，无需验证该外泌体的安全性。该递送系统可以递送各类小分子RNA，通用性强。并且病毒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。本申请提供的基于病毒载体的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。
本申请提供的基于病毒载体的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结肠炎治疗情况及RNA表达水平对比图；
图2是本申请一实施例提供的小鼠细胞因子浓度以及结肠HE染色对比图；
图3是本申请一实施例提供的小鼠结肠炎治疗情况对比图；
图4是本申请一实施例提供的小鼠疾病活动指数及多种siRNA水平对比图；
图5是本申请一实施例提供的小鼠多种siRNA、mRNA水平对比图；
图6是本申请一实施例提供的小鼠结肠HE染色情况对比图；
图7是本申请一实施例提供的基于KRAS siRNA的小鼠肺癌治疗情况图；
图8是本申请一实施例提供的基于EGFR siRNA的小鼠肺癌治疗情况图；
图9是本申请一实施例提供的小鼠多种酶含量对比图；
图10是本申请一实施例提供的小鼠羟脯氨酸含量、mRNA水平对比图；
图11是本申请一实施例提供的小鼠生存情况和肿瘤评估对比图；
图12是本申请一实施例提供的小鼠肥胖症治疗情况对比图；
图13是本申请一实施例提供的小鼠各项肥胖指标对比图；
图14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15是本申请一实施例提供的线路的构建与表征图。
图16是本申请一实施例提供的腺相关病毒载体在肝、肺、血浆、外泌体的富集效果以及EGFR基因表达量检测，图中A为携带有RNA序列的腺相关病毒载体在肝和肺中的富集效果，B为携带有RNA序列的腺相关病毒载体在血浆和外泌体中的富集效果，C和D分别为EGFR的蛋白表达量和mRNA表达量 结果。
图17是本申请一实施例提供的慢病毒载体在肝、肺、血浆、外泌体的富集效果以及EGFR基因表达量检测，图中A为携带有RNA序列的慢病毒载体在肝和肺中的富集效果，B为携带有RNA序列的慢病毒载体在血浆和外泌体中的富集效果，C和D分别为EGFR的蛋白表达量和mRNA表达量结果。
图18是本申请一实施例提供的以2种不同的腺病毒载体构建的含有不同的6种RNA序列的基因环路，静脉注射后相应蛋白和mRNA的检测结果，图中A为EGFR蛋白表达量，B为TNC蛋白表达量，C为EGFR mRNA表达量，D为TNC mRNA表达量。
图19是本申请一实施例提供的2种腺病毒载体与6种RNA序列中的任意2种或任意3种RNA序列组成的RNA片段构建后，静脉注射后的蛋白表达量，图中A为2种腺病毒载体与6种RNA序列中的任意2种RNA序列组成的RNA片段构建后，检测的EGFR和TNC的蛋白含量，B为2种腺病毒载体与6种RNA序列中的任意2种RNA序列组成的RNA片段构建后，检测的EGFR和TNC的mRNA含量，C为2种腺病毒载体与6种RNA序列中的任意3种RNA序列组成的RNA片段构建后，检测的EGFR和TNC的蛋白含量，D为2种腺病毒载体与6种RNA序列中的任意4种RNA序列组成的RNA片段构建后，检测的EGFR和TNC的mRNA含量。
图20是本申请一实施例提供的将单独的RNA片段和单独的靶向标签组合并构建进病毒载体后，静脉注射后，检测得到的EGFR siRNA和TNC siRNA在胰腺、脑中的富集效果及EGFR和TNC的表达量，图中A-D分别为AD2/AD5+PTP/RVG+siR EGFR/siR TNC的富集效果检测结果，E-H分别为AD2/AD5+PTP/RVG+siR EGFR的EGFR蛋白含量和mRNA含量检测结果，I-L分别为AD2/AD5+PTP/RVG+siR TNC的TNC蛋白含量和mRNA含量检测结果。
图21是本申请一实施例提供的将2种RNA片段和2种靶向标签任意组合并构建进病毒载体后，静脉注射后，检测得到的EGFR siRNA和TNC siRNA在胰腺、脑中的富集效果，图中A-B分别为AD2/AD5+PTP/RVG+siR EGFR+TNC的富集效果检测结果，C-D分别为AD2/AD5+(PTP-RVG)+siR EGFR的富集效果检测结果，E为AD2/AD5+(PTP-RVG)+siR EGFR+TNC的富集效果检测结果。
图22是本申请一实施例提供的将2种RNA片段和2种靶向标签任意组合并构建进病毒载体后，静脉注射后，检测得到的胰腺、脑中的EGFR和TNC的表达量，图中A-D分别为AD2/AD5+PTP/RVG+siR EGFR+TNC的EGFR蛋白含量和mRNA含量检测结果，E-H分别为TNC蛋白含量和mRNA含量检测结果。
图23是本申请另一实施例提供的将2种RNA片段和2种靶向标签任意组合并构建进病毒载体后，静脉注射后，检测得到的胰腺、脑中的EGFR和TNC的表达量，图中A-B分别为AD2/AD5+(PTP-RVG)+siR EGFR的EGFR蛋白含量和mRNA含量检测结果，C-D为AD2/AD5+(PTP-RVG)+siR EGFR+TNC的EGFR蛋白含量和mRNA含量检测结果，E-F为AD2/AD5+(PTP-RVG)+siR TNC的TNC蛋白含量和mRNA含量检测结果，G-H为AD2/AD5+(PTP-RVG)+siR EGFR+TNC的TNC蛋白含量和mRNA含量检测结果。
图24是本申请一实施例提供的以2种线路构建的递送系统静脉注射后siRNA在肝、肺、血浆、外泌体中的富集结果和EGFR蛋白及mRNA的表达量检测结果，图中A为递送系统AAV-siR E和AAV-GE11-siR E在肝、肺中的富集效果，B为递送系统AAV-siR E和AAV-GE11-siR E在血浆、外泌体中的富集效果，C为递送系统AAV-siR E和AAV-GE11-siR E的EGFR蛋白含量检测结果，D为递送系统AAV-siR E和AAV-GE11-siR E的EGFR mRNA含量检测结果。
图25是本申请一实施例提供的含有2条与5’侧翼序列/loop序列/3’侧翼序列同源性大于80％的明确 序列的腺病毒载体在肺部的富集效果，图中A为不同的5’侧翼序列的富集效果，B为不同的loop序列的富集效果，C为不同的3’侧翼序列的富集效果。
图26是本申请一实施例提供的序列4以及2条与序列4同源性大于80％的序列4-1、4-2构建进AAV载体中，静脉注射9小时后肺部组织的序列富集效果，图中A为以序列4连接时，AAV-siR E和AAV-siR T的siRNA含量检测结果，图中B为以序列4-1连接时，AAV-siR E和AAV-siR T的siRNA含量检测结果，图中C为以序列4-2连接时，AAV-siR E和AAV-siR T的siRNA含量检测结果。
图27是本申请一实施例提供的含有不同长度序列的基因环路静脉注射后的EGFR表达量检测结果，图中A为EGFR蛋白含量检测结果，B为EGFR mRNA含量检测结果。
