WO2022206788A1 - 核酸递送系统及其应用 - Google Patents

核酸递送系统及其应用 Download PDF

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WO2022206788A1
WO2022206788A1 PCT/CN2022/083876 CN2022083876W WO2022206788A1 WO 2022206788 A1 WO2022206788 A1 WO 2022206788A1 CN 2022083876 W CN2022083876 W CN 2022083876W WO 2022206788 A1 WO2022206788 A1 WO 2022206788A1
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sequence
sirna
cmv
group
mice
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French (fr)
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张辰宇
陈熹
付正
李菁
张翔
周心妍
张丽
余梦超
郭宏源
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南京大学
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Priority to EP22778970.8A priority Critical patent/EP4317433A1/en
Priority to JP2023560199A priority patent/JP2024511523A/ja
Publication of WO2022206788A1 publication Critical patent/WO2022206788A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention relates to the fields of molecular biology and medicine.
  • the present invention relates to nucleic acid molecules that can treat diseases, their delivery systems and their use in the treatment of diseases.
  • RNA interference (RNAi) therapy has been considered a promising strategy for the treatment of human diseases since its invention, but many problems have been encountered during clinical practice, and the development of this therapy has lagged far behind expectations.
  • RNA cannot exist stably outside the cell for a long time, because RNA will be degraded into fragments by RNases rich in extracellular, so it is necessary to find a method that can make RNA stable outside the cell and can enter specific tissues in a targeted manner. Highlight the effect of RNAi therapy.
  • the Chinese Patent Publication No. CN108624590A discloses a siRNA capable of inhibiting the expression of DDR2 gene; the Chinese Patent Publication No. CN108624591A discloses a siRNA capable of silencing the ARPC4 gene, and the siRNA is modified with ⁇ -phosphorus-selenium;
  • the Chinese Patent Publication No. CN108546702A discloses a siRNA targeting long-chain non-coding RNA DDX11-AS1.
  • the Chinese Patent Publication No. CN106177990A discloses a siRNA precursor that can be used for various tumor treatments. These patents design specific siRNAs to target certain diseases caused by genetic changes.
  • Chinese Patent Publication No. CN108250267A discloses a polypeptide, polypeptide-siRNA induced co-assembly, using polypeptide as a carrier of siRNA.
  • the Chinese Patent Publication No. CN108117585A discloses a polypeptide for promoting apoptosis of breast cancer cells through targeted introduction of siRNA, and the polypeptide is also used as the carrier of siRNA.
  • the Chinese Patent Publication No. CN108096583A discloses a nanoparticle carrier, which can be loaded with siRNA with breast cancer curative effect while containing chemotherapeutic drugs.
  • exosomes can deliver miRNAs to recipient cells. Powerful blocking of target gene expression.
  • 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 present invention provides an effective, safe and convenient method and medicine for assembling the RNA inhibiting gene expression in organ tissue to form a composite structure and delivering it to target tissue or its cells to treat diseases as needed.
  • an isolated nucleic acid comprising a nucleotide sequence encoding a capable, comprising:
  • RNAs A nucleotide sequence encoding one or more RNAs that inhibit gene expression, the RNAs being miRNA, shRNA, siRNA, mRNA, ncRNA, sgRNA, or a combination of any of these RNAs.
  • the above-mentioned isolated nucleic acid further comprises: (b) a nucleotide sequence encoding a targeting protein.
  • the targeting protein is a tissue-specific protein.
  • (a) is a nucleotide sequence encoding one of the RNAs that inhibit gene expression.
  • (a) is a nucleotide sequence encoding a plurality of the RNAs that inhibit gene expression.
  • the plurality of RNAs that inhibit gene expression are 2-4 RNAs that inhibit gene expression.
  • isolated refers to the separation of a substance from its original environment (in the case of a natural substance, the original environment is the natural environment).
  • the original environment is the natural environment.
  • polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances present in the natural state. .
  • the RNA that inhibits gene expression is an RNA that inhibits the expression of the following genes: EGFR gene, KRAS gene, VEGFR gene, mTOR gene, TNF- ⁇ gene, integrin- ⁇ gene, B7 gene , TGF- ⁇ 1 gene, H2-K gene, H2-D gene, H2-L gene, HLA gene, GDF15 gene, miRNA-21, miRNA-214, TNC gene, PTP1B gene, mHTT gene, Lrrk2 gene, ⁇ -synuclein Gene.
  • genes EGFR gene, KRAS gene, VEGFR gene, mTOR gene, TNF- ⁇ gene, integrin- ⁇ gene, B7 gene , TGF- ⁇ 1 gene, H2-K gene, H2-D gene, H2-L gene, HLA gene, GDF15 gene, miRNA-21, miRNA-214, TNC gene, PTP1B gene, mHTT gene, Lrrk2 gene, ⁇ -synuclein Gene.
  • siRNA also known as short interfering RNA or silencing RNA
  • siRNA is a type of double-stranded RNA molecule, generally 20-29 base pairs in length, whose double strands are 2 nucleotides at each end of the RNA beyond the other end.
  • siRNA is generally produced by mimicking the mechanism of miRNA production, and such siRNA can be processed from precursor RNA (Precursor RNA, Pre-RNA).
  • precursor RNA can be folded into a stable stem-loop (hairpin) structure, and the length of the stem-loop structure is generally between 50-100 bp. Both sides of the stem of the stem-loop structure contain two sequences that are substantially complementary.
  • the siRNA can be substantially complementary to at least a portion of the sequence of the mRNA encoding the gene.
  • “Substantially complementary” means that the sequences of nucleotides are sufficiently complementary to interact in a predictable manner, such as to form secondary structure.
  • two "substantially complementary" nucleotide sequences are at least 70% complementary to each other; preferably, at least 80% are complementary; more preferably, at least 90% of the nucleotides are complementary; further preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%.
  • siRNA interferes with post-transcriptional degradation of mRNA that expresses a specific gene with a complementary nucleotide sequence, thereby preventing translation.
  • (a) is siRNA of EGFR gene, siRNA of KRAS gene, siRNA of VEGFR gene, siRNA of mTOR gene, siRNA of TNF- ⁇ gene, siRNA of integrin- ⁇ gene, B7 Gene siRNA, TGF- ⁇ 1 gene siRNA, H2-K gene siRNA, H2-D gene siRNA, H2-L gene siRNA, HLA gene siRNA, GDF15 gene siRNA, miRNA-21 antisense strand, 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.
  • the siRNA of each of the above-mentioned genes is an RNA sequence having a function of inhibiting the expression of the gene. Any RNA sequence having a function of inhibiting the expression of the aforementioned genes can be used in the present invention. The following are some RNA sequences with better effects:
  • the siRNA of EGFR gene includes UGUUGCUUCUCUUAAUUCCU, AAAUGAUCUUCAAAAGUGCCC, UCUUUAAGAAGGAAAGAUCAU, AAUAUUCGUAGCAUUUAUGGA, UAAAAAUCCUCACAUAUACUU.
  • the siRNA of KRAS gene includes UGAUUUAGUAUUAUUUAUGGC, AAUUUGUUCUCUAUAAUGGUG, UAAUUUGUUCUCUAUAAUGGU, UUAUGUUUUCGAAUUUCUCGA, UGUAUUUACAUAAUUACACAC.
  • the siRNA of VEGFR gene includes AUUUGAAGAGUUGUAUUAGCC, UAAUAGACUGGUAACUUUCAU, ACAACUAUGUACAUAAUAGAC, UUUAAGACAAGCUUUUCUCCA, AACAAAAGGUUUUUCAUGGAC.
  • the siRNA of mTOR gene includes AGAUAGUUGGCAAAUCUGCCA, ACUAUUUCAUCCAUAUAAGGU, AAAAUGUUGUCAAAGAAGGGU, AAAAAUGUUGUCAAAGAAGGG, UGAUUUCUUCCAUUUCUUCUC.
  • the siRNA of TNF- ⁇ gene includes AAAACAUAAUCAAAAGAAGGC, UAAAAAACAUAAUCAAAAGAA, AAUAAUAAAUAAUCACAAGUG, UUUUCACGGAAAACAUGUCUG, AAACAUAAUCAAAAGAAGGCA.
  • the siRNA of integrin- ⁇ gene includes AUAAUCAUCUCCAUUAAUGUC, AAACAAUUCCUUUUUAUCUU, AUUAAAACAGGAAACUUUGAG, AUAAUGAAGGAUAUACAACAG, UUCUUUAUUCAUAAAAGUCUC.
  • the siRNA of B7 gene includes UUUUCUUUGGGUAAUCUUCAG, AGAAAAAUUCCACUUUUUCUU, AUUUCAAAGUCAGAUAUACUA, ACAAAAAUUCCAUUUACUGAG, AUUAUUGAGUUAAGUAUUCCU.
  • the siRNA of TGF- ⁇ 1 gene includes ACGGAAAUAACCUAGAUGGGC, UGAACUUGUCAUAGAUUUCGU, UUGAAGAACAUAUAUAUGCUG, UCUAACUACAGUAGUGUUCCC, UCUCAGACUCUGGGGCCUCAG.
  • the siRNA of H2-K gene includes AAAAACAAAUCAAUCAAACAA, UCAAAAAAACAAAUCAAUCAA, UAUGAGAAGACAUUGUCUGUC, AACAAUCAAGGUUACAUUCAA, ACAAAACCUCUAAGCAUUCUC.
  • siRNA of H2-D gene includes AAUCUCGGAGAGACAUUUCAG, AAUGUUGGUGUAAAGAGAACUG, AACAUCAGACAAUGUUGUGUA, UGUUAACAAUCAAGGUCACUU, AACAAAAAAACCUCUAAGCAU.
  • the siRNA of H2-L gene includes GAUCCGCUCCCAAUACUCCGG, AUCUGCGUGAUCCGCUCCCAA, UCGGAGAGACAUUUCAGAGCU, UCUCGGAGAGACAUUUCAGAG, AAUCUCGGAGAGACAUUUCAG.
  • siRNA of HLA gene AUCUGGAUGGUGUGAGAACCG, UGUCACUGCUUGCAGCCUGAG, UCACAAAGGGAAGGGCAGGAA, UUGCAGAAACAAAGUCAGGGU, ACACGAACACAGACACAUGCA.
  • the siRNA of GDF15 gene includes UAUAAAUACAGCUGUUUGGGC, AGACUUAUAUAAAUACAGCUG, AAUUAAUAAUAAAUAACAGAC, AUCUGAGGCCAUUCACCGUC, UGCAACUCCAGCUGGGGCCGU.
  • the siRNA of TNC gene included UAUGAAAUGUAAAAAAAGGGA, AAUAUAUCCUUAAAAUGGAA, UAAUCAUAUCCUUAAAAUGGA, UGAAAAAUCCUUAGUUUUCAU, AGAAGUAAAAAACUAUUGCGA.
  • the siRNA of PTP1B gene includes UGAUAUAGUCAUUAUCUUCUU, UCCAUUUUUAUCAAACUAGCG, AUUGUUUAAAUAAAUAUGGAG, AAUUUUAAUACAUUAUUGGUU, UUUAUUAUUGUACUUUUUGAU.
  • the siRNA of mHTT gene includes UAUGUUUUCACAUAUUGUCAG, AUUUAGUAGCCAACUAUAGAA, AUGUUUUUCAAUAAAUGUGCC, UAUGAAUAGCAUUCUUAUCUG, UAUUUGUUCCUCUUAAUACAA.
  • the siRNA of Lrrk2 gene includes AUUAACAUGAAAAUAUCACUU, UUAACAAUAUCAUAUAAUCUU, AUCUUUAAAAUUUGUUAACGC, UUGAUUUAAGAAAAUAGUCUC, UUUGAUAACAGUAUUUUUCUG.
  • the siRNA of ⁇ -synuclein gene includes AUAUAUUAACAAAUUUCACAA, AAGUAUUAUAUAUUAACAA, AUAACUUUUAUAUUUUUGUCCU, UAACUAAAAAAUUAUUUCGAG, UCGAAUAUUAUUUAUUGUCAG.
  • RNA sequences that can be used in the present invention also include RNA sequences with a homology greater than 80% to the aforementioned RNAs.
  • the homology is 85%, 88%, 90%, 95%, 98%, etc.
  • the nucleotide sequence encoding one or more RNAs that inhibit gene expression in the isolated nucleic acid includes an RNA fragment sequence directed against the gene.
  • the RNA fragment sequence is typically an RNA sequence complementary to the target nucleotide sequence of the gene.
  • the RNA fragment sequence is the sense strand sequence of siRNA.
  • the length of the RNA encoding one or more inhibiting gene expression in the isolated nucleic acid is 15-29 nucleotides (nt), preferably 18-22 nt, such as 18nt, 19nt, 20nt, 21nt, 22nt.
  • nt nucleotides
  • 18nt, 19nt, 20nt, 21nt, 22nt nucleotides
  • the cost of the line is greatly increased, and the effect is not better than the RNA sequence with a length of 18-22nt, and the economic benefit is poor. Therefore, when the length of the RNA sequence is 15-25nt, especially 18-22nt, the cost and the effect can be taken into consideration, and the effect is the best.
  • 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 nucleotides encoding one or more RNAs that inhibit gene expression in the isolated nucleic acid further include flanking sequences (such as 5' flanking sequences and 3' flanking sequences), One or more of a stem-loop sequence and a compensating sequence of the RNA sequence.
  • the compensation sequence is the reverse complement of the RNA fragment sequence.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment sequence, and any 1-5 bases therein are deleted.
  • the compensation sequence is the reverse complement sequence of the RNA fragment sequence and deletes any 1-3 bases therein, especially the 1-3 bases arranged consecutively.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and the 9th and/or 10th bases are deleted.
  • the RNA sequence is capable of being expressed in the target receptor, and the compensatory sequence is not expressed in the target receptor.
  • Flanking sequences are sequences used to aid in the cleavage of RNA molecules, such as siRNA molecules, into the correct final sequence.
  • the flanking structure of the natural miRNA precursor can be adopted as the flanking structure of the RNA molecule of the present invention.
  • the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with more than 80% homology to it, for example, 85%, 90%, 92%, 95% homology to the sequence %, 98%, 99% sequence, etc.
  • the 3' flanking sequence is accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence whose homology is greater than 80%, such as 85%, 90%, 92%, 95% homology to the sequence %, 98%, 99% sequence, etc.
  • the stem-loop structure is the spacer sequence encoding the hairpin structure, which maintains the stability of the RNA.
  • the sequence of the stem-loop structure is preferably gttttggccactgactgac or a sequence with a homology greater than 80%.
  • the sequence of the RNA encoding one or more inhibiting gene expression in the isolated nucleic acid has: 5' flanking sequence, RNA fragment sequence, stem-loop sequence, compensation sequence and 3' flanking sequence.
  • the RNA has a promoter 5'.
  • RNA sequence can be transcribed and cut out to the greatest extent, and its encapsulated in exosomes.
  • (a) in the nucleic acid is a nucleotide sequence encoding a plurality of the RNAs that inhibit gene expression.
  • the plurality of RNAs that inhibit gene expression are 2-4 RNAs that inhibit gene expression.
  • the plurality of RNAs are connected by a linker.
  • the structure of the linker is, for example, sequence 1-sequence 2-sequence 3.
  • sequence 1 is preferably CAGATC
  • sequence 2 can be a sequence composed of 5-80 bases, preferably a sequence composed of 10-50 bases, more preferably a sequence composed of 20-40 bases
  • sequence 3 Preferably it is TGGATC.
  • the sequence of the linker is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • the targeting protein is a target tissue-specific targeting peptide.
  • the targeting protein is a fusion protein of a target tissue-specific targeting peptide and a membrane protein.
  • the specific targeting peptide is selected from the group consisting of: RVG targeting peptide, GE11 targeting peptide, PTP targeting peptide, TCP-1 targeting peptide, and MSP targeting peptide.
  • the targeting protein is a membrane protein, for example, selected from the group consisting of: cellular receptor proteins (such as growth factor receptors), LAMP1 or LAMP2 (such as LAMP2B), antibodies or combinations thereof Fragment.
  • cellular receptor proteins such as growth factor receptors
  • LAMP1 or LAMP2 such as LAMP2B
  • the targeting protein is RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein, MSP-LAMP2B fusion protein.
  • the target tissue is brain, pineal gland, pituitary gland, eye, ear, nose, mouth, pharynx, parotid gland, tonsil, esophagus, trachea, thyroid, thymus, breast, lung, Heart, stomach, intestines, appendix, liver, gallbladder, spleen, pancreas, kidney, ureter, bladder, urethra, uterus, ovary, fallopian tube, vagina, vas deferens, prostate, penis, testis, anus, bone, muscle, connective tissue, nerve , lymph, colorectal, blood, bone marrow and/or skin, etc.
  • the target cells are cells of the above-mentioned target tissue.
  • the nucleic acid after administration to a mammal, is found in tissues (including: liver, lung, gastrointestinal tract, breast, kidney, brain, spleen, lymph, thyroid, reproductive organs, blood cells or lymphocytes) , especially the liver), and its expressed products are abundantly encapsulated in exosomes produced and secreted by cells of these tissues and delivered to target tissues to exert therapeutic effects. Therefore, on the one hand, the targeting protein encoded by the nucleic acid needs to select an available targeting tag according to the target tissue, and on the other hand, it is necessary to ensure that the targeting tag can stably appear on the surface of exosomes, so as to achieve the targeting function.
  • tissues including: liver, lung, gastrointestinal tract, breast, kidney, brain, spleen, lymph, thyroid, reproductive organs, blood cells or lymphocytes
  • Targeting peptides suitable for use in the present invention include, but are not limited to, RVG targeting peptide (nucleotide sequence shown in SEQ ID No: 1), GE11 targeting peptide (nucleotide sequence shown in SEQ ID No: 2) ), PTP targeting peptide (nucleotide sequence shown in SEQ ID No: 3), TCP-1 targeting peptide (nucleotide sequence shown in SEQ ID No: 4), MSP targeting peptide (nucleotide sequence shown in SEQ ID No: 4) The sequence is shown in SEQ ID No: 5); targeting proteins include but are not limited to RVG-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6), GE11-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6) ID No: 7), PTP-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 8), TCP-1-LAMP2B
  • 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.
  • (a) in the nucleic acid is a nucleotide sequence encoding a plurality of the RNAs that inhibit gene expression.
  • the plurality of RNAs that inhibit gene expression can be administered to the subject simultaneously or separately.
  • the multiple RNAs may be located in different plasmid vectors or viral vectors, respectively.
  • one of the plasmids or viral vectors contains a promoter and a targeting tag
  • the other plasmids contain a promoter and an RNA segment. That is, the targeting tag and the RNA fragment are loaded into different carriers, and the two or more carriers are injected into the body simultaneously or separately.
  • the vector containing the RNA sequence can be injected first, and then (for example, after 1-2 hours) the vector containing the targeting tag can be injected, In this way, a better targeting effect can be achieved.
  • the nucleic acid is enriched in mammalian liver, and its product is encapsulated in exosomes in hepatocytes.
  • a vector of RNA that inhibits gene expression comprising:
  • RNAs that inhibit gene expression
  • the RNAs being miRNA, shRNA, siRNA, mRNA, ncRNA, sgRNA, or a combination of any of these RNAs;
  • the vector comprises the isolated nucleic acid provided by the present invention described above, which encodes a nucleotide sequence of RNA capable of inhibiting gene expression.
  • the vector is a plasmid.
  • the plasmid after the plasmid is administered to a mammal, the plasmid can be expressed in tissues (including: liver, lung, gastrointestinal tract, breast, kidney, brain, spleen, lymph, thyroid, reproductive organs, blood cells, etc.) or lymphocytes, especially liver), the RNA fragments of the present invention are enriched, transcribed and/or expressed, and the RNA fragments are encapsulated in exosomes in the cells of this tissue.
  • the vector is a viral vector.
  • it can be a baculovirus expression vector, an adenovirus vector, a retrovirus vector, a herpes virus vector or a lentiviral vector and the like.
  • the vector is an adenovirus vector.
  • the adenovirus is adeno-associated virus type 5, adenovirus-associated virus type 8 or adenovirus-associated virus type 9. More preferably, the adenovirus is adeno-associated virus type 5.
  • the plasmid or viral vector after the plasmid or viral vector is administered to a mammal, it is enriched and expressed in the liver, and its product is largely encapsulated in exosomes.
  • an exosome with RNA that inhibits gene expression which comprises the nucleic acid or vector as described above.
  • the exosomes are exosomes derived from human tissue or cells.
  • the tissue includes liver, lung, gastrointestinal tract, breast, kidney, brain, spleen, lymph, thyroid, reproductive organs, blood cells or lymphocytes.
  • the exosomes are exosomes derived from liver or liver cells.
  • a pharmaceutical composition which contains the aforementioned nucleic acid, carrier or exosome.
  • the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient for delivering the nucleic acid, vector or exosome to a subject.
  • the administration modes of the drug include oral, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection. That is, the drug can be administered orally, inhaled, subcutaneously, intramuscularly or intravenously.
  • the dosage forms of the drug can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes and the like.
  • the tissue including: liver, lung, gastrointestinal tract, breast, kidney, brain, spleen, lymph, thyroid, reproductive organs, blood cells or lymphocytes, especially is enriched in the liver
  • its expressed products are largely encapsulated in exosomes in the cells of this tissue and delivered to the target tissue to play a therapeutic role.
  • the pharmaceutical composition can be used to treat various diseases, including tumors, acute and chronic infectious diseases or other acute and chronic diseases.
  • the acute and chronic infectious diseases described therein include: viral influenza, viral hepatitis, AIDS, viral diseases of SARS, bacterial diseases (such as tuberculosis, bacterial pneumonia), and acute and chronic infectious diseases caused by various other pathogenic microorganisms .
  • the other acute and chronic diseases include: diseases of the respiratory system, diseases of the immune system, diseases of the blood and hematopoietic system, diseases of the circulatory system such as cardiovascular and cerebrovascular diseases, metabolic diseases of the endocrine system, diseases of the digestive system, diseases of the nervous system, diseases of the urinary system , diseases of the reproductive system and diseases of the motor system.
  • the disease is 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 a transplant Anti-host disease.
  • a method of treating a disease comprising administering to a subject a nucleic acid, vector or exosome as previously described.
  • the diseases include tumors, acute and chronic infectious diseases or other acute and chronic diseases.
  • the actual dose administered will vary depending on factors such as the carrier, target cell or tissue, general condition of the subject to be treated, degree of transformation/modification sought, administration Route, mode of administration, type of transformation/modification sought, etc.
  • Fig. 1 is a comparison diagram of plasmid distribution and metabolism in mice provided by an embodiment of the present application
  • Fig. 2 is a comparison diagram of protein expression levels in mice provided by an embodiment of the present application.
  • FIG. 3 is a comparison diagram of related siRNA levels in mice provided by an embodiment of the present application.
  • FIG. 4 is a comparison diagram of absolute siRNA levels in various tissues of mice provided in an embodiment of the present application.
  • Figure 5 is a comparison diagram of the effect of plasmid doses on mouse siRNA levels provided by an embodiment of the present application.
  • Fig. 6 is the metabolic situation comparison diagram of the precursor and the mature body in the mouse liver after injecting the plasmid provided by an embodiment of the present application;
  • FIG. 7 is a comparison diagram of siRNA kinetics and distribution in different tissues of mice provided by an embodiment of the present application.
  • Figure 8 is a comparison diagram of the influence of different promoters on siRNA provided by an embodiment of the present application.
  • FIG. 9 is a comparison diagram of the fluorescence intensity of eGFP in different tissues of mice provided by an embodiment of the present application.
  • Figure 10 is a comparison diagram of mouse alanine aminotransferase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, serum alkaline phosphatase, creatinine content, and thymus gland weight, spleen weight, and peripheral blood cell percentage provided by an embodiment of the present application;
  • Figure 11 is a comparison diagram of the therapeutic effect of mouse EGFR mutant lung cancer tumor provided by an embodiment of the present application.
  • Figure 12 is a mouse HE staining chart, an immunohistochemical staining chart and a coloring statistics chart provided by an embodiment of the present application;
  • FIG. 13 is a comparison diagram of the therapeutic effect of mouse KRAS mutant lung cancer tumor provided by an embodiment of the present application.
  • FIG. 14 is a mouse HE staining chart, an immunohistochemical staining chart, and a coloring situation statistical chart provided by an embodiment of the present application;
  • Figure 15 is the fluorescence signal statistics of 6 different RNA plasmids provided in an embodiment of the present application after treatment of lung cancer
  • a in the figure is the fluorescence detection results of CMV-siR E and Albumin-siR E
  • B is CMV-siR T and Albumin - Fluorescence detection results of siRT
  • C is the fluorescence detection results of CMV-miR7 and Albumin-miR7
  • D is the fluorescence detection results of CMV-shRE and Albumin- shRE
  • E is the fluorescence detection results of CMV- shRT and Albumin- shRT Fluorescence detection results
  • F is the fluorescence detection results of CMV-miR133b and Albumin-miR133b.
  • Figure 16 is the fluorescence signal statistics of 4 groups of plasmids composed of any 2 RNA sequences among the 6 RNA sequences provided in an embodiment of the present application after the treatment of lung cancer, all of which are connected with CMV or Albumin RNA, and A in the figure is siRNA + shRT
  • the fluorescence detection results of B is the fluorescence detection results of shR E +miR133b
  • C is the fluorescence detection results of siRT +miR7
  • D is the fluorescence detection results of shRT +miR133b.
  • Figure 17 is the fluorescence signal statistics of 3 groups of plasmids composed of any 3 RNA sequences among the 6 RNA sequences provided in an embodiment of the present application after the treatment of lung cancer, all of which are connected with CMV or Albumin RNA, and A in the figure is siRNA + shRT +miR7 fluorescence detection results, B is the fluorescence detection results of siRT + shRE +miR7, C is the fluorescence detection results of shRE + siRT +miR133b.
  • Figure 18 shows the enrichment results of siRNA in liver, lung, plasma, and exosomes after intravenous injection of plasmid CMV-siR E and plasmid CMV-GE11-siRE E , respectively, provided in an example of the present application.
  • EGFR siRNA content in liver and lung without targeting peptide GE11 B is EGFR siRNA content in plasma and exosomes with/without targeting peptide GE11.
  • Figure 19 shows the detection results of EGFR protein and mRNA expression after intravenous injection of plasmid CMV-siR E and plasmid CMV-GE11-siR E , respectively, provided in an example of the present application, and A in the figure is the case with/without targeting peptide GE11
  • the protein content of EGFR was detected under the condition of B, and the mRNA content of EGFR was detected in the presence or absence of the targeting peptide GE11.
  • Figure 20 shows the lung enrichment effect and therapeutic effect of a plasmid containing a sequence with a 5' flanking sequence homology greater than 80% provided by an embodiment of the present application.
  • A is the case with/without targeting tag RVG 2
  • B is the fluorescence signal detection result of one 5' flanking homologous sequence with/without targeting tag RVG
  • C is the case with/without targeting tag RVG Fluorescence signal detection result of another 5' flanking homologous sequence.
  • Figure 21 shows the lung enrichment effect and therapeutic effect of a plasmid containing a sequence with a loop sequence homology greater than 80% provided by an embodiment of the present application, and A in the figure shows 2 loops with or without targeting tag RVG
  • the EGFR siRNA content of the homologous sequence B is the fluorescence signal detection result of one loop homologous sequence with/without targeting tag RVG, C is the other loop homology with/without targeting tag RVG Sequence fluorescence signal detection results.
  • Figure 22 is the lung enrichment effect and therapeutic effect of a plasmid containing a sequence with a 3' flanking sequence homology greater than 80% provided by an embodiment of the present application, and A in the figure is with/without targeting tag RVG 2 EGFR siRNA content of the 3' flanking homologous sequence, B is the fluorescence signal detection result of one 3' flanking homologous sequence with/without targeting tag RVG, C is the case with/without targeting tag RVG Fluorescence signal detection result of another 3' flanking homologous sequence.
  • Figure 23 shows the detection of EGFR siRNA content in lung tissue 9 hours after intravenous injection of plasmids containing sequence 4 and two sequences 4-1 and 4-2 with more than 80% homology to sequence 4 provided by an example of the present application
  • RNA is siRNA/ siRT
  • a in the figure is the detection result of EGFR siRNA content of sequence 4
  • B is the detection result of EGFR siRNA content of sequence 4-1
  • C is the detection result of EGFR siRNA content of sequence 4-2.
  • Figure 24 is the detection of EGFR expression after intravenous injection of three plasmids containing RNA sequences with lengths of 18, 20, and 22, respectively, provided in an embodiment of the present application.
  • A is the detection result of the protein content of EGFR
  • B is the mRNA of EGFR Content test results.
  • Figure 25 is a diagram of the treatment situation of mouse lung cancer based on KRAS siRNA provided by an embodiment of the present application.
  • Figure 26 is a diagram of the treatment situation of mouse lung cancer based on EGFR siRNA provided by an embodiment of the present application.
  • Figure 27 is a comparison diagram of the content of various enzymes in mice provided by an embodiment of the present application.
  • Figure 28 shows the enrichment effect of the lentiviral vector in liver, lung, plasma, and exosomes and the detection of EGFR gene expression provided by an example of the present application.
  • A is the expression of the lentiviral vector in the liver and lung after intravenous injection
  • the enrichment effect of EGFR siRNA in B is the enrichment effect of EGFR siRNA in plasma and exosomes after intravenous injection of lentiviral vector
  • C is the expression effect of EGFR protein after intravenous injection of lentiviral vector
  • D is the EGFR protein expression effect after intravenous injection of lentiviral vector mRNA expression effect.
  • Figure 29 shows the enrichment effect of adenovirus vector in liver, lung, plasma, and exosomes and the detection of EGFR gene expression provided by an example of the present application.
  • A is the liver and lung after intravenous injection of adenovirus vector.
  • the enrichment effect of EGFR siRNA in B is the enrichment effect of EGFR siRNA in plasma and exosomes after intravenous injection of adenoviral vector
  • C is the expression effect of EGFR protein after intravenous injection of adenoviral vector
  • D is the EGFR protein expression effect after intravenous injection of adenoviral vector.
  • mRNA expression effect is the enrichment effect of adenovirus vector in liver, lung, plasma, and exosomes and the detection of EGFR gene expression provided by an example of the present application.
  • A is the liver and lung after intravenous injection of adenovirus vector.
  • the enrichment effect of EGFR siRNA in B is the enrichment effect of EGFR siRNA in plasma
  • Figure 30 shows the statistics of fluorescence signals after 6 different RNAs are respectively constructed into adeno-associated virus vectors to treat lung cancer provided by an embodiment of the present application.
  • A is the statistics of fluorescence signals after siRNA is constructed into adeno-associated virus vectors to treat lung cancer
  • B is the fluorescence signal statistics of siRNA constructed into adeno-associated virus vector to treat lung cancer
  • C is the fluorescence signal statistics of miR-7 constructed into adeno-associated virus vector to treat lung cancer
  • D is the fluorescence signal of shRE constructed into adeno-associated virus vector to treat lung cancer
  • the fluorescence signal statistics of E is the fluorescence signal statistics after shRT is constructed into adeno-associated virus vector to treat lung cancer
  • F is the fluorescence signal statistics of miR-133b constructed into adeno-associated virus vector to treat lung cancer.
  • Figure 31 is the fluorescence signal statistics of 4 groups of RNA fragments composed of any 2 RNA sequences out of 6 different RNAs provided in an embodiment of the present application, respectively constructed into adeno-associated virus vectors after treatment of lung cancer
  • a in the figure is siR E +shR T is the fluorescence signal statistics after the adeno-associated virus vector is constructed into the adeno-associated virus vector for treatment of lung cancer
  • B is the fluorescence signal statistics after the siR T + miR-7 is constructed into the adeno-associated virus vector for the treatment of lung cancer
  • C is the shRE + miR-133b construct into the adeno-associated virus Fluorescence signal statistics after vector treatment of lung cancer
  • D is the fluorescence signal statistics after shRT + miR-133b was constructed into adeno-associated virus vector to treat lung cancer.
  • Fig. 32 is the fluorescence signal statistics of 3 groups of RNA fragments composed of any 3 kinds of RNA sequences among 6 kinds of different RNAs provided in an embodiment of the present application respectively constructed into adeno-associated virus vectors after treatment of lung cancer
  • A is siR E +shR in the figure Fluorescence signal statistics after T + miR-7 was constructed into adeno-associated virus vector for treatment of lung cancer
  • B is the fluorescence signal statistics of siR T + shR E + miR-7 constructed into adeno-associated virus vector for lung cancer treatment
  • C is shR E +siR Fluorescence signal statistics after T + miR-133b was constructed into adeno-associated virus vector for treatment of lung cancer.
  • Figure 33 shows the enrichment results of siRNA in liver, lung, plasma, and exosome species and the detection results of EGF R protein and mRNA expression after intravenous injection provided by an embodiment of the present application
  • A is AAV-siRNA E and The enrichment results of AAV-GE11- siRE in liver and lung
  • B is the enrichment results of AAV- siRE and AAV-GE11- siRE in plasma and exosomes
  • C is AAV- siRE and AAV-siRE EGFR protein expression of GE11-siRE
  • D is the EGFR mRNA expression of AAV- siRE and AAV-GE11- siRE .
  • Figure 34 shows the enrichment effect and therapeutic effect in the lungs after 2 sequences with more than 80% homology to the 5' flanking sequence provided in an embodiment of the present application are constructed into an AAV vector, and A in the figure is displayed by the content of EGFR siRNA Based on the enrichment results in the lungs, B is the therapeutic effect of one sequence, and C is the therapeutic effect of the other sequence.
  • Figure 35 shows the enrichment effect and therapeutic effect in the lung after a sequence with a homology of more than 80% to the loop sequence provided by an embodiment of the present application is constructed into an AAV vector, and A in the figure shows the EGFR siRNA content in the lung
  • the enrichment results of , B is the treatment effect of one sequence, and C is the treatment effect of the other sequence.
  • Figure 36 shows the lung enrichment effect and therapeutic effect after the sequence provided by an embodiment of the present application with a homology of more than 80% to the 3' flanking sequence is constructed into an AAV vector. Lung enrichment results, B is the treatment effect of one sequence, C is the treatment effect of the other sequence.
  • Figure 37 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 EGFR siRNA in lung tissue 9 hours after intravenous injection Content detection results, in the figure A is the detection result of sequence 4, B is the detection result of sequence 4-1, and C is the detection result of sequence 4-2.
  • Figure 38 is the detection of EGFR expression after intravenous injection of gene loops containing 3 RNA sequences of different lengths provided in an embodiment of the present application, in the figure A is the result of EGFR protein content, and B is the result of EGFR mRNA content.
  • Figure 39 is a comparison diagram of mouse renal cancer tumor images provided in an embodiment of the present application.
  • Figure 40 is a comparison diagram of the development of mouse renal cancer tumor provided by an embodiment of the present application.
  • Figure 41 shows the in vivo (plasma, exosome) enrichment, self-assembly and colorectal cancer, pancreatic cancer, glioma, lung cancer of the adeno-associated virus (AAV) vector containing RNA fragments provided in an example of the present application , The therapeutic effect of renal cancer (shown by siRNA content).
  • AAV adeno-associated virus
  • Figure 42 is an example of the application of adeno-associated virus (AAV) as a viral vector, which contains siR E , siR V , siR K and siR E+T , with in vivo enrichment, self-assembly and lung cancer, kidney cancer , pancreatic cancer, obesity and glioma treatment effect
  • AE in the figure is the fluorescence signal detection results of lung cancer, kidney cancer, pancreatic cancer, obesity and glioma, respectively.
  • Figure 43 is a lentivirus (LV) provided by another embodiment of the present application as a viral vector, when it contains siR E , siR V , siR K and siR E+T , it has in vivo enrichment, self-assembly and lung cancer, kidney
  • LV lentivirus
  • the therapeutic effects of cancer, pancreatic cancer, obesity and glioma, AE in the figure are the fluorescence signal detection results of lung cancer, kidney cancer, pancreatic cancer, obesity and glioma, respectively.
  • Figure 44 is a viral vector delivery system provided in an embodiment of the present application, in the case of carrying multiple RNA fragments, all have in vivo enrichment, self-assembly and therapeutic effects against lung cancer, A in the figure is when the RNA sequence acts alone
  • the tumor volume effect display of B is the tumor volume effect display when the RNA sequence is composed of 2-3 RNA fragments.
  • Figure 45 shows that in the viral vector delivery system provided by an example of the present application, in the case of carrying multiple RNA fragments, all have in vivo enrichment, self-assembly and therapeutic effects on renal cancer, A in the figure is the role of RNA sequence alone
  • the effect of tumor volume when B is the effect of tumor volume when 2-3 RNA fragments are composed of RNA sequences.
  • Figure 46 is a viral vector delivery system provided in an example of the present application, in the case of carrying multiple RNA fragments, all have in vivo enrichment, self-assembly and therapeutic effects on colorectal cancer, in the figure A is the RNA sequence alone The effect of tumor volume when acting is shown, and B is the effect of tumor volume when 2-3 RNA fragments are composed of RNA sequences.
  • Figure 47 shows that in the viral vector delivery system provided in an example of the present application, in the case of carrying multiple RNA fragments, all have in vivo enrichment, self-assembly and therapeutic effects on pancreatic cancer, A in the figure is the role of the RNA sequence alone
  • the effect of tumor volume when B is the effect of tumor volume when 2-3 RNA fragments are composed of RNA sequences.
  • Figure 48 shows that in the viral vector delivery system provided in an example of the present application, in the case of carrying multiple RNA fragments, all of them have in vivo enrichment, self-assembly and therapeutic effects on gliomas.
  • A is the RNA sequence alone The effect of tumor volume when acting is shown, and B is the effect of tumor volume when 2-3 RNA fragments are composed of RNA sequences.
  • Figure 49 shows that when the adenoviral vector delivery system provided in an example of the present application contains 1-2 RNA fragments and 1-2 targeting tags, it also has in vivo enrichment, self-assembly and therapeutic effects on cancer.