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。
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。
在本发明所提供的图示中，“*”表示P<0.05，“**”表示P<0.01,“***”表示P<0.005。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的试剂、材料和操作步骤均为相应领域内广泛使用的试剂、材料和常规步骤。
实施例1
本实施例提供一种基于病毒载体的RNA递送系统，该系统包含病毒载体，所述病毒载体携带所需递送的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述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。
图16-17显示了腺相关病毒载体和慢病毒载体构建的RNA递送系统具有体内富集和治疗效果，图16显示了腺相关病毒载体在肝、肺、血浆、外泌体的富集效果以及EGFR基因表达量检测，图17显示了慢病毒载体在肝、肺、血浆、外泌体的富集效果以及EGFR基因表达量检测。
在本实施例中，病毒载体还包括启动子和靶向标签。所述病毒载体包括以下任意一种线路或几种线路的组合：启动子-RNA序列、启动子-靶向标签、启动子-RNA序列-靶向标签，每一个所述病毒载体中至少包括一个RNA片段和一个靶向标签，所述RNA片段和靶向标签位于相同的线路中或位于不同的线路中。换而言之，病毒载体中可以仅包括启动子-RNA序列-靶向标签，也可以包括启动子-RNA序列、启动子-靶向标签的组合，或是启动子-靶向标签、启动子-RNA序列-靶向标签的组合。
图20-23中，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2种RNA片段和2种靶向标签单独使用或任意组合并构建进病毒载体后，尾静脉注射进入小鼠体内，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，所述病毒载体还可以包括能够使所述线路折叠成正确结构并表达的侧翼序列、补偿序列和loop序列，所述侧翼序列包括5’侧翼序列和3’侧翼序列；所述病毒载体包括以下任意一种线路或几种线路的组合：5’-启动子-5’侧翼序列-RNA片段-loop序列-补偿序列-3’侧翼序列、5’-启动子-靶向标签、5’-启动子-靶向标签-5’侧翼序列-RNA片段-loop序列-补偿序列-3’侧翼序列。
其中，所述5’侧翼序列优选为ggatcctggaggcttgctgaaggctgtatgctgaattc或与其同源性大于80％的序列，包括与ggatcctggaggcttgctgaaggctgtatgctgaattc同源性为85％、90％、92％、95％、98％、99％的序列等。
所述loop序列优选为gttttggccactgactgac或与其同源性大于80％的序列，包括与gttttggccactgactgac同源性为85％、90％、92％、95％、98％、99％的序列等。
所述3’侧翼序列优选为accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag或与其同源性大于80％的序列，包括与accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag同源性为85％、90％、92％、95％、98％、99％的序列等。
所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-5位碱基。在RNA片段中仅包含一个RNA序列时，所述补偿序列可以为该RNA序列的删除其中任意1-5位碱基的反向互补序列。
优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-3位碱基。在RNA片段 中仅包含一个RNA序列时，所述补偿序列可以为该RNA序列的删除其中任意1-3位碱基的反向互补序列。
更为优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-3位连续排列的碱基。在RNA片段中仅包含一个RNA序列时，所述补偿序列可以为该RNA序列的删除其中任意1-3位连续排列的碱基的反向互补序列。
最为优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中的第9位和/或第10位碱基。在RNA片段中仅包含一个RNA序列时，所述补偿序列可以为该RNA序列的删除其中第9位和/或第10位的反向互补序列。删除第9位和第10位碱基效果最优。
需要说明的是，上述侧翼序列、补偿序列、loop序列均不是随意选择的，而是基于大量的理论研究和试验确定的，在上述特定侧翼序列、补偿序列、loop序列的配合下，能够最大程度的提高RNA片段的表达率。
图24中，5’-启动子-5’侧翼序列-RNA序列-loop序列-补偿序列-3’侧翼序列对应递送载体名称为AAV-siR E，5’-启动子-靶向标签-5’侧翼序列-RNA序列-loop序列-补偿序列-3’侧翼对应递送载体名称为AAV-GE11-siR E，图24为静脉注射后siRNA在肝、肺、血浆、外泌体种的富集结果和EGFR蛋白及mRNA的表达量检测结果。
图25中，显示了一组腺病毒载体在肺部的富集效果数据，腺病毒载体中的序列为：2条与5’侧翼序列/loop序列/3’侧翼序列同源性大于80％的明确序列。
上述各序列具体如下表1所示。
名称 序列
5'侧翼序列-1 CTGGAGGCTTGCTGAAGGCTGTATGCTGAATTCG
5'侧翼序列-2 CTGGAGGCTTGCTCGGAGGCTGTATGCTGGCTTCG
loop-1 GTTTTGGCCACTGACTGAC
loop-2 GTGGCGGCCACTGACTGAC
3'侧翼序列-1 CACCGGTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC
3'侧翼序列-2 CAGAGGTCAGGACACAAGGCCTACGACTAGCACTCACATGTCTCAAATGGCC
在病毒载体携带两个或多个线路的情况下，相邻的线路之间可以通过序列1-序列2-序列3相连；其中，序列1优选为CAGATC，序列2可以为由5-80个碱基组成的序列，比如10个、15个、20个、25个、30个、35个、40个、45个、50个、55个、60个、65个、70个、75个碱基组成的序列均可，优选为10-50个碱基组成的序列，更优选为20-40个碱基组成的序列，序列3优选为TGGATC。
序列2具体如下表2所示。
Figure PCTCN2022083601-appb-000001
Figure PCTCN2022083601-appb-000002