  • A is the therapeutic effect of the delivery system on pancreatic cancer
  • the carrier is AAV
  • the line carried is siR K or PTP-siR K
  • B is the therapeutic effect of the delivery system on glioma
  • the carrier is AAV
  • the line carried is siR E +T or RVG-siR E+T .
  • Figure 50 shows that when the adenovirus vector provided in an embodiment of the present application carries multiple RNA fragments with different 5' flanking sequences/loop sequences/3' flanking sequences, it has in vivo enrichment, self-assembly and targeting of lung cancer, kidney cancer, The therapeutic effect of pancreatic cancer and glioma.
  • A shows that two different 5' flanking sequences/loop sequences/3' flanking sequences are connected with siRNA E sequence, and in the case of connecting with or without RVG, The effect on the volume of lung cancer tumors
  • B shows that two different 5' flanking sequences/loop sequences/3' flanking sequences are connected with the siR V sequence, and in the case of connecting with or without RVG, on renal cancer tumors
  • the effect of volume C shows the effect of connecting two different 5' flanking sequences/loop sequences/3' flanking sequences with siRNA P sequence, and with or without RVG connected, on pancreatic cancer tumor volume Effect
  • D shows the effect of connecting two different 5' flanking sequences/loop sequences/3' flanking sequences with siR E+T sequence, and with or without RVG connected, on glioma tumor volume Effect.
  • Figure 51 shows that the adenoviral vector provided in an embodiment of the present application carries multiple RNA fragments with different 5' flanking sequences/loop sequences/3' flanking sequences, and it has in vivo enrichment, self-assembly and treatment for colorectal cancer Effect, the figure shows the effect of connecting two different 5' flanking sequences/loop sequences/3' flanking sequences with siR V sequence, and in the case of connecting with or without RVG, on the effect of colorectal cancer tumor volume .
  • Figure 52 shows that when the connecting sequence provided by an embodiment of the present application is sequence 4 and sequence 4-1 and sequence 4-2 with more than 80% homology to sequence 4, the delivery system containing the above sequence also has corresponding enrichment, Self-assembly and cancer treatment effect, the figure shows the detection results of EGFR siRNA content of sequence 4/4-1/4-2, and the connected RNAs are siRNA and siRNA respectively .
  • Figure 53 shows that when the lengths of the RNA sequences provided in an embodiment of the present application are 18, 20, and 22, respectively, the delivery system containing the RNA sequences also has corresponding enrichment, self-assembly and cancer treatment effects, and A in the figure represents three lengths
  • B is the EGFR mRNA content detected after the injection of the delivery system constructed by the three lengths of RNA sequences.
  • Figure 54 is a comparison diagram of the development of colitis in mice provided by an embodiment of the present application.
  • Figure 55 is a comparison diagram of the HE staining situation of mouse colon provided by an embodiment of the present application.
  • Figure 56 is a comparison diagram of the development of colitis in mice provided by an embodiment of the present application.
  • Figure 57 is a comparison diagram of HE staining of mouse colon provided by an embodiment of the present application.
  • Figure 58 is a comparison diagram of the treatment of colitis in mice and RNA expression levels provided by an embodiment of the present application.
  • Figure 59 is a comparison diagram of mouse cytokine concentration and colon HE staining provided by an embodiment of the present application.
  • Figure 60 is a comparison diagram of the treatment of colitis in mice provided by an embodiment of the present application.
  • Figure 61 is a comparison diagram of the mouse disease activity index and various siRNA levels provided by an embodiment of the present application.
  • Figure 62 is a comparison diagram of various siRNA and mRNA levels in mice provided in an embodiment of the present application.
  • Figure 63 is a comparison diagram of the HE staining situation of mouse colon provided by an embodiment of the present application.
  • Figure 64 is a graph showing the results of in vivo enrichment of TNF- ⁇ siRNA by injecting mice with TNF- ⁇ siRNA-lentivirus provided in an example of the present application.
  • A is the liver enrichment result
  • B is the plasma enrichment result
  • Figure 65 is a graph showing the results of injecting mice with TNF- ⁇ siRNA-lentivirus, and detecting TNF- ⁇ siRNA in plasma exosomes to determine the spontaneous formation of complex structures provided in an example of the present application.
  • Figure 66 is a graph showing the results of specific curative effects after injecting mice with TNF- ⁇ siRNA-lentivirus according to an embodiment of the present application.
  • A is the disease index score
  • B is the detection result of inflammatory factors
  • C is the detection of target gene mRNA result.
  • Figure 67 is a graph of the enrichment results in vivo after mice are injected with viral vectors containing 6 different RNAs provided in an example of the present application.
  • the 6 RNAs are: miR-19a (target gene TNF- ⁇ ), miR -124-3p (target gene TNF- ⁇ ), B7-siRNA-1, B7-siRNA-2, integrin ⁇ 4 shRNA-1, integrin ⁇ 4 shRNA-2, A is the result of intrahepatic detection of small RNA expression, B is The results of small RNA expression in plasma, and C is the result of small RNA expression in colon.
  • Fig. 68 is a graph showing the specific therapeutic effects of mice injected with viral vectors containing 6 different RNAs provided in an example of the present application.
  • the 6 RNAs are: miR-19a (target gene TNF- ⁇ ), miR- 124-3p (target gene TNF- ⁇ ), B7-siRNA-1, B7-siRNA-2, integrin ⁇ 4 shRNA-1, integrin ⁇ 4 shRNA-2, A is the disease index score in the figure, and B is the detection result of inflammatory factors.
  • Figure 69 is a graph of in vivo enrichment results provided by an embodiment of the present application after injecting mice with viral vectors containing 4 groups of different RNA fragments, respectively.
  • the 4 groups of RNA fragments respectively contain any two RNA sequences, specifically: miR -19a (target gene TNF- ⁇ )+B7-siRNA-1, miR-124-3p (target gene TNF- ⁇ )+B7-siRNA-2, B7-siRNA-1+integrin ⁇ 4 shRNA-1, B7-siRNA -2+integrin ⁇ 4 shRNA-2, in the figure
  • A is the expression result of small RNA (sequence 1) detected in liver
  • B is the expression result of small RNA (sequence 1) detected in plasma
  • C is the result of small RNA detected in colon (sequence 1) Express the result.
  • Figure 70 is a graph of in vivo enrichment results provided by another embodiment of the present application after injecting mice with viral vectors containing 4 groups of different RNA fragments, respectively.
  • the 4 groups of RNA fragments respectively contain any two RNA sequences, specifically: miR-19a (target gene TNF- ⁇ )+B7-siRNA-1, miR-124-3p (target gene TNF- ⁇ )+B7-siRNA-2, B7-siRNA-1+integrin ⁇ 4 shRNA-1, B7- siRNA-2+integrin ⁇ 4 shRNA-2, in the figure A is the expression result of small RNA (sequence 2) detected in liver, B is the expression result of small RNA (sequence 2) detected in plasma, and C is the result of small RNA detected in colon (sequence 2). ) to express the result.
  • miR-19a target gene TNF- ⁇
  • miR-124-3p target gene TNF- ⁇
  • B7-siRNA-1+integrin ⁇ 4 shRNA-1 B7
  • Figure 71 is a graph showing the results of specific curative effects after injecting mice with viral vectors containing 4 groups of different RNA fragments provided in an embodiment of the present application.
  • the 4 groups of RNA fragments respectively contain any two RNA sequences, specifically: miR -19a (target gene TNF- ⁇ )+B7-siRNA-1, miR-124-3p (target gene TNF- ⁇ )+B7-siRNA-2, B7-siRNA-1+integrin ⁇ 4 shRNA-1, B7-siRNA -2+integrin ⁇ 4 shRNA-2, A is the disease index score in the figure, and B is the detection result of inflammatory factors.
  • Figure 72 is a graph of the in vivo enrichment results provided by an embodiment of the present application after injecting mice with viral vectors containing 3 groups of different RNA fragments.
  • the 3 groups of RNA fragments respectively contain any three kinds of RNA sequences, specifically: miR -19a (target gene TNF- ⁇ )+B7-siRNA-1+integrin ⁇ 4 shRNA-1, miR-124-3p (target gene TNF- ⁇ )+B7-siRNA-2+integrin ⁇ 4 shRNA-2, miR-19a (target gene TNF- ⁇ )+B7-siRNA-1+integrin ⁇ 4 shRNA-2, in the figure A is the expression result of small RNA (sequence 1) detected in liver, B is the expression result of small RNA (sequence 1) detected in plasma, C is the expression result of small RNA (sequence 1) detected in colon.
  • A is the expression result of small RNA (sequence 1) detected in liver
  • B is the expression result of small RNA (sequence
  • Figure 73 is a graph of the in vivo enrichment results provided by another embodiment of the present application after injecting mice with viral vectors containing 3 groups of different RNA fragments, respectively.
  • the 3 groups of RNA fragments respectively contain any 3 kinds of RNA sequences, specifically: miR-19a (target gene TNF- ⁇ )+B7-siRNA-1+integrin ⁇ 4 shRNA-1, miR-124-3p (target gene TNF- ⁇ )+B7-siRNA-2+integrin ⁇ 4 shRNA-2, miR- 19a (target gene TNF- ⁇ )+B7-siRNA-1+integrin ⁇ 4shRNA-2, in the figure
  • A is the expression result of small RNA (sequence 2) detected in liver
  • B is the expression result of small RNA (sequence 2) detected in plasma
  • C is the expression result of small RNA (sequence 2) detected in colon.
  • Figure 74 is a graph of the enrichment results in vivo after injecting mice with viral vectors containing 3 groups of different RNA fragments respectively provided by another embodiment of the present application, and the 3 groups of RNA fragments respectively contain any 3 kinds of RNA sequences, specifically: miR-19a (target gene TNF- ⁇ )+B7-siRNA-1+integrin ⁇ 4shRNA-1, miR-124-3p (target gene TNF- ⁇ )+B7-siRNA-2+integrin ⁇ 4 shRNA-2, miR-19a (target gene TNF- ⁇ )+B7-siRNA-1+integrin ⁇ 4 shRNA-2, in the figure A is the expression result of small RNA (SEQ ID NO: 3) detected in liver, B is the expression result of small RNA (SEQ ID NO: 3) detected in plasma, C is the expression result of small RNA (sequence 3) detected in colon.
  • A is the expression result of small RNA (SEQ ID NO: 3) detected in liver
  • B is the expression result of
  • Figure 75 is a graph showing the results of specific curative effects after injecting mice with viral vectors containing 3 groups of different RNA fragments according to an example of the present application.
  • the 3 groups of RNA fragments respectively contain any 3 kinds of RNA sequences, specifically: miR -19a (target gene TNF- ⁇ )+B7-siRNA-1+integrin ⁇ 4 shRNA-1, miR-124-3p (target gene TNF- ⁇ )+B7-siRNA-2+integrin ⁇ 4 shRNA-2, miR-19a (target gene TNF- ⁇ )+B7-siRNA-1+integrin ⁇ 4 shRNA-2, A is the disease index score in the figure, and B is the detection result of inflammatory factors.
  • Figure 76 is a graph showing the results of in vivo enrichment after injection into mice of viral vectors containing RNA sequences of different lengths provided in an example of the present application.
  • the RNA sequences of different lengths are: TNF- ⁇ -siRNA-1 (siRNA length 18bp), TNF- ⁇ -siRNA-1 (siRNA length 18bp), - ⁇ -siRNA-2 (siRNA length 20bp), TNF- ⁇ -siRNA-3 (siRNA length 22bp), in the figure A is the result of intrahepatic detection of siRNA expression, B is the result of siRNA expression detected in plasma, and C is the detection of siRNA in colon siRNA expression results.
  • Figure 77 is a graph showing the specific curative effect of virus vectors containing RNA sequences of different lengths provided by another embodiment of the present application after injection into mice.
  • the RNA sequences of different lengths are: TNF- ⁇ -siRNA-1 (siRNA length 18bp), TNF- ⁇ -siRNA-1 (siRNA length 18bp), - ⁇ -siRNA-2 (siRNA length 20bp), TNF- ⁇ -siRNA-3 (siRNA length 22bp),
  • A is the disease index score in the figure
  • B is the detection result of inflammatory factors
  • C is the detection result of target gene mRNA.
  • Figure 78 is a graph showing the results of in vivo enrichment of viral vectors containing 3 homologous TNF- ⁇ -siRNA sequences provided in an example of the present application after injection into mice.
  • the homologous TNF- ⁇ -siRNA sequences are: TNF- ⁇ -siRNA-4, TNF- ⁇ -siRNA-5, TNF- ⁇ -siRNA-6, in the figure A is the expression result of TNF- ⁇ -siRNA detected in liver, B is the expression result of TNF- ⁇ -siRNA detected in plasma, C The expression results of TNF- ⁇ -siRNA were detected in colon, and D was the expression results of TNF- ⁇ -siRNA detected in plasma exosomes.
  • Figure 79 is a graph showing the results of in vivo enrichment of viral vectors containing 3 homologous B7-siRNA sequences provided in another embodiment of the present application after injection into mice.
  • the homologous B7-siRNA sequences are: B7-siRNA-1, B7-siRNA-2, B7-siRNA-3, in the figure A is the expression result of B7-siRNA detected in liver, B is the expression result of B7-siRNA detected in plasma, C is the expression result of B7-siRNA detected in colon, D is the expression result of plasma B7-siRNA expression results were detected in exosomes.
  • Figure 80 is a graph showing the results of in vivo enrichment of viral vectors containing 3 homologous integrin ⁇ 4 siRNA sequences respectively provided in another embodiment of the present application after injection into mice.
  • the homologous integrin ⁇ 4 siRNA sequences are: integrin ⁇ 4 siRNA-1, integrin ⁇ 4 siRNA-2, integrin ⁇ 4 siRNA-3, in the figure A is the result of detecting integrin ⁇ 4 siRNA expression in liver, B is the result of detecting integrin ⁇ 4 siRNA expression in plasma, C is the result of detecting integrin ⁇ 4 siRNA expression in colon, D is the result of plasma detection Detection of integrin ⁇ 4 siRNA expression results in exosomes.
  • Figure 81 is a graph showing the specific curative effect results of virus vectors containing 9 homologous siRNA sequences provided in an example of the present application after injection into mice.
  • the homologous siRNA sequences are: TNF- ⁇ -siRNA-4, TNF- ⁇ - siRNA-5, TNF- ⁇ -siRNA-6, B7-siRNA-1, B7-siRNA-2, B7-siRNA-3, integrin ⁇ 4 siRNA-1, integrin ⁇ 4 siRNA-2, integrin ⁇ 4 siRNA-3,
  • Figure A is the disease index score
  • B is the detection result of inflammatory factors.
  • Figure 82 is a graph showing the results of in vivo enrichment of mice injected with viral vectors containing RNA fragments with different flanking sequences, loop sequences and reverse complementary sequences provided in an embodiment of the present application.
  • Definite 5' flanking sequences with more than 80% homology, 2 unambiguous sequences with more than 80% homology to the identified loop sequence, and 2 clear sequences with more than 80% homology to the identified 3' flanking sequences A clear sequence, a reverse complementary sequence of a normal sequence, and a clear reverse complementary sequence of a clear sequence whose 5' flanking sequence homology is greater than 80%,
  • a in the figure is the intrahepatic detection of TNF- ⁇ -siRNA Expression results
  • B is the expression result of TNF- ⁇ -siRNA in plasma
  • C is the expression result of TNF- ⁇ -siRNA in colon
  • D is the expression result of TNF- ⁇ -siRNA in plasma exosome.
  • Figure 83 is a graph showing the specific therapeutic effects of mice injected with viral vectors containing RNA fragments with different flanking sequences, loop sequences and reverse complementary sequences provided in an embodiment of the present application.
  • the 5' flanking sequence homology is greater than 80% of the clear sequence, 2 clear sequences with the identified loop sequence homology greater than 80%, 2 clear sequences with the identified 3' flanking sequence homology greater than 80%
  • A is the disease index score
  • B is the detection of inflammatory factors
  • C is the detection result of target gene mRNA.
  • Figure 84 shows that when the adenovirus vector provided by an embodiment of the present application carries multiple lines, adjacent lines are connected by sequence 1-sequence 2-sequence 3, and wherein sequence 2 is 5 bases, 10 bases, In the case of 20 bases, 30 bases, 40 bases, 50 bases, and 80 bases, the enrichment results of mice in vivo, the figure A is the expression of TNF- ⁇ -siRNA detected in the liver Results, B is the result of detecting the expression of TNF- ⁇ -siRNA in plasma, and C is the result of detecting the expression of TNF- ⁇ -siRNA in colon.
  • Figure 85 shows that when the adenovirus vector provided by another embodiment of the present application carries multiple lines, adjacent lines are connected by sequence 1-sequence 2-sequence 3, wherein sequence 2 is 5 bases and 10 bases respectively , 20 bases, 30 bases, 40 bases, 50 bases, 80 bases, the in vivo enrichment results of mice, the figure A is the intrahepatic detection of B7-1-siRNA Expression results, B is the expression result of B7-1-siRNA detected in plasma, C is the expression result of B7-1-siRNA detected in colon.
  • Figure 86 shows that when the adenovirus vector provided by another embodiment of the present application carries multiple lines, adjacent lines are connected by sequence 1-sequence 2-sequence 3, wherein sequence 2 is 5 bases and 10 bases respectively , 20 bases, 30 bases, 40 bases, 50 bases, and 80 bases, the enrichment results of mice in vivo, in the figure A is the expression of integrin ⁇ 4-siRNA detected in the liver Results, B is the result of detecting the expression of integrin ⁇ 4-siRNA in plasma, and C is the result of detecting the expression of integrin ⁇ 4-siRNA in colon.
  • Figure 87 is a graph of the enrichment results in mice in vivo when the adenovirus vector provided in an example of the present application carries multiple lines, and the connecting sequence is sequence 4 and two sequences with more than 80% homology to sequence 4 , in the figure, A is the result of detecting TNF- ⁇ -siRNA expression in the liver, B is the result of detecting the expression of TNF- ⁇ -siRNA in the plasma, and C is the result of detecting the expression of TNF- ⁇ -siRNA in the colon.
  • Figure 88 shows the in vivo enrichment results of mice when the adenoviral vector provided in another embodiment of the present application carries multiple lines, and the connecting sequence is sequence 4 and two sequences with more than 80% homology to sequence 4
  • Figure A is the result of B7-1-siRNA expression detected in liver
  • B is the result of B7-1-siRNA expression detected in plasma
  • C is the result of B7-1-siRNA expression detected in colon.
  • Figure 89 shows the in vivo enrichment results of mice when the adenovirus vector provided in another embodiment of the present application carries multiple lines, and the connecting sequence is sequence 4 and two sequences with a homology of more than 80% to sequence 4
  • Figure A is the result of detecting the expression of integrin ⁇ 4-siRNA in the liver
  • B is the result of detecting the expression of integrin ⁇ 4-siRNA in the plasma
  • C is the result of detecting the expression of integrin ⁇ 4-siRNA in the colon.
  • Fig. 90 shows that when the adenoviral vector provided by an embodiment of the present application carries multiple lines, and the connecting sequence is sequence 4 and two sequences with more than 80% homology to sequence 4, the small The specific curative effect results of mice, A is the disease index score, B is the target gene mRNA detection result.
  • Figure 91 is a graph showing the enrichment results of TNF- ⁇ -siRNA loaded in mice when adenovirus-associated virus types 2, 7 and 8 are used as viral vectors provided in an example of the present application, and A in the figure is the liver
  • the expression results of TNF- ⁇ -siRNA were detected in B, the expression results of TNF- ⁇ -siRNA in plasma were detected in B, and the expression results of TNF- ⁇ -siRNA in colon were detected in C.
  • Figure 92 is a graph showing the specific therapeutic effect of adenovirus-associated virus type 2, type 7 and type 8 provided by an example of the present application after the TNF- ⁇ -siRNA loaded thereon is expressed in mice.
  • A is the disease index score
  • B is the detection result of inflammatory factors
  • C is the detection result of target gene mRNA.
  • Figure 93 is a comparison diagram of mouse hydroxyproline content provided by an embodiment of the application.
  • Figure 94 is a fluorescent staining diagram of the mouse lung provided by an embodiment of the present application.
  • Figure 95 is a Masson's trichrome staining diagram of the mouse lung provided by an embodiment of the present application.
  • Figure 96 is the HE staining diagram of mouse lung provided by an embodiment of the present application.
  • Figure 97 is a comparison diagram of mouse partial protein and mRNA levels provided by an embodiment of the present application.
  • Figure 98 is a data diagram of the treatment effect of pulmonary fibrosis of the plasmid delivery system containing RNA fragments provided by an embodiment of the present application, in the figure A is an RNA fragment containing 6 kinds of RNA sequences, any two of the 6 kinds of RNA sequences, and 6 kinds of RNA sequences.
  • A is an RNA fragment containing 6 kinds of RNA sequences, any two of the 6 kinds of RNA sequences, and 6 kinds of RNA sequences.
  • Figure 99 shows the metabolic distribution results of CMV-siRNA-1+2 after intravenous injection provided in an example of the present application.
  • A is the enrichment effect in the lungs
  • B is the enrichment effect in the blood.
  • Figure 100 shows the metabolic distribution results after intravenous injection of CMV-GE11-siRNA-1+2 and (CMV-GE11-siRNA-1+CMV-GE11-siRNA-2) with targeting tag GE11 provided in an example of the present application
  • a and C are the enrichment effects of CMV-GE11-siRNA-1+2 in the lung and plasma, respectively
  • B and D are the enrichment effects of CMV-GE11-siRNA-1+CMV-GE11-siRNA-2 in the lung, respectively The enrichment effect in parts and plasma.
  • Figure 101 shows CMV-GE11-siRNA-1, CMV-GE11-siRNA-1+2 and CMV-GE11-siRNA-1+CMV-GE11-siRNA-2 veins with targeting tag GE11 provided in an example of this application
  • Pulmonary fibrosis treatment effect data chart after injection A and C in the figure are the results of the protein content and mRNA content of TGFb1 of CMV-GE11-siRNA-1 and CMV-GE11-siRNA-1+2, respectively, B and D are respectively The results of protein content and mRNA content of TGFb1 in CMV-GE11-siRNA-1 and CMV-GE11-siRNA-1+CMV-GE11-siRNA-2.
  • Figure 102 is a graph of the enrichment effect (represented as siRNA content) in blood after injection of a plasmid containing sequence fragments containing 3 different 5' flanking sequences, loop sequences and 3' flanking sequences provided in an embodiment of the present application.
  • Figure 103 is a graph showing the enrichment effect (represented as siRNA content) in blood after injection of the plasmid delivery system containing multiple linker sequences (SEQ ID NO: 2) provided by an embodiment of the present application.
  • Figure 104 is a graph of the enrichment effect (indicated as siRNA content) in blood after injection of a plasmid delivery system containing multiple linker sequences (sequence 4) with a homology greater than 80% provided by an embodiment of the present application.
  • the abscissa sequence 4-1 is the basic sequence 4, and the sequences 4-2/4-3/4-4 are the homologous sequences with the homology of sequence 4-1 (sequence 4) greater than 80%, respectively.
  • Figure 105 is the therapeutic effect of pulmonary fibrosis when the RNA sequence lengths in the plasmid delivery system provided by an embodiment of the present application are 18, 19, and 21 respectively, in the figure A is the mRNA content result of TGFb1, and B is the protein content result of TGFb1 .
  • Figure 106 is the result of the detected hydroxyproline content when the gene circuit provided in an embodiment of the present application includes the antisense strand of miRNA-21 and five TGF- ⁇ 1 gene siRNAs.
  • Figure 107 is a comparison diagram of mouse hydroxyproline content and mRNA level provided by an embodiment of the present application.
  • Figure 108 is a detection diagram of in vivo enrichment, self-assembly and therapeutic effects of pulmonary fibrosis when RNA fragments are loaded using adenovirus and lentivirus as viral vectors provided in an embodiment of the present application, and the viral vectors are adenovirus/lentivirus , the enrichment results are displayed in terms of siRNA content.
  • A is the enrichment detection map in the lung after delivery system injection (siRNA-1)
  • B is the enrichment detection map in the lung after delivery system injection (siRNA-1).
  • C is the enrichment detection chart in blood after delivery system injection (siRNA-1)
  • D is the enrichment detection chart in blood after delivery system injection (siRNA-2).
  • Figure 109 is a detection diagram of in vivo enrichment, self-assembly and therapeutic effects of pulmonary fibrosis when RNA fragments are loaded with adenovirus and lentivirus as viral vectors provided by another embodiment of the present application, and the viral vectors are adenovirus/lentivirus Virus, the enrichment results are displayed in terms of siRNA content.
  • A is the enrichment detection chart in the lungs after injection of the delivery system without targeting peptide (GE11), and B is the delivery system with targeting peptide (GE11) after injection.
  • the enrichment detection map in the lungs C is the enrichment detection map in the blood (siRNA-1) after injection of the delivery system without targeting peptide (GE11), D is the delivery system with targeting peptide (GE11) After injection, the enrichment profile (siRNA-2) in blood.
  • Figure 110 shows the detection results of in vivo enrichment, self-assembly and therapeutic effect on pulmonary fibrosis when the viral vector system provided by an embodiment of the present application carries a variety of different RNA fragments
  • a in the figure is the mRNA of PTP1B
  • the relative amount detection results, B is the relative amount detection results of PTP1B protein.
  • Figure 111 shows that when the viral vector delivery system provided by an embodiment of the present application contains multiple RNA fragments and multiple targeting tags (CMV-siRNA-1+2), after intravenous injection, it has in vivo enrichment, self-assembly and lung The detection results of the fibrosis treatment effect.
  • A is the enrichment effect in the lungs (displayed by the siRNA content)
  • B is the enrichment effect in the blood (displayed by the siRNA content).
  • Fig. 112 shows when the viral vector delivery system provided by another embodiment of the present application contains multiple RNA fragments and multiple targeting tags (CMV-GE11-siRNA-1+2, CMV-GE11-siRNA-1+CMV- GE11-siRNA-2), after intravenous injection, it has the detection results of in vivo enrichment, self-assembly and treatment effect of pulmonary fibrosis. Enrichment effect in plasma (shown as siRNA content).
  • Fig. 113 shows when the viral vector delivery system provided by another embodiment of the present application contains multiple RNA fragments and multiple targeting tags (CMV-GE11-siRNA-1+2, CMV-GE11-siRNA-1+CMV- GE11-siRNA-2), the detection results of pulmonary fibrosis treatment effect after intravenous injection, A and B in the figure are the detection results of the protein content of TGFb1, and C and D are the detection results of the mRNA content of TGFb1.
  • CMV-GE11-siRNA-1+2, CMV-GE11-siRNA-1+CMV- GE11-siRNA-2 the detection results of pulmonary fibrosis treatment effect after intravenous injection
  • a and B in the figure are the detection results of the protein content of TGFb1
  • C and D are the detection results of the mRNA content of TGFb1.
  • Figure 114 is a graph showing the detection results of in vivo enrichment when three 5' flanking sequences/loop sequences/3' flanking sequences with homology greater than 80% are included in the adenoviral vector delivery system provided in an example of the present application (shown as siRNA content in blood).
  • Figure 115 shows the structure constructed when the adenovirus vector provided in an embodiment of the present application carries multiple lines and adjacent lines are connected by sequence 1-sequence 2-sequence 3, wherein sequence 2 contains multiple bases
  • the delivery system has a graph of in vivo enrichment assays (shown as siRNA levels in blood).
  • Figure 116 is a graph showing the detection results of the in vivo enrichment of the delivery system constructed by the linker sequence provided by an example of the present application, which is sequence 4 and a sequence with more than 80% homology to sequence 4 (based on the content of siRNA in blood). show).
  • Figure 117 is a diagram showing the detection results of the delivery system constructed when the lengths of the RNA sequences provided by an embodiment of the present application are respectively 18, 20, and 21, and has the therapeutic effect of pulmonary fibrosis, in the figure, A is the PTP1B mRNA of RNA sequences of different lengths The relative amount detection results, B is the relative amount detection results of PTP1B protein of different length RNA sequences.
  • Figure 118 is the result of the detected hydroxyproline content in the gene circuit provided by an embodiment of the present application when the antisense strand of miRNA-21 and five siRNAs of TGF- ⁇ 1 gene are included.
  • Figure 119 is a comparison diagram of mouse siRNA-related expression provided by an embodiment of the present application.
  • Figure 120 is a comparison diagram of the treatment of glioblastoma in mice provided in an embodiment of the present application.
  • Figure 121 is a comparison diagram of immunohistochemical staining of mouse brain provided in an embodiment of the present application.
  • Figure 122 is the effect verification that the plasmid delivery system provided by an embodiment of the present application has in vivo enrichment and spontaneous formation of a composite structure when carrying a single RNA fragment; wherein A is the in vivo enrichment of plasmids containing different RNA fragments The effect of aggregation, B is the in vivo self-assembly effect shown by the expression levels of different RNA fragments.
  • Figure 123 is the effect verification that the plasmid delivery system provided by an embodiment of the present application has in vivo enrichment and spontaneous formation of composite structures in the case of carrying any two RNA fragments; wherein A is the in vivo effect of plasmids containing different combinations of RNA fragments The effect of enrichment, B is the in vivo self-assembly effect shown by the expression levels of different combined RNA fragments.
  • Figure 124 is the effect verification that the plasmid delivery system provided by an embodiment of the present application has in vivo enrichment and spontaneous formation of composite structures in the case of carrying any three RNA fragments; wherein A is the plasmid containing different combinations of RNA fragments in vivo The effect of enrichment, B is the in vivo self-assembly effect shown by the expression levels of different combined RNA fragments.
  • Figure 125 is the effect verification that the plasmid delivery system provided by another embodiment of the present application has in vivo enrichment and spontaneous formation of composite structure in the case of carrying any two RNA fragments; wherein A is the plasmid containing different combinations of RNA fragments in The effect of enrichment in vivo, B is the effect of self-assembly in vivo shown by the expression levels of different combinations of RNA fragments.
  • Figure 126 is a verification of the effect of in vivo enrichment of the plasmid delivery system provided by an embodiment of the present application when it carries random 1-2 RNA fragments and 1-2 targeting tags and the two are located in the same route.
  • Figure 19 is a verification of the effect of in vivo enrichment of the plasmid delivery system provided by another embodiment of the present application when it carries random 1-2 RNA fragments and 1-2 targeting tags and the two are located in different routes.
  • Figure 20 shows that the plasmid delivery system provided by an embodiment of the present application has in vivo enrichment and spontaneous formation of a composite structure under the condition that it carries a definite 5' flanking sequence and at least 2 definite sequences whose homology is greater than 80% The effect verification of ; where A is the enrichment effect of plasmids containing different 5' flanking sequences in vivo, and B is the in vivo self-assembly effect shown by the expression levels of RNA fragments of different 5' flanking sequences.
  • Figure 21 shows that the plasmid delivery system provided in an embodiment of the present application has the effect of in vivo enrichment and spontaneous formation of composite structures when it carries a defined loop sequence and at least two defined sequences with a homology greater than 80%. Verification; where A is the enrichment effect of plasmids containing different loop sequences in vivo, and B is the in vivo self-assembly effect shown by the expression levels of RNA fragments of different loop sequences.
  • Figure 22 shows that the plasmid delivery system provided by an embodiment of the present application has in vivo enrichment and spontaneous formation of a composite structure when it carries a definite 3' flanking sequence and at least 2 definite sequences with a homology greater than 80%.
  • A is the enrichment effect of plasmids containing different 3' flanking sequences in vivo
  • B is the in vivo self-assembly effect shown by the expression levels of RNA fragments with different 3' flanking sequences.
  • Figure 23 is an RNA sequence of the plasmid delivery system provided by an embodiment of the present application carrying the reverse complementary sequence after deletion of any of the 1, 2, 3, 4, and 5 bases, with in vivo enrichment and spontaneous formation of a composite structure The effect is verified; where A is the enrichment effect of plasmids containing different compensation sequences in vivo, and B is the in vivo self-assembly effect shown by the expression levels of RNA fragments of different compensation sequences.
  • Figure 24 is a verification of the effect of spontaneously forming a composite structure when the plasmid delivery system provided in an embodiment of the present application carries four of the lines, and adjacent lines are connected by sequence 1-sequence 2-sequence 3.
  • Figure 25 shows that the plasmid delivery system provided by an embodiment of the present application carries four said lines, and adjacent lines are connected by sequence 1-sequence 2-sequence 3, and sequence 2 is 5 bases and 10 bases respectively The effect of spontaneous formation of complex structure was verified when the composition of base, 20 bases, 30 bases, 40 bases, 50 bases and 80 bases.
  • Figure 26 is a verification of the effect of spontaneously forming a composite structure when the plasmid delivery system provided in an embodiment of the present application contains sequence 4 and at least two sequences with more than 80% homology to sequence 4.
  • FIG. 27 is a verification of the effect of in vivo enrichment when the plasmid delivery system provided in an embodiment of the present application only contains a targeting peptide tag.
  • FIG. 28 is a verification of the effect of in vivo enrichment when the plasmid delivery system provided in an embodiment of the present application only contains a targeting protein tag.
  • Figure 29 is the verification of the effect of in vivo enrichment and spontaneous formation of composite structure when the siRNA containing EGFR gene in the gene circuit provided in an embodiment of the present application; wherein A is the enrichment of different gene circuits containing EGFR gene siRNA sequences in vivo The effect of B is the in vivo self-assembly effect shown by different expression levels of EGFR gene-containing siRNA sequences.
  • Figure 30 is the verification of the effect of in vivo enrichment and spontaneous formation of complex structures when the siRNA containing TNC gene in the gene circuit provided by an embodiment of the present application; wherein A is the enrichment of different gene circuits containing TNC gene siRNA sequences in vivo The effect of B is the in vivo self-assembly effect shown by different expression levels of siRNA sequences containing TNC gene.
  • Figure 31 shows the effect verification of in vivo enrichment and spontaneous formation of composite structures when the delivery system provided by an example of the application contains two different ribose-modified RNA sequences; wherein A is the delivery system of different ribose-modified RNAs in In vivo enrichment effect, B is the in vivo self-assembly effect shown by the expression levels of different ribose-modified RNAs.
  • Figure 39 is a comparison chart of mouse survival and tumor assessment provided by an embodiment of the present application.
  • Fig. 2 is a verification of the effect of in vivo enrichment and self-assembly of three other viral vectors provided in an embodiment of the present application.
  • A is the in vivo enrichment result of other viral vectors 1
  • B is the in vivo enrichment results of other viral vectors 2.
  • C is the in vivo enrichment result of other viral vectors 3
  • D is the in vivo self-assembly result of three other viral vectors.
  • A is the vector containing different RNA fragments in the In vivo enrichment effect
  • B is the in vivo self-assembly effect shown by different RNA fragment expression levels.
  • FIG. 4 is a verification of the effect of in vivo enrichment and self-assembly of the viral vector provided by an embodiment of the present application in the case of carrying 4 groups of RNA fragments containing any two RNA sequences respectively; wherein A is a vector containing different RNA fragments.
  • the effect of vector enrichment in vivo, B is the in vivo self-assembly effect shown by the expression levels of different RNA fragments.
  • A is a vector containing different RNA fragments.
  • B is the in vivo self-assembly effect shown by the expression levels of different RNA fragments.
  • FIG. 6 is the verification of the effect of in vivo enrichment and self-assembly of the viral vector provided by another embodiment of the present application in the case of carrying two groups of RNA fragments containing any two other RNA sequences respectively; wherein A is a sample containing different RNAs The effect of fragment vector enrichment in vivo, B is the in vivo self-assembly effect shown by different RNA fragment expression levels.
  • A is the in vivo enrichment effect of vectors containing different RNA fragments and targeting tags
  • B is the in vivo self-assembly effect shown by the expression levels of different RNA fragments.
  • Fig. 8 shows that the viral vector provided by another embodiment of the present application has the effect of in vivo enrichment and self-assembly when it carries random 1-2 RNA fragments and 1-2 targeting tags and the two are located in different lines Verification; where A is the enrichment effect of vectors containing different RNA fragments and targeting tags in vivo, and B is the in vivo self-assembly effect shown by the expression levels of different RNA fragments.
  • FIG. 9 is a verification of the effect of in vivo enrichment and self-assembly of the viral vector provided in an example of the present application when it carries a definite 5' flanking sequence and at least 2 definite sequences with a homology greater than 80%. ; where A is the enrichment effect of vectors containing different 5' flanking sequences in vivo, and B is the in vivo self-assembly effect shown by the expression levels of RNA fragments of different 5' flanking sequences.
  • 10 is a verification of the effect of in vivo enrichment and self-assembly of the viral vector provided in an embodiment of the present application when it carries a defined loop sequence and at least two defined sequences with a homology greater than 80%; wherein A is the enrichment effect of vectors containing different loop sequences in vivo, and B is the in vivo self-assembly effect shown by the expression levels of RNA fragments of different loop sequences.
  • Figure 11 is a verification of the effect of in vivo enrichment and self-assembly of the viral vector provided in an example of the present application when it carries a definite 3' flanking sequence and at least 2 definite sequences with a homology greater than 80%. ; where A is the enrichment effect of vectors containing different 3' flanking sequences in vivo, and B is the in vivo self-assembly effect shown by the expression levels of RNA fragments of different 3' flanking sequences.
  • Figure 12 is the RNA sequence of the viral vector provided in an embodiment of the present application carrying the reverse complementary sequence after deleting any of the 1, 2, 3, 4, and 5 bases, and has the effect of enrichment and self-assembly in vivo. ; where A is the enrichment effect of vectors containing different compensation sequences in vivo, and B is the in vivo self-assembly effect shown by the expression levels of RNA fragments of different compensation sequences.
  • 13 is a verification of the effect of self-assembly of the viral vector provided in an embodiment of the present application when it carries four lines and adjacent lines are connected by sequence 1-sequence 2-sequence 3.
  • Figure 14 shows that the viral vector provided by an embodiment of the present application carries four lines, and adjacent lines are connected by sequence 1-sequence 2-sequence 3, and sequence 2 is 5 bases, 10 bases, 20 bases, respectively. When composed of 1 base, 30 bases, 40 bases, 50 bases and 80 bases, the effect of self-assembly is verified.