更为优选地，在病毒载体携带两个或多个线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
图26，是将序列4以及2条与序列4同源性大于80％的序列4-1、4-2构建进AAV载体中，静脉注射9小时后肺部组织的EGFR siRNA含量检测结果。
序列具体如下表3所示。
名称 序列
序列4 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-1 CAGATCTGCTCTAACTCGATTTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGTTTCACTCATTCTAGTGAGTCGACCAGTGGATC
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。
图27分别显示了不同长度的RNA序列所构建的基因环路静脉注射后的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AAV-siR E(18)、AAV-siR E(20)、AAV-siR E(22)。
不同长度的RNA序列如下表4所示。
名称 序列
siRE(18) ACCTATTCCGTTACACACT
siRE(20) ATACCTATTCCGTTACACAC
siRE(22) ATACCTATTCCGTTACACACTT
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，则可以在同一个病毒载体载体上联合使用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。
以在同一个病毒载体上联合使用“siRNA1”和“siRNA2”为例，该病毒载体的功能结构区可以表示为：(启动子-siRNA1)-连接序列-(启动子-siRNA2)-连接序列-(启动子-靶向标签)，或(启动子-靶向标签-siRNA1)-连接序列-(启动子-靶向标签-siRNA2)，或(启动子-siRNA1)-连接序列-(启动子-靶向标签-siRNA2)等。
更加具体地，该病毒载体的功能结构区可以表示为：(5’-启动子-5’侧翼序列-siRNA1-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-5’侧翼序列-siRNA2-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-靶向标签)，或(5’-启动子-靶向标签-5’侧翼序列-siRNA1-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-靶向标签-5’侧翼序列-siRNA2-loop序列-补偿序列-3’侧翼序列)，或(5’-启动子-5’侧翼序列-siRNA1-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-靶向标签-5’侧翼序列-siRNA2-loop序列-补偿序列-3’侧翼序列)、(5’-启动子-靶向标签-5’侧翼序列-siRNA1-siRNA2-loop序列-补偿序列-3’侧翼序列)等。其他情况均可以此类推，在此不再赘述。以上连接序列可以为“序列1-序列2-序列3”或“序列4”，一个括号表示一个完整的线路(circuit)。
优选地，上述RNA还可以通过对其中的RNA序列(siRNA、shRNA或miRNA)进行核糖修饰得到，优选2’氟嘧啶修饰。2’氟嘧啶修饰是将siRNA、shRNA或miRNA上嘧啶核苷酸的2’-OH用2’-F替代，2’-F能够使人体内的RNA酶不易识别siRNA、shRNA或miRNA，如此能够增加RNA在体内传输的稳定性。
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。
本实施例中所述的宿主体内的器官，优选肝脏。肝脏是所有哺乳动物组织器官中最活跃的细胞开合组织。
通过特异性地选择感染肝脏后，病毒载体会释放出RNA片段(siRNA、shRNA或miRNA)，肝脏组织能够自发地将RNA片段包裹进外泌体内部，这些外泌体就变成了RNA输送机构(含有所需递送RNA的并具有靶向结构的复合结构)。
为了使该外泌体具有“精确制导”的能力，在注入体内的病毒中可以设计靶向标签，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，如此能够大大提高本发明所述的RNA输送机构的精准性，大大提高了潜在药物递送的效率。
靶向标签的选择是需要创造性劳动的过程，一方面需要根据目标组织选取可用的靶向标签，另一方面还需要保证该靶向标签能够在稳定地出现在外泌体的表面，从而达到靶向功能。目前已经筛选出的靶向标签包括靶向肽、靶向蛋白、抗体。其中，靶向肽包括但不限于RVG靶向肽(核苷酸序列如SEQ ID No：1所示)、GE11靶向肽(核苷酸序列如SEQ ID No：2所示)、PTP靶向肽(核苷酸序列如SEQ ID No：3所示)、TCP-1靶向肽(核苷酸序列如SEQ ID No：4所示)、MSP靶向肽(核苷酸序列如SEQ ID No：5所示)；靶向蛋白包括但不限于RVG-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：6所示)、GE11-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：7所示)、PTP-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：8所示)、TCP-1-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：9所示)、MSP-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：10所示)。
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。
病毒载体的选取和结构设计是本发明所公开的技术的核心内容之一，经研究，病毒载体优选为腺病毒相关病毒(AAV)，更优选为腺病毒相关病毒5型(AAV-5)、腺病毒相关病毒8型(AAV-8)或腺病毒相关病毒9型(AAV-9)。
另外，为了达到精准递送的目的，我们实验了多种病毒载体搭载的方案，得出另一优化的方案：所述病毒载体还可以由具有不同结构的多种病毒构成，其中一种病毒包含启动子和靶向标签，其他病毒包含启动子和RNA片段。即将靶向标签与RNA片段装载到不同的病毒载体中，将两种病毒载体注入体内，其靶向效果不差于将相同的靶向标签与RNA片段装载在一个病毒载体中产生的靶向效果。
更优选地，两种不同的病毒载体注入宿主体内时，可以先将装有RNA序列的病毒载体注入，在1-2小时后再注入含有靶向标签的病毒载体，如此能够达到更优的靶向效果。