  • 15 is a verification of the effect of self-assembly of the viral vector provided in an example of the present application when it contains sequence 4 and at least two sequences with a homology greater than 80% to sequence 4.
  • FIG. 16 is a verification of the in vivo enrichment effect of the viral vector provided in an example of the present application when it contains different targeting peptide tags.
  • FIG. 17 is a verification of the in vivo enrichment effect of the viral vector provided in an embodiment of the present application when it contains different targeting protein tags.
  • Figure 18 is the verification of the effect of enrichment and self-assembly in the gene circuit provided by an example of the present application when the siRNA containing the EGFR gene has in vivo enrichment; wherein A is the enrichment effect of different gene circuits containing the EGFR gene siRNA sequence in vivo , B is the in vivo self-assembly effect shown by different expression levels of EGFR gene-containing siRNA sequences.
  • Figure 19 is the verification of the effect of enrichment and self-assembly in vivo when the siRNA containing TNC gene in the gene circuit provided in an embodiment of the present application; wherein A is the enrichment effect of different gene circuits containing TNC gene siRNA sequences in vivo , B is the in vivo self-assembly effect shown by different expression levels of TNC gene-containing siRNA sequences.
  • Figure 20 is a verification of the effect of in vivo enrichment and self-assembly of the viral vector delivery system provided by an embodiment of the present application when it contains two different ribose modified RNA sequences; wherein A is the viral vector of different ribose modified RNAs The enrichment effect of the delivery system in vivo, B is the in vivo self-assembly effect shown by the expression levels of different ribose-modified RNAs.
  • Figure 40 is a fluorescence microscope image of the mouse hypothalamus and liver provided by an embodiment of the present application.
  • Figure 41 is a comparison diagram of the treatment of obesity in mice provided by an embodiment of the present application.
  • Figure 42 is a comparison diagram of the treatment of obesity fatty liver in mice provided by an embodiment of the present application.
  • Figure 43 is a comparison diagram of the treatment of obesity in mice provided by an embodiment of the present application.
  • Figure 44 is a comparison diagram of various obesity indicators in mice provided by an embodiment of the present application.
  • Fig. 3 is an RNA delivery system constructed with adenovirus and lentivirus as viral vectors provided in an embodiment of the present application, and has a detection diagram of in vivo enrichment effect.
  • A is the detection of siRNA content in blood after injection of the delivery system
  • B is the detection result of the siRNA content in the hypothalamus after injection of the delivery system.
  • FIG. 4 is a detection diagram of an RNA delivery system constructed with adenovirus and lentivirus as viral vectors provided by another embodiment of the present application, and has an enrichment effect in vivo. The figure shows that after injection of the delivery system, blood exosomes Detection results of siRNA content.
  • Fig. 5 is an RNA delivery system constructed with adenovirus and lentivirus as viral vectors provided by an embodiment of the present application, and has a detection diagram of in vivo self-assembly effect and obesity treatment effect, and A in the figure is the detection result of the mRNA content of PTP1B , B is the detection result of protein content of PTP1B, C is the change value of body weight with the increase of days.
  • Fig. 6 is the detection result of in vivo enrichment, self-assembly and treatment effect for obesity when the viral vector system provided by an embodiment of the present application carries a variety of different RNA fragments
  • a in the figure is the relative mRNA of PTP1B Quantitative detection results
  • B is the detection results of the relative amount of PTP1B protein.
  • FIG. 7 is a graph showing the detection results of in vivo enrichment, self-assembly and obesity treatment effects when the adenoviral vector delivery system provided by an embodiment of the present application contains multiple RNA fragments and multiple targeting tags, wherein targeting The label is RVG, and the RNA fragments are siRNA-1, siRNA-2, siRNA-1+siRNA-2.
  • A is the detection result of the relative amount of mRNA of PTP1B
  • B is the detection result of the relative amount of protein of PTP1B
  • C is the number of days Change in weight gain.
  • FIG. 8 is a graph showing the detection results of in vivo enrichment when three 5' flanking sequences/loop sequences/3' flanking sequences with homology greater than 80% are included in the adenovirus vector delivery system provided in an example of the present application (shown as siRNA content in blood).
  • Fig. 9 is a structure constructed when the adenovirus vector provided in an embodiment of the present application carries multiple lines, and adjacent lines are connected by sequence 1-sequence 2-sequence 3, wherein sequence 2 contains multiple bases.
  • the delivery system has a graph of in vivo enrichment assays (shown as siRNA levels in blood).
  • Fig. 10 is a graph showing the detection results of in vivo enrichment of the delivery system constructed by the linker sequence provided by an embodiment of the present application as sequence 4 and a sequence with more than 80% homology to sequence 4 (based on the siRNA content in blood show).
  • Figure 11 is a graph of the detection results of the delivery system constructed when the lengths of RNA sequences provided in an embodiment of the present application are respectively 18, 20, and 21, and has the effect of treating obesity.
  • A is the relative PTP1B mRNA of RNA sequences of different lengths.
  • Quantitative detection results B is the relative detection results of PTP1B protein of different length RNA sequences.
  • Figure 45 is a comparison diagram of the treatment of Huntington's disease in mice provided by an embodiment of the present application.
  • Figure 46 is a comparison diagram of siRNA and protein in mouse liver, cortex, and striatum provided by an example of the present application; RNA that inhibits gene expression
  • Figure 47 is a comparison diagram of the treatment of mouse Huntington's disease provided by an embodiment of the present application.
  • Figure 48 is a comparison diagram of mHTT protein and toxic aggregates in mouse striatum and cortex provided by an embodiment of the present application;
  • Figure 49 is a comparison diagram of the treatment of mouse Huntington's disease provided by an embodiment of the present application.
  • FIG. 3 is an effect diagram of in vivo enrichment of the siRNA-loaded lentiviral vector provided in an embodiment of the present application.
  • FIG. 4 is an effect diagram of in vivo self-assembly of the siRNA-loaded lentiviral vector provided in an embodiment of the present application.
  • Fig. 5 is a graph showing the electrophoresis results of the therapeutic effect of the siRNA-loaded lentiviral vector provided in an embodiment of the present application on Huntington's disease.
  • FIG. 6 is a graph showing the effect of in vivo enrichment of adenovirus-associated virus type 1, type 4, and type 7 vectors loaded with siRNA provided in an example of the present application.
  • FIG. 7 is a graph showing the electrophoresis results of the therapeutic effect of the siRNA-loaded adeno-associated virus type 1, type 2, and type 7 vectors provided in an embodiment of the present application for Huntington's disease.
  • FIG. 8 is a data diagram showing the therapeutic effect of siRNA-loaded adeno-associated virus type 1, type 2, and type 7 vectors for Huntington's disease provided by an embodiment of the present application, and the data in the diagram is based on HTT mRNA levels for comparison.
  • Figure 9 is an effect diagram of in vivo enrichment of the adenovirus-associated virus type 8 vector loaded with 6 individual RNA sequences provided in an embodiment of the present application.
  • FIG. 10 is a graph showing the effect of in vivo enrichment of the adenovirus-associated virus type 9 vector loaded with 6 individual RNA sequences provided in an example of the present application.
  • FIG. 11 is a graph showing the electrophoresis results of the therapeutic effect of the adenovirus-associated virus type 9 vector loaded with 6 individual RNA sequences provided in an example of the present application on Huntington's disease.
  • Figure 12 is a data diagram showing that the adenovirus-associated virus type 8 vector loaded with RNA fragments of any two RNA sequences provided in an embodiment of the present application has the therapeutic effect of Huntington's disease, and the data diagram is based on HTT mRNA levels for comparison.
  • FIG. 13 is a graph showing the electrophoresis results of the therapeutic effect of Huntington's disease on the adenovirus-associated virus type 9 vector loaded with RNA fragments of any two RNA sequences provided in an example of the present application.
  • Figure 14 is a data diagram showing that the adenovirus-associated virus type 8 vector loaded with RNA fragments of any three RNA sequences provided in an example of the present application has the therapeutic effect of Huntington's disease, and the data diagram is based on HTT mRNA levels for comparison.
  • Figure 15 is a graph showing the electrophoresis results of the therapeutic effect of Huntington's disease on the adenovirus-associated virus type 9 vector loaded with RNA fragments of any three RNA sequences provided in an example of the present application.
  • FIG. 16 is a graph of the in vivo enrichment data of the adeno-associated virus type 9 sequence provided in an example of the present application when siRNA and RVG are included.
  • Fig. 17 is a graph showing the effect of HTT mRNA expression data on the treatment of Huntington's disease when the sequence in the adeno-associated virus type 9 provided in an example of the present application includes siRNA and RVG.
  • Figure 18 is a data comparison diagram of the therapeutic effect of RVG-LAMP2B fusion protein and other fusion proteins on adenovirus vectors for Huntington's disease provided by an example of the present application, and the data is reflected by the relative level of HTT mRNA.
  • FIG. 19 is a graph of in vivo enrichment data of the adenovirus vector system provided in an example of the present application when three RNA sequences with more than 80% homology to the siRNA sequence of the HTT gene are included.
  • Figure 20 is a data comparison diagram of the therapeutic effect on Huntington's disease when the adenovirus vector system provided by an embodiment of the present application includes 3 RNA sequences with a homology of more than 80% to the siRNA sequence of the HTT gene. relative levels of mRNA.
  • Figure 50 is a comparison diagram of the Parkinson's treatment situation of transgenic mice provided in an embodiment of the present application.
  • FIG. 12 is a graph of in vivo enrichment data when plasmid 1 provided in an example of the present application contains 6 kinds of RNA alone.
  • FIG. 13 is a graph of in vivo enrichment data when plasmid 2 provided by another embodiment of the present application contains 6 kinds of RNAs alone.
  • FIG. 14 is a graph of the in vivo self-assembly data of the exosomes provided in an example of the present application when each of the exosomes individually contains 6 kinds of RNAs.
  • Fig. 15 is a graph showing the therapeutic effect on Parkinson's disease reflected by the expression results of LRRK2 gene when the plasmids provided in an example of the present application contain 6 kinds of RNA alone.
  • FIG. 16 is a graph of in vivo enrichment data when plasmid 1 provided in an example of the present application contains any two RNA sequences respectively.
  • Figure 17 is a graph of the in vivo self-assembly data of exosomes provided in an embodiment of the present application when they contain any two RNA sequences.
  • Figure 18 is a graph showing the effect of treating Parkinson's disease through the expression data of LRRK2 mRNA when plasmid 1 provided in an embodiment of the present application contains any two RNA sequences respectively.
  • Fig. 19 is a graph showing the therapeutic effect on Parkinson's disease reflected by the expression result of LRRK2 gene when plasmid 2 provided by another embodiment of the present application contains any two RNA sequences respectively.
  • Figure 20 is a graph showing the effect of treating Parkinson's disease through the expression data of LRRK2 mRNA when the plasmid 1 provided in an embodiment of the present application contains any three RNA sequences respectively.
  • Fig. 21 is a diagram showing the therapeutic effect on Parkinson's disease reflected by the expression result of LRRK2 gene when plasmid 2 provided in another embodiment of the present application contains any three RNA sequences respectively.
  • Figure 22 is a graph of the in vivo enrichment data of the plasmid provided in an example of the present application when the sequences in the plasmid contain siRNA and RVG.
  • Figure 23 is a diagram showing the effect of treating Parkinson's disease through the expression data of LRRK2 mRNA when the sequences in the plasmid provided in an example of the present application contain siRNA and RVG.
  • Figure 24 is a data comparison diagram of the therapeutic effect of RVG-LAMP2B fusion protein and other fusion proteins on a plasmid vector for Parkinson's disease provided by an embodiment of the present application, and the data is reflected by the relative level of LRRK2 mRNA.
  • FIG. 25 is a graph of in vivo enrichment data of the gene circuit provided in an example of the present application when three RNA sequences with more than 80% homology to the siRNA sequence of the LRRK2 gene are included.
  • Figure 26 is a data comparison diagram of the therapeutic effect on Parkinson's disease when the gene circuit provided by an embodiment of the present application includes 3 RNA sequences with a homology of more than 80% to the siRNA sequence of the LRRK2 gene. relative level.
  • FIG. 4 is an effect diagram of in vivo enrichment of the siRNA-loaded lentiviral vector provided in an embodiment of the present application.
  • FIG. 5 is an effect diagram of in vivo self-assembly of the siRNA-loaded lentiviral vector provided in an embodiment of the present application.
  • FIG. 6 is a graph of the data results of the Parkinson’s treatment effect of the lentiviral vector loaded with siRNA provided in an embodiment of the present application, and the data graph is based on the LRRK2 mRNA level for comparison.
  • FIG. 7 is a graph showing the effect of in vivo enrichment of adeno-associated virus type 1, type 4, and type 7 vectors loaded with siRNA provided in an example of the present application.
  • FIG. 8 is a diagram showing the effect of in vivo self-assembly of the siRNA-loaded adeno-associated virus type 1, type 4, and type 7 vectors provided in an embodiment of the present application.
  • Figure 9 is a graph of the data results of the Parkinson's treatment effect of adenovirus-associated virus type 1, type 4, and type 7 vectors loaded with siRNA provided in an example of the present application, and the LRRK2 mRNA level is compared in the data figure.
  • Figure 10 is an effect diagram of in vivo enrichment of adenovirus-associated virus type 8 vectors loaded with 6 individual RNA sequences provided by an embodiment of the present application.
  • FIG. 11 is an effect diagram of in vivo self-assembly of adeno-associated virus type 8 and type 9 vectors loaded with 6 individual RNA sequences provided in an example of the present application.
  • FIG. 12 is a diagram showing the effect of in vivo enrichment of an adeno-associated virus type 9 vector loaded with RNA fragments of any two RNA sequences provided in an example of the present application.
  • FIG. 13 is a diagram showing the effect of in vivo self-assembly of adeno-associated virus type 8 and type 9 vectors loaded with RNA fragments of any two RNA sequences provided in an example of the present application.
  • FIG. 14 is an electrophoresis result of the Parkinson's treatment effect of an adenovirus vector loaded with RNA fragments of any three RNA sequences provided in an example of the present application.
  • FIG. 15 is a graph showing the in vivo enrichment data of the adeno-associated virus type 9 sequences provided in an example of the present application when siRNA and RVG are included.
  • Figure 16 is a graph showing the effect of Parkinson's treatment according to the expression data of LRRK2 mRNA when the sequence in the adeno-associated virus type 9 provided in an example of the present application contains siRNA and RVG.
  • FIG. 17 is a data diagram of in vivo enrichment of adenovirus vectors loaded with RVG-LAMP2B fusion proteins and other fusion proteins provided in an example of the present application.
  • Figure 18 is a data comparison diagram of the therapeutic effect of RVG-LAMP2B fusion protein and other fusion proteins on adenovirus vectors for Parkinson's disease provided in an example of the present application, and the data is reflected by the relative level of LRRK2 mRNA.
  • Figure 19 is a graph of in vivo enrichment data of the adenoviral vector system provided in an example of the present application when three RNA sequences with more than 80% homology to the siRNA sequence of the LRRK2 gene are included.
  • 20 is a data comparison diagram of the therapeutic effect on Parkinson’s disease when the adenovirus vector system provided by an embodiment of the present application includes 3 RNA sequences with a homology of more than 80% to the siRNA sequence of the LRRK2 gene, and the data is obtained by LRRK2 relative levels of mRNA.
  • Figure 51 is a graph of changes in siRNA concentration in cynomolgus monkey whole blood provided by an embodiment of the present application.
  • Figure 52 is a comparison diagram of the effect of chemical modification on the level of mouse siRNA provided in an example of the present application.
  • FIG. 53 is a schematic diagram of a system for delivering RNA that inhibits gene expression provided by the present invention, and a verification experiment result diagram of one of its embodiments.
  • Figure 54 is a schematic structural diagram of an exemplary plasmid CMV- siRE provided by the present invention.
  • Figure 55 is a schematic structural diagram of an exemplary plasmid U6- siRE provided by the present invention.
  • Hematoxylin-eosin staining referred to as HE staining.
  • the hematoxylin staining solution is alkaline and can stain the basophilic structure of the tissue (such as ribosome, nucleus and ribonucleic acid in the cytoplasm) into blue-violet;
  • eosin is an acid dye, which can stain the eosinophilic structure of the tissue ( Such as intracellular and intercellular proteins, including Lewy bodies, alcohol bodies, and most of the cytoplasm) stained pink, making the morphology of the entire cell organization clearly visible.
  • HE staining include: sample tissue fixation and sectioning; tissue sample dewaxing; tissue sample hydration; tissue section hematoxylin staining, differentiation and anti-blue; tissue section eosin staining and dehydration; tissue sample section air-drying and sealing; Observe and photograph under the microscope.
  • Masson staining renders collagen fibers blue (stained by aniline blue) or green (stained by bright green) and muscle fibers red (stained by acid fuchsin and Ponceau).
  • the fixed tissue is stained sequentially or mixed with a series of anionic water-soluble dyes. It can be found that red blood cells are stained with the smallest molecular anionic dyes, muscle fibers and cytoplasm are stained with medium-sized anionic dyes, and collagen fibers are stained with macromolecular anionic dyes. Dyeing with anionic dyes. This shows that red blood cells have the least permeability to anionic dyes, followed by muscle fibers and cytoplasm, and collagen fibers have the largest permeability. Type I and III collagens are green (GBM, TBM, mesangial matrix and renal interstitium are green), and erythropoietin, tubular cytoplasm, and erythrocytes are red.
  • Masson staining include: fix the tissue in Bouin's solution, rinse with running water overnight, and routinely dehydrate and embed; deparaffinize the sections to water (deparaffinize in xylene for 10 min ⁇ 3 times, blot the liquid with absorbent paper; 100% ethanol 5min ⁇ 2 times, dry the liquid with absorbent paper; 95% ethanol 5min ⁇ 2 times, dry the liquid with absorbent paper; running water for 2min, dry the water with absorbent paper); Weiger’s iron hematoxylin staining for 5-10min; ; 0.5% hydrochloric acid alcohol differentiation for 15s; running water for 3 minutes; Ponceau red acid fuchsin solution for 8 minutes; slightly rinsed with distilled water; 5 min; 1% glacial acetic acid treatment for 1 min; 95% ethanol dehydration for 5 min ⁇ 2 times, blotting the liquid with absorbent paper; 100% ethanol 5 min ⁇ 2 times, blotting the liquid with absorbent paper; transparent in xylene for 5 min ⁇
  • Western blotting is to transfer the protein to the membrane, and then use the antibody to detect.
  • 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 antibody of the fusion part.
  • Western blotting 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 the application of antigen-antibody reaction, through the chemical reaction to make the colorant (fluorescein, enzyme, metal ion, isotope) of the labeled antibody develop color to determine the antigen (polypeptide and protein) in tissue cells, to locate and qualitatively And relative quantitative research, called immunohistochemistry (immunohistochemistry) or immunocytochemistry (immunocytochemistry).
  • immunohistochemistry immunohistochemistry
  • immunocytochemistry immunocytochemistry
  • 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 inventors designed a method to deliver RNA that inhibits gene expression to organs and tissues as needed, enrich and assemble in cells to form cellular microvesicles including exosomes, and then deliver them to target tissues to treat diseases after being released by cells.
  • a system comprising one or more RNAs that inhibit gene expression, and proteins that target tissue of interest.
  • Figure 53a is a schematic diagram of one of the exemplary systems provided by the present invention for delivering RNA that inhibits gene expression.
  • the system is a vector (plasmid in this embodiment) comprising nucleic acid expressing RNA that inhibits gene expression and/or protein that targets tissue of interest, which allows the free combination of different functional modules.
  • the core part consists of a promoter part and an siRNA expression part (such as siRNA-1 backbone in Figure 53a), which is designed to generate and organize siRNA as the payload of exosomes.
  • siRNA expression part such as siRNA-1 backbone in Figure 53a
  • Other composable parts can be integrated into the system framework containing the core gene circuit for plug-and-play functionality.
  • combinable components include two types of siRNA that can be optimized for action: one that modifies membrane-anchored proteins of exosomes to achieve tissue selectivity (eg, Guiding Tag in Figure 53a); another that can co-express a second siRNA (such as siRNA-2 backbone in Figure 53a) to inhibit two molecular targets simultaneously.
  • tissue selectivity eg, Guiding Tag in Figure 53a
  • second siRNA such as siRNA-2 backbone in Figure 53a
  • the inventors optimized the portion of the siRNA expression backbone encoded under the control of the promoter portion to maximize guide strand expression while minimizing undesired passenger strands )Express.
  • epidermal growth factor receptor is used as the siRNA target of the core circuit.
  • EGFR is an oncogene that is frequently mutated and highly expressed in a variety of human tumors, such as lung cancer and glioblastoma.
  • human embryonic kidney 293t cells HEK293T
  • mouse hepatoma cells hep1-6 were selected as cell chassis for siRNA assembly in vitro.
  • the other is to use the U6 promoter to drive the expression of short hairpin RNA (shRNA), thus constructing a plasmid EGFR shRNA (U6-siR E ) (see Figure 55 for the plasmid structure, synthesized and provided by Realgene Biotech Company in Nanjing, China).
  • shRNA short hairpin RNA
  • CMV- siRE CMV-directed pre-miRNAs encoding EGFR siRNA
  • U6-directed EGFR shRNA U6-directed EGFR shRNA
  • CMV-scr R has the same type and number of nucleotides encoding siRNA sequence as in CMV-siR E but different nucleotide arrangement to serve as a blank control encoding CMV-siR E.
  • HEK293T cells were transfected with CMV-scr R 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.
  • enrichment for specific exon markers CD63, TSG101 and CD9 was only detected in purified exosomes, but not in cell culture medium.
  • an RVG-Lamp2b fusion protein (an RVG targeting peptide linked to the N-terminus of the Lamp2b protein) is used as an anchor protein to introduce exosomes into the brain.
  • Rabies virus glycoprotein (RVG) is a neurotropic protein that can bind to acetylcholine receptors expressed by nerve cells. RVG has been shown to help exosomes cross the blood-brain barrier into nerve cells.
  • CMV-RVG- siRE the sequence encoding the RVG-Lamp2b fusion protein was inserted downstream of the CMV promoter and upstream of the siRNA.
  • the amino acid sequence of RVG is shown in SEQ ID NO.:17.
  • the amino acid sequence of the entire RVG-Lamp2b fusion protein is shown in SEQ ID NO.:18.
  • the efficiency of the promoter in promoting the expression of the RVG-Lamp2b fusion protein was assessed.
  • the CMV promoter produces RVG-Lamp2b mRNA and marker protein eGFP in HEK293T cells. Immunoprecipitation was then used to verify the correct expression of the guide targeting tag on the exosome surface.
  • the Flag-tag was used in the experiments to temporarily replace RVG, and after transfection of HEK293T and Hepa1-6 cells with CMV-directed Flag-Lamp2b, intact exosomes were successfully immunoprecipitated with anti-Flag beads, see Figure 53f, demonstrating targeting Precise positioning of labels.
  • tenascin-C (TNC), a key oncogene associated with many cancers, especially glioblastoma, is used as the second siRNA target.
  • TNC-siRNA includes the CMV promoter and is expressly linked to an siRNA sequence that inhibits TNC, and is also embedded in the pre-miR-155 backbone to obtain the plasmid CMV- siRT .
  • the siRNA of TNC gene has the following nucleotide sequence: UAUGAAAUGUAAAAAAAGGGA (SEQ ID NO.5)
  • an EGFR-inhibiting siRNA and a TNC-inhibiting siRNA were constructed in series on the same plasmid, and TNC-siRNA was inserted downstream of EGFR-siRNA, with cagatctggccgcactcgaggtagtgagtcgaccagtggatc (SEQ ID NO.: 6) as the sequence encoding EGFR-siRNA and TNC-siRNA. the linker to obtain the plasmid CMV siR E+T . It was found in the experiment that EGFR and TNC siRNAs were detected almost indistinguishably regardless of single (CMV siR E or CMV siR T ) or tandem (CMV siR E+T ) transcription, see Figure 53g.
  • the code CMV-siR gene is used to shorten or initialize plasmids that have the construction structure of CMV-siR E as described above but differ only in the RNA sequence of the encoded suppressor gene.
  • plasmid CMV- siRT or "CMV- siRT” refers to a plasmid expressingly linked a TNC-inhibiting siRNA sequence behind the CMV promoter, as previously described, in which ggatcctggaggcttgctgaaggctgtatgctgaattc (SEQ ID NO.: 2) be the 5' flanking sequence, take gttttggccactgactgac (SEQ ID NO.: 4) as the stem-loop sequence, take accggtcaggacacaaggcctgttactagcactcacatggaacaaaatggcccagatctggccgcactcgag (SEQ ID NO.
  • the first letter of the first gene of the code name CMV-siR + the first letter of the second gene represents a plasmid that simultaneously carries the siRNA sequence that suppresses the first gene and the siRNA that suppresses the siRNA of the second gene, which has the construction of CMV-siR E+T A plasmid with a different structure but only the RNA sequence encoding the suppressor gene, and wherein the siRNA sequence suppressing the second gene is inserted downstream of the siRNA sequence suppressing the first gene, with cagatctggccgcactcgaggtagtgagtcgaccagtggatc (SEQ ID NO.: 6) as the encoding TNC-siRNA and Linker between sequences of EGFR-siRNA.
  • the code CMV-target peptide abbreviation or acronym-siR gene abbreviation or acronym indicates that there is a target peptide, such as RVG, downstream of the CMV promoter and upstream of the siRNA.
  • FIG. 1A in order to understand the distribution of the plasmid in the body, a plate test was performed on mice. 168h, 720h) sampling, use the plasmid extracted by spectinomycin to transform, observe the number of clones in liver, plasma, lung, brain, kidney, spleen, the results are shown in Figure 1B, Figure 1C, Figure 1D, it can be seen that , the plasmid was most distributed in the mouse liver, and reached the peak at about 3h after injection, and was basically metabolized at 12h after injection.
  • C57BL/6J mice were intravenously injected with CMV eGFP siRNA co-expressing eGFP protein and EGFR siRNA.
  • the results are shown in Figure 2.
  • the eGFP fluorescence in the mouse liver gradually increased over time, reaching a peak at about 12 hours. 48 After hours, it dropped to the background level, and no obvious eGFP signal was seen in other tissues.
  • CMV-scrR The control plasmid
  • CMV-siR E the plasmid expressing EGFR siRNA
  • Figure 3A The related siRNA levels in exosomes, the results are shown in Figure 3A, it can be seen that there is siRNA expression in the exosomes of mouse hepatocytes injected with CMV-siRNA.
  • Input represents the sample in which the exosomes were directly lysed and detected without immunoprecipitation, representing the positive control.
  • FIG. 4A After intravenous injection of plasmids into mice, the distribution of mature siRNA in different tissues is shown in Figure 4. It can be seen from Figure 4A that the levels of EGFR-siRNA in plasma, exosomes, and exosome-free plasma show time-dependent changes; from Figure 4B, it can be seen that mouse EGFR-siRNAs in the liver, lung, pancreas, and spleen , The accumulation in the kidney is time-dependent.
  • mice were injected with control plasmid (CMV-scrR), 0.05mg/kg CMV-siR E plasmid, 0.5mg/kg CMV-siR E plasmid, 5mg/kg CMV-siR E plasmid, and detected the liver, Absolute siRNA (EGFR siRNA) levels in spleen, heart, lung, kidney, pancreas, brain, skeletal muscle, CD4+ cells, the results are shown in Figure 5A, it can be seen that there is no siRNA expression in the tissue of mice injected with the control plasmid, In each tissue of mice injected with CMV-siR E plasmid, the level of siRNA expression was positively correlated with the concentration of CMV-siR E plasmid.
  • CMV-scrR control plasmid
  • EGFR siRNA Absolute siRNA
  • fluorescence in situ hybridization assay FISH also confirmed that the level of siRNA expression was positively correlated with the concentration of CMV-siR E plasmid, that is, the tissue distribution of EGFR siRNA was dose-dependent.
  • the plasmid After the plasmid enters the body, it will express the precursor (Precursor), and then process it into the mature body (siRNA). The metabolism of the precursor (Precursor) and the mature body (siRNA) in the liver after the plasmid was injected into mice was detected. The results As shown in Figure 6. It can be seen that the expression levels of precursor (Precursor) and mature body (siRNA) in the mouse liver reached a peak at the time point of 6 hours after the injection of the plasmid. Metabolism of the precursor (siRNA) was complete, and the metabolism of the precursor (Precursor) in the mouse liver was complete 48 hours after the injection of the plasmid.
  • siRNA with albumin ALB as the promoter siRNA with CMV as the promoter
  • siRNA without any promoter were injected into mice intravenously.
  • the absolute siRNA levels in the mice were detected at 48 h, and the results are shown in Figure 8. It can be seen that the level of siRNA with CMV as the promoter in mice is the highest, that is, the effect of CMV as the promoter is the best.
  • mice were intravenously injected with PBS or 5 mg/kg CMV-siR G or CMV-RVG-siR G plasmid, and after 24 hours of treatment Mice were sacrificed and their eGFP fluorescence levels were detected in cryosections.
  • Figure 9A shows a representative fluorescence microscope image, in which green indicates positive eGFP signal, blue indicates DAPI-stained nuclei, scale bar: 100 ⁇ m, CMV-
  • the inhibitory effect of RVG-siRG plasmid on mouse eGFP was more obvious; eGFP transgenic mice were intravenously injected with PBS or CMV-scrR or CMV-siR E plasmid, and the mice were sacrificed after 24 hours of treatment, and their eGFP fluorescence was detected in cryosections level
  • Figure 9B is a column comparison of the fluorescence intensity of the heart, lung, kidney, pancreas, brain, and skeletal muscle of mice injected with PBS, CMV- siRE , and CMV-RVG- siRE . It can be seen that in the liver , spleen, lung and kidney parts of mice fluorescence intensity contrast is more obvious.
  • mice injected with PBS, CMV-scrR, and CMV-siR E their alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), blood urea nitrogen (BUN), serum alkaline phosphatase (ALP), creatinine (CREA) content, thymus weight, spleen weight, and peripheral blood cell percentage were detected.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • TBIL total bilirubin
  • BUN blood urea nitrogen
  • ALP serum alkaline phosphatase
  • CREA creatinine
  • FIG. 10G is a comparison chart of mouse liver, lung, spleen, and kidney tissue
  • Figure 10H-I is a mouse thymus
  • the RNA delivery system uses a plasmid as a carrier and the plasmid as a mature injectable substance, and its safety and reliability have been fully verified, and its druggability is very good.
  • the final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of plasmids is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • RNA delivery system provided in this example can be tightly combined with AGO2 and enriched into a composite structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation and maintain its stability in circulation, but also facilitates Receptor cell uptake, intracytoplasmic release and lysosomal escape require low doses.
  • mice were selected, and mice were injected with mouse lung cancer cells (LLC cells), and then the mice were injected with PBS buffer/CMV-scrR/gefitinib/CMV-siR E every two days.
  • LLC cells mouse lung cancer cells
  • mice were subjected to survival analysis and tumor assessment, respectively, starting on day 30 and ending on day 44.
  • CMV-scrR is a control plasmid
  • CMV-siR E is a plasmid carrying the EGFR siRNA gene.
  • the horizontal axis represents time
  • the vertical axis represents survival rate
  • FIG. 11C the figure shows the 3D modeling of mouse lung tissue based on CT images before and after treatment in mice injected with PBS buffer/CMV-scrR/gefitinib/CMV-siR E .
  • the tumors of mice injected with CMV- siRE were significantly reduced.
  • FIG. 11D is a comparison chart of the tumor volume (mm 3 ) of mice injected with PBS buffer/CMV-scrR/gefitinib/CMV- siRE before and after treatment. It can be seen that, The tumor volume of mice injected with CMV- siRE was significantly reduced. The tumor volume of mice injected with PBS buffer/CMV-scrR/gefitinib not only did not decrease, but also increased to varying degrees.
  • this figure is a comparison chart of western blot of normal mice and mice injected with PBS buffer/CMV-scrR/gefitinib/CMV-siR E. It can be seen that PBS buffer/CMV-scrR/ Gefitinib-treated mice had significantly higher levels of the EGFR gene.
  • FIG. 11F the figure shows the comparison of EGFR miRNA levels in normal mice and mice injected with PBS buffer/CMV-scrR/gefitinib/CMV-siR E. It can be seen that PBS buffer/CMV-scrR was injected /Gefitinib-treated mice had relatively higher levels of EGFR-related miRNAs.
  • CMV-siR E has a significant therapeutic effect on EGFR-mutated lung cancer.
  • mice HE staining and immunohistochemical staining were performed on mice injected with PBS buffer/CMV-scrR/gefitinib/CMV-siR E , respectively.
  • the results are shown in Figure 12A- Figure 12B. Gefitinib was more expressed in mice. The staining areas of EGFR and PCNA in the mice were counted.
  • the results are shown in Figure 12C- Figure 12D. It can be seen that the mice injected with CMV-siRE have the least staining areas of EGFR and PCNA, which proves that it has the most therapeutic effect on EGFR-mutated lung cancer tumors. it is good.
  • CMV-siR K represents a plasmid carrying a KRAS-inhibiting siRNA sequence (the siRNA of the KRAS gene has the following nucleotide sequence: UGAUUUAGUAUUAUUUAUGGC (SEQ ID NO.: 7).
  • the horizontal axis represents the time after infection, and the vertical axis represents the survival rate, and it can be seen from this figure that the mice injected with CMV-siR K have a higher survival rate.
  • the figure shows the 3D modeling of mouse lung tissue before and after treatment in mice injected with CMV-scrR/CMV-siR K. It can be seen that the injection of CMV-siRK can Significantly inhibits the growth of lung cancer tumors.
  • the figure is a comparison of the number of tumors (mm 3 ) in mice injected with CMV-scrR/CMV-siR K before and after treatment. It can be seen that the tumors in mice injected with CMV-siR K Volume growth is slow. The tumor volume of mice injected with CMV-scrR increased significantly.
  • FIG. 13G which is a comparison of the related KRAS mRNA levels in mice injected with CMV-scrR/CMV-siR K , it can be seen that the related KRAS mRNA levels in mice injected with CMV-scrR are relatively high.
  • CMV-siR K has a significant therapeutic effect on KRAS-mutated lung cancer tumors.
  • HE staining and immunohistochemical staining were performed on mice injected with CMV-scrR/CMV-siR K respectively.
  • the results are shown in Figure 14A, Figure 14D, and Figure 14E. It can be seen that KRAS and p-AKT in CMV-scrR-injected mice , p-ERK has more expression and higher percentage of staining.
  • Western blot was used to detect the expression levels of related proteins in mice. The results were shown in Figure 14B and Figure 14C, and the related proteins were more expressed in CMV-scrR-injected mice. This also shows that CMV-siR K has a significant inhibitory effect on KRAS-mutated lung cancer tumors.
  • the 5' flanking sequence is preferably ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with more than 80% homology to it, including sequences with 85%, 90%, 92%, 95%, 98%, 99% homology to ggatcctggaggcttgctgaaggctgtatgctgaattc, etc.
  • the loop sequence is preferably gttttggccactgactgac or a sequence with more than 80% homology thereto, including sequences with 85%, 90%, 92%, 95%, 98%, 99% homology with gttttggccactgactgac, and the like.
  • the 3' flanking sequence is preferably accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag, etc.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-5 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-3 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
  • the compensation sequence may be the reverse complementary sequence of the RNA sequence by deleting any 1-3 consecutively arranged bases.
  • the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
  • the compensation sequence may be the reverse complementary sequence of the 9th position and/or the 10th position in the deletion of the RNA sequence. Deleting bases 9 and 10 works best.
  • flanking sequences are not randomly selected, but are determined based on a large number of theoretical studies and experiments. increase the expression rate of RNA fragments.
  • sequence 1 is preferably CAGATC
  • sequence 2 can be composed of 5-80 bases
  • Sequences of composition such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases
  • Any sequence may be used, preferably a sequence consisting of 10-50 bases, more preferably a sequence consisting of 20-40 bases, and sequence 3 is preferably TGGATC.
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • Sequence 4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
  • the nucleic acid construct fragment of siRNA expressing suppressor gene is encapsulated by AAV-5 adeno-associated virus with high affinity in liver.
  • the adenovirus packaging kit and services provided by Hanheng Biotechnology (Shanghai) Co., Ltd. are used to construct the required virus.
  • the packaging method steps include:
  • the vector plasmid pAAV-RC vector plasmid and pHelper vector plasmid provided by the target Hanheng Biotechnology (Shanghai) Co., Ltd.
  • the vector plasmid AAV051 carrying the target nucleic acid construct fragment wherein the nucleic acid construct fragment is from CMV-siR E or CMV-
  • the clones in the siR K plasmid respectively include the nucleic acid fragments encoding siRNA initiated by CMV as described previously, wherein the CMV promoter is operably linked to an EGFR-inhibiting siRNA sequence (which has a nucleus as shown in SEQ ID NO.: 1).
  • nucleotide sequence UGUGGCUUCUCUUAACUCCU
  • KRAS-inhibiting siRNA sequence which has the nucleotide sequence shown in SEQ ID NO.: 7: UGAUUUAGUAUUAUUUAUGGC
  • taking the nucleotide sequence shown in SEQ ID NO.: 3 as the 3' flanking sequence and having the nucleotide sequence of the RNA fragment
  • the reverse complement wherein the 9th and/or 10th base of the RNA fragment is deleted.
  • AAV-5 viral vector carrying EGFR siRNA or KRAS-siRNA was named AAV-CMV-EGFR siRNA or AAV-CMV-KRAS siRNA.
  • AAV-CMV-gene siRNA or AAV-CMV-siR gene abbreviations or acronyms are used to denote the viral vectors with the structure of AAV-CMV-EGFR siRNA as previously described, but only carry The RNA sequences of the suppressor genes differ.
  • mice 100 ⁇ L of AAV solution with a titer of 1012 V.g/ml was injected into the mice through the 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 selected in each group, mouse lung cancer cells (LLC cells) were injected into the mice, and CT scanning technology was used to observe the progress of mouse model construction.
  • LLC cells mouse lung cancer cells
  • 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 administrations.