以上所述的递送系统均可用于包括人在内的哺乳动物。
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15a，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15a。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15b。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15b。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15c。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15d、图15e，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15a。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15f，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15。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15g。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15h。
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15i、图15j。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。
因此，本实施例提供的基于病毒载体的RNA递送系统以病毒作为载体，病毒载体作为成熟的注入物，其安全性和可靠性已被充分验证，成药性非常好。最终发挥效果的RNA序列由内源性外泌体包裹输送，不存在任何免疫反应，无需验证该外泌体的安全性。该递送系统可以递送各类小分子RNA，通用性强。并且病毒载体的制备要比外泌体或是蛋白质、多肽等物质的制备便宜地多，经济性好。本实施例提供的基于病毒载体的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。该药物包括该系统包括病毒载体，所述病毒载体携带有所需递送的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述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。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。
图18-19中，2种腺病毒分别表示为ADV1、ADV2，6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)、siR T(靶基因为TNC)、miR-7(靶基因为EGFR)、miR-133b(靶基因为EGFR)。图18表示2种腺病毒载体构建的12种基因环路静脉注射后相应蛋白的表达量，图19表示2种腺病毒载体与6种RNA序列中的任意2种或任意3种RNA序列组成的RNA片段构建后，静脉注射后的蛋白表达量。
RNA序列具体如下表5所示。
Figure PCTCN2022083601-appb-000003
Figure PCTCN2022083601-appb-000004
可选地，所述病毒载体包括启动子和靶向标签，所述靶向标签能够在宿主的器官组织中形成所述复合结构的靶向结构，所述靶向结构位于复合结构的表面，所述复合结构能够通过所述靶向结构寻找并结合目标组织，将所述RNA片段递送进入目标组织。
关于本实施例中上述病毒载体、RNA片段、靶向标签等的解释说明均可以参考实施例1，在此不再赘述。
该药物可以通过口服、吸入、皮下注射、肌肉注射或静脉注射的方式进入人体后，通过实施例1所述的基于病毒载体的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病毒作为载体，病毒载体作为成熟的注入物，其安全性和可靠性已被充分验证，成药性非常好。最终发挥效果的RNA序列由内源性外泌体包裹输送，不存在任何免疫反应，无需验证该外泌体的安全性。该药物可以递送各类小分子RNA，通用性强。并且病毒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，本实施例提供一种基于病毒载体的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基于病毒载体的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，我们设置三个试验组和两个对照组，三个试验组分别为AAV-CMV-siR TNF-α(low)组、AAV-CMV-siR TNF-α(medium)组、AAV-CMV-siR TNF-α(high)组；对照组分别为Normal组、AAV-CMV-scrR组。
试验过程如图1A所示，AAV-CMV-siR TNF-α(low)组、AAV-CMV-siR TNF-α(medium)组、AAV-CMV-siR TNF-α(high)组分别利用肝脏高亲和的AAV-5型腺相关病毒包裹TNF-αsiRNA系统(AAV-CMV-siR TNF-α)，通过尾静脉注射滴度为10 12V.g/ml的AAV溶液，25μL、50μL、100μL至小鼠体内。
通过小动物活体监测AAV系统的体内表达情况，结果如图1B所示，3周后可见AAV系统在体内尤其是肝脏，稳定表达，其中AAV-CMV-siR TNF-α(high)组，平均辐亮度(Average Radiance)达到8.42*105(p/sec/cm 2/sr)，且表达部位在肝脏，这说明AAV系统的表达具有剂量依赖效应。
随即开始构建DSS诱导的慢性结肠炎模型，期间每两天进行称重记录，结果如图1C所示，可以看出AAV包裹的CMV-siR TNF-α系统能够减轻慢性结肠炎小鼠的体重下降情况，并且3个试验组的小鼠 在炎症缓解期体重回复的速度也显著快于AAV-CMV-scrR组。
第十周模型构建结束，通过小动物活体监测AAV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1D所示，可以看出AAV-CMV-siR TNF-α(low)组、AAV-CMV-siR TNF-α(medium)组、AAV-CMV-siR TNF-α(high)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，其中AAV-CMV-siR TNF-α(high)组炎症情况的改善最为显著。
分别对各组小鼠的疾病指数进行评分统计，结果如图1E所示，可以看出AAV-CMV-siR TNF-α(high)组的小鼠疾病指数低于AAV-CMV-siR TNF-α(low)组、AAV-CMV-siR TNF-α(medium)组和AAV-CMV-scrR组。
分别检测各组小鼠体内的TNF-αsiRNA水平，结果如图1F所示，可以看出三个试验组小鼠体内TNF-αsiRNA水平均较高，而对照组AAV-CMV-scrR组小鼠体内几乎无TNF-αsiRNA的表达，这说明上述的AAV系统能够产生一定量的TNF-αsiRNA。
分别检测各组小鼠体内的TNF-αmRNA水平，结果如图1G所示，可以看出Normal组和三个试验组的小鼠体内TNF-αmRNA水平均比较低，而AAV-CMV-scrR组小鼠体内TNF-αmRNA水平则较高，这说明AAV系统能够降低结肠TNF-α的表达及分泌。
对小鼠结肠内的促炎细胞因子IL-6、IL-12、IL-23进行检测，结果如图2A所示，可见Normal组和AAV-CMV-siR TNF-α(high)组小鼠促炎细胞因子的分泌最少，AAV-CMV-scrR组小鼠促炎细胞因子的分泌最多。
对小鼠结肠切片进行HE染色以及病理评分统计，结果如图2B、图2C所示，可以看出试验组，尤其是AAV-CMV-siR TNF-α(high)组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AAV-CMV-scrR组。
以上试验说明，利用亲和肝脏的AAV包裹CMV-siR TNF-α回路，能够实现长期的TNF-αsiRNA表达以及长期的TNF-α沉默，并且能够在一定程度上缓解结肠炎，具有极大的成药潜力以及临床研究价值。
在第二个试验中，我们设置三个试验组和两个对照组。其中，试验组分别为AAV-CMV-siR T+B+I(low)组、AAV-CMV-siR T+B+I(medium)组、AAV-CMV-siR T+B+I(high)组；对照组分别为Normal组、AAV-CMV-scrR组。
AAV-CMV-siR T+B+I(low)组、AAV-CMV-siR T+B+I(medium)组、AAV-CMV-siR T+B+I(high)组分别利用肝脏高亲和的AAV-5型腺相关病毒包裹TNF-αsiRNA，B7-siRNA以及Integrinα4 siRNA元件串联递药系统(AAV-CMV-siR T+B+I)，通过尾静脉注射滴度为10 12V.g/ml的AAV溶液25μL、50μL、100μL至小鼠体内。
通过小动物活体监测AAV系统的体内表达情况，结果如图3A所示，3周后可见AAV系统在体内尤其是肝脏，稳定表达，并且AAV系统的表达具有剂量依赖效应。
随即开始构建DSS诱导的慢性结肠炎模型，期间每两天进行称重记录，结果如图3B所示，可以看出AAV包裹的CMV-siR T+B+I系统能够减轻慢性结肠炎小鼠的体重下降情况，并且三个试验组的小鼠在炎症缓解期体重回复的速度也显著快于AAV-CMV-scrR组。
第十周模型构建结束，通过小动物活体监测AAV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3C所示，可以看出AAV-CMV-siR T+B+I(low)组、AAV-CMV-siR T+B+I(medium)组、AAV-CMV-siR T+B+I(high)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，其中AAV-CMV-siR T+B+I(high)组炎症情况的改善最为显著。
分别对各组小鼠的疾病指数进行评分统计，结果如图4A所示，可以看出AAV-CMV-siR T+B+I(high)组的小鼠疾病指数低于AAV-CMV-siR T+B+I(low)组、AAV-CMV-siR T+B+I(medium)组和AAV-CMV-scrR组。
对小鼠血浆中TNF-αsiRNA、B7 siRNA以及integrinα4 siRNA进行检测，结果如图4B、图4C、图4D所示，可见AAV包裹的CMV-siR T+B+I系统在小鼠血浆中生成了一定量的稳定表达的siRNA，并且呈现剂量依赖效应。
对小鼠肝脏中TNF-αsiRNA、B7 siRNA以及integrinα4 siRNA进行检测，结果如图4E、图4F、图4G所示，可见AAV包裹的CMV-siR T+B+I系统在小鼠肝脏中生成了一定量的稳定表达的siRNA，并且呈现剂量依赖效应。
对小鼠结肠中TNF-αsiRNA、B7 siRNA以及integrinα4 siRNA进行检测，结果如图5A、图5B、图5C所示，可见AAV包裹的CMV-siR T+B+I系统在小鼠结肠中生成了一定量的稳定表达的siRNA，并且呈现剂量依赖效应。
对小鼠结肠中TNF-αmRNA、B7mRNA以及integriα4 mRNA进行检测，结果如图5D、图5E、图5F所示，可见AAV包裹的CMV-siR T+B+I系统显著降低了小鼠结肠中TNF-α、B7以及integrinα4 mRNA表达。
对小鼠结肠切片进行HE染色以及病理评分统计，结果如图6A和图6B所示。可见试验组，尤其是AAV-CMV-siR T+B+I(high)组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AAV-CMV-scrR组。
以上试验说明，利用亲和肝脏的AAV包裹CMV-siR T+B+I回路，能够实现长期的TNF-αsiRNA、B7 siRNA以及integrinα4 siRNA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4
在实施例1或2的基础上，本实施例提供一种基于病毒载体的RNA递送系统在药物中的应用，该药物为治疗肺癌的药物，本实施例将通过以下四个试验对基于病毒载体的RNA递送系统在肺癌治疗方面的效果进行具体说明。
在第一个试验中，我们利用肝脏高亲和的AAV-5型腺相关病毒包裹EGFR siRNA系统(AAV-CMV-EGFR siRNA)和KRAS siRNA系统(AAV-CMV-KRAS siRNA)，尾静脉注射100μL滴度为10 12V.g/ml的AAV溶液至小鼠体内。通过小动物活体监测AAV系统的体内表达情况，3周后可见AAV系统在体内尤其是肝脏，稳定表达。
在第二个试验中，设置1个试验组和2个对照组，其中，试验组为AAV-CMV-KRAS-siRNA组，对照组为PBS组和AAV-CMV-scrR组。
各组选取相同数量的小鼠，向小鼠体内注射小鼠肺癌细胞(LLC细胞),采用CT扫描技术观察小鼠模型构建进展。30天后对构建成功的小鼠进行给药，两天给药一次，即每两日向PBS组/AAV-CMV-scrR组/AAV-CMV-KRAS siRNA组小鼠注射一次PBS缓冲液/AAV-CMV-scrR/AAV-CMV-KRAS siRNA进行治疗，分别对小鼠进行生存分析和肿瘤评估，给药7次后停止治疗。