  • FIG. 15B “PBS pre” indicates the PBS group before administration, “PBS post” indicates the PBS group after administration; “AAV-CMV-scrRpre” 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-CMVKRASsiRNA group before administration, “AAV-CMV-KRAS-siRNA post” indicates the AAV-CMVKRASsiRNA group after drug.
  • 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/AAVCMV-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 16E.
  • 3D modeling of the mouse lung tissue was performed, and the tumor volume was calculated.
  • the results are shown in Figure 16B. .
  • 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-EGFRsiRNA pre indicates the AAV-CMV-EGFR siRNA 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-CMVEGFRsiRNA group was significantly reduced 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 varying degrees of tumor volume. Increase.
  • the experimental groups were AAV-CMV-KRAS siRNA group and AAV-CMV-EGFR siRNA group, and the control groups were PBS group and AAVCMV-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
  • the viral vector can also include flanking sequences, compensation sequences and loop sequences that can fold the circuit into a correct structure and express, and the flanking sequences include 5' flanking sequences and 3' flanking sequences; the viral vector includes any one of the following Line or combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag, 5'-promoter-target To tag - 5' flanking sequence - RNA fragment - loop sequence - compensation sequence - 3' flanking sequence.
  • the 5' flanking sequence is preferably ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with ggatcctggaggcttgctgaaggctgtatgctgaattc, etc.
  • the loop sequence is preferably gttttggccactgactgac or a sequence with more than 80% homology thereto, including sequences with 85%, 90%, 92%, 95%, 98%, 99% homology with gttttggccactgactgac, and the like.
  • the 3' flanking sequence is preferably accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag, etc.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-5 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-3 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
  • the compensation sequence may be the reverse complementary sequence of the RNA sequence by deleting any 1-3 consecutively arranged bases.
  • the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
  • the compensation sequence may be the reverse complementary sequence of the 9th position and/or the 10th position in the deletion of the RNA sequence. Deleting bases 9 and 10 works best.
  • flanking sequences are not randomly selected, but are determined based on a large number of theoretical studies and experiments. increase the expression rate of RNA fragments.
  • 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.
  • Sequence 4 and two sequences 4-1 and 4-2 with more than 80% homology to sequence 4 were constructed into AAV vectors, and the EGFR siRNA content in lung tissue was detected 9 hours after intravenous injection.
  • Sequence 4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
  • the plasmid CMV-siR V carrying VEGFR siRNA that inhibits VEGFR expression or the plasmid CMV-siR mT carrying mTOR siRNA that inhibits mTOR expression was constructed according to the method described in Example 2.
  • the siRNA of the VEGFR gene has the following nucleotide sequence: AUUUGAAGAGUUGUAUUAGCC (SEQ ID NO.: 8).
  • the siRNA of the mTOR gene has the following nucleotide sequence: AGAUAGUUGGCAAAUCUGCCA (SEQ ID NO. 9).
  • mice Different mice were injected with PBS buffer/control plasmid/VEGFR siRNA plasmid/mTOR siRNA plasmid/MIX siRNA plasmid (the combination of VEGFR siRNA and mTOR siRNA)/Sunitinib/Everolimus, The development of renal cancer tumors in mice was observed, and the results are shown in Figure 18 and Figure 19 . It can be seen that the development of kidney cancer in mice injected with MIX siRNA plasmid was most significantly inhibited, while the development of kidney cancer in mice injected with PBS buffer/control plasmid was more rapid.
  • VEGFR siRNA and mTOR siRNA has a significant therapeutic effect on renal cancer tumors.
  • 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.
  • the connecting sequence is the above sequence 4 and the sequence 4-1 and sequence 4-2 which have more than 80% homology with the sequence 4, after the delivery system containing the above sequence is injected, it is detected in the lung tissue 9 hours later, and there is also a corresponding enrichment. , self-assembly and cancer therapeutic effects.
  • Sequence 4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
  • TNF- ⁇ siRNA has the following nucleotide sequence: AAAACAUAAUCAAAAGAAGGC (SEQ ID NO. 10).
  • the integrin- ⁇ siRNA has the following nucleotide sequence AUAAUCAUCUCCAUUAAUGUC (SEQ ID NO. 11).
  • B7 siRNA has the following nucleotide sequence UUUUCUUUGGGUAAUCUUCAG (SEQ ID NO. 12).
  • Example 2 a plasmid CMV-si mix carrying siRNA of TNF- ⁇ siRNA, integrin- ⁇ siRNA and B7 siRNA at the same time was constructed, namely CMV-siR TNF- ⁇ +integrin- ⁇ +B7 .
  • the experimental groups were anti-TNF- ⁇ (0.5) group, anti-TNF- ⁇ (5) group, and anti-TNF- ⁇ (20) group.
  • the control group was the mock group, the scr-RNA group and the IFX group, respectively.
  • anti-TNF- ⁇ (0.5) group, anti-TNF- ⁇ (5) group and anti-TNF- ⁇ (20) group were plasmids carrying TNF- ⁇ siRNA (CMV-siR TNF- ⁇ ), tail 0.5 ⁇ L, 5 ⁇ L and 20 ⁇ L of CMV-siR TNF- ⁇ solutions were intravenously injected into mice.
  • CMV-siR TNF- ⁇ TNF- ⁇ siRNA
  • the mock group was the negative control group, and the scr-RNA plasmid and IFX (infliximab) were injected into the tail vein of mice in the scr-RNA group and the IFX group, respectively.
  • the in vivo expression of the plasmid system was monitored by small animals, and then the mice were sacrificed to observe the colon.
  • 0.5) group, anti-TNF- ⁇ (5) group and anti-TNF- ⁇ (20) group had relatively longer colon lengths, and the higher the TNF- ⁇ siRNA injection dose, the longer the mice colon lengths were. This indicates that the plasmid-encapsulated TNF- ⁇ siRNA system has different degrees of improvement in the shortening of colon length caused by chronic inflammation.
  • the disease activity index (Disease activity index) of mice was evaluated, and the results are shown in Figure 20C.
  • the disease activity index of mice was higher, while the disease activity index of mice in anti-TNF- ⁇ (20) group and IFX group was lower.
  • TNF- ⁇ mRNA was detected in the colon of mice, and the results are shown in Figure 20D. It can be seen that the CMV-siR TNF- ⁇ system can reduce the expression and secretion of TNF- ⁇ in the colon; TNF- ⁇ was detected in the colon of mice, and the results are as follows As shown in Figure 17E, it can be seen that the AAV system can produce a certain amount of TNF- ⁇ ; the pro-inflammatory factors IL-6, IL-12p70, IL-17A, and IL-23 in the colon were detected, and the results were shown in Figure 20F , it can be seen that the secretion of inflammatory factors in the high-dose group is generally lower than that in the control group.
  • mice in the (20) group especially the mice in the anti-TNF- ⁇ (20) group, had higher colonic mucosal integrity, less infiltration of immune cells, colonic crypt abscesses, and colonic congestion and hemorrhage. significantly lighter than the control group.
  • the treatment using the plasmid-encapsulated TNF- ⁇ siRNA system provided by the present invention is more effective than IFX.
  • the experimental groups were anti-TNF- ⁇ group, anti-integrin- ⁇ group, anti-B7 group, and anti-mix group, respectively.
  • mock group PBS group, scr-RNA group.
  • the anti-TNF- ⁇ group, anti-integrin- ⁇ group, anti-B7 group and anti-mix group used plasmids to carry TNF- ⁇ siRNA (CMV-siR TNF- ⁇ ) and integrin- ⁇ siRNA (CMVsiR integrin- ⁇ ) respectively , B7 siRNA (CMV-siR B7 ), siRNA carrying both TNF- ⁇ siRNA), integrin- ⁇ siRNA and B7 siRNA (CMV-si mix is CMV-siR TNF- ⁇ +integrin- ⁇ +B7 ).
  • 20 ⁇ L was injected into mice by tail vein, and the in vivo expression of the system was monitored by small animals. It can be seen that the above system is stably expressed in vivo, especially in the liver.
  • the mock group was the negative control group, and the scr-RNA plasmid and PBS solution (phosphate buffered saline) were injected into the tail vein of mice in the scr-RNA group and the PBS group, respectively.
  • PBS solution phosphate buffered saline
  • the disease activity index of mice was evaluated, and the results are shown in Figure 22C. It can be seen that the disease activity index of mice in the scr-RNA group and PBS group was higher, while the anti-TNF- ⁇ group, anti-integrin- The disease activity index of mice in ⁇ group, anti-B7 group and anti-mix group decreased in turn.
  • TNF- ⁇ mRNA, integrin mRNA and B7 mRNA were detected in mouse plasma, liver and colon.
  • the results are shown in Figure 22D- Figure 22F. It can be seen that the system produced a certain amount of stably expressed mRNA in plasma, liver and colon. RNA, and the system significantly reduced colonic TNF- ⁇ , integrin and B7 mRNA expression.
  • mice in the 4 experimental groups especially the mice in the anti-mix group, had higher colonic mucosal integrity and less infiltration of immune cells. Colonic crypt abscesses and colonic congestion and hemorrhage were also significantly lighter than controls.
  • the virus AAV-CMV-siRTNF- ⁇ carrying the TNF- ⁇ siRNA that inhibits the expression of TNF- ⁇ or the virus AAV-CMVsiR integrin- ⁇ carrying the anti-integrin- ⁇ siRNA that inhibits the expression of integrin- ⁇ was constructed according to the method described in Example 5 Or viral AAV-CMV-siR B7 carrying B7 siRNA that inhibits B7 expression.
  • TNF- ⁇ siRNA has the following nucleotide sequence AAAACAUAAUCAAAAGAAGGC (SEQ ID NO. 10).
  • the integrin- ⁇ siRNA has the following nucleotide sequence AUAAUCAUCUCCAUUAAUGUC (SEQ ID NO. 11).
  • B7 siRNA has the following nucleotide sequence UUUUCUUUGGGUAAUCUUCAG (SEQ ID NO. 12).
  • a virus AAV-CMV-si mix , CMV-siR TNF- ⁇ +integrin- ⁇ +B7 ) carrying TNF- ⁇ siRNA, integrin- ⁇ siRNA and B7 siRNA at the same time was constructed.
  • the three experimental groups were AAV-CMV-siRTNF- ⁇ (low) group, AAV-CMV-siRTNF- ⁇ (medium) group, AAV -CMV-siRTNF- ⁇ (high) group; control group were Normal group and AAV-CMV-scrR group.
  • the experimental process is shown in Figure 24A.
  • the AAV-CMV-siRTNF- ⁇ (low) group, the AAV-CMV-siRTNF- ⁇ (medium) group, and the AAV-CMV-siRTNF- ⁇ (high) group used high-affinity liver AAV-5 adeno-associated virus-encapsulated TNF- ⁇ siRNA system (AAV-CMV-siRTNF- ⁇ ) was injected into mice with AAV solution with a titer of 1012V.g/ml, 25 ⁇ L, 50 ⁇ L, and 100 ⁇ L through the tail vein.
  • the in vivo expression of the AAV system was monitored by small animals. The results are shown in Figure 24B. After 3 weeks, it can be seen that the AAV system is stably expressed in vivo, especially in the liver. Among them, the AAV-CMV-siRTNF- ⁇ (high) group, the average radiance (Average Radiance) reached 8.42*105 (p/sec/cm2/sr), and the expression site was in the liver, which indicated that the expression of AAV system had a dose-dependent effect.
  • the disease index of each group of mice was scored and counted, and the results are shown in Figure 24E. It can be seen that the disease index of the mice in the AAV-CMV-siRTNF- ⁇ (high) group was lower than that of the AAV-CMV-siRTNF- ⁇ (low) group. ) group, AAV-CMV-siRTNF- ⁇ (medium) group and AAV-CMV-scrR group.
  • TNF- ⁇ siRNA The levels of TNF- ⁇ siRNA in the mice of each group were detected respectively, and the results are shown in Figure 24F. 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 was smaller. There is almost no expression of TNF- ⁇ siRNA in mice, which indicates that the above-mentioned AAV system can produce a certain amount of TNF- ⁇ siRNA.
  • TNF- ⁇ mRNA levels in the mice of each group were detected respectively.
  • the results are shown in Figure 24G. It can be seen that the TNF- ⁇ mRNA levels in the mice of the Normal group and the three experimental groups were relatively low, while the AAV-CMV-scrR The level of TNF- ⁇ mRNA in the mice in the group was higher, which indicated that the AAV system could reduce the expression and secretion of TNF- ⁇ in the colon.
  • mice in the Normal group and the AAV-CMV-siRTNF- ⁇ (high) group are pro-inflammatory
  • the secretion of cytokines was the least, and the secretion of proinflammatory cytokines was the most in the mice in the AAV-CMV-scrR group.
  • HE staining and pathological score statistics were performed on mouse colon sections. The results are shown in Figure 25B and Figure 25C. It can be seen that the colonic mucosa of the experimental group, especially the AAV-CMV-siRTNF- ⁇ (high) group, has higher integrity. , and the infiltration of immune cells was shallower, colonic crypt abscesses, and colonic congestion and hemorrhage were significantly lighter than those in the AAV-CMV-scrR group.
  • liver-friendly AAV to encapsulate CMV-siRTNF- ⁇ can achieve long-term TNF- ⁇ siRNA expression and long-term TNF- ⁇ silencing, and can relieve colitis to a certain extent, which has great drug potential and clinical research value.
  • the experimental groups were AAV-CMV-siRT+B+I(low) group, AAV-CMV-siRT+B+I(medium) group, AAV-CMV-siRT+B+I(high) group; control group They are Normal group and AAV-CMV-scrR group, respectively.
  • the AAV-CMV-siRT+B+I(low) group, the AAV-CMV-siRT+B+I(medium) group, and the AAV-CMV-siRT+B+I(high) group used the liver high-affinity AAV- Adeno-associated virus type 5 encapsulated TNF- ⁇ siRNA, B7-siRNA and Integrin ⁇ 4 siRNA element tandem drug delivery system (AAV-CMV-siRT+B+I), AAV solution with a titer of 1012V.g/ml was injected through the tail vein 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 26A. 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, and the results are shown in Figure 27A. It can be seen that the disease index of mice in the AAV-CMV-siRT+B+I(high) group was lower than that of AAV-CMV-siRT+B +I(low) group, AAV-CMV-siRT+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 27B, Figure 27C, and Figure 27D. It can be seen that the CMV-siR T+B+I system encapsulated by AAV is in the mouse plasma. A certain amount of stably expressed siRNA was generated and showed a dose-dependent effect.
  • the system generated a certain amount of stably expressed siRNA in mouse liver, and showed a dose-dependent effect.
  • liver-friendly AAV to encapsulate CMV-siR T+B+I 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. It has great drug potential and clinical research value.
  • the plasmid CMV-siR miR-21 was or the plasmid CMVsiR TGF- ⁇ 1 carrying TGF- ⁇ 1 siRNA that inhibited the expression of TGF- ⁇ 1 was constructed according to the method described in Example 2.
  • miR-21 siRNA has the antisense strand of miR-21.
  • TGF- ⁇ 1 siRNA has the following nucleotide sequence: ACGGAAAUAACCUAGAUGGGC (SEQ ID NO. 13).
  • the plasmid CMV-siR miR-21+TGF- ⁇ 1 carrying both miR-21 siRNA and TGF- ⁇ 1 siRNA was constructed according to the method described in Example 2.
  • the experimental groups were Anti-miR-21(1mg/kg) group, Anti-miR-21(5mg/kg) group, Anti-miR-21(10mg/kg) group, TGF- ⁇ 1 siRNA(1mg/kg) group , TGF- ⁇ 1 siRNA (5mg/kg) group, TGF- ⁇ 1 siRNA (10mg/kg) group, Anti-miR-21+TGF- ⁇ 1 siRNA (10mg/kg) group, Pirfenidone (300mg/kg) group, control group They are Normal group, PBS group and scrRNA group, respectively.
  • Anti-miR-21 (1mg/kg) group, Anti-miR-21 (5mg/kg) group and Anti-miR-21 (10mg/kg) group were injected into the tail vein of mice with pulmonary fibrosis respectively 1mg/kg, 5mg/kg, 10mg/kg miR-21 siRNA plasmid, TGF- ⁇ 1 siRNA (1mg/kg) group, TGF- ⁇ 1 siRNA (5mg/kg) group, TGF- ⁇ 1 siRNA (10mg/kg) group
  • Mice with pulmonary fibrosis were injected with 1 mg/kg, 5 mg/kg and 10 mg/kg of TGF- ⁇ 1 siRNA plasmids into the tail vein, and the Anti-miR-21+TGF- ⁇ 1 siRNA (10 mg/kg) group was injected with 1 mg/kg, 5 mg/kg and 10 mg/kg.
  • mice with pulmonary fibrosis were injected with 10 mg/kg Anti-miR-21 and TGF- ⁇ 1 siRNA plasmids in the tail vein, and the Pirfenidone (300 mg/kg) group was injected with 300 mg/kg of pirfenidone in the tail vein of mice with pulmonary fibrosis , Normal group was the normal control group, PBS group and scrRNA group were injected with PBS solution and control plasmid into the tail vein of mice with pulmonary fibrosis, respectively.
  • hydroxyproline is the main component of collagen, and its content reflects the degree of pulmonary fibrosis.
  • Anti-miR-21 (5mg/kg) group, Anti-miR-21 (10mg/kg) group, TGF- ⁇ 1 siRNA (10mg/kg) group, Anti-miR-21+TGF - ⁇ 1 siRNA (10mg/kg) group had relatively lower hydroxyproline content and suppressed pulmonary fibrosis.
  • H&E staining was performed on the lungs of mice in each group, and the results are shown in Figure 33. It can be seen that the alveolar space of the mice in the PBS group and the scrRNA group was widened, the inflammatory cells were infiltrated, and the alveolar structure was damaged, while the lung tissue in the experimental group was relatively normal.
  • Normal group, PBS group, scrRNA group, TGF- ⁇ 1 siRNA (1mg/kg) group, TGF- ⁇ 1 siRNA (5mg/kg) group, TGF- ⁇ 1 siRNA (10mg/kg) group, Pirfenidone (300mg/kg) group were detected by western blot.
  • TGF- ⁇ 1 protein level and TGF- ⁇ 1 mRNA level of mice in the TGF- ⁇ 1 siRNA (10 mg/kg) group the results are shown in Figure 34A- Figure 34C. mRNA levels were lowest. This indicated that TGF- ⁇ 1 could be successfully delivered to the lungs to function after the corresponding siRNA expression plasmid was injected into the tail vein.
  • mice in the Normal group, PBS group, scrRNA group, Anti-miR-21 (1mg/kg) group, Anti-miR-21 (5mg/kg) group and Anti-miR-21 (10mg/kg) group were detected respectively.
  • miR-21 level the results are shown in Figure 34D, it can be seen that the anti-miR-21 (10 mg/kg) group has the highest relative miR-21 level. This indicated that the antisense strand of miR-21 could be successfully delivered to the lungs to function after the corresponding antisense strand expression plasmid was injected into the tail vein.
  • liver-friendly plasmids to encapsulate CMV-siR miR-21 , CMVsiR TGF- ⁇ 1 , CMV-siR miR-21+TGF- ⁇ 1 can significantly alleviate the degree of pulmonary fibrosis, and has great drug potential, and Clinical research value.
  • Plasmids containing 3 different 5' flanking sequences, loop sequences and sequence fragments of 3' flanking sequences also have in vivo enrichment, self-assembly and therapeutic effects on pulmonary fibrosis.
  • the sequences are:
  • flanking sequence -1 CTGGAGGCTTGCTGAAGGCTGTATGCTGAATTCG 5' flanking sequence-2 CTGGAGGCTTGCTGAAGGCTGTATGCTGTTAACG 5' flanking sequence -3 CTGGAGGCTTGCTGAAGGCTGTATGCTGGCAACG loop-1 GTTTTGGCCACTGACTGAC loop-2 GTTAAGGCCACTGACTGAC loop-3 GAATTGGCCACTGACTGAC 3' flanking sequence -1 CACCGGTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC 3' flanking sequence-2 CAGGCCTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC 3' flanking sequence-2 CAGGCCTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC 3' flanking sequence -3 CAGCGCTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC 3' flanking sequence -3 CAGCGCTCAGGACACAAGGCCTGTT
  • the connecting sequence between adjacent lines can be composed of multiple bases.
  • the plasmid also has an enrichment effect after injection.
  • the sequence 2 is shown in the following table. .
  • sequence 4-1 is the above-mentioned sequence 4
  • sequences 4-2/4-3/4-4 are the homologous sequences with a homology greater than 80% of the sequence 4-1, respectively.
  • Sequence 4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
  • RNA sequence lengths in the plasmid delivery system were 18, 19, and 21, respectively.
  • RNA fragments In the plasmid delivery system, in the case of carrying RNA fragments, it has in vivo enrichment, spontaneous formation of composite structures and therapeutic effects on pulmonary fibrosis.
  • the grouping of RNA fragments includes but is not limited to:
  • RNA fragment that comprises any 2 kinds of RNA sequences
  • RNA fragment comprising any three kinds of RNA sequences.
  • the virus AAV-CMV-siR miR-21 carrying the miR-21 siRNA that inhibits the expression of miR-21 or the virus AAV-CMVsiR carrying the TGF- ⁇ 1 siRNA that inhibits the expression of TGF- ⁇ 1 was constructed according to the methods described in Examples 5 and 9 TGF- ⁇ 1 .
  • miR-21 siRNA has the antisense strand of miR-21.
  • TGF- ⁇ 1 siRNA has the following nucleotide sequence ACGGAAAUAACCUAGAUGGGC (SEQ ID NO. 13).
  • a virus AAV-CMV-MIX carrying both miR-21 siRNA and TGF- ⁇ 1 siRNA was constructed, namely AAV-CMV-siR miR-21+TGF- ⁇ 1 .
  • AAV solution 100 ⁇ L of AAV solution with a titer of 1012 V.g/ml was injected into the mice through the tail vein.
  • the in vivo expression of the AAV system was monitored by small animals. After 3 weeks, it was found that the AAV system was stably expressed in vivo, especially in the liver.
  • mice were then selected for modeling. After successful modeling, mice were injected with PBS buffer/AAV-scrR/AAV-anti-miR21/AAV-TGF- ⁇ 1 siRNA/AAV-MIX (10 mg/kg) to form PBS group/AAV-scrR group/AAV-anti-miR21 group/AAV-TGF- ⁇ 1 siRNA group/AAV-MIX group.
  • 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, and the results are shown in Figure 35B. It can be seen that the relative TGF- ⁇ 1 mRNA level of mice in the AAV-TGF- ⁇ 1 siRNA group was relatively lower.
  • mice in each group were detected respectively, and the results are shown in Figure 35A. It can be seen that the hydroxyproline content of mice 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.
  • the viral vector may also include a flanking sequence, a compensation sequence and a loop sequence that can make the circuit fold into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence; the viral vector Including any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag, 5' - Promoter - Targeting Tag - 5' Flanking Sequence - RNA Fragment - Loop Sequence - Compensation Sequence - 3' Flanking Sequence.
  • the 5' flanking sequence is preferably ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with ggatcctggaggcttgctgaaggctgtatgctgaattc, etc.
  • the loop sequence is preferably gttttggccactgactgac or a sequence with more than 80% homology thereto, including sequences with 85%, 90%, 92%, 95%, 98%, 99% homology with gttttggccactgactgac, and the like.
  • the 3' flanking sequence is preferably accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag, etc.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-5 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-3 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
  • the compensation sequence may be the reverse complementary sequence of the RNA sequence by deleting any 1-3 consecutively arranged bases.
  • the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
  • the compensation sequence may be the reverse complementary sequence of the 9th position and/or the 10th position in the deletion of the RNA sequence. Deleting bases 9 and 10 works best.
  • flanking sequences are not randomly selected, but are determined based on a large number of theoretical studies and experiments. increase the expression rate of RNA fragments.
  • adenovirus vectors containing three homologous sequences they also have in vivo enrichment, self-assembly and therapeutic effects on pulmonary fibrosis.
  • the sequences are grouped as follows:
  • sequence 1 is preferably CAGATC
  • sequence 2 can be composed of 5-80 bases
  • Sequence of bases such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases
  • sequence of 10-50 bases is preferable, and the sequence of 20-40 bases is more preferable.
  • Sequence 3 is preferably TGGATC.
  • the adjacent lines are connected by sequence 1-sequence 2-sequence 3, where sequence 2 contains multiple bases, and the constructed delivery system also has in vivo enrichment, self-assembly and lung Fibrosis treatment effect.
  • Sequence 2 is specifically shown in Table 3 below.
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • Sequence 4-1 is the sequence 4.
  • 4-2/4-3/4-4 are the homologous sequences of sequence 4-1 respectively, and the specific sequences are shown in Table 4 below.
  • Sequence 4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
  • RNA fragments comprise one, two or more specific RNA sequences of medical significance, the RNA sequences can be expressed in the target receptor, and the compensatory sequence cannot be expressed in the target receptor.
  • the RNA sequence can be an siRNA sequence, a shRNA sequence or a miRNA sequence, preferably an siRNA sequence.
  • the length of an RNA sequence is 15-25 nucleotides (nt), preferably 18-22nt, such as 18nt, 19nt, 20nt, 21nt, and 22nt. This range of sequence lengths was not chosen arbitrarily, but was determined through trial and error. A large number of experiments have proved that when the length of the RNA sequence is less than 18nt, especially less than 15nt, the RNA sequence is mostly invalid and will not play a role. The cost of the line is greatly increased, and the effect is not better than the RNA sequence with a length of 18-22nt, and the economic benefit is poor. Therefore, when the length of the RNA sequence is 15-25nt, especially 18-22nt, the cost and the effect can be taken into consideration, and the effect is the best.
  • nt nucleotides
  • RNA sequence lengths were 18, 20, and 21, respectively, the constructed delivery system also had the therapeutic effects of in vivo enrichment, self-assembly and pulmonary fibrosis.
  • the specific sequences are shown in Table 5.
  • RNA fragments When the viral vector system carries a variety of different RNA fragments, it has in vivo enrichment, self-assembly and therapeutic effects on pulmonary fibrosis, as shown in Figure 5, where the RNA fragments are grouped as follows:
  • RNA-1 alone siRNA-2 alone, shRNA-1 alone, shRNA-2 alone, miRNA-1 alone, miRNA-2 alone;
  • RNA-1+siRNA-2 siRNA-1+siRNA-2, shRNA-1+shRNA-2, miRNA-1+miRNA-2;
  • RNA-1+siRNA-2+shRNA-1 siRNA-1+siRNA-2+shRNA-2
  • siRNA-1+siRNA-2+miRNA- 1 siRNA-1+siRNA-2+miRNA-2.
  • RNA sequences are specifically shown in Table 1 below.
  • the experimental groups are CMV-siR E group, CMV-siR T group, CMV-RVG-siR E+T group, CMV-siR E+T group, CMV-Flag-siR E+T group, where "E” stands for EGFR , "T” represent TNC, and the control groups are the PBS group, the CMV-scrR group, and the CMV-Flag-scrR group, respectively.
  • E stands for EGFR
  • T represent TNC
  • the control groups are the PBS group, the CMV-scrR group, and the CMV-Flag-scrR group, respectively.
  • the specific experimental process is shown in Figure 36A.
  • the experimental groups were CMV-RVG-siR E group and CMV-RVG-siR E+T group, respectively, and the control group were PBS group and CMV-scrR group, respectively.
  • mice were selected, and glioblastoma cells (U-87MG-Luc cells) were injected into the mice. From the 7th day to the 21st day, the mice were injected every two days. One treatment with PBS buffer/CMV-scrR/CMV-RVG-siR E /CMV-RVG-siR E+T (5 mg/kg), mice were subjected to survival analysis and tumor assessment, respectively. On the 7th, 14th, 28th, and 35th days, the mice were detected by BLI in vivo imaging, respectively.
  • this figure is a comparison chart of BLI in vivo imaging detection of mice on the 7th, 14th, 28th, and 35th days. It can be seen that the mice in the CMV-RVG-siR E+T group have glioblastoma The tumor inhibition effect was the most significant.
  • FIG. 37C which is a comparison chart of the survival rate of mice in each group, it can be seen that the mice in the CMV-RVG-siR E+T group have the longest survival time.
  • the graph is a fluorescence comparison graph of each group of mice, which was obtained by luciferase in vivo imaging, and the ordinate reflects the intensity of the lucifer fluorescence signal. Since the gene has been artificially integrated into the implanted tumor, the map reflects tumor progression. It can be seen that the tumors of the mice in the control group developed rapidly, while the tumors of the mice in the experimental group were suppressed to a great extent. As shown in Figure 37E, this figure is the relative siRNA comparison chart of each group of mice.
  • mice in the CMV-RVG-siR E group have higher levels of EGFR siRNA
  • mice in the CMV-RVG-siR E+T group have higher levels of EGFR siRNA. and TNC siRNA levels were higher.
  • the figure is a western blot comparison chart of mice in each group. It can be seen that the mice in the PBS group, the CMV-scrR group, and the CMV-RVG-siR E group have higher EGFR and TNC gene contents.
  • CMV-RVG-siR E plasmid can inhibit the expression of EGFR and PCNA in the brain
  • CMV-RVG-siR E+T plasmid can inhibit the expression of EGFR, TNC and PCNA in the brain.
  • the plasmid does have the effect of enriching in vivo and spontaneously forming a composite structure containing RNA fragments.
  • the present invention randomly provides a set of plasmids carrying four of the lines, and adjacent lines are connected by sequence 1-sequence 2-sequence 3, and Sequence 2 is the experimental data consisting of 5 bases, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases and 80 bases respectively. Set and self-assemble effects, .
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • the present invention randomly provides a set of plasmids containing the connecting sequence as sequence 4 and at least two corresponding sequences with more than 80% homology to sequence 4.
  • Experimental data, and the enrichment and self-assembly effects of plasmids were verified by experiments.
  • Sequence 4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
  • Viruses carrying RVG targeting peptides prepared as described in Examples 2 and 5 were tested for their effect on glioblastoma.
  • AAV-CMV-RVG-siR E and AAV-CMV-RVG-siR E+T 100 ⁇ L of AAV solution with a titer of 1012 V.g/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 and injected with glioblastoma cells (U-87MG-Luc cells) into the mice.
  • mice were injected every two days with PBS buffer/AAV-CMV-scrR/AAV-CMV-RVG-siR E /AAV-CMV-RVG-siR E+T (5 mg/ kg) were treated to form a PBS group/AAV-scrR group/AAV-CMV-RVG-siRE group/AAV-CMV-RVG-siRE+T group.
  • the present invention randomly provides a set of viral vectors carrying four of the lines, the adjacent lines are connected by sequence 1-sequence 2-sequence 3, and the sequence 2 are the experimental data consisting of 5 bases, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases and 80 bases respectively. Set and self-assemble effects.
  • Sequence 2 is specifically shown in Table 3 below.
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • the present invention randomly provides a set of viral vectors containing the corresponding experimental data of the connecting sequence as sequence 4 and at least two sequences with more than 80% homology to sequence 4, The enrichment and self-assembly effects of viral vectors were verified by experiments.
  • Sequence 4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
  • the plasmid CMV- siRP carrying the PTP1B siRNA that inhibits the expression of PTP1B, and the plasmid CMV-RVG- siRP carrying the targeting peptides RVG and PTP1B siRNA were constructed according to the method described in Example 2.
  • the virus AAV-CMV- siRP carrying the PTP1B siRNA that inhibits the expression of PTP1B and the virus AAV-CMV-RVG- siRP carrying the targeting peptide RVG were constructed according to the method described in Example 5.
  • the siRNA of the PTP1B gene has the following nucleotide sequence: UGAUAUAGUCAUUAUCUUCUU (SEQ ID NO. 14).
  • the experimental groups were the CMV- siRP group and the CMV-RVG-siRP group, and the control group was the CMV-scrR group, where "P" represented PTP1B.
  • the CMV- siRP group, CMV-RVG- siRP group, and CMV-scrR group were injected with 5 mg/kg of CMV- siRP plasmid, CMV-RVG- siRP plasmid, and CMV-scrR plasmid, respectively, and then obtained each
  • the hypothalamus and liver fluorescence microscope images of the mice in the group are shown in Figure 40. The results show that PTP1B siRNA can be delivered to the hypothalamus.
  • mice in the second experiment were set up.
  • the experimental groups were the weight comparison chart of mice in the CMV- siRP group. It can be seen that the weight of the mice in the CMV-RVG- siRP group was the most stable. .
  • mice 41D- Figure 41G The results showed that the CMV-RVG-siR P plasmid can effectively increase the oxygen consumption of mice, which means that this group of mice is in a state of high energy metabolism compared with other groups of mice. Normal mice mainly use glucose as their own energy source. CMV-RVG-siRP plasmid can reduce the respiratory exchange ratio of mice, which means that this group of mice is more inclined to use protein as its own energy source compared to other groups of mice. . The activity of mice injected with CMV-RVG- siRP plasmid was significantly increased. Moreover, the mice in the CMV-RVG- siRP group also had significantly increased thermogenesis.
  • the figure is a comparison chart of the initial body weight curves of mice in each group. It can be seen that the mice in the CMV-RVG- siRP group had the lightest body weight.
  • the figure is a comparison chart of the initial food intake curves of mice in each group. As can be seen, the mice in the CMV-RVG- siRP group had the least food intake.
  • FIG 41J the figure is a comparison chart of serum leptin content of mice in each group. It can be seen that the serum leptin content of mice in the CMV-RVG- siRP group was the lowest.
  • FIG 41K the figure is the western blot comparison chart of mice in each group. It can be seen that the CMVRVG- siRP group mice had the lowest PTP1B protein content.
  • the figure is a comparison chart of blood glucose change curves of mice in each group. It can be seen that the mice in the CMV-RVG- siRP group had the lowest blood glucose levels.
  • the figure is a comparison chart of the basal glucose change curves of mice in each group. It can be seen that the mice in the CMV-RVG- siRP group had the lowest basal glucose content.
  • mice in each group were measured, and the results are shown in Figure 42A .
  • Mouse TC, TG and LDL were the lowest.
  • the liver tissue was collected from the mice in each group after treatment, and compared with the normal control. The results are shown in Figure 31D.
  • the liver tissue pathological sections of the mice in the PBS group and the CMV-scrR group showed obvious pathological characteristics of fatty liver.
  • the fatty liver of the mice in the CMV- siRP group was lighter.
  • mice were selected and injected with PBS buffer/AAV-CMV-scrR/AAV-CMV- siRP /AAV-CMV-RVG- siRP after 12 weeks to form a 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 43A 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.
  • 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 43D 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 43E 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. 44D which is a comparison of the body lengths of mice in each group, it can be seen that the body lengths of the four groups of mice are almost the same.
  • FIG. 44E which is a comparison chart of the HFD food intake of mice in each group, it can be seen that the HFD food intake of the four groups of mice is also similar.
  • adenovirus vectors containing 3 homologous sequences they also have in vivo enrichment, self-assembly and obesity treatment effects, as shown in Figure 8, the sequences are grouped as follows:
  • sequence 1 is preferably CAGATC
  • sequence 2 can be composed of 5-80 bases
  • Sequence of bases such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases
  • sequence of 10-50 bases is preferable, and the sequence of 20-40 bases is more preferable.
  • Sequence 3 is preferably TGGATC.
  • the adjacent lines are connected by sequence 1-sequence 2-sequence 3, wherein sequence 2 contains multiple bases, and the constructed delivery system also has in vivo enrichment, self-assembly and obesity. disease treatment effect.
  • Sequence 2 is specifically shown in Table 3 below.
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • Sequence 4-1 is the sequence 4
  • sequence 4- 2/4-3/4-4 are the homologous sequences of sequence 4-1 respectively, and the specific sequences are shown in Table 4 below.
  • Sequence 4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
  • RNA fragments comprise one, two or more specific RNA sequences of medical significance, the RNA sequences can be expressed in the target receptor, and the compensatory sequence cannot be expressed in the target receptor.
  • the RNA sequence can be an siRNA sequence, a shRNA sequence or a miRNA sequence, preferably an siRNA sequence.
  • the length of an RNA sequence is 15-25 nucleotides (nt), preferably 18-22nt, such as 18nt, 19nt, 20nt, 21nt, and 22nt. This range of sequence lengths was not chosen arbitrarily, but was determined through trial and error. A large number of experiments have proved that when the length of the RNA sequence is less than 18nt, especially less than 15nt, the RNA sequence is mostly invalid and will not play a role. The cost of the line is greatly increased, and the effect is not better than the RNA sequence with a length of 18-22nt, and the economic benefit is poor. Therefore, when the length of the RNA sequence is 15-25nt, especially 18-22nt, the cost and the effect can be taken into consideration, and the effect is the best.
  • nt nucleotides
  • the constructed delivery system also has the therapeutic effects of in vivo enrichment, self-assembly and obesity.
  • the specific sequences are shown in Table 5.
  • RNA fragments When the viral vector system carries a variety of different RNA fragments, it has in vivo enrichment, self-assembly and therapeutic effects on obesity, as shown in Figure 6, where the RNA fragments are grouped as follows:
  • RNA-1 alone siRNA-2 alone, shRNA-1 alone, shRNA-2 alone, miRNA-1 alone, miRNA-2 alone;
  • RNA-1+siRNA-2 siRNA-1+siRNA-2, shRNA-1+shRNA-2, miRNA-1+miRNA-2;
  • RNA-1+siRNA-2+shRNA-1 siRNA-1+siRNA-2+shRNA-2
  • siRNA-1+siRNA-2+miRNA- 1 siRNA-1+siRNA-2+miRNA-2.
  • RNA sequences are specifically shown in Table 1 below.
  • the plasmid CMV-siR mHTT carrying the mHTT siRNA that inhibits mHTT expression, and the plasmid CMV-RVG- siRP carrying the targeting peptide RVG and mHTT siRNA were constructed according to the method described in Example 2.
  • virus AAV-CMV-siRmHTT carrying mHTT siRNA that inhibits mHTT expression and virus AAV-CMV-RVG- siRmHTT carrying targeting peptide RVG were constructed according to the method described in Example 5.
  • the siRNA of mHTT gene has the following nucleotide sequence: UAUGUUUUCACAUAUUGUCAG (SEQ ID NO. 15).
  • the experimental groups were CMV-siR mHTT group and CMV-RVG-siR mHTT group, respectively, and the control group were PBS group and CMV-scrR group, respectively.
  • the experimental process is shown in Figure 45A.
  • the mice in the CMV-siR mHTT group, the CMV-RVG-siR mHTT group, the PBS group, and the CMV-scrR group were injected with the CMV-siR mHTT plasmid and CMV-RVG into the tail vein of mice with Huntington's disease.
  • plasma exosomes were isolated, labeled with PKH26 dye, and co-cultured with cells to observe the absorption of exosomes by cells.
  • Figure 45B is a comparison chart of siRNA levels in plasma exosomes of mice in each group, it can be seen that the levels of siRNA in plasma exosomes of mice in the two experimental groups are higher.
  • Plasma exosomes extracted from mice in each group after injection of plasmid/solution were labeled with PKH26, co-cultured with cells, and photographed with a confocal microscope. The results are shown in Figure 45C, showing that the siRNA-encapsulated exosomes entered the cells.
  • siR mHTT can reduce the level of HTT protein, indicating that siRNA assembled into exosomes can still exert gene silencing function.
  • this figure is the in situ hybridization map of the liver, cortex, and striatum of the mice in each group. It can be seen that the liver tissue sections of the CMV-siR mHTT group and the CMV-RVG-siR mHTT group have obvious The fluorescence of the mice in the CMV-RVG-siR mHTT group showed obvious fluorescence in the cortex and striatum tissue sections. This indicates that RVG can guide exosomal siRNA to enter and function through the blood-brain barrier.
  • the experimental groups were the CMV-siR GFP group and the CMV-RVG-siR GFP group, and the control groups were the PBS group and the CMV-scrR group, respectively.
  • the figures are the slices of the liver, cortex and striatum of mice in each group,
  • the experimental groups were the CMV-siR mHTT group, the CMV-RVG-siR mHTT group, and the control group was the CMV-scrR group.
  • mice 8-week-old N17182Q mice were selected, and they were CMV-siR mHTT group, CMV-RVG-siR mHTT group, and CMV-scrR group.
  • Mice with Huntington's disease were injected with CMV-siR in the tail vein.
  • mHTT plasmid, CMV-RVG-siR mHTT plasmid, CMV-scrR plasmid, spin assays were performed on days 0 and 14, and mice were sacrificed for analysis after 14 days.
  • this figure is a comparison chart of the descending latency of mice in wild-type mice, CMV-scrR group, and CMV-RVG-siR mHTT group. It can be seen that on day 0, CMV-scrR group, CMV-RVG- The descending latency of mice in the siR mHTT group was relatively consistent, and on day 14, the descending latency of the mice in the CMV-scrR group was the shortest.
  • Figure 47C is the western bolt image of the striatum of mice in the CMV-scrR group and the CMV-RVG-siR mHTT group
  • Figure 47D is the small mouse in the CMV-scrR group and the CMV-RVG-siR mHTT group.
  • the comparison of the relative mHTT mRNA levels in the striatum of the mice showed that the N171-mHTT protein content and the relative mHTT mRNA level were also higher in the striatum of the CMV-scrR group mice.
  • the experimental group was the CMV-RVG-siR mHTT group
  • the control group was the CMV-scrR group.
  • mice in the CMV-RVGsiR mHTT group and the CMV-scrR group with Huntington's disease were injected into the tail vein of the CMV-RVG-siR mHTT plasmid, CMV-scrR Plasmids, mice were sacrificed 14 days later for analysis.
  • Figure 47F is the western bolt image of the cortex and striatum of mice in the CMV-scrR group and CMV-RVG-siR mHTT group. It can be seen that the cortex and striatum of mice in the CMV-RVG-siR mHTT group The contents of mutant HTT (Mutant HTT) and endogenous HTT (Endogenous HTT) were lower.
  • Figure 47G is a comparison chart of the relative mHTT protein levels in the cortex and striatum of mice in the CMV-scrR group and the CMV-RVG-siR mHTT group. Relative mHTT protein levels were lower in mice in the RVG-siR mHTT group.
  • Figure 47H shows the immunofluorescence images of mice in the CMV-scrR group and CMV-RVG-siR mHTT group
  • Figure 47I shows the cortex and The comparison of the relative mHTT mRNA levels in the striatum shows that the relative mHTT mRNA levels of the mice in the CMV-RVG-siR mHTT group were lower in both the mouse cortex and the striatum.
  • the experimental group was the CMV-RVG-siR mHTT group
  • the control group was the CMV-scrR group.
  • mice 6-week-old YAC128 mice were selected, and mice in the CMV-RVG-siR mHTT group and the CMV-scrR group with Huntington's disease were injected into the tail vein of the CMV-RVG-siR mHTT plasmid and CMV.
  • -scrR plasmid, spin assays were performed on day 0, week 4, and week 8 of the experiment, and mice were sacrificed for analysis.
  • the figure is a comparison chart of the descending latency of wild-type mice, CMV-RVG-siR mHTT group, and CMV-scrR group mice. It can be seen that on day 0, CMV-RVG-siR mHTT group, CMV- The descending latencies of the mice in the scrR group were relatively consistent. At the fourth and eighth weeks, the mice in the CMV-scrR group had the shortest descending latencies.
  • the figure is the western bolt image of the cortex and striatum of mice in the CMV-RVG-siR mHTT group and CMV-scrR group. It can be seen that the mutant HTT in the cortex of the CMV-RVG-siR mHTT group mice and endogenous HTT contents were lower, the striatal mutant had lower HTT content, and striatum endogenous HTT content was higher.
  • this figure is the immunofluorescence image of the cortex and striatum of mice in the CMV-RVG-siR mHTT group and CMV-scrR group. It can be seen that the expressions of NeuN and EM48 in the CMV-RVG-siRmHTT group mice lower than the CMV-scrR group.
  • 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.
  • Figure 49A is a comparison chart of siRNA levels in plasma exosomes of mice in each group, it can be seen that the plasma exosomes of mice in AAV-CMV-siR mHTT group and AAV-CMV-RVG-siR mHTT group siRNA levels were 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 and cells. It can be seen that the AAV-CMV-siR mHTT group and AAV-CMV-RVGsiR The relative mHTT mRNA level of the mice in the mHTT group was lower, which indicated that AAV-CMV-siR mHTT and AAV-CMV-RVG-siR mHTT could reduce the HTT mRNA level, that is, the siRNA assembled into exosomes could still exert gene silencing function.
  • the figure is a comparison chart of the descending latency of wild-type mice (WT), AAV-CMV-siR mHTT group and AAV-CMV-RVG-siR mHTT group. It can be seen that at 0 weeks, The descending latencies of the three groups of mice 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 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.
  • 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 2 is specifically shown in Table 3 below.
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • Sequence 4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
  • the plasmid CMV-siR LRRK2 carrying the LRRK2 siRNA that inhibits the expression of LRRK2, and the plasmid CMV-RVG-siR LRRK2 carrying the targeting peptide RVG and LRRK2 siRNA were constructed according to the method described in Example 2.
  • the siRNA of LRRK2 gene is AUUAACAUGAAAAUAUCACUU (SEQ ID NO. 16).
  • LRRK2R1441G transgenic mice were selected for the experiment when they were 3 months old, and the experiment set up LPS intervention group and LPS non-intervention group.
  • the LPS intervention group was treated with CMV-scrR/CMV-RVG-siR LRRK2 after 7 days of LPS intervention.
  • Figure 50A is a western bolt image of LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2
  • Figure 50B is a LRRK2R1441G transgenic mouse injected with CMV-scrR/CMV-RVG-siR LRRK2
  • the grayscale analysis of the protein shows that the levels of LRRK2 protein and S935 protein in mice injected with CMV-RV G-siR LRRK2 decreased, indicating that CMV-RVG-siR LRRK2 can pass through the blood brain after the liver releases siRNA and assembles into exosomes
  • the barrier reduces the expression of proteins deep in the brain.
  • FIG. 50C is an immunofluorescence image of TH+ neurons in the substantia nigra of LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2 .
  • the results showed that mice injected with CMV-RVG-siR LRRK2 rescued TH
  • the loss of neurons indicates that CMV-RVG-siR LRRK2 can cross the blood-brain barrier and enter the deep brain to function after the liver releases siRNA and assembles into exosomes.
  • this figure is an immunofluorescence image of the activation level of microglia in LRRK2R1441G transgenic mice injected with CMV-scrR/CMV-RVG-siR LRRK2 .
  • the results show that injection of CMV-RVG-siR LRRK2 mice can inhibit microglia
  • the activation of CMV-RVG-siR LRRK2 indicates that CMV-RVG-siR LRRK2 can cross the blood-brain barrier and enter the deep brain to function after the liver releases siRNA and assembles into exosomes.
  • sequence 1 is preferably CAGATC
  • sequence 2 can be composed of 5-80 bases
  • Sequences of composition such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases
  • Any sequence may be used, preferably a sequence consisting of 10-50 bases, more preferably a sequence consisting of 20-40 bases, and sequence 3 is preferably TGGATC.
  • Sequence 2 is specifically shown in Table 3 below.
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
  • 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 2 is specifically shown in Table 3 below.
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • Sequence 4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
  • Embodiment 16 Security Detection
  • RNA delivery vector system provided by the present invention has been studied in more detail in cynomolgus monkeys (Macaca fascicularis).
  • the cynomolgus macaque is a well-known non-human primate model for safety assessment studies.
  • Figure 51A is a graph of siRNA concentration changes in cynomolgus monkey whole blood injected with a single injection
  • Figure 51B is a graph of siRNA concentration changes in cynomolgus monkey whole blood injected with multiple injections. It can be seen that a single injection of cynomolgus monkeys The siRNA concentration peaked at 6 hours after intravenous injection and then decreased, the multi-injected cynomolgus monkey siRNA concentration peaked at 3 hours after intravenous injection and then decreased, and the multi-injected cynomolgus monkey siRNA concentration decreased more slowly .

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Abstract

一种分离的核酸、其质粒或病毒载体、药物组合物以及治疗疾病的方法。所述核酸和载体包括至少一个能够抑制基因表达的RNA片段和/或具有靶向功能的靶向标签，能够被递送至宿主体内，并在宿主的器官组织中富集，自组装形成和分泌外泌体和靶向目标组织，从而对疾病进行治疗。

Description

核酸递送系统及其应用
本申请要求以下中国专利申请的优先权：2021年3月29日提交的、申请号为202110335617.9、发明名称为“一种RNA质粒递送系统及其应用”；2021年3月29日提交的、申请号为202110336982.1、发明名称为“一种基于病毒载体的RNA递送系统及其应用”；和2021年3月29日提交的、申请号为202110336983.6、发明名称为“一种基因线路、RNA递送系统及其应用”，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及分子生物学和药物领域。具体的，本发明涉及可治疗疾病的核酸分子、其递送系统和其在疾病治疗中的应用。
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相关的研究很多，主要聚焦在以下几个方面：1、设计具有治疗效果的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、设计各种人工载体(如脂质纳米粒子、阳离子聚合物和质粒)，以提高siRNA在体内传递的效率。其中第3方面的专利很多，其根本原因是研究人员们已经意识到目前缺乏合适的siRNA传递系统，将siRNA安全地、精确地、高效地输送到目标组织，该问题已经成为制约RNAi疗法的核心问题。
公开号为CN108624590A的中国专利公开了一种能够抑制DDR2基因表达的siRNA；公开号为CN108624591A的中国专利公开了一种能够沉默ARPC4基因的siRNA，并且对该siRNA进行了α-磷-硒修饰；公开号为CN108546702A的中国专利公开了一种靶向长链非编码RNA DDX11-AS1的siRNA。公开号为CN106177990A的中国专利公开了一种可以用于多种肿瘤治疗的siRNA前体。这些专利均设计了特定的siRNA并且来针对某些由基因变化引起的疾病。
公开号为CN108250267A的中国专利公开了一种多肽、多肽-siRNA诱导共组装体，使用多肽作为siRNA的载体。公开号为CN108117585A的中国专利公开了一种靶向导入siRNA促进乳腺癌细胞凋亡的多肽，同样使用多肽作为siRNA的载体。公开号为CN108096583A的中国专利公开了一种纳米粒子载体，该载体在包含化疗药物的同时还可以装载具有乳腺癌疗效的siRNA。这些专利均为在siRNA载体方面的发明创造，但是其技术方案具有一个共同特征，那就是载体和siRNA均在体外预先组装，然后再引入宿主体内。事实上，目前绝大部分设计的传递技术均是如此。然而这类传递体系具有共同的问题，那就是这些人工合成的外源性传递体系很容易被宿主的循环系统清除，也有可能引起免疫原性反应，甚至可能对特定的细胞类型和组织有毒。
本发明的研究团队发现内源性细胞可以将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
本发明提供了一种有效、安全和方便地按照需要将抑制基因表达的RNA在器官组织中组装形成复合结构并递送到靶组织或其细胞来治疗疾病的方法和药物。
具体的，在本发明的其中一个方面，提供了一种分离的核酸，其包括编码能够的核苷酸序列，其包含：
(a)编码一个或多个抑制基因表达的RNA的核苷酸序列，所述RNA为miRNA、shRNA、siRNA、mRNA、ncRNA、sgRNA或任意这些RNA的组合。
在本发明的其中一种实施方式中，上述分离的核酸还包括：(b)编码靶向蛋白的核苷酸序列。在本发明的其中一种实施方式中，所述靶向蛋白是组织特异性的蛋白。
在本发明的其中一种实施方式中，(a)为编码一个所述抑制基因表达的RNA的核苷酸序列。
在本发明的其中一种实施方式中，(a)为编码多个所述抑制基因表达的RNA的核苷酸序列。例如，所述多个抑制基因表达的RNA为2-4个抑制基因表达的RNA。
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的，但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
在本发明的其中一种实施方式中，所述抑制基因表达的RNA为抑制以下基因表达的RNA：EGFR基因，KRAS基因，VEGFR基因，mTOR基因，TNF-α基因,integrin-α基因，B7基因，TGF-β1基因，H2-K基因，H2-D基因，H2-L基因，HLA基因，GDF15基因，miRNA-21，miRNA-214，TNC基因，PTP1B基因，mHTT基因，Lrrk2基因，α-synuclein基因。
在本发明的其中一种实施方式中，(a)为siRNA。siRNA也称为短干扰RNA或沉默RNA，是一类双链RNA分子，长度一般为20-29个碱基对，其双链分别在RNA的两端超出另一端2个核苷酸。siRNA一般通过模拟miRNA产生机制来产生的，这样的siRNA就可从前体RNA(Precursor RNA，Pre-RNA)加工而来。前体RNA可折叠成稳定的茎环(发夹)结构，所述的茎环结构长度一般在50-100bp之间。茎环结构的茎部两侧包含基本上互补的两条序列。
siRNA可与编码基因的mRNA的至少一部分序列基本上互补。“基本上互补”是指核苷酸的序列是足够互补的，可以以一种可预见的方式发生相互作用，如形成二级结构。通常，两条“基本上互补”的核苷酸序列互相之间至少有70％的核苷酸是互补的；优选的，至少有80％的核苷酸是互补的；更优选的，至少有90％的核苷酸是互补的；进一步优选的，至少有95％的核苷酸是互补的；如98％、99％或100％。在功能上，siRNA干扰了表达与互补的核苷酸序列的特定基因的转录后降解的mRNA，从而防止翻译。
在本发明的其中一种实施方式中，(a)为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。
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。具有抑制前述基因表达的 功能的RNA序列均可用于本发明。以下为部分效果较优的RNA序列：
EGFR基因的siRNA包括UGUUGCUUCUCUUAAUUCCU、AAAUGAUCUUCAAAAGUGCCC、UCUUUAAGAAGGAAAGAUCAU、AAUAUUCGUAGCAUUUAUGGA、UAAAAAUCCUCACAUAUACUU。
KRAS基因的siRNA包括UGAUUUAGUAUUAUUUAUGGC、AAUUUGUUCUCUAUAAUGGUG、UAAUUUGUUCUCUAUAAUGGU、UUAUGUUUUCGAAUUUCUCGA、UGUAUUUACAUAAUUACACAC。
VEGFR基因的siRNA包括AUUUGAAGAGUUGUAUUAGCC、UAAUAGACUGGUAACUUUCAU、ACAACUAUGUACAUAAUAGAC、UUUAAGACAAGCUUUUCUCCA、AACAAAAGGUUUUUCAUGGAC。
mTOR基因的siRNA包括AGAUAGUUGGCAAAUCUGCCA、ACUAUUUCAUCCAUAUAAGGU、AAAAUGUUGUCAAAGAAGGGU、AAAAAUGUUGUCAAAGAAGGG、UGAUUUCUUCCAUUUCUUCUC。
TNF-α基因的siRNA包括AAAACAUAAUCAAAAGAAGGC、UAAAAAACAUAAUCAAAAGAA、AAUAAUAAAUAAUCACAAGUG、UUUUCACGGAAAACAUGUCUG、AAACAUAAUCAAAAGAAGGCA。
integrin-α基因的siRNA包括AUAAUCAUCUCCAUUAAUGUC、AAACAAUUCCUUUUUUAUCUU、AUUAAAACAGGAAACUUUGAG、AUAAUGAAGGAUAUACAACAG、UUCUUUAUUCAUAAAAGUCUC。
B7基因的siRNA包括UUUUCUUUGGGUAAUCUUCAG、AGAAAAAUUCCACUUUUUCUU、AUUUCAAAGUCAGAUAUACUA、ACAAAAAUUCCAUUUACUGAG、AUUAUUGAGUUAAGUAUUCCU。
TGF-β1基因的siRNA包括ACGGAAAUAACCUAGAUGGGC、UGAACUUGUCAUAGAUUUCGU、UUGAAGAACAUAUAUAUGCUG、UCUAACUACAGUAGUGUUCCC、UCUCAGACUCUGGGGCCUCAG。
H2-K基因的siRNA包括AAAAACAAAUCAAUCAAACAA、UCAAAAAAACAAAUCAAUCAA、UAUGAGAAGACAUUGUCUGUC、AACAAUCAAGGUUACAUUCAA、ACAAAACCUCUAAGCAUUCUC。
H2-D基因的siRNA包括AAUCUCGGAGAGACAUUUCAG、AAUGUUGUGUAAAGAGAACUG、AACAUCAGACAAUGUUGUGUA、UGUUAACAAUCAAGGUCACUU、AACAAAAAAACCUCUAAGCAU。
H2-L基因的siRNA包括GAUCCGCUCCCAAUACUCCGG、AUCUGCGUGAUCCGCUCCCAA、UCGGAGAGACAUUUCAGAGCU、UCUCGGAGAGACAUUUCAGAG、AAUCUCGGAGAGACAUUUCAG。
HLA基因的siRNA、AUCUGGAUGGUGUGAGAACCG、UGUCACUGCUUGCAGCCUGAG、UCACAAAGGGAAGGGCAGGAA、UUGCAGAAACAAAGUCAGGGU、ACACGAACACAGACACAUGCA。
GDF15基因的siRNA包括UAUAAAUACAGCUGUUUGGGC、AGACUUAUAUAAAUACAGCUG、AAUUAAUAAUAAAUAACAGAC、AUCUGAGAGCCAUUCACCGUC、UGCAACUCCAGCUGGGGCCGU。
TNC基因的siRNA包括UAUGAAAUGUAAAAAAAGGGA、AAUCAUAUCCUUAAAAUGGAA、UAAUCAUAUCCUUAAAAUGGA、UGAAAAAUCCUUAGUUUUCAU、AGAAGUAAAAAACUAUUGCGA。
PTP1B基因的siRNA包括UGAUAUAGUCAUUAUCUUCUU、UCCAUUUUUAUCAAACUAGCG、AUUGUUUAAAUAAAUAUGGAG、 AAUUUUAAUACAUUAUUGGUU、UUUAUUAUUGUACUUUUUGAU。
mHTT基因的siRNA包括UAUGUUUUCACAUAUUGUCAG、AUUUAGUAGCCAACUAUAGAA、AUGUUUUUCAAUAAAUGUGCC、UAUGAAUAGCAUUCUUAUCUG、UAUUUGUUCCUCUUAAUACAA。
Lrrk2基因的siRNA包括AUUAACAUGAAAAUAUCACUU、UUAACAAUAUCAUAUAAUCUU、AUCUUUAAAAUUUGUUAACGC、UUGAUUUAAGAAAAUAGUCUC、UUUGAUAACAGUAUUUUUCUG。
α-synuclein基因的siRNA包括AUAUAUUAACAAAUUUCACAA、AAGUAUUAUAUAUAUUAACAA、AUAACUUUAUAUUUUUGUCCU、UAACUAAAAAAUUAUUUCGAG、UCGAAUAUUAUUUAUUGUCAG。
本领域的技术人员可以理解，可用于本发明的RNA序列也同时包括与前述RNA的同源性大于80％的RNA序列。例如同源性为85％、88％、90％、95％、98％等。
在本发明的其中一种实施方式中，所述分离的核酸中所述编码一个或多个抑制基因表达的RNA的核苷酸序列包括针对所述基因的RNA片段序列。所述RNA片段序列通常为与所述基因的目标核苷酸序列互补的RNA序列。当所述RNA为siRNA时，所述RNA片段序列为siRNA的正义链序列。
在本发明的其中一种实施方式中，所述分离的核酸中所述编码一个或多个抑制基因表达的RNA的长度为15-29个核苷酸(nt)，优选为18-22nt，比如18nt、19nt、20nt、21nt、22nt。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。
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。
在本发明的其中一种实施方式中，所述分离的核酸中所述编码一个或多个抑制基因表达的RNA的核苷酸还包括侧翼序列(如5’侧翼序列和3’侧翼序列)、茎环序列和所述RNA序列的补偿序列中的一个或多个。
所述补偿序列为所述RNA片段序列的反向互补序列。在本发明的其中一种实施方式中，所述补偿序列为所述RNA片段序列的反向互补序列并删除其中任意1-5位碱基。在本发明的其中又一种实施方式中，所述补偿序列为所述RNA片段序列的反向互补序列并删除其中任意1-3位碱基，特别是其中1-3位连续排列的碱基。最优选的，所述补偿序列为为所述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。通常而言，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。
侧翼序列是用于帮助RNA分子如siRNA分子剪切成正确的最终序列的序列。在本发明中，可采用天然miRNA前体的侧翼结构作为本发明的RNA分子的侧翼结构。例如，采用pre-miR-155的侧翼结构。
在本发明的其中一种实施方式中，所述5’侧翼序列为ggatcctggaggcttgctgaaggctgtatgctgaattc或与其同源性大于80％的序列，例如为与所述序列同源性为85％、90％、92％、95％、98％、99％的序 列等。
在本发明的其中一种实施方式中，所述3’侧翼序列为accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag或与其同源性大于80％的序列，例如为与所述序列同源性为85％、90％、92％、95％、98％、99％的序列等。
茎环结构是编码发卡结构的间隔序列，其可维持所述RNA的稳定性。在本发明的其中一种实施方式中，所述茎环结构的序列优选为gttttggccactgactgac或与其同源性大于80％的序列。
在本发明的其中一种实施方式中，所述分离的核酸中所述编码一个或多个抑制基因表达的RNA的依序具有：5’侧翼序列、RNA片段序列、茎环序列、补偿序列和3’侧翼序列。在本发明的其中一种实施方式中，所述RNA的5’具有启动子。
本申请的发明人出乎意料地发现并通过实验证明，在上述特定侧翼序列、补偿序列、茎环结构序列的配合下，能够最大程度的转录和剪切出所需的RNA序列，并将其包裹在外泌体中。
在本发明的其中一种实施方式中，所述核酸中的(a)为编码多个所述抑制基因表达的RNA的核苷酸序列。例如，所述多个抑制基因表达的RNA为2-4个抑制基因表达的RNA。当(a)为编码多个所述抑制基因表达的RNA的核苷酸序列时，所述多个RNA之间通过连接子相连。所述连接子的结构例如为序列1-序列2-序列3。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，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。在本发明的其中一种实施方式中，所述连接子的序列为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
在本发明的其中一种实施方式中，所述靶向蛋白为靶组织特异性靶向肽。
在本发明的其中一种实施方式中，所述靶向蛋白为靶组织特异性靶向肽和膜蛋白的融合蛋白。
在本发明的其中一种实施方式中，所述特异性靶向肽选自：RVG靶向肽、GE11靶向肽、PTP靶向肽、TCP-1靶向肽、MSP靶向肽。
在本发明的其中一种实施方式中，所述靶向蛋白为膜蛋白，其例如选自：细胞受体蛋白(例如生长因子受体)、LAMP1或LAMP2(例如为LAMP2B)、抗体或其结合片段。
在本发明的其中一种实施方式中，所述靶向蛋白为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。
在本发明的其中一种实施方式中，所述靶组织为脑、松果体、垂体、眼、耳、鼻、口、咽、腮腺、扁桃体、食道、气管、甲状腺、胸腺、乳腺、肺、心脏、胃、肠、阑尾、肝脏、胆囊、脾脏、胰腺、肾脏、输尿管、膀胱、尿道、子宫、卵巢、输卵管、阴道、输精管、前列腺、阴茎、睾丸、肛门、骨骼、肌肉、结缔组织、神经、淋巴、结直肠、血液、骨髓和/或皮肤等。在本发明的其中一种实施方式中，所述靶细胞为上述靶组织的细胞。
在本发明的其中一个方面，所述核酸在给药哺乳动物后，在组织(包括：肝脏、肺脏、胃肠道、乳腺、肾脏、脑、脾脏、淋巴、甲状腺、生殖器官、血细胞或淋巴细胞，特别是肝脏)中富集，并且其表达的产物被大量包裹在这些组织的细胞产生和分泌的外泌体中，并递送至目标组织，发挥治疗作用。因此，所述核酸编码的靶向蛋白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。
适于用于本发明的靶向肽包括但不限于RVG靶向肽(核苷酸序列如SEQ ID No：1所示)、GE11靶向肽(核苷酸序列如SEQ ID No：2所示)、PTP靶向肽(核苷酸序列如SEQ ID No：3所示)、TCP-1靶向肽(核苷酸序列如SEQ ID No：4所示)、MSP靶向肽(核苷酸序列如SEQ ID No：5所示)；靶向蛋白包括但不限于RVG-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：6所示)、GE11-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：7所示)、PTP-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：8所示)、TCP-1-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：9所示)、MSP-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：10所示)。
其中，RVG靶向肽、RVG-LAMP2B融合蛋白可以精准靶向脑组织；GE11靶向肽、GE11-LAMP2B融合蛋白可以精准靶向EGFR高表达的器官组织，比如EGFR突变的肺癌组织；PTP靶向肽、 PTP-LAMP2B融合蛋白可以精准靶向胰腺，特别是人源及鼠源胰腺癌组织中特异性表达的plectin-1蛋白；TCP-1靶向肽、TCP-1-LAMP2B融合蛋白可以精准靶向结肠；MSP靶向肽、MSP-LAMP2B融合蛋白可以精准靶向肌肉组织。
在实际应用中，靶向蛋白可以搭配各种不同的抑制基因表达的RNA，在不同的组织中对其特异性目标基因进行抑制。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。
在本发明的其中一种实施方式中，所述核酸中的(a)为编码多个所述抑制基因表达的RNA的核苷酸序列。所述多个抑制基因表达的RNA可同时或分开给与治疗的对象。在本发明的其中一种实施方式中，所述多个RNA可分别位于不同的质粒载体或病毒载体。例如其中一种质粒或病毒载体包含启动子和靶向标签，其他质粒包含启动子和RNA片段。即将靶向标签与RNA片段装载到不同的载体中，将两种载体或更多种同时或分开注入体内。更优选地，所述两种或更多种不同的载体注入宿主体内时，可以先将装有RNA序列的载体注入，然后(如在1-2小时后)再注入含有靶向标签的载体，如此能够达到更优的靶向效果。
在本发明的其中一种实施方式中，所述核酸在哺乳动物的肝脏中富集，其产物在肝细胞中被包裹在外泌体中。
在本发明的其中一个方面，提供了一种抑制基因表达的RNA的载体，其包含：
(a)编码一个或多个抑制基因表达的RNA的核苷酸序列，所述RNA为miRNA、shRNA、siRNA、mRNA、ncRNA、sgRNA或任意这些RNA的组合；
任选的，(b)编码靶向蛋白的核苷酸序列。
在本发明的其中一种实施方式中，所述载体中包含前面描述的本发明提供的分离的核酸，其编码能够抑制基因表达的RNA的核苷酸序列。
在本发明的其中一种实施方式中，所述载体为质粒。在本发明的其中一种实施方式中，所述质粒在给予哺乳动物后，可在组织(包括：肝脏、肺脏、胃肠道、乳腺、肾脏、脑、脾脏、淋巴、甲状腺、生殖器官、血细胞或淋巴细胞，特别是肝脏)中富集，转录和/或表达出本发明RNA片段，并且所述RNA片段在该组织的细胞中被包裹在外泌体中。
在本发明的其中一种实施方式中，所述载体为病毒载体。例如可为杆状病毒表达载体，腺病毒载体，逆转录病毒载体，孢疹病毒载体或慢病毒载体等。
在本发明的其中一种实施方式中，所述载体为腺病毒载体。优选的，所述腺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。更优选的，所述腺病毒为腺病毒相关病毒5型。
在本发明的其中一种实施方式中，所述质粒或病毒载体在给药哺乳动物后，在肝脏中富集和表达，其产物被大量包裹在外泌体中。
在本发明的其中一个方面，提供了一种具有抑制基因表达的RNA的外泌体，其包含如前所述的核酸或载体。在本发明的其中一种实施方式中，所述外泌体为人体组织或细胞来源的外泌体。所述组织包括肝脏、肺脏、胃肠道、乳腺、肾脏、脑、脾脏、淋巴、甲状腺、生殖器官、血细胞或淋巴细胞。在本发明的其中一种实施方式中，所述外泌体为肝脏或肝脏细胞来源的外泌体。
在本发明的其中一个方面，提供了一种药物组合物，其含有如前所述的核酸、载体或外泌体。所述药物组合物还包括将所述核酸、载体或外泌体递送给对象的可药用载体或赋形剂。
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。即所述药物可以通过口服、吸入、皮下注射、肌肉注射或静脉注射的方式。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。所述药物中的质粒或病毒载体在给药哺乳动物后，在组织(包括：肝脏、肺脏、胃肠道、乳腺、肾脏、脑、脾脏、淋巴、甲状腺、 生殖器官、血细胞或淋巴细胞，特别是肝脏)中富集，其表达的产物在该组织的细胞中被大量包裹在外泌体中并递送至目标组织，发挥治疗作用。
所述药物组合物可用于治疗各种疾病，包括肿瘤、急慢性传染病或其它急慢性疾病。其中所述的急慢性传染病包括：病毒性流感、病毒性肝炎、艾滋病、SARS的病毒性疾病，细菌性疾病(例如结核、细菌性肺炎)，以及其它各种病原微生物导致的急慢性传染病。所述的其它急慢性疾病包括：呼吸系统疾病，免疫系统疾病，血液与造血系统疾病，如心脑血管疾病的循环系统疾病，内分泌系统代谢性疾病，消化系统疾病，神经系统疾病，泌尿系统疾病，生殖系统疾病和运动系统疾病。例如，所述疾病为癌症、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。
在本发明的其中一个方面，提供了治疗疾病的方法，其中包括向对象施用如前所述的核酸、载体或外泌体。所述疾病包括肿瘤、急慢性传染病或其它急慢性疾病。
本领域技术人员理解的是，给药的实际剂量取决于多种因素而变化，如载体、靶细胞或组织、有待治疗的受试者的一般状况、所寻求的转化/修饰的程度、给药途径、给药方式、所寻求的转化/修饰的类型，等等。
附图说明
图1是本申请一实施例提供的小鼠体内质粒分布与代谢情况对比图；
图2是本申请一实施例提供的小鼠体内蛋白表达水平对比图；
图3是本申请一实施例提供的小鼠体内相关siRNA水平对比图；
图4是本申请一实施例提供的小鼠各组织中绝对siRNA水平对比图；
图5是本申请一实施例提供的质粒剂量对小鼠siRNA水平的影响对比图；
图6是本申请一实施例提供的注射质粒后的小鼠肝脏内前体及成熟体的代谢情况对比图；
图7是本申请一实施例提供的小鼠不同组织中siRNA动力学和分布情况对比图；
图8是本申请一实施例提供的不同启动子对siRNA的影响对比图；
图9是本申请一实施例提供的小鼠不同组织中eGFP荧光强度对比图；
图10是本申请一实施例提供的小鼠谷丙转氨酶、谷草转氨酶、总胆红素、血尿素氮、血清碱性磷酸酶、肌酐含量以及胸腺重量、脾脏重量、外周血细胞百分比对比图；
图11是本申请一实施例提供的小鼠EGFR突变肺癌肿瘤治疗效果对比图；
图12是本申请一实施例提供的小鼠HE染色图、免疫组织染色图以及着色情况统计图；
图13是本申请一实施例提供的小鼠KRAS突变肺癌肿瘤治疗效果对比图；
图14是本申请一实施例提供的小鼠HE染色图、免疫组织染色图以及着色情况统计图；
图15是本申请一实施例提供的6种不同RNA的质粒治疗肺癌后的荧光信号统计，图中A为CMV-siR E和Albumin-siR E的荧光检测结果，B为CMV-siR T和Albumin-siR T的荧光检测结果，C为CMV-miR7和Albumin-miR7的荧光检测结果，D为CMV-shR E和Albumin-shR E的荧光检测结果，E为CMV-shR T和Albumin-shR T的荧光检测结果，F为CMV-miR133b和Albumin-miR133b的荧光检测结果。
图16是本申请一实施例提供的6种RNA中任意2种RNA序列组成的4组质粒治疗肺癌后的荧光信号统计，均是以CMV或Albumin连接RNA，图中A为siR E+shR T的荧光检测结果，B为shR E+miR133b的荧光检测结果，C为siR T+miR7的荧光检测结果，D为shR T+miR133b的荧光检测结果。