统计各组小鼠在治疗后的100天内的生存情况，结果如图7A所示，可以看出，PBS组和AAV-CMV-scrR组小鼠存活率相差无几，而AAV-CMV-KRAS siRNA组小鼠存活率最高。
给药前后分别对各组小鼠进行CT扫描，根据CT影像图对小鼠肺组织进行3D建模，并计算肿瘤体 积大小，结果如图7B所示。在图7B中，“PBS pre”表示给药前的PBS组，“PBS post”表示给药后的PBS组；“AAV-CMV-scrR pre”表示给药前的AAV-CMV-scrR组，“AAV-CMV-scrR post”表示给药后的AAV-CMV-scrR组；“AAV-CMV-KRAS-siRNA pre”表示给药前的AAV-CMV-KRAS siRNA组，“AAV-CMV-KRAS-siRNA post”表示给药后的AAV-CMV-KRAS siRNA组。可以看出，AAV-CMV-KRAS siRNA组的小鼠在给药后肿瘤体积显著减小，而PBS组和AAV-CMV-scrR组的小鼠在给药后肿瘤体积不仅没有减小，还呈现不同程度的增加。
分别通过RT-qPCR和Western blotting检测各组小鼠肺部KRAS蛋白和mRNA表达水平，结果如图7C、图7D所示。结果显示AAV-CMV-KRAS siRNA组的小鼠肺部KRAS蛋白和mRNA表达量相对于对照组有所降低。
以上试验说明，AAV-CMV-KRAS siRNA对小鼠肺癌肿瘤具有显著的治疗效果。
在第三个试验中，设置1个试验组和2个对照组，其中，试验组为AAV-CMV-EGFR siRNA组，对照组为PBS组和AAV-CMV-scrR组。
构建EGFR-DEL19小鼠模型，饲喂强力霉素饲料诱导肿瘤产生，30天后对构建成功的小鼠进行给药，两天给药一次，即每两日向PBS组/AAV-CMV-scrR组/AAV-CMV-EGFR siRNA组小鼠注射一次PBS缓冲液/AAV-CMV-scrR/AAV-CMV-EGFR siRNA进行治疗，分别对小鼠进行生存分析和肿瘤评估，给药7次后停止治疗。
统计各组小鼠在治疗后的100天内的生存情况，结果如图8A所示，可以看出，PBS组和AAV-CMV-scrR组小鼠存活率相差无几，而AAV-CMV-EGFR siRNA组小鼠存活率最高。
给药前后分别对各组小鼠进行CT扫描，CT影像如图8E所示，根据图8E的CT影像图对小鼠肺组织进行3D建模，并计算肿瘤体积大小，结果如图8B所示。在图8B中，“PBS pre”表示给药前的PBS组，“PBS post”表示给药后的PBS组；“AAV-CMV-scrR pre”表示给药前的AAV-CMV-scrR组，“AAV-CMV-scrR post”表示给药后的AAV-CMV-scrR组；“AAV-CMV-EGFR siRNA pre”表示给药前的AAV-CMV-EGFRsiRNA组，“AAV-CMV-EGFR siRNA post”表示给药后的AAV-CMV-EGFR siRNA组。可以看出，AAV-CMV-EGFR siRNA组的小鼠在给药后肿瘤体积显著减小，而PBS组和AAV-CMV-scrR组的小鼠在给药后肿瘤体积不仅没有减小，还呈现不同程度的增加。
分别通过RT-qPCR、Western blotting检测各组小鼠肺部EGFR蛋白和mRNA表达水平，结果如图8C、图8D所示。结果显示AAV-CMV-EGFR siRNA组的小鼠肺部EGFR蛋白和mRNA表达量相对于对照组有所降低。
以上试验说明，AAV-CMV-EGFR siRNA对EGFR突变型小鼠肺癌肿瘤具有显著的治疗效果。
在第四个试验中，设置2个试验组和2个对照组，其中，试验组为AAV-CMV-KRAS siRNA组、AAV-CMV-EGFR siRNA组，对照组为PBS组和AAV-CMV-scrR组。
构建EGFR-DEL19小鼠模型，饲喂强力霉素饲料诱导肿瘤产生，30天后对构建成功的小鼠进行给药，两天给药一次，即每两日向PBS组/AAV-CMV-scrR组/AAV-CMV-EGFR siRNA组/AAV-CMV-KRAS siRNA组小鼠注射一次PBS缓冲液/AAV-CMV-scrR/AAV-CMV-EGFR siRNA/AAV-CMV-KRAS siRNA进行治疗。
在治疗后分别检测各组小鼠中谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TBIL)、血清碱性磷酸酶(ALP)、肌酐(CREA)、血尿素氮(BUN)的含量，结果如图9A-图9F所示，可见，PBS组、AAV-CMV-scrR组、AAV-CMV-EGFR siRNA组、AAV-CMV-KRAS siRNA组小鼠中上述酶的含量均 相差无几。
以上试验可以说明，用肝脏高亲和的AAV-5型腺相关病毒包裹EGFR siRNA系统(AAV-CMV-EGFR siRNA)和KRAS siRNA系统(AAV-CMV-KRAS siRNA)安全性好，可靠性高，不会产生负面作用，适于大规模推广和应用。
实施例5
在实施例1或2的基础上，本实施例提供一种基于病毒载体的RNA递送系统在药物中的应用，该药物为治疗肺纤维化的药物，本实施例将通过以下试验对基于病毒载体的RNA递送系统在肺纤维化治疗方面的效果进行具体说明。
在此试验中，利用肝脏高亲和的AAV-5型腺相关病毒包裹抗-miR-21/TGF-β1 siRNA/抗-miR-21+TGF-β1 siRNA系统，分别得到AAV-anti-miR21/AAV-TGF-β1 siRNA/AAV-MIX，尾静脉注射100μL滴度为10 12V.g/ml的AAV溶液至小鼠体内。通过小动物活体监测AAV系统的体内表达情况，3周后可见AAV系统在体内尤其是肝脏，稳定表达。
随即选取小鼠进行造模，造模成功后，分别向小鼠注射注射PBS缓冲液/AAV-scrR/AAV-anti-miR21/A AV-TGF-β1 siRNA/AAV-MIX(10mg/kg)，形成PBS组/AAV-scrR组/AAV-anti-miR21组/AAV-TGF-β1 si RNA组/AAV-MIX组。
分别检测正常小鼠、PBS组小鼠、AAV-scrR组小鼠、AAV-TGF-β1 siRNA组小鼠相对TGF-β1 mRN A水平，结果如图10B所示，可见，AAV-TGF-β1 siRNA组小鼠相对TGF-β1 mRNA水平相对较低。
分别检测正常小鼠、PBS组小鼠、AAV-scrR组小鼠、AAV-anti-miR21组小鼠相对miR21 mRNA水平，结果如图10C所示，可见，AAV-anti-miR21组小鼠相对miR21 mRNA水平相对较低。
羟脯氨酸是胶原的主要成分，其含量反映了肺纤维化的程度。分别检测各组小鼠羟脯氨酸含量，结果如图10A所示，可见PBS组、AAV-scrR组小鼠体内羟脯氨酸含量最高，AAV-anti-miR21组、AAV-TGF-β1 siRNA组、AAV-MIX组小鼠体内羟脯氨酸含量均较低，说明AAV-anti-miR21组、AAV-TGF-β1 siRN A组、AAV-MIX组小鼠的肺纤维化得到抑制。