图17是本申请一实施例提供的6种RNA中任意3种RNA序列组成的3组质粒治疗肺癌后的荧光信号统计，均是以CMV或Albumin连接RNA，图中A为siR E+shR T+miR7的荧光检测结果，B为siR T+shR E+miR7的荧光检测结果，C为shR E+siR T+miR133b的荧光检测结果。
图18是本申请一实施例提供的分别静脉注射质粒CMV-siR E和质粒CMV-GE11-siR E后siRNA在 肝、肺、血浆、外泌体中的富集结果，图中A为有/无靶向肽GE11的情况下在肝、肺中的EGFR siRNA含量，B为有/无靶向肽GE11的情况下在血浆、外泌体中的EGFR siRNA含量。
图19是本申请一实施例提供的分别静脉注射质粒CMV-siR E和质粒CMV-GE11-siR E后EGFR蛋白及mRNA的表达量检测结果，图中A为有/无靶向肽GE11的情况下检测EGFR的蛋白含量，B为有/无靶向肽GE11的情况下检测EGFR的mRNA含量。
图20是本申请一实施例提供的含有5’侧翼序列同源性大于80％的序列的质粒在肺部富集效果以及治疗效果，图中A为有/无靶向标签RVG的情况下2条5’侧翼同源序列的EGFR siRNA含量，B为有/无靶向标签RVG的情况下1条5’侧翼同源序列的荧光信号检测结果，C为有/无靶向标签RVG的情况下另1条5’侧翼同源序列的荧光信号检测结果。
图21是本申请一实施例提供的含有loop序列同源性大于80％的序列的质粒在肺部富集效果以及治疗效果，图中A为有/无靶向标签RVG的情况下2条loop同源序列的EGFR siRNA含量，B为有/无靶向标签RVG的情况下1条loop同源序列的荧光信号检测结果，C为有/无靶向标签RVG的情况下另1条loop同源序列的荧光信号检测结果。
图22是本申请一实施例提供的含有3’侧翼序列同源性大于80％的序列的质粒在肺部富集效果以及治疗效果，图中A为有/无靶向标签RVG的情况下2条3’侧翼同源序列的EGFR siRNA含量，B为有/无靶向标签RVG的情况下1条3’侧翼同源序列的荧光信号检测结果，C为有/无靶向标签RVG的情况下另1条3’侧翼同源序列的荧光信号检测结果。
图23是本申请一实施例提供的分别含有序列4以及2条与序列4同源性大于80％的序列4-1、4-2的质粒静脉注射9小时后肺部组织的EGFR siRNA含量检测结果，RNA为siR E/siR T，图中A为序列4的EGFR siRNA含量检测结果，B为序列4-1的EGFR siRNA含量检测结果，C为序列4-2的EGFR siRNA含量检测结果。
图24是本申请一实施例提供的分别含有长度为18、20、22的RNA序列的3种质粒静脉注射后EGFR表达量检测，图中A为EGFR的蛋白含量检测结果，B为EGFR的mRNA含量检测结果。
图25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26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27是本申请一实施例提供的小鼠多种酶含量对比图；
图28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为静脉注射慢病毒载体后在肝和肺中的EGFR siRNA富集效果，B静脉注射慢病毒载体后在血浆和外泌体中的EGFR siRNA富集效果，C静脉注射慢病毒载体后的EGFR蛋白表达效果，D静脉注射慢病毒载体后的EGFR mRNA表达效果。
图29是本申请一实施例提供的腺病毒载体在肝、肺、血浆、外泌体的富集效果以及EGFR基因表达量检测，图中，A为静脉注射腺病毒载体后在肝和肺中的EGFR siRNA富集效果，B静脉注射腺病毒载体后在血浆和外泌体中的EGFR siRNA富集效果，C静脉注射腺病毒载体后的EGFR蛋白表达效果，D静脉注射腺病毒载体后的EGFR mRNA表达效果。
图30是本申请一实施例提供的将6种不同RNA分别构建进腺相关病毒载体治疗肺癌后的荧光信号统计，图中A为siR E构建进腺相关病毒载体治疗肺癌后的荧光信号统计，B为siR T构建进腺相关病毒载体治疗肺癌后的荧光信号统计，C为miR-7构建进腺相关病毒载体治疗肺癌后的荧光信号统计，D为shR E构建进腺相关病毒载体治疗肺癌后的荧光信号统计，E为shR T构建进腺相关病毒载体治疗肺癌后的荧光信号统计，F为miR-133b构建进腺相关病毒载体治疗肺癌后的荧光信号统计。
图31是本申请一实施例提供的将6种不同RNA中任意2种RNA序列组成的4组RNA片段分别构建进腺相关病毒载体治疗肺癌后的荧光信号统计，图中A为siR E+shR T构建进腺相关病毒载体治疗肺癌后的荧光信号统计，B为siR T+miR-7构建进腺相关病毒载体治疗肺癌后的荧光信号统计，C为shR E+miR-133b构建进腺相关病毒载体治疗肺癌后的荧光信号统计，D为shR T+miR-133b构建 进腺相关病毒载体治疗肺癌后的荧光信号统计。
图32是本申请一实施例提供的将6种不同RNA中任意3种RNA序列组成的3组RNA片段分别构建进腺相关病毒载体治疗肺癌后的荧光信号统计，图中A为siR E+shR T+miR-7构建进腺相关病毒载体治疗肺癌后的荧光信号统计，B为siR T+shR E+miR-7构建进腺相关病毒载体治疗肺癌后的荧光信号统计，C为shR E+siR T+miR-133b构建进腺相关病毒载体治疗肺癌后的荧光信号统计。
图33是本申请一实施例提供的静脉注射后siRNA在肝、肺、血浆、外泌体种的富集结果和EGF R蛋白及mRNA的表达量检测结果，图中A为AAV-siR E和AAV-GE11-siR E在肝、肺中的富集结果，B为AAV-siR E和AAV-GE11-siR E在血浆、外泌体中的富集结果，C为AAV-siR E和AAV-GE11-siR E的EGFR蛋白表达量，D为AAV-siR E和AAV-GE11-siR E的EGFR mRNA表达量。
图34是本申请一实施例提供的与5’侧翼序列同源性大于80％的2条序列构建进AAV载体后，在肺部富集效果及治疗效果，图中A为通过EGFR siRNA含量显示出在肺部的富集结果，B为其中1条序列的治疗效果，C为另一条序列的治疗效果。
图35是本申请一实施例提供的与loop序列同源性大于80％的序列构建进AAV载体后，在肺部富集效果及治疗效果，图中A为通过EGFR siRNA含量显示出在肺部的富集结果，B为其中1条序列的治疗效果，C为另一条序列的治疗效果。
图36是本申请一实施例提供的与3’侧翼序列同源性大于80％的序列构建进AAV载体后，在肺部富集效果及治疗效果，图中A为通过EGFR siRNA含量显示出在肺部的富集结果，B为其中1条序列的治疗效果，C为另一条序列的治疗效果。
图37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EGFR siRNA含量检测结果，图中A为序列4的检测结果，B为序列4-1的检测结果，C为序列4-2的检测结果。
图38是本申请一实施例提供的含有3种不同长度RNA序列的基因环路静脉注射后EGFR表达量检测，图中A为EGFR蛋白含量结果，B为EGFR mRNA含量结果。
图39是本申请一实施例提供的小鼠肾癌肿瘤影像对比图；
图40是本申请一实施例提供的小鼠肾癌肿瘤发展情况对比图；
图41是本申请一实施例提供的含有RNA片段的腺相关病毒(AAV)载体所具有的体内(血浆、外泌体)富集、自组装和结直肠癌、胰腺癌、胶质瘤、肺癌、肾癌的治疗效果图(以siRNA含量显示)。
图42是本申请一实施例提供的腺相关病毒(AAV)做为病毒载体，其中含有siR E、siR V、siR K和siR E+T时，具有体内富集、自组装及肺癌、肾癌、胰腺癌、肥胖症和胶质瘤的治疗效果，图中A-E分别为肺癌、肾癌、胰腺癌、肥胖症和胶质瘤的荧光信号检测结果。
图43是本申请另一实施例提供的慢病毒(LV)做为病毒载体，其中含有siR E、siR V、siR K和si R E+T时，具有体内富集、自组装及肺癌、肾癌、胰腺癌、肥胖症和胶质瘤的治疗效果，图中A-E分别为肺癌、肾癌、胰腺癌、肥胖症和胶质瘤的荧光信号检测结果。
图44是本申请一实施例提供的病毒载体递送系统中，携带有多个RNA片段的情况下，均具有体内富集、自组装及针对肺癌的治疗效果，图中A为RNA序列单独作用时的肿瘤体积效果显示，B为2-3个RNA片段组成RNA序列作用时的肿瘤体积效果显示。
图45是本申请一实施例提供的病毒载体递送系统中，携带有多个RNA片段的情况下，均具有体内富集、自组装及针对肾癌的治疗效果，图中A为RNA序列单独作用时的肿瘤体积效果显示，B为2-3个RNA片段组成RNA序列作用时的肿瘤体积效果显示。
图46是本申请一实施例提供的病毒载体递送系统中，携带有多个RNA片段的情况下，均具有体内富集、自组装及针对结直肠癌的治疗效果，图中A为RNA序列单独作用时的肿瘤体积效果显示， B为2-3个RNA片段组成RNA序列作用时的肿瘤体积效果显示。
图47是本申请一实施例提供的病毒载体递送系统中，携带有多个RNA片段的情况下，均具有体内富集、自组装及针对胰腺癌的治疗效果，图中A为RNA序列单独作用时的肿瘤体积效果显示，B为2-3个RNA片段组成RNA序列作用时的肿瘤体积效果显示。
图48是本申请一实施例提供的病毒载体递送系统中，携带有多个RNA片段的情况下，均具有体内富集、自组装及针对胶质瘤的治疗效果，图中A为RNA序列单独作用时的肿瘤体积效果显示，B为2-3个RNA片段组成RNA序列作用时的肿瘤体积效果显示。
图49是本申请一实施例提供的腺病毒载体递送系统中包含有1-2个RNA片段和1-2个靶向标签时，其也具有体内富集、自组装及癌症的治疗效果，图中A为递送系统对胰腺癌的治疗效果，载体为AAV，携带的线路为siR K或PTP-siR K，B为递送系统对胶质瘤的治疗效果，载体为AAV，携带的线路为siR E+T或RVG-siR E+T。
图50是本申请一实施例提供的腺病毒载体携带有多个不同5’侧翼序列/loop序列/3’侧翼序列的RNA片段时，其具有体内富集、自组装及针对肺癌、肾癌、胰腺癌和胶质瘤的治疗效果，图中A显示为以siR E序列连接不同的2条5’侧翼序列/loop序列/3’侧翼序列，且在连接有或不连接有RVG的情况下，对肺癌肿瘤体积的影响效果，B显示为以siR V序列连接不同的2条5’侧翼序列/loop序列/3’侧翼序列，且在连接有或不连接有RVG的情况下，对肾癌肿瘤体积的影响效果，C显示为以siR P序列连接不同的2条5’侧翼序列/loop序列/3’侧翼序列，且在连接有或不连接有RVG的情况下，对胰腺癌肿瘤体积的影响效果，D显示为以siR E+T序列连接不同的2条5’侧翼序列/loop序列/3’侧翼序列，且在连接有或不连接有RVG的情况下，对胶质瘤肿瘤体积的影响效果。
图51是本申请一实施例提供的腺病毒载体携带有多个不同5’侧翼序列/loop序列/3’侧翼序列的RNA片段时，其具有体内富集、自组装及针对结直肠癌的治疗效果，图中显示为以siR V序列连接不同的2条5’侧翼序列/loop序列/3’侧翼序列，且在连接有或不连接有RVG的情况下，对结直肠癌肿瘤体积的影响效果。
图52是本申请一实施例提供的连接序列为序列4以及与序列4同源性大于80％的序列4-1和序列4-2时，含有以上序列的递送系统也具有相应的富集、自组装及癌症治疗效果，图中分别显示了序列4/4-1/4-2的EGFR siRNA含量检测结果，所连接的RNA分别为siR E和siR T。
图53是本申请一实施例提供的RNA序列的长度分别为18、20、22时，含有RNA序列的递送系统也具有相应的富集、自组装和癌症治疗效果，图中A为3种长度的RNA序列构建的递送系统注射后检测的EGFR蛋白含量，B为3种长度的RNA序列构建的递送系统注射后检测的EGFR mRNA含量。
图54是本申请一实施例提供的小鼠结肠炎发展情况对比图；
图55是本申请一实施例提供的小鼠结肠HE染色情况对比图；
图56是本申请一实施例提供的小鼠结肠炎发展情况对比图；
图57是本申请一实施例提供的小鼠结肠HE染色对比图；
图58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59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60是本申请一实施例提供的小鼠结肠炎治疗情况对比图；
图61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62是本申请一实施例提供的小鼠多种siRNA、mRNA水平对比图；
图63是本申请一实施例提供的小鼠结肠HE染色情况对比图；
图64是本申请一实施例提供的以TNF-α siRNA-慢病毒注射小鼠，体内富集TNF-α siRNA的结果图，图中A为肝富集结果，B为血浆富集结果，C为结肠富集结果。
图65是本申请一实施例提供的以TNF-α siRNA-慢病毒注射小鼠，通过血浆外泌体里检测TNF-α siRNA确定自发形成复合结构的结果图。
图66是本申请一实施例提供的以TNF-α siRNA-慢病毒注射小鼠后，具体疗效的结果图，图中A为疾病指数评分，B为炎症因子检测结果，C为靶基因mRNA检测结果。
图67是本申请一实施例提供的分别以包含有6种不同RNA的病毒载体注射小鼠后的体内富集结果图，6种RNA分别为：miR-19a(靶基因TNF-α)，miR-124-3p(靶基因TNF-α)，B7-siRNA-1，B7-siRNA-2，integrin α4 shRNA-1，integrin α4 shRNA-2，图中A为肝内检测small RNA表达结果，B为血浆内检测small RNA表达结果，C为结肠内检测small RNA表达结果。
图68是本申请一实施例提供的分别以包含有6种不同RNA的病毒载体注射小鼠后的具体疗效结果图，6种RNA分别为：miR-19a(靶基因TNF-α)，miR-124-3p(靶基因TNF-α)，B7-siRNA-1，B7-siRNA-2，integrin α4 shRNA-1，integrin α4 shRNA-2，图中A为疾病指数评分，B为炎症因子检测结果。
图69是本申请一实施例提供的分别以包含有4组不同RNA片段的病毒载体注射小鼠后的体内富集结果图，4组RNA片段分别包含有任意2种RNA序列，具体为：miR-19a(靶基因TNF-α)+B7-siRNA-1，miR-124-3p(靶基因TNF-α)+B7-siRNA-2，B7-siRNA-1+integrin α4 shRNA-1，B7-siRNA-2+integrin α4 shRNA-2，图中A为肝内检测small RNA(序列1)表达结果，B为血浆内检测small RNA(序列1)表达结果，C为结肠内检测small RNA(序列1)表达结果。
图70是本申请另一实施例提供的分别以包含有4组不同RNA片段的病毒载体注射小鼠后的体内富集结果图，4组RNA片段分别包含有任意2种RNA序列，具体为：miR-19a(靶基因TNF-α)+B7-siRNA-1，miR-124-3p(靶基因TNF-α)+B7-siRNA-2，B7-siRNA-1+integrin α4 shRNA-1，B7-siRNA-2+integrin α4 shRNA-2，图中A为肝内检测small RNA(序列2)表达结果，B为血浆内检测small RNA(序列2)表达结果，C为结肠内检测small RNA(序列2)表达结果。
图71是本申请一实施例提供的分别以包含有4组不同RNA片段的病毒载体注射小鼠后，具体疗效的结果图，4组RNA片段分别包含有任意2种RNA序列，具体为：miR-19a(靶基因TNF-α)+B7-siRNA-1，miR-124-3p(靶基因TNF-α)+B7-siRNA-2，B7-siRNA-1+integrin α4 shRNA-1，B7-siRNA-2+integrin α4 shRNA-2，图中A为疾病指数评分，B为炎症因子检测结果。
图72是本申请一实施例提供的分别以包含有3组不同RNA片段的病毒载体注射小鼠后的体内富集结果图，3组RNA片段分别包含有任意3种RNA序列，具体为：miR-19a(靶基因TNF-α)+B7-siRNA-1+integrin α4 shRNA-1，miR-124-3p(靶基因TNF-α)+B7-siRNA-2+integrin α4 shRNA-2，miR-19a(靶基因TNF-α)+B7-siRNA-1+integrin α4 shRNA-2，图中A为肝内检测small RNA(序列1)表达结果，B为血浆内检测small RNA(序列1)表达结果，C为结肠内检测small RNA(序列1)表达结果。
图73是本申请另一实施例提供的分别以包含有3组不同RNA片段的病毒载体注射小鼠后的体内富集结果图，3组RNA片段分别包含有任意3种RNA序列，具体为：miR-19a(靶基因TNF-α)+B7-siRNA-1+integrin α4 shRNA-1，miR-124-3p(靶基因TNF-α)+B7-siRNA-2+integrin α4 shRNA-2，miR-19a(靶基因TNF-α)+B7-siRNA-1+integrin α4shRNA-2，图中A为肝内检测small RNA(序列2)表达结果，B为血浆内检测small RNA(序列2)表达结果，C为结肠内检测small RNA(序列2)表达结果。
图74是本申请再一实施例提供的分别以包含有3组不同RNA片段的病毒载体注射小鼠后的体内富集结果图，3组RNA片段分别包含有任意3种RNA序列，具体为：miR-19a(靶基因TNF-α)+B7-siRNA-1+integrin α4shRNA-1，miR-124-3p(靶基因TNF-α)+B7-siRNA-2+integrin α4 shRNA-2，miR-19a(靶基因TNF-α)+B7-siRNA-1+integrin α4 shRNA-2，图中A为肝内检测small RNA(序列3)表达结果，B为血浆内检测small RNA(序列3)表达结果，C为结肠内检测small RNA(序列3) 表达结果。
图75是本申请一实施例提供的分别以包含有3组不同RNA片段的病毒载体注射小鼠后，具体疗效的结果图，3组RNA片段分别包含有任意3种RNA序列，具体为：miR-19a(靶基因TNF-α)+B7-siRNA-1+integrin α4 shRNA-1，miR-124-3p(靶基因TNF-α)+B7-siRNA-2+integrin α4 shRNA-2，miR-19a(靶基因TNF-α)+B7-siRNA-1+integrin α4 shRNA-2，图中A为疾病指数评分，B为炎症因子检测结果。
图76是本申请一实施例提供的含有不同长度RNA序列的病毒载体注射小鼠后的体内富集结果图，不同长度RNA序列分别为：TNF-α-siRNA-1(siRNA长度18bp)，TNF-α-siRNA-2(siRNA长度20bp)，TNF-α-siRNA-3(siRNA长度22bp)，图中A为肝内检测siRNA表达结果，B为血浆内检测siRNA表达结果，C为结肠内检测siRNA表达结果。
图77是本申请另一实施例提供的含有不同长度RNA序列的病毒载体注射小鼠后的具体疗效结果图，不同长度RNA序列分别为：TNF-α-siRNA-1(siRNA长度18bp)，TNF-α-siRNA-2(siRNA长度20bp)，TNF-α-siRNA-3(siRNA长度22bp)，图中A为疾病指数评分，B为炎症因子检测结果，C为靶基因mRNA检测结果。
图78是本申请一实施例提供的分别含有3个同源TNF-α-siRNA序列的病毒载体注射小鼠后的体内富集结果图，同源TNF-α-siRNA序列分别为：TNF-α-siRNA-4，TNF-α-siRNA-5，TNF-α-siRNA-6，图中A为肝内检测TNF-α-siRNA表达结果，B为血浆内检测TNF-α-siRNA表达结果，C为结肠内检测TNF-α-siRNA表达结果，D为血浆外泌体内检测TNF-α-siRNA表达结果。
图79是本申请另一实施例提供的分别含有3个同源B7-siRNA序列的病毒载体注射小鼠后的体内富集结果图，同源B7-siRNA序列分别为：B7-siRNA-1，B7-siRNA-2，B7-siRNA-3，图中A为肝内检测B7-siRNA表达结果，B为血浆内检测B7-siRNA表达结果，C为结肠内检测B7-siRNA表达结果，D为血浆外泌体内检测B7-siRNA表达结果。
图80是本申请再一实施例提供的分别含有3个同源integrin α4 siRNA序列的病毒载体注射小鼠后的体内富集结果图，同源integrin α4 siRNA序列分别为：integrin α4 siRNA-1，integrin α4 siRNA-2，integrin α4 siRNA-3，图中A为肝内检测integrin α4 siRNA表达结果，B为血浆内检测integrin α4 siRNA表达结果，C为结肠内检测integrin α4 siRNA表达结果，D为血浆外泌体内检测integrin α4 siRNA表达结果。
图81是本申请一实施例提供的分别含有9个同源siRNA序列的病毒载体注射小鼠后的具体疗效结果图，同源siRNA序列分别为：TNF-α-siRNA-4，TNF-α-siRNA-5，TNF-α-siRNA-6，B7-siRNA-1，B7-siRNA-2，B7-siRNA-3，integrin α4 siRNA-1，integrin α4 siRNA-2，integrinα4 siRNA-3，图中A为疾病指数评分，B为炎症因子检测结果。
图82是本申请一实施例提供的分别以包含有不同侧翼序列、loop序列及反向互补序列的RNA片段的病毒载体注射小鼠后的体内富集结果图，序列分别为：2条与已经明确的5’侧翼序列同源性大于80％的明确序列、2条与已经明确的loop序列同源性大于80％的明确序列、2条与已经明确的3’侧翼序列同源性大于80％的明确序列、1条正常序列的反向互补序列、1条已经明确的5’侧翼序列同源性大于80％的明确序列的反向互补序列，图中A为肝内检测TNF-α-siRNA表达结果，B为血浆内检测TNF-α-siRNA表达结果，C为结肠内检测TNF-α-siRNA表达结果，D为血浆外泌体内检测TNF-α-siRNA表达结果。
图83是本申请一实施例提供的分别以包含有不同侧翼序列、loop序列及反向互补序列的RNA片段的病毒载体注射小鼠后的具体疗效结果图，序列分别为：2条与已经明确的5’侧翼序列同源性大于80％的明确序列、2条与已经明确的loop序列同源性大于80％的明确序列、2条与已经明确的3’侧翼序列同源性大于80％的明确序列、1条正常序列的反向互补序列、1条已经明确的5’侧翼序列同源性大于80％的明确序列的反向互补序列，图中A为疾病指数评分，B为炎症因子检测结果，C为靶基因 mRNA检测结果。
图84是本申请一实施例提供的腺病毒载体携带多个线路时，相邻线路之间以序列1-序列2-序列3相连且其中序列2分别为5个碱基、10个碱基、20个碱基、30个碱基、40个碱基、50个碱基、80个碱基的情况下，小鼠的体内富集结果图，图中A为肝内检测TNF-α-siRNA表达结果，B为血浆内检测TNF-α-siRNA表达结果，C为结肠内检测TNF-α-siRNA表达结果。
图85是本申请另一实施例提供的腺病毒载体携带多个线路时，相邻线路之间以序列1-序列2-序列3相连且其中序列2分别为5个碱基、10个碱基、20个碱基、30个碱基、40个碱基、50个碱基、80个碱基的情况下，小鼠的体内富集结果图，图中A为肝内检测B7-1-siRNA表达结果，B为血浆内检测B7-1-siRNA表达结果，C为结肠内检测B7-1-siRNA表达结果。
图86是本申请再一实施例提供的腺病毒载体携带多个线路时，相邻线路之间以序列1-序列2-序列3相连且其中序列2分别为5个碱基、10个碱基、20个碱基、30个碱基、40个碱基、50个碱基、80个碱基的情况下，小鼠的体内富集结果图，图中A为肝内检测integrin α4-siRNA表达结果，B为血浆内检测integrin α4-siRNA表达结果，C为结肠内检测integrin α4-siRNA表达结果。
图87是本申请一实施例提供的腺病毒载体携带多个线路时，连接序列为序列4以及2条与序列4同源性大于80％的序列的情况下，小鼠的体内富集结果图，图中A为肝内检测TNF-α-siRNA表达结果，B为血浆内检测TNF-α-siRNA表达结果，C为结肠内检测TNF-α-siRNA表达结果。
图88是本申请另一实施例提供的腺病毒载体携带多个线路时，连接序列为序列4以及2条与序列4同源性大于80％的序列的情况下，小鼠的体内富集结果图，图中A为肝内检测B7-1-siRNA表达结果，B为血浆内检测B7-1-siRNA表达结果，C为结肠内检测B7-1-siRNA表达结果。
图89是本申请再一实施例提供的腺病毒载体携带多个线路时，连接序列为序列4以及2条与序列4同源性大于80％的序列的情况下，小鼠的体内富集结果图，图中A为肝内检测integrin α4-siRNA表达结果，B为血浆内检测integrin α4-siRNA表达结果，C为结肠内检测integrin α4-siRNA表达结果。
图90是本申请一实施例提供的本申请一实施例提供的腺病毒载体携带多个线路时，连接序列为序列4以及2条与序列4同源性大于80％的序列的情况下，小鼠的具体疗效结果图，图中A为疾病指数评分，B为靶基因mRNA检测结果。
图91是本申请一实施例提供的以腺病毒相关病毒2型、7型和8型为病毒载体时，其负载的TNF-α-siRNA在小鼠体内富集结果图，图中A为肝内检测TNF-α-siRNA表达结果，B为血浆内检测TNF-α-siRNA表达结果，C为结肠内检测TNF-α-siRNA表达结果。
图92是本申请一实施例提供的以腺病毒相关病毒2型、7型和8型为病毒载体时，其负载的TNF-α-siRNA在小鼠体内表达后的具体疗效结果图，图中A为疾病指数评分，B为炎症因子检测结果，C为靶基因mRNA检测结果。
图93是本申请一实施例提供的小鼠羟脯氨酸含量对比图；
图94是本申请一实施例提供的小鼠肺部荧光染色图；
图95是本申请一实施例提供的小鼠肺部Masson三色染色图；
图96是本申请一实施例提供的小鼠肺部HE染色图；
图97是本申请一实施例提供的小鼠部分蛋白、mRNA水平对比图；
图98是本申请一实施例提供的含有RNA片段的质粒递送系统的肺纤维化治疗效果数据图，图中A为含有6种RNA序列、6种RNA序列中任意2种组成的RNA片段、6种RNA序列中任意3种组成的RNA片段的质粒递送系统注射后，PTP1B的mRNA相对量检测结果，B为含有6种RNA序列、6种RNA序列中任意2种组成的RNA片段、6种RNA序列中任意3种组成的RNA片段的质粒递送系统注射后，PTP1B的蛋白相对量检测结果。
图99是本申请一实施例提供的CMV-siRNA-1+2静脉注射后的代谢分布结果，图中A为肺部的富集效果，B为血液中的富集效果。
图100是本申请一实施例提供的具有靶向标签GE11的CMV-GE11-siRNA-1+2和(CMV-GE11-siRNA-1+CMV-GE11-siRNA-2)静脉注射后的代谢分布结果，图中A和C分别为CMV-GE11-siRNA-1+2在肺部和血浆中的富集效果，B和D分别为CMV-GE11-siRNA-1+CMV-GE11-siRNA-2在肺部和血浆中的富集效果。
图101是本申请一实施例提供的具有靶向标签GE11的CMV-GE11-siRNA-1、CMV-GE11-siRNA-1+2和CMV-GE11-siRNA-1+CMV-GE11-siRNA-2静脉注射后的肺纤维化治疗效果数据图，图中A和C分别为CMV-GE11-siRNA-1和CMV-GE11-siRNA-1+2的TGFb1的蛋白含量和mRNA含量结果，B和D分别为CMV-GE11-siRNA-1和CMV-GE11-siRNA-1+CMV-GE11-siRNA-2的TGFb1的蛋白含量和mRNA含量结果。
图102是本申请一实施例提供的含有3条不同5’侧翼序列、loop序列和3’侧翼序列的序列片段的质粒注射后，在血液中的富集效果(体现为siRNA含量)图。
图103是本申请一实施例提供的含有多个不同数量碱基的连接序列(序列2)的质粒递送系统注射后，在血液中的富集效果(体现为siRNA含量)图。
图104是本申请一实施例提供的含有多个同源性大于80％的连接序列(序列4)的质粒递送系统注射后，在血液中的富集效果(体现为siRNA含量)图，图中的横坐标序列4-1即为基础的序列4，序列4-2/4-3/4-4分别为序列4-1(序列4)同源性大于80％的同源序列。
图105是本申请一实施例提供的质粒递送系统中的RNA序列长度分别为18、19、21时的肺纤维化治疗效果，图中A为TGFb1的mRNA含量结果，B为TGFb1的蛋白含量结果。
图106是本申请一实施例提供的基因线路在包括有miRNA-21的反义链及5条TGF-β1基因siRNA的情况下，所检测到的羟脯氨酸含量结果。
图107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108是本申请一实施例提供的以腺病毒和慢病毒做为病毒载体，负载RNA片段时具有体内富集、自组装及肺纤维化治疗效果的检测图，病毒载体为腺病毒/慢病毒，富集结果以siRNA含量显示，图中A为递送系统注射后，在肺部的富集检测图(siRNA-1)，B为递送系统注射后，在肺部的富集检测图(siRNA-2)，C为递送系统注射后，在血液中的富集检测图(siRNA-1)，D为递送系统注射后，在血液中的富集检测图(siRNA-2)。
图109是本申请另一实施例提供的以腺病毒和慢病毒做为病毒载体，负载RNA片段时具有体内富集、自组装及肺纤维化治疗效果的检测图，病毒载体为腺病毒/慢病毒，富集结果以siRNA含量显示，图中A为无靶向肽(GE11)的递送系统注射后，在肺部的富集检测图，B为有靶向肽(GE11)的递送系统注射后，在肺部的富集检测图，C为无靶向肽(GE11)的递送系统注射后，在血液中的富集检测图(siRNA-1)，D为有靶向肽(GE11)递送系统注射后，在血液中的富集检测图(siRNA-2)。
图110是本申请一实施例提供的病毒载体系统中携带有多种不同RNA片段的情况下，具有体内富集、自组装及针对肺纤维化治疗效果的检测结果，图中A为PTP1B的mRNA相对量检测结果，B为PTP1B的蛋白相对量检测结果。
图111是本申请一实施例提供的病毒载体递送系统中包含有多个RNA片段和多个靶向标签时(CMV-siRNA-1+2)，静脉注射后具有体内富集、自组装及肺纤维化治疗效果的检测结果，图中A为肺部的富集效果(以siRNA含量显示)，B为血液中的富集效果(以siRNA含量显示)。
图112是本申请另一实施例提供的病毒载体递送系统中包含有多个RNA片段和多个靶向标签时(CMV-GE11-siRNA-1+2、CMV-GE11-siRNA-1+CMV-GE11-siRNA-2)，静脉注射后具有体内富集、自组装及肺纤维化治疗效果的检测结果，图中A和B为肺部的富集效果(以siRNA含量显示)，C和 D为血浆中的富集效果(以siRNA含量显示)。
图113是本申请再一实施例提供的病毒载体递送系统中包含有多个RNA片段和多个靶向标签时(CMV-GE11-siRNA-1+2、CMV-GE11-siRNA-1+CMV-GE11-siRNA-2)，静脉注射后的肺纤维化治疗效果检测结果，图中A和B为TGFb1的蛋白含量检测结果，C和D为TGFb1的mRNA含量检测结果。
图114是本申请一实施例提供的腺病毒载体递送系统中，包含有同源性大于80％的3条5’侧翼序列/loop序列/3’侧翼序列时，具有体内富集的检测结果图(以血液中的siRNA含量显示)。
图115是本申请一实施例提供的腺病毒载体携带多个线路时，相邻线路之间以序列1-序列2-序列3相连，其中序列2含有多个碱基的情况下，所构建的递送系统具有体内富集的检测结果图(以血液中的siRNA含量显示)。
图116是本申请一实施例提供的连接序列为序列4以及与序列4同源性大于80％的序列时，其构建的递送系统也具有体内富集的检测结果图(以血液中的siRNA含量显示)。
图117是本申请一实施例提供的RNA序列长度分别为18、20、21时，所构建的递送系统，具有肺纤维化治疗效果的检测结果图，图中A为不同长度RNA序列的PTP1B mRNA相对量检测结果，B为不同长度RNA序列的PTP1B蛋白相对量检测结果。
图118是本申请一实施例提供的基因线路在包括有miRNA-21的反义链及5条TGF-β1基因siRNA的情况下，所检测到的羟脯氨酸含量结果。
图119是本申请一实施例提供的小鼠siRNA相关表达对比图；
图120是本申请一实施例提供的小鼠胶质母细胞瘤治疗情况对比图；
图121是本申请一实施例提供的小鼠脑部免疫组织染色对比图；
图122是本申请一实施例提供的质粒递送系统在携带有单独1种RNA片段的情况下，具有体内富集、自发形成复合结构的效果验证；其中A为含有不同RNA片段的质粒在体内富集的效果，B为通过不同RNA片段表达水平显示出的体内自组装效果。
图123是本申请一实施例提供的质粒递送系统在携带有任意2种RNA片段的情况下，具有体内富集、自发形成复合结构的效果验证；其中A为含有不同组合RNA片段的质粒在体内富集的效果，B为通过不同组合RNA片段表达水平显示出的体内自组装效果。
图124是本申请一实施例提供的质粒递送系统在携带有任意3种RNA片段的情况下，具有体内富集、自发形成复合结构的效果验证；其中A为含有不同组合RNA片段的质粒在体内富集的效果，B为通过不同组合RNA片段表达水平显示出的体内自组装效果。
图125是本申请另一实施例提供的质粒递送系统在携带有任意2种RNA片段的情况下，具有体内富集、自发形成复合结构的效果验证；其中A为含有不同组合RNA片段的质粒在体内富集的效果，B为通过不同组合RNA片段表达水平显示出的体内自组装效果。
图126是本申请一实施例提供的质粒递送系统在携带有随机1-2个RNA片段和1-2个靶向标签且二者位于相同线路的情况下，具有体内富集的效果验证。图19是本申请另一实施例提供的质粒递送系统在携带有随机1-2个RNA片段和1-2个靶向标签且二者位于不同线路的情况下，具有体内富集的效果验证。
图20是本申请一实施例提供的质粒递送系统在携带有已经明确的5’侧翼序列以及至少2条与其同源性大于80％的明确序列的情况下，具有体内富集、自发形成复合结构的效果验证；其中A为含有不同5’侧翼序列的质粒在体内富集的效果，B为通过不同5’侧翼序列RNA片段表达水平显示出的体内自组装效果。
图21是本申请一实施例提供的质粒递送系统在携带有已经明确的loop序列以及至少2条与其同源性大于80％的明确序列的情况下，具有体内富集、自发形成复合结构的效果验证；其中A为含有 不同loop序列的质粒在体内富集的效果，B为通过不同loop序列RNA片段表达水平显示出的体内自组装效果。
图22是本申请一实施例提供的质粒递送系统在携带有已经明确的3’侧翼序列以及至少2条与其同源性大于80％的明确序列的情况下，具有体内富集、自发形成复合结构的效果验证；其中A为含有不同3’侧翼序列的质粒在体内富集的效果，B为通过不同3’侧翼序列RNA片段表达水平显示出的体内自组装效果。
图23是本申请一实施例提供的质粒递送系统在携带有删除其中任意1、2、3、4、5位碱基后的反向互补序列的RNA序列，具有体内富集、自发形成复合结构的效果验证；其中A为含有不同补偿序列的质粒在体内富集的效果，B为通过不同补偿序列RNA片段表达水平显示出的体内自组装效果。
图24是本申请一实施例提供的质粒递送系统在携带四个所述线路，相邻线路之间以序列1-序列2-序列3相连时，具有自发形成复合结构的效果验证。
图25是本申请一实施例提供的质粒递送系统在携带四个所述线路，相邻线路之间以序列1-序列2-序列3相连，且序列2分别为5个碱基、10个碱基、20个碱基、30个碱基、40个碱基、50个碱基以及80个碱基组成时，具有自发形成复合结构的效果验证。
图26是本申请一实施例提供的质粒递送系统含有连接序列为序列4以及至少2条与序列4同源性大于80％的序列时，具有自发形成复合结构的效果验证。
图27是本申请一实施例提供的质粒递送系统仅含有靶向肽标签时，具有体内富集的效果验证。
图28是本申请一实施例提供的质粒递送系统仅含有靶向蛋白标签时，具有体内富集的效果验证。
图29是本申请一实施例提供的基因线路中含有EGFR基因的siRNA时，具有体内富集、自发形成复合结构的效果验证；其中A为不同的含有EGFR基因siRNA序列的基因线路在体内富集的效果，B为通过不同的含有EGFR基因siRNA序列的表达水平显示出的体内自组装效果。
图30是本申请一实施例提供的基因线路中含有TNC基因的siRNA时，具有体内富集、自发形成复合结构的效果验证；其中A为不同的含有TNC基因siRNA序列的基因线路在体内富集的效果，B为通过不同的含有TNC基因siRNA序列的表达水平显示出的体内自组装效果。
图31是本申请一实施例提供的递送系统含有2种不同核糖修饰后的RNA序列时，具有体内富集、自发形成复合结构的效果验证；其中A为不同核糖修饰后的RNA的递送系统在体内富集的效果，B为通过不同核糖修饰后的RNA的表达水平显示出的体内自组装效果。
图39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2是本申请一实施例提供的3种其它病毒载体具有体内富集和自组装的效果验证，图中A为其它病毒载体1的体内富集结果，B为其它病毒载体2的体内富集结果，C为其它病毒载体3的体内富集结果，D为三种其它病毒载体在体内自组装结果。
图3是本申请一实施例提供的病毒载体在分别单独携带有6种中的一种RNA片段的情况下，具有体内富集、自组装的效果验证；其中A为含有不同RNA片段的载体在体内富集的效果，B为通过不同RNA片段表达水平显示出的体内自组装效果。
图4是本申请一实施例提供的病毒载体在分别携带有4组含有任意2种RNA序列的RNA片段的情况下，具有体内富集、自组装的效果验证；其中A为含有不同RNA片段的载体在体内富集的效果，B为通过不同RNA片段表达水平显示出的体内自组装效果。
图5是本申请一实施例提供的病毒载体在分别携带有3组含有任意3种RNA序列的RNA片段的情况下，具有体内富集、自组装的效果验证；其中A为含有不同RNA片段的载体在体内富集的效果，B为通过不同RNA片段表达水平显示出的体内自组装效果。
图6是本申请另一实施例提供的病毒载体在分别携带有2组含有其它任意2种RNA序列的RNA 片段的情况下，具有体内富集、自组装的效果验证；其中A为含有不同RNA片段的载体在体内富集的效果，B为通过不同RNA片段表达水平显示出的体内自组装效果。
图7是本申请一实施例提供的病毒载体在携带有随机1-2个RNA片段和1-2个靶向标签且二者位于相同线路的情况下，具有体内富集、自组装的效果验证；其中A为含有不同RNA片段和靶向标签的载体在体内富集的效果，B为通过不同RNA片段表达水平显示出的体内自组装效果。
图8是本申请另一实施例提供的病毒载体在携带有随机1-2个RNA片段和1-2个靶向标签且二者位于不同线路的情况下，具有体内富集、自组装的效果验证；其中A为含有不同RNA片段和靶向标签的载体在体内富集的效果，B为通过不同RNA片段表达水平显示出的体内自组装效果。
图9是本申请一实施例提供的病毒载体在携带有已经明确的5’侧翼序列以及至少2条与其同源性大于80％的明确序列的情况下，具有体内富集、自组装的效果验证；其中A为含有不同5’侧翼序列的载体在体内富集的效果，B为通过不同5’侧翼序列RNA片段表达水平显示出的体内自组装效果。
图10是本申请一实施例提供的病毒载体在携带有已经明确的loop序列以及至少2条与其同源性大于80％的明确序列的情况下，具有体内富集、自组装的效果验证；其中A为含有不同loop序列的载体在体内富集的效果，B为通过不同loop序列RNA片段表达水平显示出的体内自组装效果。
图11是本申请一实施例提供的病毒载体在携带有已经明确的3’侧翼序列以及至少2条与其同源性大于80％的明确序列的情况下，具有体内富集、自组装的效果验证；其中A为含有不同3’侧翼序列的载体在体内富集的效果，B为通过不同3’侧翼序列RNA片段表达水平显示出的体内自组装效果。
图12是本申请一实施例提供的病毒载体在携带有删除其中任意1、2、3、4、5位碱基后的反向互补序列的RNA序列，具有体内富集、自组装的效果验证；其中A为含有不同补偿序列的载体在体内富集的效果，B为通过不同补偿序列RNA片段表达水平显示出的体内自组装效果。
图13是本申请一实施例提供的的病毒载体在携带四个线路且相邻线路之间以序列1-序列2-序列3相连时，具有自组装的效果验证。
图14是本申请一实施例提供的病毒载体在携带四个线路，相邻线路之间以序列1-序列2-序列3相连，且序列2分别为5个碱基、10个碱基、20个碱基、30个碱基、40个碱基、50个碱基以及80个碱基组成时，具有自组装的效果验证。
图15是本申请一实施例提供的病毒载体在含有连接序列为序列4以及至少2条与序列4同源性大于80％的序列时，具有自组装的效果验证。
图16是本申请一实施例提供的病毒载体在含有不同靶向肽标签时，具有体内富集的效果验证。
图17是本申请一实施例提供的病毒载体在含有不同靶向蛋白标签时，具有体内富集的效果验证。
图18是本申请一实施例提供的基因线路中含有EGFR基因的siRNA时，具有体内富集、自组装的效果验证；其中A为不同的含有EGFR基因siRNA序列的基因线路在体内富集的效果，B为通过不同的含有EGFR基因siRNA序列的表达水平显示出的体内自组装效果。
图19是本申请一实施例提供的基因线路中含有TNC基因的siRNA时，具有体内富集、自组装的效果验证；其中A为不同的含有TNC基因siRNA序列的基因线路在体内富集的效果，B为通过不同的含有TNC基因siRNA序列的表达水平显示出的体内自组装效果。
图20是本申请一实施例提供的病毒载体递送系统在含有2种不同核糖修饰后的RNA序列时，具有体内富集、自组装的效果验证；其中A为不同核糖修饰后的RNA的病毒载体递送系统在体内富集的效果，B为通过不同核糖修饰后的RNA的表达水平显示出的体内自组装效果。
图40是本申请一实施例提供的小鼠下丘脑、肝脏的荧光显微镜图像；
图41是本申请一实施例提供的小鼠肥胖症治疗情况对比图；
图42是本申请一实施例提供的小鼠肥胖症脂肪肝治疗情况对比图；
图43是本申请一实施例提供的小鼠肥胖症治疗情况对比图；
图44是本申请一实施例提供的小鼠各项肥胖指标对比图；
图3是本申请一实施例提供的以腺病毒和慢病毒做为病毒载体构建的RNA递送系统，具有体内富集效果的检测图，图中A为注射递送系统后，血液中siRNA含量的检测结果，B为注射递送系统后，下丘脑中siRNA含量的检测结果。
图4是本申请另一实施例提供的以腺病毒和慢病毒做为病毒载体构建的RNA递送系统，具有体内富集效果的检测图，图中显示了注射递送系统后，血液外泌体中siRNA含量的检测结果。
图5是本申请一实施例提供的以腺病毒和慢病毒做为病毒载体构建的RNA递送系统，具有体内自组装效果和肥胖症治疗效果的检测图，图中A为PTP1B的mRNA含量检测结果，B为PTP1B的蛋白含量检测结果，C为随天数增长体重的变化值。
图6是本申请一实施例提供的病毒载体系统中携带有多种不同RNA片段的情况下，具有体内富集、自组装及针对肥胖症治疗效果的检测结果，图中A为PTP1B的mRNA相对量检测结果，B为PTP1B的蛋白相对量检测结果。
图7是本申请一实施例提供的腺病毒载体递送系统中包含有多个RNA片段和多个靶向标签时，具有体内富集、自组装及肥胖症治疗效果的检测结果图，其中靶向标签为RVG，RNA片段分别为siRNA-1、siRNA-2、siRNA-1+siRNA-2，图中A为PTP1B的mRNA相对量检测结果，B为PTP1B的蛋白相对量检测结果，C为随天数增长体重的变化值。
图8是本申请一实施例提供的腺病毒载体递送系统中，包含有同源性大于80％的3条5’侧翼序列/loop序列/3’侧翼序列时，具有体内富集的检测结果图(以血液中的siRNA含量显示)。
图9是本申请一实施例提供的腺病毒载体携带多个线路时，相邻线路之间以序列1-序列2-序列3相连，其中序列2含有多个碱基的情况下，所构建的递送系统具有体内富集的检测结果图(以血液中的siRNA含量显示)。
图10是本申请一实施例提供的连接序列为序列4以及与序列4同源性大于80％的序列时，其构建的递送系统也具有体内富集的检测结果图(以血液中的siRNA含量显示)。
图11是本申请一实施例提供的RNA序列长度分别为18、20、21时，所构建的递送系统，具有肥胖症治疗效果的检测结果图，图中A为不同长度RNA序列的PTP1B mRNA相对量检测结果，B为不同长度RNA序列的PTP1B蛋白相对量检测结果。
图45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46是本申请一实施例提供的小鼠肝脏、皮质、纹状体中siRNA、蛋白对比图；抑制基因表达的RNA
图47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48是本申请一实施例提供的小鼠纹状体和皮层mHTT蛋白和毒性聚集体情况对比图；
图49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3是本申请一实施例提供的负载有siRNA的慢病毒载体具有体内富集的效果图。
图4是本申请一实施例提供的负载有siRNA的慢病毒载体具有体内自组装的效果图。
图5是本申请一实施例提供的负载有siRNA的慢病毒载体具有亨廷顿病治疗效果的电泳结果图。
图6是本申请一实施例提供的负载有siRNA的腺病毒相关病毒1型、4型、7型载体具有体内富集的效果图。
图7是本申请一实施例提供的负载有siRNA的腺病毒相关病毒1型、2型、7型载体具有亨廷顿病治疗效果的电泳结果图。
图8是本申请一实施例提供的负载有siRNA的腺病毒相关病毒1型、2型、7型载体具有亨廷顿病治疗效果的数据图，数据图中是以HTT mRNA水平进行对比。
图9是本申请一实施例提供的负载有6种单独RNA序列的腺病毒相关病毒8型载体具有体内富 集的效果图。
图10是本申请一实施例提供的负载有6种单独RNA序列的腺病毒相关病毒9型载体具有体内富集的效果图。
图11是本申请一实施例提供的负载有6种单独RNA序列的腺病毒相关病毒9型载体具有亨廷顿病治疗效果的电泳结果图。
图12是本申请一实施例提供的负载有任意2种RNA序列的RNA片段的腺病毒相关病毒8型载体具有亨廷顿病治疗效果的数据图，数据图中是以HTT mRNA水平进行对比。
图13是本申请一实施例提供的负载有任意2种RNA序列的RNA片段的腺病毒相关病毒9型载体具有亨廷顿病治疗效果的电泳结果图。
图14是本申请一实施例提供的负载有任意3种RNA序列的RNA片段的腺病毒相关病毒8型载体具有亨廷顿病治疗效果的数据图，数据图中是以HTT mRNA水平进行对比。
图15是本申请一实施例提供的负载有任意3种RNA序列的RNA片段的腺病毒相关病毒9型载体具有亨廷顿病治疗效果的电泳结果图。
图16是本申请一实施例提供的腺病毒相关病毒9型中的序列包含有siRNA和RVG的情况下，其所具有的体内富集数据图。
图17是本申请一实施例提供的腺病毒相关病毒9型中的序列包含有siRNA和RVG的情况下，通过HTT mRNA的表达数据所体现出的针对亨廷顿病治疗的效果图。
图18是本申请一实施例提供的RVG-LAMP2B融合蛋白及其它融合蛋白在腺病毒载体上针对亨廷顿病治疗效果的数据对比图，数据通过HTT mRNA的相对水平来体现。
图19是本申请一实施例提供的腺病毒载体系统中包括有与HTT基因的siRNA序列同源性大于80％的3条RNA序列时，其所具有的体内富集数据图。
图20是本申请一实施例提供的腺病毒载体系统中包括有与HTT基因的siRNA序列同源性大于80％的3条RNA序列时，其针对亨廷顿病治疗效果的数据对比图，数据通过HTT mRNA的相对水平来体现。
图50是本申请一实施例提供的转基因小鼠帕金森治疗情况对比图；
图12是本申请一实施例提供的质粒1中分别单独含有6种RNA的情况下，所具有的体内富集数据图。
图13是本申请另一实施例提供的质粒2中分别单独含有6种RNA的情况下，所具有的体内富集数据图。
图14是本申请一实施例提供的外泌体分别单独含有6种RNA的情况下，所具有的体内自组装数据图。
图15是本申请一实施例提供的质粒中分别单独含有6种RNA的情况下，通过LRRK2基因的表达结果所体现出的针对帕金森的治疗效果图。
图16是本申请一实施例提供的质粒1中分别含有任意2种RNA序列的情况下，所具有的体内富集数据图。
图17是本申请一实施例提供的外泌体分别含有任意2种RNA序列的情况下，所具有的体内自组装数据图。
图18是本申请一实施例提供的质粒1中分别含有任意2种RNA序列的情况下，通过LRRK2 mRNA的表达数据所体现出的针对帕金森治疗的效果图。
图19是本申请另一实施例提供的质粒2中分别含有任意2种RNA序列的情况下，通过LRRK2基因的表达结果体现出的针对帕金森的治疗效果图。
图20是本申请一实施例提供的质粒1中分别含有任意3种RNA序列的情况下，通过LRRK2 mRNA的表达数据所体现出的针对帕金森治疗的效果图。
图21是本申请另一实施例提供的质粒2中分别含有任意3种RNA序列的情况下，通过LRRK2基因的表达结果体现出的针对帕金森的治疗效果图。
图22是本申请一实施例提供的质粒中的序列包含有siRNA和RVG的情况下，其所具有的体内富集数据图。
图23是本申请一实施例提供的质粒中的序列包含有siRNA和RVG的情况下，通过LRRK2 mRNA的表达数据所体现出的针对帕金森治疗的效果图。
图24是本申请一实施例提供的RVG-LAMP2B融合蛋白及其它融合蛋白在质粒载体上针对帕金森治疗效果的数据对比图，数据通过LRRK2 mRNA的相对水平来体现。
图25是本申请一实施例提供的基因线路中包括有与LRRK2基因的siRNA序列同源性大于80％的3条RNA序列时，其所具有的体内富集数据图。
图26是本申请一实施例提供的基因线路中包括有与LRRK2基因的siRNA序列同源性大于80％的3条RNA序列时，其针对帕金森治疗效果的数据对比图，数据通过LRRK2 mRNA的相对水平来体现。
图4是本申请一实施例提供的负载有siRNA的慢病毒载体具有体内富集的效果图。
图5是本申请一实施例提供的负载有siRNA的慢病毒载体具有体内自组装的效果图。
图6是本申请一实施例提供的负载有siRNA的慢病毒载体具有帕金森治疗效果的数据结果图，数据图中是以LRRK2 mRNA水平进行对比。
图7是本申请一实施例提供的负载有siRNA的腺病毒相关病毒1型、4型、7型载体具有体内富集的效果图。
图8是本申请一实施例提供的负载有siRNA的腺病毒相关病毒1型、4型、7型载体具有体内自组装的效果图。
图9是本申请一实施例提供的负载有siRNA的腺病毒相关病毒1型、4型、7型载体具有帕金森治疗效果的数据结果图，数据图中是以LRRK2 mRNA水平进行对比。
图10是本申请一实施例提供的负载有6种单独RNA序列的腺病毒相关病毒8型载体具有体内富集的效果图.
图11是本申请一实施例提供的负载有6种单独RNA序列的腺病毒相关病毒8型和9型载体具有体内自组装的效果图。
图12是本申请一实施例提供的负载有任意2种RNA序列的RNA片段的腺病毒相关病毒9型载体具有体内富集的效果图。
图13是本申请一实施例提供的负载有任意2种RNA序列的RNA片段的腺病毒相关病毒8型和9型载体具有体内自组装的效果图。
图14是本申请一实施例提供的负载有任意3种RNA序列的RNA片段的腺病毒载体具有帕金森治疗效果的电泳结果图。
图15是本申请一实施例提供的腺病毒相关病毒9型中的序列包含有siRNA和RVG的情况下，其所具有的体内富集数据图。
图16是本申请一实施例提供的腺病毒相关病毒9型中的序列包含有siRNA和RVG的情况下，通过LRRK2 mRNA的表达数据所体现出的针对帕金森治疗的效果图。
图17是本申请一实施例提供的负载有RVG-LAMP2B融合蛋白及其它融合蛋白在腺病毒载体具有体内富集的数据图。
图18是本申请一实施例提供的RVG-LAMP2B融合蛋白及其它融合蛋白在腺病毒载体上针对帕金森治疗效果的数据对比图，数据通过LRRK2 mRNA的相对水平来体现。
图19是本申请一实施例提供的腺病毒载体系统中包括有与LRRK2基因的siRNA序列同源性大于80％的3条RNA序列时，其所具有的体内富集数据图。
图20是本申请一实施例提供的腺病毒载体系统中包括有与LRRK2基因的siRNA序列同源性大于80％的3条RNA序列时，其针对帕金森治疗效果的数据对比图，数据通过LRRK2 mRNA的相对水平来体现。
图51是本申请一实施例提供的食蟹猕猴全血siRNA浓度变化图；
图52是本申请一实施例提供的化学修饰对小鼠siRNA水平的影响对比图；
图53是本发明提供的递送抑制基因表达的RNA的系统的示意图以及其中一种实施方式的验证实验结果图。
图54是本发明提供的一种示例性质粒CMV-siR E的结构示意图。
图55是本发明提供的一种示例性质粒U6-siR E的结构示意图。
具体实施方式
下面将结合实施例进一步说明本发明的实质内容和有益效果，该实施例仅用于说明本发明而非对本发明的限制。
实施例1材料和方法
苏木精-伊红染色法(hematoxylin-eosin staining)，简称HE染色。苏木精染液为碱性，可以将组织的嗜碱性结构(如核糖体、细胞核及细胞质中的核糖核酸等)染成蓝紫色；伊红为酸性染料，可以将组织的嗜酸性结构(如细胞内及细胞间的蛋白质，包括路易体、酒精小体以及细胞质的大部分)染成粉红色，使整个细胞组织的形态清晰可见。
HE染色的具体步骤包括：样本组织固定与切片；组织样本脱蜡；组织样本水化；组织切片苏木素染色、分化与反蓝；组织切片伊红染色与脱水；组织样本切片风干封片；最后在显微镜下观察并拍照。
Masson染色使胶原纤维呈蓝色(被苯胺蓝所染)或绿色(被亮绿所染)，肌纤维呈红色(被酸性品红和丽春红所染)。已固定的组织用一系列阴离子水溶性染料先后或混合染色，则可发现红细胞被最小分子的阴离子染料着染，肌纤维与胞质被中等大小的阴离子染料着染，而胶原纤维则被大分子的阴离子染料着染。由此说明了红细胞对阴离子染料的渗透性最小，肌纤维与胞质次之，而胶原纤维具有最大的渗透性。I型、III型胶原呈绿色(GBM、TBM、系膜基质及肾间质呈绿色)，嗜复红蛋白、肾小管胞质、红细胞呈红色。
Masson染色的具体步骤包括：组织固定于Bouin氏液，流水冲洗一晚，常规脱水包埋；切片脱蜡至水(二甲苯中脱蜡10min×3次，用吸水纸吸干液体；100％乙醇5min×2次，用吸水纸吸干液体；95％乙醇5min×2次，用吸水纸吸干液体；流水2min，用吸水纸吸干水分)；Weiger氏铁苏木素染5-10min；流水稍洗；0.5％盐酸酒精分化15s；流水冲洗3min；丽春红酸性品红液染8min；蒸馏水稍冲洗；1％磷钼酸水溶液处理约5min；不用水洗，直接用苯胺蓝液或亮绿液复染5min；1％冰醋酸处理1min；95％乙醇脱水5min×2次，用吸水纸吸干液体；100％乙醇5min×2次，用吸水纸吸干液体；二甲苯中透明5min×2次，用吸水纸吸干液体；中性树胶封片。
Western免疫印迹(Western Blot)是将蛋白质转移到膜上,然后利用抗体进行检测。对已知表达蛋白,可用相应抗体作为一抗进行检测,对新基因的表达产物，可通过融合部分的抗体检测。
Western免疫印迹(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，通过化学反应使标记抗体的显色剂(荧光素、酶、金属离子、同位素)显色来确定组织细胞内抗原(多肽和蛋白质)，对其进行定位、定性及相对定量的研究，称为免疫组织化学技术(immunohistochemistry)或免疫细胞化学技术(immunocytochemistry)。
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。
实施例2
发明人设计了一种按照需要将抑制基因表达的RNA递送到器官组织，在细胞中富集、组装形成包括外泌体等细胞微囊泡，在被细胞释放后递送到目的组织来治疗疾病的系统，所述系统包括一个或多个抑制基因表达的RNA，以及靶向目标组织的蛋白。
图53a为示例性的本发明提供的递送抑制基因表达的RNA的其中一种系统的示意图。所述系统为包括表达抑制基因表达的RNA和/或靶向目标组织的蛋白的核酸的载体(在本实施方式中为质粒)，其允许不同功能模块的自由组合。在系统中，核心线路(core part)由启动子部分和siRNA表达部分(如图53a中的siRNA-1 backbone)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(composable part)可以集成到包含核心基因线路的系统框架中，以实现即插即用功能。可组合部件的例子包括两种可优化siRNA的作用类型：一种是修饰外显体的膜锚定蛋白以实现组织选择性(如图53a中的Guiding Tag)；另一种能够共同表达第二个siRNA(如图53a中的siRNA-2 backbone)，以同时抑制两个分子靶标。
对于核心基因线路构建体，发明人对其在启动子部分的控制下编码的siRNA表达骨架部分进行了优化，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。
在一种实施方式中，将表皮生长因子受体(Epidermal growth factor receptor，EGFR)作为核心线路的siRNA靶点。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。另外，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，比较了两种设计方案：一种是由CMV启动子驱动表达miRNA前体(pre-miRNA)并用siRNA替换miRNA序列，因此构建了质粒EGFR siRNA(CMV-siR E)(质粒结构图见图54，中国南京锐真生物技术有限公司合成和提供)，其在CMV启动子后可操作地连接抑制EGFR的siRNA序列(其具有如SEQ ID NO.：1所示的核苷酸序列：UGUGGCUUCUCUUAACUCCU)，其以ggatcctggaggcttgctgaaggctgtatgctgaattc(SEQ ID NO.：2)为5’侧翼序列，以gttttggccactgactgac(SEQ ID NO.：4)为茎环序列，以accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag(SEQ ID NO.：3)为3’侧翼序列，并具有所述RNA片段的反向互补序列，其中删除所述RNA片段的第9位和/或第10位碱基。
另一种是使用U6启动子驱动表达短发夹RNA(shRNA)，因此构建了质粒EGFR shRNA(U6-siR E)(质粒结构图见图55，中国南京Realgene Biotech Company合成和提供)。
比较编码EGFR siRNA(CMV-siR E)和U6定向EGFR shRNA(U6-siR E)的CMV引导的pre-miRNA的siRNA产生效率，参见图53b。两种方案在驱动EGFR siRNA导向链转录方面具有相似的效率；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，乘客链被降解)，参见图53b。因此，在后续实验选择CMV驱动的pre-miRNA设计来避免脱靶效应。
检查核心基因线路是否能够引导siRNA自主加载到外泌体。
构建CMV-scr R。CMV-scr R具有与CMV-siR E中的编码siRNA序列的核苷酸种类和数量相同但核 苷酸排列不同，以作为编码CMV-siR E的空白对照。
采用CMV-scr R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，基因线路转染并不影响HEK293T细胞产生的外泌体的大小、结构或数量。最后，在CMV-siR E基因线路转染的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53c。当这些外泌体与小鼠Lewis 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53d、图53e，表明外泌体siRNA具有生物学功能。
在一种实施方式中，以RVG-Lamp2b融合蛋白(Lamp2b蛋白的N末端连接RVG靶向肽)为锚定蛋白将将外泌体导入大脑。狂犬病毒糖蛋白(rabies virus glycoprotein,RVG)是一种嗜神经性的蛋白质，能够与神经细胞表达的乙酰胆碱受体相结合。RVG已被证明有助于外泌体穿过血脑屏障进入神经细胞。在CMV-RVG-siR E中，将编码RVG-Lamp2b融合蛋白的序列插入CMV启动子的下游和SiRNA的上游。其中，RVG的氨基酸序列如SEQ ID NO.：17所示。整个RVG-Lamp2b融合蛋白的氨基酸序列如SEQ ID NO.：18所示。
评估启动子启动RVG-Lamp2b融合蛋白表达的效率。CMV启动子在HEK293T细胞中产生RVG-Lamp2b mRNA和标记蛋白eGFP。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。在实验中采用Flag标签暂时代替RVG，在用CMV引导的Flag-Lamp2b转染HEK293T和Hepa1-6细胞后，用抗Flag珠成功地免疫沉淀完整的外泌体，参见图53f，证明了靶向标签的精确定位。
在一种实施方式中，以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包括CMV启动子后可表达地连接抑制TNC的siRNA序列，也被嵌入前miR-155骨架中，得到质粒CMV-siR T。其中，TNC基因的siRNA具有以下核苷酸序列：UAUGAAAUGUAAAAAAAGGGA(SEQ ID NO.5)
另外构建将抑制EGFR的siRNA和抑制TNC的siRNA串联在同一质粒，TNC-siRNA插入EGFR-siRNA的下游，以cagatctggccgcactcgaggtagtgagtcgaccagtggatc(SEQ ID NO.：6)作为编码EGFR-siRNA和TNC-siRNA的序列之间的连接子，得到质粒CMV siR E+T。在实验发现，无论单个(CMV siR E或CMV siR T)或串联(CMV siR E+T)转录，均检测到了相差无几的EGFR和TNC siRNA，参见图53g。
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基因线路转染的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53i、图53j。此外发现，外泌体表面的RVG标签不影响EGFR和TNC siRNA对靶标的沉默效果。
在本文中，如无特别说明，用代号CMV-siR 基因缩小或首字母表示具有如前描述的CMV-siR E的构建结构而只是编码的抑制基因的RNA序列不同的质粒。例如，“质粒CMV-siR T”或“CMV-siR T”是指如前描述的在CMV启动子后可表达地连接抑制TNC的siRNA序列的质粒，其中以ggatcctggaggcttgctgaaggctgtatgctgaattc(SEQ ID NO.：2)为5’侧翼序列，以gttttggccactgactgac(SEQ ID NO.：4)为茎环序列，以accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag(SEQ ID NO.：3)为3’侧翼序列，并具有所述RNA片段的反向互补序列，其中删除所述RNA片段的第9位和/或第10位碱基。另外，代号CMV-siR 第一基因首字母+第二基因首字母表示同时携带抑制第一基因的siRNA序列和抑制第二基因的siRNA的siRNA的质粒，其具有如CMV-siR E+T的构建结构而只是编码的抑制基因的RNA序列不同的质粒，并且其中抑制第二基因的siRNA序列插入抑制第一基因的siRNA序列的下游，以cagatctggccgcactcgaggtagtgagtcgaccagtggatc(SEQ ID NO.：6)作为编码 TNC-siRNA和EGFR-siRNA的序列之间的连接子。以此类推，可表示表示同时携带抑制多个基因的siRNA序列的质粒。另外，代号CMV-导向肽缩写或首字母-siR 基因缩小或首字母表示在CMV启动子的下游和SiRNA的上游具有导向肽，如RVG。
实施例3
如图1A所示，为了了解质粒在体内的分布情况，对小鼠进行了平板试验，对小鼠注射质粒CMV siR E后按时间点(1h、3h、6h、9h、12h、24h、72h、168h、720h)取样，采用大观霉素提取的质粒进行转化，观测肝脏、血浆、肺、大脑、肾脏、脾脏中克隆体的数量，结果如图1B、图1C、图1D所示，可以看出，质粒在小鼠肝脏中分布最多，并且在注射后3h左右达到峰值，注射后12h已基本代谢。
向C57BL/6J小鼠静脉注射共表达eGFP蛋白和EGFR siRNA的CMV eGFP siR E，结果如图2所示，随着时间的推移，小鼠肝脏内eGFP荧光逐渐增强，约12小时达到峰值，48小时降至背景水平，其他组织未见明显的eGFP信号。
分别向小鼠注射对照质粒(CMV-scrR)、表达EGFR siRNA的质粒(CMV-siR E)，并建立小鼠肝细胞体外模型，分别检测注射CMV-scrR和CMV-siR E的小鼠肝细胞外泌体中相关siRNA水平，结果如图3A所示，可见在注射CMV-siR E的小鼠肝细胞外泌体中存在siRNA的表达。
进行Ago2免疫沉淀实验，结果如图3B、图3C所示。其中，Input代表不经过免疫沉淀，直接将外泌体裂解并进行检测的样品，代表阳性对照。
对小鼠静脉注射质粒后，成熟siRNA在不同组织中的分布如图4所示。从图4A可以看出，血浆、外泌体、无外泌体的血浆中EGFR-siRNA水平呈时间依赖性变化；从图4B可以看出，小鼠EGFR-siRNA在肝、肺、胰腺、脾脏、肾脏中的积累具有时间依赖性。
分别对小鼠注射对照质粒(CMV-scrR)、0.05mg/kg的CMV-siR E质粒、0.5mg/kg的CMV-siR E质粒、5mg/kg的CMV-siR E质粒，检测小鼠肝脏、脾脏、心脏、肺、肾脏、胰腺、脑、骨骼肌、CD4+细胞中绝对siRNA(EGFR siRNA)水平，结果如图5A所示，可以看出，注射对照质粒的小鼠组织中无siRNA的表达，注射CMV-siR E质粒的小鼠各组织中，siRNA表达的水平与CMV-siR E质粒浓度呈正相关。如图5B所示，荧光原位杂交试验(FISH)同样证实了siRNA表达的水平与CMV-siR E质粒浓度呈正相关，即EGFR siRNA的组织分布具有剂量依赖性。
由于质粒进入体内后，会表达前体(Precursor)，再加工成成熟体(siRNA)，对小鼠注射质粒之后肝脏中前体(Precursor)和成熟体(siRNA)的代谢情况进行了检测，结果如图6所示。可以看出，在注射质粒后6个小时的时间节点，小鼠肝脏中前体(Precursor)和成熟体(siRNA)的表达水平达到峰值，在注射质粒后36个小时，小鼠肝脏中的成熟体(siRNA)代谢完成，在注射质粒后48个小时，小鼠肝脏中前体(Precursor)代谢完成。
对小鼠进行胆总管注射外源性siRNA后分别检测小鼠无外泌体血浆(exosome-free)、外泌体(exosome)、血浆中绝对siRNA水平，结果如图7A所示。对小鼠进行胆总管注射外源性siRNA后分别检测小鼠脾脏、心脏、肺、肾脏、胰腺、脑、骨骼肌、CD4+细胞中siRNA的水平，结果如图7B所示。这两张图反映出siRNA在不同组织中动力学几乎相同，在不同组织中siRNA的分布有显著差别。
分别向小鼠体内静脉注射以白蛋白ALB为启动子的siRNA、以CMV为启动子的siRNA、不含任何启动子的siRNA，在注射后0h、3h、6h、9h、12h、24h、36h、48h分别检测小鼠体内的绝对siRNA水平，结果如图8所示。可见，小鼠体内以CMV为启动子的siRNA的水平最高，即以CMV作为启动子效果最优。
通过荧光试验观察自组装的eGFP siRNA对小鼠体内eGFP水平的抑制，过程如下：对eGFP转基因小鼠静脉注射PBS或5mg/kg CMV-siR G或CMV-RVG-siR G质粒，治疗24小时后处死小鼠，在冷冻切片中检测其eGFP荧光水平，图9A所示为具有代表性的荧光显微镜图像，其中绿色表示阳性eGFP信号，蓝色显表示DAPI染色的细胞核，比例尺：100μm，可见CMV-RVG-siRG质粒对 小鼠eGFP的抑制效果更为明显；对eGFP转基因小鼠静脉注射PBS或CMV-scrR或CMV-siR E质粒，治疗24小时后处死小鼠，在冷冻切片中检测其eGFP荧光水平，图9B为注射PBS、CMV-siR E、CMV-RVG-siR E的小鼠心脏、肺、肾脏、胰腺、脑、骨骼肌的荧光强度(Fluorescence intensity)柱形对比图，可见，在肝脏、脾脏、肺、肾脏部位小鼠荧光强度对比更为明显。
分别对于注射PBS、CMV-scrR、CMV-siR E的小鼠其谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TBIL)、血尿素氮(BUN)、血清碱性磷酸酶(ALP)、肌酐(CREA)含量以及胸腺重量、脾脏重量、外周血细胞百分比进行检测，结果如图10所示，图10A-F为分别注射PBS、小鼠CMV-scrR、CMV-siR E的谷丙转氨酶、谷草转氨酶、总胆红素、血尿素氮、血清碱性磷酸酶、肌酐含量对比图，图10G为小鼠肝脏、肺、脾脏、肾脏组织对比图，图10H-I为小鼠胸腺、脾脏结果显示注射PBS、CMV-scrR、CMV-siR E的小鼠ALT、AST等含量以及胸腺重量、脾脏重量、外周血细胞百分比均相差无几，注射CMV-siR E的小鼠与注射PBS的小鼠相比，其肝脏、肺、脾脏、肾脏也无组织损伤。
因此，本实施例提供的RNA递送系统以质粒作为载体，质粒作为成熟的注入物，其安全性和可靠性已被充分验证，成药性非常好。最终发挥效果的RNA序列由内源性外泌体包裹输送，不存在任何免疫反应，无需验证该外泌体的安全性。该递送系统可以递送各类小分子RNA，通用性强。并且质粒的制备要比外泌体或是蛋白质、多肽等物质的制备便宜地多，经济性好。本实施例提供的RNA递送系统在体内自组装后能够与AGO2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4
如图11A所示，选取小鼠，向小鼠体内注射小鼠肺癌细胞(LLC细胞)，而后每两日向小鼠注射一次PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR E进行治疗，分别对小鼠进行生存分析和肿瘤评估，第30天治疗开始，第44天治疗结束。其中，CMV-scrR为对照质粒，CMV-siR E为携带EGFR siRNA基因的质粒。
如图11B所示，横轴表示时间，纵轴表示生存率，从该图中可以看出，注射CMV-siR E的小鼠生存率最高。
如图11C所示，该图为注射PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR E的小鼠在治疗之前和治疗之后根据CT影像图对小鼠肺组织进行3D建模，可以看出，注射CMV-siR E的小鼠肿瘤显著减小。
如图11D所示，该图为注射PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR E的小鼠在治疗之前和治疗之后的肿瘤体积(mm 3)对比图，可以看出，注射CMV-siR E的小鼠肿瘤体积显著减小。而注射PBS缓冲液/CMV-scrR/吉非替尼的小鼠肿瘤体积不仅没有减小，还呈现不同程度的增加。
如图11E所示，该图为正常小鼠、注射PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR E的小鼠的western blot对比图，可见注射PBS缓冲液/CMV-scrR/吉非替尼的小鼠其EGFR基因含量明显较高。
如图11F所示，该图为正常小鼠、注射PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR E的小鼠相关EGFR miRNA水平对比图，可见注射PBS缓冲液/CMV-scrR/吉非替尼的小鼠其相关EGFR miRNA水平相对较高。
综上，CMV-siR E对EGFR突变的肺癌肿瘤具有显著的治疗效果。
分别对注射PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR E的小鼠进行HE染色和免疫组织染色，结果如图12A-图12B所示，EGFR在注射PBS缓冲液/CMVscrR/吉非替尼的小鼠中具有更多的表达。统计小鼠中EGFR和PCNA的着色面积，结果如图12C-图12D所示，可见注射CMV-siRE的小鼠EGFR和PCNA的着色面积均最少，证明其对EGFR突变的肺癌肿瘤的治疗效果最好。
如图13A所示，选取KRAS G12D p53 -/-小鼠，使小鼠吸入Adv-Cre后的第50天至第64天，每两日向小鼠注射一次PBS缓冲液/CMV-scrR/吉非替尼/CMV-siR K进行治疗，分别对小鼠进行生存分析和肿瘤评估。其中，CMV-siR K表示携带抑制KRAS的siRNA序列(KRAS基因的siRNA具有以下 核苷酸序列：UGAUUUAGUAUUAUUUAUGGC(SEQ ID NO.：7)的质粒。
如图13B所示，横轴表示感染后的时间，纵轴表示生存率，从该图中可以看出，注射CMV-siR K的小鼠生存率更高。
如图13C所示，该图为注射CMV-scrR/CMV-siR K的小鼠在治疗之前和治疗之后根据CT影像图对小鼠肺组织进行3D建模，可以看出，注射CMV-siRK能够显著抑制肺癌肿瘤的增长。
如图13D所示，该图为注射CMV-scrR/CMV-siR K的小鼠在治疗之前和治疗之后的肿瘤数量对比图，可以看出，注射CMV-siR K的小鼠肿瘤数量增长显著更少。
如图13E所示，该图为注射CMV-scrR/CMV-siR K的小鼠在治疗之前和治疗之后的肿瘤数量(mm 3)对比图，可以看出，注射CMV-siR K的小鼠肿瘤体积增长缓慢。而注射CMV-scrR的小鼠肿瘤体积增长显著。
如图13F所示，该图为注射CMV-scrR/CMV-siR K的小鼠的western blot对比图，可见注射CMV-scrR的小鼠其KRAS基因含量明显较高。
如图13G所示，该图为注射CMV-scrR/CMV-siR K的小鼠相关KRAS mRNA水平对比图，可见注射CMV-scrR的小鼠其相关KRAS mRNA水平相对较高。
综上，CMV-siR K对KRAS突变的肺癌肿瘤具有显著的治疗效果。
分别对注射CMV-scrR/CMV-siR K的小鼠进行HE染色和免疫组织染色，结果如图14A、图14D、图14E所示，可见在注射CMV-scrR的小鼠中KRAS、p-AKT、p-ERK具有更多的表达，着色百分比更高。采用western blot免疫印迹法检测小鼠体内相关蛋白的表达水平，结果如图14B、图14C所示，在注射CMV-scrR的小鼠中相关蛋白具有更多的表达。这也说明CMV-siR K对KRAS突变的肺癌肿瘤具有显著的抑制作用。
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。
任选的2条5’侧翼同源序列、2条loop同源序列和2条3’侧翼同源序列具体如下表所示。
名称 序列
5flank1 ggataatggaggcttgctgcaggctgtatgctgaattc
5flank2 ggatactggacgcttgcttaaggctgtatggtgaattc
Loop1 gacttggccactgactgac
Loop2 gttttggccactggctgtc
3flank1 agccgtcaggacatgaggcctgttactagcactcacgtggctcaaatggcagagatctggctacactccag
3flank2 actggtcacgacacaaggcctattactagcagtcacattgaacaaatggccaagatctcgccgcactgtag
在质粒携带两个或多个线路的情况下，相邻的线路之间可以通过序列1-序列2-序列3相连；其中，序列1优选为CAGATC，序列2可以为由5-80个碱基组成的序列，比如10个、15个、20个、25个、30个、35个、40个、45个、50个、55个、60个、65个、70个、75个碱基组成的序列均可，优选为10-50个碱基组成的序列，更优选为20-40个碱基组成的序列，序列3优选为TGGATC。
序列2具体如下表所示。
Figure PCTCN2022083876-appb-000001
更为优选地，在质粒携带两个或多个线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
序列4以及2条与序列4同源性大于80％的序列4-1、4-2静脉注射9小时后肺部组织的EGFR siRNA含量检测结果。
序列具体如下表所示。
序列4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
实施例5
携带EGFR siRNA/KRAS-siRNA的病毒构建。
利用肝脏高亲和的AAV-5型腺相关病毒包裹表达抑制基因的siRNA的核酸构建体片段。
采用汉恒生物科技(上海)有限公司提供的腺病毒包装试剂盒和服务构建需要的病毒，包装方法步骤包括：
1.合成或克隆目的核酸片段；
2.选用合适的限制性内切酶进行酶切载体，琼脂糖凝胶回收得到纯化的线性化载体；
3.根据设计的引物进行目的片段进行酶切，琼脂糖凝胶回收得到正确大小的目的片段；
4.将线性化载体和目的片段按照同源重组或者T4连接的方法进行连接；
5.转化感受态DH5a或者stbl3，菌液涂板，培养12-16h；
6.挑选单克隆行进菌落验证；
7.选择菌落验证正确的阳性克隆进行测序；
8.测序正确的克隆样品进行质粒抽提。
采用目的汉恒生物科技(上海)有限公司提供的载体质粒pAAV-RC载体质粒和pHelper载体质粒，以及携带目的核酸构建体片段的载体质粒AAV051，其中核酸构建体片段从CMV-siR E或CMV-siR K质粒中克隆，分别包括如前描述的编码由CMV启动的siRNA的核酸片段，其中CMV启动子后可操作地连接抑制EGFR的siRNA序列(其具有如SEQ ID NO.：1所示的核苷酸序列：UGUGGCUUCUCUUAACUCCU)或抑制KRAS的siRNA序列(其具有如SEQ ID NO.：7所示的核苷酸序列：UGAUUUAGUAUUAUUUAUGGC)，其以SEQ ID NO.：2所示的核苷酸序列为5’侧翼序列，以SEQ ID NO.：4所示的核苷酸序列为茎环序列，以SEQ ID NO.：3所示的核苷酸序列为3’侧翼序列，并具有所述RNA片段的反向互补序列，其中删除所述RNA片段的第9位和/或第10位碱基。
由此获得的携带EGFR siRNA或KRAS-siRNA的AAV-5病毒载体，命名为AAV-CMV-EGFR siRNA或AAV-CMV-KRAS siRNA。
在本文中，如无特别说明，用代号AAV-CMV-基因siRNA或AAV-CMV-siR 基因缩写或首字母表示具有如前描述的AAV-CMV-EGFR siRNA的结构的病毒载体，而只是携带的抑制基因的RNA序列不同。
实验：
尾静脉注射100μL滴度为1012V.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15A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-KRASsiRNA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15B所示。在图15B中，“PBS pre”表示给药前的PBS组，“PBS post”表示给药后的PBS组；“AAV-CMV-scrRpre”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-CMVKRASsiRNA组，“AAV-CMV-KRAS-siRNA post”表示给药后的AAV-CMVKRASsiRNA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15C、图15D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/AAVCMV-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16A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-EGFRsiRNA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16E所示，根据图16E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16B所示。在图16B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-EGFRsiRNA pre”表示给药前的AAV-CMV-EGFR siRNA组，“AAV-CMV-EGFR siRNA post”表示给药后的AAV-CMV-EGFR siRNA组。可以看出，AAV-CMVEGFRsiRNA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16C、图16D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AAVCMV-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17A-图17F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-KRASsiRNA)安全性好，可靠性高，不会产生负面作用。
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。
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具体如下表所示。
Figure PCTCN2022083876-appb-000002
Figure PCTCN2022083876-appb-000003
更为优选地，在病毒载体携带两个或多个线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
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。
序列具体如下表所示。
序列4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
实施例6
根据实施例2描述的方法构建携带抑制VEGFR表达的VEGFR siRNA的质粒CMV-siR V或携带抑制mTOR表达的mTOR siRNA的质粒CMV-siR mT。其中，VEGFR基因的siRNA具有以下核苷酸序列：AUUUGAAGAGUUGUAUUAGCC(SEQ ID NO.：8)。mTOR基因的siRNA具有以下核苷酸序列：AGAUAGUUGGCAAAUCUGCCA(SEQ ID NO.9)。
分别对不同小鼠注射PBS缓冲液/对照质粒/VEGFR siRNA质粒/mTOR siRNA质粒/MIX siRNA质粒(VEGFR siRNA和mTOR siRNA联合使用)/舒尼替尼(Sunitinib)/依维莫司(Everolimus)，观察小鼠肾癌肿瘤的发展情况，结果如图18、图19所示。可见，注射MIX siRNA质粒的小鼠其肾癌的发展得到了最为显著的抑制，而注射PBS缓冲液/对照质粒的小鼠肾癌发展则较为迅速。
综上，VEGFR siRNA和mTOR siRNA联合使用对肾癌肿瘤具有显著的治疗效果。
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具体如下表所示。
Figure PCTCN2022083876-appb-000004
Figure PCTCN2022083876-appb-000005
更为优选地，在病毒载体携带两个或多个线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
连接序列为上述序列4以及与序列4同源性大于80％的序列4-1和序列4-2时，含有以上序列的递送系统注射后，在肺组织9h后检测，也具有相应的富集、自组装及癌症治疗效果。
序列具体如下表所示。
序列4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
实施例7
根据实施例2描述的方法构建携带抑制TNF-α表达的TNF-α siRNA的质粒CMV-siR TNF-α或携带抑制integrin-α表达的anti-integrin-α siRNA的质粒CMVsiR integrin-α或携带抑制B7表达的B7siRNA的质粒CMV-siR B7。其中，TNF-α siRNA具有以下核苷酸序列：AAAACAUAAUCAAAAGAAGGC(SEQ ID NO.10)。其中，integrin-α siRNA具有以下核苷酸序列AUAAUCAUCUCCAUUAAUGUC(SEQ ID NO.11)。其中B7 siRNA具有以下核苷酸序列UUUUCUUUGGGUAAUCUUCAG(SEQ ID NO.12)。
另外，根据实施例2描述的方法构建同时携带TNF-α siRNA、integrin-α siRNA和B7 siRNA的siRNA的质粒CMV-si mix，即CMV-siR TNF-α+integrin-α+B7。
在第一个试验中，设置3组试验组和3组对照组，试验组分别为anti-TNF-α(0.5)组、anti-TNF- α(5)组、anti-TNF-α(20)组；对照组分别为mock组、scr-RNA组、IFX组。
其中，anti-TNF-α(0.5)组、anti-TNF-α(5)组、anti-TNF-α(20)组分别为携带TNF-α siRNA的质粒(CMV-siR TNF-α)，尾静脉注射0.5μL、5μL、20μL的CMV-siR TNF-α的溶液至小鼠体内。
mock组为阴性对照组，scr-RNA组、IFX组分别向小鼠尾静脉注射scr-RNA质粒和IFX(英夫利西单抗)。
随即开始构建DSS诱导的慢性结肠炎模型，其间每天进行称重记录，结果如图20A所示，可见scr-RNA组的小鼠体重下降最快，anti-TNF-α(0.5)组、anti-TNF-α(5)组、anti-TNF-α(20)组的小鼠体重下降较缓，并且TNF-α siRNA溶液的剂量越高，小鼠体重下降越缓，这说明质粒包裹的TNF-α siRNA系统能够减轻结肠炎小鼠的体重下降情况。
模型构建结束，通过小动物活体监测质粒系统的体内表达情况，而后处死小鼠进行结肠的观察，结果如图20B所示，可见scr-RNA组的小鼠结肠长度最短，anti-TNF-α(0.5)组、anti-TNF-α(5)组、anti-TNF-α(20)组的小鼠结肠长度相对较长，并且TNF-α siRNA注射剂量越高，小鼠结肠长度相对越长。这说明质粒包裹的TNF-α siRNA系统对慢性炎症导致的结肠长度缩短有着不同程度的改善。
对小鼠疾病活动指数(Disease activity index)进行评估，结果如图20C所示，可见，注射scr-RNA组、anti-TNF-α(0.5)组、anti-TNF-α(5)组的小鼠疾病活动指数较高，而anti-TNF-α(20)组、IFX组的小鼠疾病活动指数则较低。
对小鼠结肠进行TNF-α mRNA检测，结果如图20D所示，可见该CMV-siR TNF-α系统能够降低结肠TNF-α的表达及分泌；对小鼠结肠进行TNF-α检测，结果如图17E所示，可见，该AAV系统能够产生一定量的TNF-α；对结肠内的促炎因子IL-6、IL-12p70、IL-17A、IL-23进行检测，结果如图20F所示，可见高剂量组的炎症因子分泌整体较低于对照组。
对小鼠结肠切片进行HE染色，以及病理评分统计，结果如图21A和图21B所示，可见anti-TNF-α(0.5)组、anti-TNF-α(5)组、anti-TNF-α(20)组的小鼠，尤其是anti-TNF-α(20)组的小鼠结肠黏膜完整性更高，且免疫细胞的浸润程度更浅，结肠隐窝脓肿以及结肠的充血和出血情况也显著轻于对照组。
以上试验可证明，在改善结肠炎的表现方面，本发明提供的使用质粒包裹TNF-α siRNA系统治疗比IFX更有效。
在第二个试验中，设置4组试验组和3组对照组，试验组分别为anti-TNF-α组、anti-integrin-α组、anti-B7组、anti-mix组，对照组分别为mock组、PBS组、scr-RNA组。
anti-TNF-α组、anti-integrin-α组、anti-B7组、anti-mix组分别利用质粒携带TNF-α siRNA(CMV-siR TNF-α)、integrin-α siRNA(CMVsiR integrin-α)、B7 siRNA(CMV-siR B7)、同时携带TNF-α siRNA)、integrin-α siRNA和B7 siRNA的siRNA(CMV-si mix即CMV-siR TNF-α+integrin-α+B7)。尾静脉注射20μL至小鼠体内，通过小动物活体监测该系统的体内表达情况，可见上述系统在体内尤其是肝脏稳定表达。