实施例6
在实施例1或2的基础上，本实施例提供一种基于病毒载体的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，本实施例将通过以下试验对基于病毒载体的RNA递送系统在胶质母细胞瘤治疗方面的效果进行具体说明。
在此试验中，利用肝脏高亲和的AAV-5型腺相关病毒包裹EGFR siRNA系统(AAV-CMV-RVG-siR E)和EGFR siRNA和TNC siRNA系统(AAV-CMV-RVG-siRE+T)，尾静脉注射100μL滴度为10 12V.g/ml的AAV溶液至小鼠体内。通过小动物活体监测AAV系统的体内表达情况，3周后可见AAV系统在体内尤其是肝脏，稳定表达。
随即选取小鼠，向小鼠体内注射胶质母细胞瘤细胞(U-87 MG-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/AAV-CMV-scrR/AAV-CMV-RVG-siR E/AAV-CMV-RVG-siR E+T(5m g/kg)进行治疗，形成PBS组/AAV-scrR组/AAV-CMV-RVG-siR E组/AAV-CMV-RVG-siR E+T组。
分别对各组小鼠进行生存分析，统计各组小鼠在接受治疗后20天、40天、60天、80天的存活率，结果如图11A所示，可以看出AAV-CMV-RVG-siR E+T组小鼠的生存时间最长，AAV-CMV-RVG-siR E组次之。
分别对各组小鼠进行肿瘤评估，即在第7天、14天、28天、35天分别对小鼠进行BLI活体成像检测，结果如图11B所示，可以看出AAV-CMV-RVG-siR E+T组小鼠其胶质母细胞瘤的抑制效果最为显著。
实施例7
在实施例1或2的基础上，本实施例提供一种基于病毒载体的RNA递送系统在药物中的应用，该药物为治疗肥胖症的药物，本实施例将通过以下试验对基于病毒载体的RNA递送系统在肥胖症治疗方面的效果进行具体说明。
利用肝脏高亲和的AAV-5型腺相关病毒包裹PTP1B siRNA系统(AAV-CMV-siR P/AAV-CMV-RVG-siR P)，尾静脉注射100μL滴度为10 12V.g/ml的AAV溶液至小鼠体内。通过小动物活体监测AAV系统的体内表达情况，3周后可见AAV系统在体内尤其是肝脏，稳定表达。
选取C57BL/6小鼠，12周后分别注射PBS缓冲液/AAV-CMV-scrR/AAV-CMV-siR P/AAV-CMV-RVG-siR P，形成PBS组/AAV-CMV-scrR组/AAV-CMV-siR P组/AAV-CMV-RVG-siR P组，并在24天内每两天注射一次。分别对各组小鼠进行体重变化、覆盖脂肪垫重量、初始食物摄入量、血清瘦素含量、血糖含量、基础葡萄糖含量、血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)、体长、食物摄入量的检测和统计，结果如下。
如图12A所示，该图为各组小鼠体重对比图，可以看出，AAV-CMV-RVG-siR P组的小鼠体重最为稳定。
如图12B所示，该图为各组小鼠的附睾脂肪垫重量对比图，可以看出，AAAV-CMV-RVG-siR P组的小鼠附睾脂肪垫重量最轻。
如图12C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，AAV-CMV-RVG-siR P组的小鼠食物摄入量最少。
如图12D所示，该图为各组小鼠血清瘦素含量对比图。可以看出，AAV-CMV-RVG-siR P组的小鼠血清瘦素含量最低。
如图12E所示，该图为各组小鼠血糖变化曲线对比图。可以看出，AAV-CMV-RVG-siR P组的小鼠血糖含量最低。
如图12F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，AAV-CMV-RVG-siR P组的小鼠基础葡萄糖含量最低。
如图13A-图13C所示，此三幅图分别为各组小鼠血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)对比图，可以看出，AAV-CMV-RVG-siR P组的小鼠TC、TG、LDL最低。
如图13D所示，该图为各组小鼠的体长对比图，可以看出，四组小鼠体长相差无几。
如图13E所示，该图为各组小鼠HFD食物摄入量对比图，可以看出，四组小鼠HFD食物摄入量同样相差无几。
以上试验可以说明，AAV-CMV-siR P、AAV-CMV-RVG-siR P对肥胖具有一定程度的抑制作用。
实施例8
在实施例1或2的基础上，本实施例提供一种基于病毒载体的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，本实施例将通过以下试验对基于病毒载体的RNA递送系统在亨廷顿病治疗方面的效果进行具体说明。
在此试验中，利用肝脏高亲和的AAV-5型腺相关病毒包裹HTT siRNA系统(AAV-CMV-siR mHTT/AA V-CMV-RVG-siR mHTT)，尾静脉注射100μL滴度为10 12V.g/ml的AAV溶液至小鼠体内。通过小动物活体监测AAV系统的体内表达情况，3周后可见AAV系统在体内尤其是肝脏，稳定表达。
随即选取小鼠进行造模，造模完成后向小鼠注射PBS缓冲液/AAV-CMV-scrR/AAV-CMV-siR mHTT/AAV-CMV-RVG-siR mHTT,形成PBS组/AAV-CMV-scrR组/AAV-CMV-siR mHTT组/AAV-CMV-RVG-siR mHTT组。尾静脉注射上述溶液后，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，结果如下。
如图14A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，AAV-CMV-siR mHTT组和AAV-CMV-RVG-siR mHTT组小鼠血浆的外泌体中siRNA水平较高。
如图14B所示，该图为小鼠血浆外泌体与细胞共培后，各组小鼠的相对mHTT mRNA水平对比图，可以看出，AAV-CMV-siR mHTT组和AAV-CMV-RVG-siR mHTT组小鼠相对mHTT mRNA水平较低，这说明AAV-CMV-siRmHTT与AAV-CMV-RVG-siRmHTT可以降低HTT mRNA水平，即组装进入外泌体的siRNA仍然可以发挥基因沉默功能。
如图14C所示，该图为小鼠肝脏siRNA绝对水平对比图，可以看出，AAV-CMV-siR mHTT组和AAV-CMV-RVG-siR mHTT组的小鼠绝对siRNA水平较高。
如图14D所示，该图为小鼠血浆siRNA绝对水平对比图，可以看出，AAV-CMV-siR mHTT组和AAV-CMV-RVG-siR mHTT组的小鼠绝对siRNA水平较高。
如图14E所示，该图为野生型小鼠(WT)、AAV-CMV-scrR组、AAV-CMV-RVG-siR mHTT组的小鼠下降潜伏期对比图，可以看出在0周时，三组小鼠的下降潜伏期比较一致，在第4周和第8周时，CMV-scrR组的小鼠下降潜伏期最短。
如图14F所示，该图为AAV-CMV-scrR组、AAV-CMV-RVG-siR mHTT组小鼠的皮质和纹状体中相对mHTT mRNA水平对比图，可以看出，不论是在皮质还是在纹状体中，AAV-CMV-RVG-siR mHTT组小鼠的mHTT mRNA水平均低于AAV-CMV-scrR组。
以上试验可以说明，静脉注射AAV-CMV-RVG-siR mHTT有助于纹状体和皮层mHTT蛋白和毒性聚集体减少，从而发挥对亨廷顿舞蹈症的治疗作用。
在本文中，“上”、“下”、“前”、“后”、“左”、“右”等仅用于表示相关部分之间的相对位置关系，而非限定这些相关部分的绝对位置。
在本文中，“第一”、“第二”等仅用于彼此的区分，而非表示重要程度及顺序、以及互为存在的前提等。
在本文中，“相等”、“相同”等并非严格的数学和/或几何学意义上的限制，还包含本领域技术人员可以理解的且制造或使用等允许的误差。
除非另有说明，本文中的数值范围不仅包括其两个端点内的整个范围，也包括含于其中的若干子范围。
上面结合附图对本申请优选的具体实施方式和实施例作了详细说明，但是本申请并不限于上述实施方式和实施例，在本领域技术人员所具备的知识范围内，还可以在不脱离本申请构思的前提下做出各种变化。

Claims (19)

  1. 一种基于病毒载体的RNA递送系统，其特征在于，该系统包括病毒载体，所述病毒载体携带有所需递送的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述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。
  2. 如权利要求1所述的基于病毒载体的RNA递送系统，其特征在于，所述病毒载体为腺病毒相关病毒。
  3. 如权利要求2所述的基于病毒载体的RNA递送系统，其特征在于，所述腺病毒相关病毒为腺病毒相关病毒5型、腺病毒相关病毒8型或腺病毒相关病毒9型。
  4. 如权利要求1所述的基于病毒载体的RNA递送系统，其特征在于，所述RNA片段包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列是具有医学意义的siRNA、shRNA或miRNA。
  5. 如权利要求1所述的基于病毒载体的RNA递送系统，其特征在于，所述病毒载体包括启动子和靶向标签，所述靶向标签能够在宿主的器官组织中形成所述复合结构的靶向结构，所述靶向结构位于复合结构的表面，所述复合结构能够通过所述靶向结构寻找并结合目标组织，将所述RNA片段递送进入目标组织。
  6. 如权利要求5所述的基于病毒载体的RNA递送系统，其特征在于，所述病毒载体中包括以下任意一种线路或几种线路的组合：启动子-RNA片段、启动子-靶向标签、启动子-RNA片段-靶向标签；每一个所述病毒载体中至少包括一个RNA片段和一个靶向标签，所述RNA片段和靶向标签位于相同的线路中 或位于不同的线路中。
  7. 如权利要求6所述的基于病毒载体的RNA递送系统，其特征在于，所述病毒载体还包括能够使所述线路折叠成正确结构并表达的侧翼序列、补偿序列和loop序列，所述侧翼序列包括5’侧翼序列和3’侧翼序列；
    所述病毒载体中包括以下任意一种线路或几种线路的组合：5'-启动子-5'侧翼序列-RNA片段-loop序列-补偿序列-3'侧翼序列、5'-启动子-靶向标签、5'-启动子-靶向标签-5'侧翼序列-RNA片段-loop序列-补偿序列-3'侧翼序列。
  8. 如权利要求7所述的基于病毒载体的RNA递送系统，其特征在于，所述5’侧翼序列为ggatcctggaggcttgctgaaggctgtatgctgaattc或与其同源性大于80％的序列；
    所述loop序列为gttttggccactgactgac或与其同源性大于80％的序列；
    所述3’侧翼序列为accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag或与其同源性大于80％的序列；
    所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-5位碱基。
  9. 如权利要求6所述的基于病毒载体的RNA递送系统，其特征在于，在病毒载体中存在至少两种线路的情况下，相邻的线路之间通过序列1-3组成的序列相连；
    其中，序列1为CAGATC，序列2是由5-80个碱基组成的序列，序列3为TGGATC。
  10. 如权利要求9所述的基于病毒载体的RNA递送系统，其特征在于，在病毒载体中存在至少两种线路的情况下，相邻的线路之间通过序列4或与序列 4同源性大于80％的序列相连；
    其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
  11. 如权利要求1所述的基于病毒载体的RNA递送系统，其特征在于，所述器官组织为肝脏，所述复合结构为外泌体。
  12. 如权利要求5所述的基于病毒载体的RNA递送系统，其特征在于，所述靶向标签选自具有靶向功能的靶向肽或靶向蛋白。
  13. 如权利要求12所述的基于病毒载体的RNA递送系统，其特征在于，所述靶向肽包括RVG靶向肽、GE11靶向肽、PTP靶向肽、TCP-1靶向肽、MSP靶向肽；
    所述靶向蛋白包括RVG-LAMP2B融合蛋白、GE11-LAMP2B融合蛋白、PTP-LAMP2B融合蛋白、TCP-1-LAMP2B融合蛋白、MSP-LAMP2B融合蛋白。
  14. 如权利要求5所述的基于病毒载体的RNA递送系统，其特征在于，所述RNA序列的长度为15-25个核苷酸。
  15. 如权利要求14所述的基于病毒载体的RNA递送系统，其特征在于，所述RNA序列选自以下RNA中的任意一种或几种：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。
  16. 如权利要求1所述的基于病毒载体的RNA递送系统，其特征在于，所述递送系统为用于包括人在内的哺乳动物中的递送系统。
  17. 一种权利要求1-16任意一项所述的基于病毒载体的RNA递送系统在药物中的应用。
  18. 如权利要求17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。
  19. 如权利要求17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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