mock组为阴性对照组，scr-RNA组、PBS组分别向小鼠尾静脉注射scr-RNA质粒和PBS溶液(磷酸缓冲盐溶液)。
随即开始构建DSS诱导的慢性结肠炎模型，其间每天进行称重记录，结果如图22A所示，可见anti-mix组小鼠的体重增长最为平稳，即质粒包裹的CMV-siR TNF-α+integrin-α+B7基因线路能够显著减轻慢性结肠炎小鼠的体重下降情况，anti-TNF-α组、anti-integrin-α组、anti-B7组的小鼠在炎症缓解期体重回复的速度也显著快于scr-RNA组和PBS组。模型构建结束，通过小动物活体监测质粒系统的体内表达情况，而后处死小鼠进行结肠的观察，结果如图22B所示，可见4个试验组小鼠结肠的红肿情况有不同程度的减轻，慢性炎症导致的结肠长度缩短也有不同程度的改善。
对小鼠疾病活动指数(Disease activity index)进行评估，结果如图22C所示，可见，scr-RNA组和PBS组小鼠疾病活动指数较高，而anti-TNF-α组、anti-integrin-α组、anti-B7组、anti-mix组的小鼠疾病活动指数则依次降低。
对小鼠血浆、肝脏以及结肠进行TNF-α mRNA、integrin mRNA以及B7mRNA检测，结果如图22D-图22F所示，可见，该系统在血浆、肝脏以及结肠内，生成了一定量的稳定表达的RNA，同时该系统显著降低了结肠TNF-α，integrin以及B7mRNA表达。
对小鼠结肠切片进行HE染色，结果如图23所示，可见4个试验组的小鼠，尤其是anti-mix组的小鼠结肠黏膜完整性更高，且免疫细胞的浸润程度更浅，结肠隐窝脓肿以及结肠的充血和出血情况也显著轻于对照组。
实施例8
根据实施例5描述的方法构建携带抑制TNF-α表达的TNF-α siRNA的病毒AAV-CMV-siRTNF-α或携带抑制integrin-α表达的anti-integrin-α siRNA的病毒AAV-CMVsiR integrin-α或携带抑制B7表达的B7 siRNA的病毒AAV-CMV-siR B7。其中，TNF-α siRNA具有以下核苷酸序列AAAACAUAAUCAAAAGAAGGC(SEQ ID NO.10)。其中，integrin-α siRNA具有以下核苷酸序列AUAAUCAUCUCCAUUAAUGUC(SEQ ID NO.11)。其中B7 siRNA具有以下核苷酸序列UUUUCUUUGGGUAAUCUUCAG(SEQ ID NO.12)。另外，构建同时携带TNF-αsiRNA、integrin-α siRNA和B7 siRNA的病毒(AAV-CMV-si mix即CMV-siR TNF-α+integrin-α+B7)。
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-siRTNF-α(low)组、AAV-CMV-siRTNF-α(medium)组、AAV-CMV-siRTNF-α(high)组；对照组分别为Normal组、AAV-CMV-scrR组。
试验过程如图24A所示，AAV-CMV-siRTNF-α(low)组、AAV-CMV-siRTNF-α(medium)组、AAV-CMV-siRTNF-α(high)组分别利用肝脏高亲和的AAV-5型腺相关病毒包裹TNF-α siRNA系统(AAV-CMV-siRTNF-α)，通过尾静脉注射滴度为1012V.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24B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-siRTNF-α(high)组，平均辐亮度(Average Radiance)达到8.42*105(p/sec/cm2/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24C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24D所示，可以看出AAV-CMV-siRTNF-α(low)组、AAV-CMV-siRTNF-α(medium)组、AAV-CMV-siRTNF-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-siRTNF-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24E所示，可以看出AAV-CMV-siRTNF-α(high)组的小鼠疾病指数低于AAV-CMV-siRTNF-α(low)组、AAV-CMV-siRTNF-α(medium)组和AAV-CMV-scrR组。
分别检测各组小鼠体内的TNF-α siRNA水平，结果如图24F所示，可以看出三个试验组小鼠体内TNF-α siRNA水平均较高，而对照组AAV-CMV-scrR组小鼠体内几乎无TNF-α siRNA的表达，这说明上述的AAV系统能够产生一定量的TNF-α siRNA。
分别检测各组小鼠体内的TNF-α mRNA水平，结果如图24G所示，可以看出Normal组和三个试验组的小鼠体内TNF-α mRNA水平均比较低，而AAV-CMV-scrR组小鼠体内TNF-α mRNA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25A所示，可见Normal组和AAV-CMV-siRTNF-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25B、图25C所示，可以看出试验 组，尤其是AAV-CMV-siRTNF-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-siRTNF-α，能够实现长期的TNF-α siRNA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-siRT+B+I(low)组、AAV-CMV-siRT+B+I(medium)组、AAV-CMV-siRT+B+I(high)组；对照组分别为Normal组、AAV-CMV-scrR组。
AAV-CMV-siRT+B+I(low)组、AAV-CMV-siRT+B+I(medium)组、AAV-CMV-siRT+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-α siRNA，B7-siRNA以及Integrin α4 siRNA元件串联递药系统(AAV-CMV-siRT+B+I)，通过尾静脉注射滴度为1012V.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26A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26B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26C所示，可以看出AAV-CMV-siRT+B+I(low)组、AAV-CMV-siRT+B+I(medium)组、AAV-CMV-siRT+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-siRT+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27A所示，可以看出AAV-CMV-siRT+B+I(high)组的小鼠疾病指数低于AAV-CMV-siRT+B+I(low)组、AAV-CMV-siRT+B+I(medium)组和AAV-CMV-scrR组。
对小鼠血浆中TNF-α siRNA、B7 siRNA以及integrin α4 siRNA进行检测，结果如图27B、图27C、图27D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-α siRNA、B7 siRNA以及integrin α4 siRNA进行检测，结果如图27E、图27F、图27G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-α siRNA、B7 siRNA以及integrin α4 siRNA进行检测，结果如图28A、图28B、图28C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-α mRNA、B7 mRNA以及integri α4 mRNA进行检测，结果如图28D、图28E、图28F所示，可见AAV包裹的CMV-siR T+B+I系统对小鼠结肠切片进行HE染色以及病理评分统计，结果如图29A和图29B所示。可见试验组，尤其是AAV-CMV-siRT+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，能够实现长期的TNF-α siRNA、B7 siRNA以及integrin α4 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。
上述所涉及的序列具体如下表所示。
Figure PCTCN2022083876-appb-000006
Figure PCTCN2022083876-appb-000007
实施例9
根据实施例2描述的方法构建携带抑制miR-21表达的miR-21 siRNA的质粒CMV-siR miR-21为或携带抑制TGF-β1表达的TGF-β1 siRNA的质粒CMVsiR TGF-β1。其中，miR-21 siRNA具有miR-21的反义链。其中，TGF-β1 siRNA具有以下核苷酸序列：ACGGAAAUAACCUAGAUGGGC(SEQ ID NO.13)。
另外，根据实施例2描述的方法构建同时携带miR-21 siRNA和TGF-β1 siRNA的质粒CMV-siR miR-21+TGF-β1。
本实施例设置8组试验组和3组对照组。试验组分别为Anti-miR-21(1mg/kg)组、Anti-miR-21(5mg/kg)组、Anti-miR-21(10mg/kg)组、TGF-β1 siRNA(1mg/kg)组、TGF-β1 siRNA(5mg/kg)组、TGF-β1 siRNA(10mg/kg)组、Anti-miR-21+TGF-β1 siRNA(10mg/kg)组、Pirfenidone(300mg/kg)组，对照组分别为Normal组、PBS组、scrRNA组。
其中，Anti-miR-21(1mg/kg)组、Anti-miR-21(5mg/kg)组、Anti-miR-21(10mg/kg)组分别向患有肺纤维化的小鼠尾静脉注射1mg/kg、5mg/kg、10mg/kg的miR-21 siRNA质粒，TGF-β1 siRNA(1mg/kg)组、TGF-β1 siRNA(5mg/kg)组、TGF-β1 siRNA(10mg/kg)组分别向患有肺纤维化的小鼠尾静脉注射1mg/kg、5mg/kg、10mg/kg的TGF-β1 siRNA质粒，Anti-miR-21+TGF-β1 siRNA(10mg/kg)组向患有肺纤维化的小鼠尾静脉注射10mg/kgAnti-miR-21和TGF-β1 siRNA质粒，Pirfenidone(300mg/kg)组向患有肺纤维化的小鼠尾静脉注射300mg/kg的吡非尼酮，Normal组为正常对照组，PBS组、scrRNA组分别向患有肺纤维化的小鼠尾静脉注射PBS溶液和对照质粒。
分别检测各组小鼠的羟脯氨酸含量，结果如图30所示。羟脯氨酸是胶原的主要成分，其含量反映了肺纤维化程度。从图30中可以看出，Anti-miR-21(5mg/kg)组、Anti-miR-21(10mg/kg)组、TGF-β1 siRNA(10mg/kg)组、Anti-miR-21+TGF-β1 siRNA(10mg/kg)组小鼠的羟脯氨酸含量相对较低，其肺纤维化得到抑制。
分别对各组小鼠肺部进行荧光染色，结果如图31所示，图中绿色部分表示I型胶原(Collagen I)，红色部分表示α-SMA，蓝色部分表示DAPI。可以看出，PBS组、scrRNA组小鼠I型胶原、α-SMA含量较多，而试验组小鼠的I型胶原、α-SMA含量均相对较少，尤其是Anti-miR-21(5mg/kg)组、Anti-miR-21(10mg/kg)组、Anti-miR-21+TGF-β1 siRNA(10mg/kg)组几乎无I型胶原、α-SMA的表达。
分别对各组小鼠肺部进行Masson三色染色，结果如图32所示。可以看出PBS组和scrRNA组小鼠肺泡间隙被严重破坏，造成肺间质胶原，而试验组则显著减轻了这些现象。
分别对各组小鼠肺部进行H&E染色，结果如图33所示。可以看出PBS组和scrRNA组小鼠肺泡间隙增宽、炎性细胞被浸润、肺泡结构被损害，而试验组肺组织则较为正常。通过western blot分别检测Normal组、PBS组、scrRNA组、TGF-β1 siRNA(1mg/kg)组、TGF-β1 siRNA(5mg/kg)组、TGF-β1 siRNA(10mg/kg)组、Pirfenidone(300mg/kg)组小鼠TGF-β1蛋白水平、TGF-β1 mRNA水平，结果如图34A-图34C所示，可见TGF-β1 siRNA(10mg/kg)组小鼠TGF-β1蛋白水平以及TGF-β1 mRNA水平最低。这说明了尾静脉注射相应siRNA表达质粒后，TGF-β1能够成功递送至肺部发挥功能。
分别检测Normal组、PBS组、scrRNA组、Anti-miR-21(1mg/kg)组、Anti-miR-21(5mg/kg)组、Anti-miR-21(10mg/kg)组小鼠的相对miR-21水平，结果如图34D所示，可见Anti-miR-21(10mg/kg)组小鼠的相对miR-21水平最高。这说明了尾静脉注射相应反义链表达质粒后，miR-21的 反义链能够成功递送至肺部发挥功能。
以上试验说明，利用亲和肝脏的质粒包裹CMV-siR miR-21、CMVsiR TGF-β1、CMV-siR miR-21+TGF-β1，能够显著缓解肺纤维化程度，具有极大的成药潜力，以及临床研究价值。
含有3条不同5’侧翼序列、loop序列和3’侧翼序列的序列片段的质粒同样具有体内富集、自组装及针对肺纤维化的治疗效果，序列分别为：
1、3条同源性大于80％的5’侧翼序列；
2、3条同源性大于80％的loop序列；
3、3条同源性大于80％的3’侧翼序列。序列具体如下表所示。
5'侧翼序列-1 CTGGAGGCTTGCTGAAGGCTGTATGCTGAATTCG
5'侧翼序列-2 CTGGAGGCTTGCTGAAGGCTGTATGCTGTTAACG
5'侧翼序列-3 CTGGAGGCTTGCTGAAGGCTGTATGCTGGCAACG
loop-1 GTTTTGGCCACTGACTGAC
loop-2 GTTAAGGCCACTGACTGAC
loop-3 GAATTGGCCACTGACTGAC
3'侧翼序列-1 CACCGGTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC
3'侧翼序列-2 CAGGCCTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC
3'侧翼序列-3 CAGCGCTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC
RNA质粒递送系统携带多个线路时，其相邻线路之间的连接序列，即序列2可以为多个碱基组成，此时的质粒注射后也具有富集效果，序列2具体如下表所示。
Figure PCTCN2022083876-appb-000008
Figure PCTCN2022083876-appb-000009
具体序列如下表所示，其中序列4-1即为上述的序列4，序列4-2/4-3/4-4分别为序列4-1同源性大于80％的同源序列。
序列4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
质粒递送系统中的RNA序列长度分别为18、19、21时的体内富集、自组装及针对肺纤维化治疗效果。
具体序列如下表所示。
21nt序列 TATCTTTGCTGTCACAAGAGC
19nt序列 TAAAGTCAATGTACAGCTG
18nt序列 TTCATGTCATGGATGGTG
质粒递送系统中，在携带有RNA片段的情况下，具有体内富集、自发形成复合结构及针对肺纤维化的治疗效果，RNA片段分组情况包括但不限于：
1)siRNA1单独、siRNA2单独、shRNA1单独、shRNA2单独、miRNA1单独、miRNA2单独；
2)上述1)中，包含有任意2种RNA序列的RNA片段；
3)上述1)中，包含有任意3种RNA序列的RNA片段。
具体序列如下表所示。
Figure PCTCN2022083876-appb-000010
Figure PCTCN2022083876-appb-000011
实施例10
根据实施例5和实施例9描述的方法构建携带抑制miR-21表达的miR-21 siRNA的病毒AAV-CMV-siR miR-21或携带抑制TGF-β1表达的TGF-β1 siRNA的病毒AAV-CMVsiR TGF-β1。其中，miR-21 siRNA具有miR-21的反义链。其中，TGF-β1 siRNA具有以下核苷酸序列ACGGAAAUAACCUAGAUGGGC(SEQ ID NO.13)。另外，构建同时携带miR-21 siRNA和TGF-β1 siRNA的病毒AAV-CMV-MIX即AAV-CMV-siR miR-21+TGF-β1。
尾静脉注射100μL滴度为1012V.g/ml的AAV溶液至小鼠体内。通过小动物活体监测AAV系统的体内表达情况，3周后可见AAV系统在体内尤其是肝脏，稳定表达。
随即选取小鼠进行造模，造模成功后，分别向小鼠注射注射PBS缓冲液/AAV-scrR/AAV-anti-miR21/AAV-TGF-β1 siRNA/AAV-MIX(10mg/kg)，形成PBS组/AAV-scrR组/AAV-anti-miR21组/AAV-TGF-β1 siRNA组/AAV-MIX组。
分别检测正常小鼠、PBS组小鼠、AAV-scrR组小鼠、AAV-TGF-β1 siRNA组小鼠相对TGF-β1 mRNA水平，结果如图35B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35C所示，可见，AAV-anti-miR21组小鼠相对miR21 mRNA水平相对较低。
分别检测各组小鼠羟脯氨酸含量，结果如图35A所示，可见PBS组、AAV-scrR组小鼠体内羟脯氨酸含量最高，AAV-anti-miR21组、AAV-TGF-β1 siRNA组、AAV-MIX组小鼠体内羟脯氨酸含量均较低，说明AAV-anti-miR21组、AAV-TGF-β1 siRNA组、AAV-MIX组小鼠的肺纤维化得到抑制。
进一步地，所述病毒载体还可以包括能够使所述线路折叠成正确结构并表达的侧翼序列、补偿序列和loop序列，所述侧翼序列包括5’侧翼序列和3’侧翼序列；所述病毒载体包括以下任意一种线路或几种线路的组合：5’-启动子-5’侧翼序列-RNA片段-loop序列-补偿序列-3’侧翼序列、5’-启动子-靶向标签、5’-启动子-靶向标签-5’侧翼序列-RNA片段-loop序列-补偿序列-3’侧翼序列。
其中，所述5’侧翼序列优选为ggatcctggaggcttgctgaaggctgtatgctgaattc或与其同源性大于80％的序列，包括与ggatcctggaggcttgctgaaggctgtatgctgaattc同源性为85％、90％、92％、95％、98％、99％的序列等。
所述loop序列优选为gttttggccactgactgac或与其同源性大于80％的序列，包括与gttttggccactgactgac同源性为85％、90％、92％、95％、98％、99％的序列等。
所述3’侧翼序列优选为accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag或与其同源性大于80％的序列，包括与accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag同源性为85％、90％、92％、95％、98％、99％的序列等。
所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-5位碱基。在RNA片段中仅包含一个RNA序列时，所述补偿序列可以为该RNA序列的删除其中任意1-5位碱基的反向互补序列。
优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-3位碱基。在RNA片段中仅包含一个RNA序列时，所述补偿序列可以为该RNA序列的删除其中任意1-3位碱基的反向互补序列。
更为优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-3位连续排列的碱基。在RNA片段中仅包含一个RNA序列时，所述补偿序列可以为该RNA序列的删除其中任意1-3位连续排列的碱基的反向互补序列。
最为优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中的第9位和/或第10位碱基。在RNA片段中仅包含一个RNA序列时，所述补偿序列可以为该RNA序列的删除其中第9位和/或第10位的反向互补序列。删除第9位和第10位碱基效果最优。
需要说明的是，上述侧翼序列、补偿序列、loop序列均不是随意选择的，而是基于大量的理论研究和试验确定的，在上述特定侧翼序列、补偿序列、loop序列的配合下，能够最大程度的提高RNA片段的表达率。
腺病毒载体中，含有3种同源序列的情况下，也具有体内富集、自组装及肺纤维化治疗效果，序列分组如下：
1、3条同源性大于80％的5’侧翼序列；
2、3条同源性大于80％的loop序列；
3、3条同源性大于80％的3’侧翼序列。
序列具体如下表2所示。
Figure PCTCN2022083876-appb-000012
在病毒载体携带两个或多个线路的情况下，相邻的线路之间可以通过序列1-序列2-序列3相连； 其中，序列1优选为CAGATC，序列2可以为由5-80个碱基组成的序列，比如10个、15个、20个、25个、30个、35个、40个、45个、50个、55个、60个、65个、70个、75个碱基组成的序列均可，优选为10-50个碱基组成的序列，更优选为20-40个碱基组成的序列，序列3优选为TGGATC。
腺病毒载体携带多个线路时，相邻线路之间以序列1-序列2-序列3相连，其中序列2含有多个碱基，所构建的递送系统也同样具有体内富集、自组装和肺纤维化治疗效果。
序列2具体如下表3所示。
Figure PCTCN2022083876-appb-000013
更为优选地，在病毒载体携带两个或多个线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
连接序列为序列4以及与序列4同源性大于80％的序列时，构建的递送系统也具有体内富集、自组装和肺纤维化治疗效果，序列4-1即为所述序列4，序列4-2/4-3/4-4分别为序列4-1的同源序列，序列具体如下表4所示。
序列4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
以上所述的RNA片段包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列能够在目标受体中被表达，所述补偿序列在目标受体中不能被表达。RNA序列可以为siRNA序列、shRNA序列或miRNA序列，优选为siRNA序列。
一个RNA序列的长度为15-25个核苷酸(nt)，优选为18-22nt，比如18nt、19nt、20nt、21nt、22nt均可。此序列长度的范围并不是随意选择的，而是经过反复的试验后确定的。大量试验证明，在RNA序列的长度小于18nt，特别是小于15nt的情况下，该RNA序列大多无效，不会发挥作用，而在RNA序列的长度大于22nt，特别是大于25nt的情况下，则不仅线路的成本大大提高，而且效果也并未优于长度为18-22nt的RNA序列，经济效益差。因此，在RNA序列的长度为15-25nt，特别是18-22nt时，能够兼顾成本与作用的发挥，效果最好。
RNA序列长度分别为18、20、21时，所构建的递送系统，也具有体内富集、自组装和肺纤维化的治疗效果，具体序列如表5所示。
21nt序列 TATCTTTGCTGTCACAAGAGC
19nt序列 TAAAGTCAATGTACAGCTG
18nt序列 TTCATGTCATGGATGGTG
病毒载体系统中携带有多种不同RNA片段的情况下，具有体内富集、自组装及针对肺纤维化治疗效果，如图5所示，其中RNA片段分组如下所示：
1、6种RNA单独使用：siRNA-1单独、siRNA-2单独、shRNA-1单独、shRNA-2单独、miRNA-1单独、miRNA-2单独；
2、6种RNA序列中的任意2种组合成RNA片段：siRNA-1+siRNA-2、shRNA-1+shRNA-2、miRNA-1+miRNA-2；
3、6种RNA序列中的任意3种组合成RNA片段：siRNA-1+siRNA-2+shRNA-1、siRNA-1+siRNA-2+shRNA-2、siRNA-1+siRNA-2+miRNA-1、siRNA-1+siRNA-2+miRNA-2。
RNA序列具体如下表1所示。
Figure PCTCN2022083876-appb-000014
实施例11
检测根据实施例2制备的携带RVG导向肽的质粒对胶质母细胞瘤的作用。
在第一个试验中，设置5个试验组和3个对照组。试验组分别为CMV-siR E组、CMV-siR T组、CMV-RVG-siR E+T组、CMV-siR E+T组、CMV-Flag-siR E+T组，其中“E”表示EGFR、“T”表示TNC，对照组分别为PBS组、CMV-scrR组、CMV-Flag-scrR组，具体试验过程参见图36A。
分别检测不同组别小鼠的CD63蛋白表达含量、siRNA表达水平，结果如图36B-图36D所示，这表明静脉注射CMV-RVG-siR E+T可将siRNA传递至大脑。
在第二个试验中，设置2个试验组和2个对照组。试验组分别为CMV-RVG-siR E组、CMV-RVG-siR E+T组，对照组分别为PBS组、CMV-scrR组。
具体试验过程如图37A所示，选取小鼠，向小鼠体内注射胶质母细胞瘤细胞(U-87MG-Luc细胞)，自第7天开始至第21天，期间每两日向小鼠注射一次PBS缓冲液/CMV-scrR/CMV-RVG-siR E/CMV-RVG-siR E+T(5mg/kg)进行治疗，分别对小鼠进行生存分析和肿瘤评估。在第7天、14天、28天、35天分别对小鼠进行BLI活体成像检测。
如图37B所示，该图为第7天、14天、28天、35天小鼠BLI活体成像检测对比图，可以看出，CMV-RVG-siR E+T组的小鼠其胶质母细胞瘤抑制效果最为显著。
如图37C所示，该图为各组小鼠生存率对比图，可见，CMV-RVG-siR E+T组的小鼠其生存时间最长。
如图37D所示，该图为各组小鼠的荧光对比图，该图通过luciferase生物活体成像得到，纵坐标反应lucifer荧光信号强弱。由于种植的肿瘤中已经人工整合了该基因，因此，该图能够反应肿瘤的进展情况。可以看出对照组小鼠肿瘤发展均比较迅速，而试验组小鼠的肿瘤则得到了很大程度的抑制。如图37E所示，该图为各组小鼠的相对siRNA对比图，可见CMV-RVG-siR E组的小鼠EGFR siRNA水平较高，CMV-RVG-siR E+T组的小鼠EGFR siRNA和TNC siRNA水平均较高。
如图37F所示，该图为各组小鼠的western blot对比图，可见PBS组、CMV-scrR组、CMV-RVG-siR E组的小鼠其EGFR、TNC基因含量较高。
以上试验数据说明了静脉注射CMV-RVG-siR E+T质粒能够将siRNA传递到大脑并抑制胶质母细胞瘤的生长。
分别对各组小鼠脑部进行免疫组织染色处理，并统计每视野中EGFR、TNC、PCNA着色比例，结果如图38所示。可以看出，CMV-RVG-siR E+T组的小鼠脑部EGFR、TNC、PCNA含量最低，CMV-RVG-siR E组的小鼠脑部EGFR、PCNA含量较低。可见注射CMV-RVG-siR E质粒能够抑制脑部EGFR、PCNA的表达，注射CMV-RVG-siR E+T质粒能够抑制脑部EGFR、TNC、PCNA的表达。
质粒确实具有体内富集并自发形成含有RNA片段复合结构的效果，本发明随机提供了一组质粒携带四个所述线路时，相邻线路之间以序列1-序列2-序列3相连，且序列2分别为5个碱基、10个碱基、20个碱基、30个碱基、40个碱基、50个碱基以及80个碱基组成的实验数据，通过实验验证了质粒的富集和自组装效果，。
序列2具体如下表所示。
Figure PCTCN2022083876-appb-000015
Figure PCTCN2022083876-appb-000016
更为优选地，在质粒携带两个或多个线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
为了证明质粒确实具有体内富集并自发形成含有RNA片段复合结构的效果，本发明随机提供了一组质粒含有连接序列为序列4以及至少2条与序列4同源性大于80％的序列的相应实验数据，并通过实验验证了质粒的富集和自组装效果。
序列具体如下表所示。
序列4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
实施例12
检测根据实施例2和5的描述制备的携带RVG导向肽的病毒对胶质母细胞瘤的作用。
材料：AAV-CMV-RVG-siR E和AAV-CMV-RVG-siR E+T，尾静脉注射100μL滴度为1012V.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-87MG-Luc细胞)，
自第7天开始至第21天，期间每两日向小鼠注射一次PBS缓冲液/AAV-CMV-scrR/AAV-CMV-RVG-siR E/AAV-CMV-RVG-siR E+T(5mg/kg)进行治疗，形成PBS组/AAV-scrR组/AAV-CMV-RVG-siRE组/AAV-CMV-RVG-siRE+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39A所示，可以看出AAV-CMV-RVG-siRE+T组小鼠的生存时间最长，AAV-CMV-RVG-siRE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39B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。
为了证明病毒载体确实具有体内富集和自组装的效果，本发明随机提供了一组病毒载体携带四个所述线路时，相邻线路之间以序列1-序列2-序列3相连，且序列2分别为5个碱基、10个碱基、20个碱基、30个碱基、40个碱基、50个碱基以及80个碱基组成的实验数据，通过实验验证了病毒载体的富集和自组装效果。
序列2具体如下表3所示。
Figure PCTCN2022083876-appb-000017
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。
为了证明病毒载体确实具有体内富集和自组装的效果，本发明随机提供了一组病毒载体含有连接序列为序列4以及至少2条与序列4同源性大于80％的序列的相应实验数据，并通过实验验证了病毒载体的富集和自组装效果。
序列具体如下表4所示。
序列4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
实施例13
根据实施例2描述的方法构建携带抑制PTP1B表达的PTP1B siRNA的质粒CMV-siR P，以及携带靶向肽RVG和PTP1B siRNA的质粒CMV-RVG-siR P。类似的，根据实施例5描述的方法构建携带抑制PTP1B表达的PTP1B siRNA的病毒AAV-CMV-siR P和携带靶向肽RVG的病毒AAV-CMV-RVG-siR P。其中，PTP1B基因的siRNA具有以下核苷酸序列：UGAUAUAGUCAUUAUCUUCUU(SEQ ID NO.14)。
在第一个试验中，设置2个试验组和1个对照组。试验组分别为CMV-siR P组、CMV-RVG-siRP组，对照组为CMV-scrR组，其中“P”表示PTP1B。
CMV-siR P组、CMV-RVG-siR P组、CMV-scrR组分别对小鼠注射5mg/kg的CMV-siR P质粒、CMV-RVG-siR P质粒、CMV-scrR质粒，而后分别获取各组小鼠的下丘脑、肝脏荧光显微镜图像，结果如图40所示，结果显示PTP1B siRNA能够向下丘脑传递。
在第二个试验中，设置2个试验组和2个对照组，试验组分别为CMV-siR P组小鼠体重对比图，可以看出，CMV-RVG-siR P组的小鼠体重最为稳定。
如图41C所示，该图为各组小鼠附睾脂肪垫重量对比图，可以看出，CMV-RVG-siR P组的小鼠附睾脂肪垫重量最轻。
利用代谢笼对不同处理小鼠的氧气消耗量、呼吸交换比、活动量、产热量进行连续72小时的检测，再取平均值绘图进行统计分析，结果如图41D-图41G所示。结果表明CMV-RVG-siR P质粒可以有效提高小鼠的氧气消耗量，这意味着这组小鼠相比于其他组小鼠处于高能量代谢状态。正常小鼠主要以葡萄糖作为自己的能量来源，CMV-RVG-siRP质粒可以降低小鼠的呼吸交换比，这意味着这组小鼠相对于其他组小鼠更倾向于利用蛋白质作为自己的能量来源。注射CMV-RVG-siR P质粒的小鼠活动量明显增加。而且，CMV-RVG-siR P组的小鼠产热也明显提高。
如图41H所示，该图为各组小鼠初始体重曲线对比图。可以看出，CMV-RVG-siR P组的小鼠体重最轻。
如图41I所示，该图为各组小鼠初始食物摄入量曲线对比图。可以看出，CMV-RVG-siR P组的小鼠食物摄入量最少。
如图41J所示，该图为各组小鼠血清瘦素含量对比图。可以看出，CMV-RVG-siR P组的小鼠血清瘦素含量最低。
如图41K所示，该图为各组小鼠的western blot对比图。可以看出，CMVRVG-siR P组小鼠的PTP1B蛋白含量最低。
如图41L所示，该图为各组小鼠血糖变化曲线对比图。可以看出，CMV-RVG-siR P组的小鼠血糖含量最低。
如图41M所示，该图为各组小鼠基础葡萄糖变化曲线对比图。可以看出，CMV-RVG-siR P组的小鼠基础葡萄糖含量最低。
以上试验可以得出，静脉注射CMV-RVG-siR P质粒能够降低肥胖小鼠模型的肥胖。
对各组小鼠的血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)进行测定，结果如图42A所示，可以看出，CMV-RVG-siR P组的小鼠TC、TG、LDL最低。
对各组小鼠的体长进行测定，结果如图42B所示，可以看出，四组小鼠体长相差无几。
统计各组小鼠的HFD食物摄入量，结果如图42C所示，可以看出，四组小鼠HFD食物摄入量相差无几。
对各组小鼠分别进行治疗后取肝组织，与正常对照进行对比，结果如图31D所示，PBS组、CMV-scrR组小鼠的肝组织病理切片中可见有明显的脂肪肝病理特征，CMV-siR P组小鼠的脂肪肝较轻。
以上试验说明静脉注射CMV-RVG-siR P质粒能够减轻肥胖小鼠的脂肪肝。
另外，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-siRP组/AAV-CMV-RVG-siRP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43A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43B所示，该图为各组小鼠的附睾脂肪垫重量对比图，可以看出，AAV-CMV-RVG-siR P组的小鼠附睾脂肪垫重量最轻。
如图43C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43D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43E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43F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44A-图44C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44D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44E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对肥胖具有抑制作用。
腺病毒载体中，含有3种同源序列的情况下，也具有体内富集、自组装及肥胖症治疗效果，如图8所示，序列分组如下：
1、3条同源性大于80％的5’侧翼序列；
2、3条同源性大于80％的loop序列；
3、3条同源性大于80％的3’侧翼序列。
序列具体如下表2所示。
Figure PCTCN2022083876-appb-000018
Figure PCTCN2022083876-appb-000019
在病毒载体携带两个或多个线路的情况下，相邻的线路之间可以通过序列1-序列2-序列3相连；其中，序列1优选为CAGATC，序列2可以为由5-80个碱基组成的序列，比如10个、15个、20个、25个、30个、35个、40个、45个、50个、55个、60个、65个、70个、75个碱基组成的序列均可，优选为10-50个碱基组成的序列，更优选为20-40个碱基组成的序列，序列3优选为TGGATC。
腺病毒载体携带多个线路时，相邻线路之间以序列1-序列2-序列3相连，其中序列2含有多个碱基，所构建的递送系统也同样具有体内富集、自组装和肥胖症治疗效果，。
序列2具体如下表3所示。
Figure PCTCN2022083876-appb-000020
更为优选地，在病毒载体携带两个或多个线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
连接序列为序列4以及与序列4同源性大于80％的序列时，构建的递送系统也具有体内富集、自组装和肥胖症治疗效果序列4-1即为所述序列4，序列4-2/4-3/4-4分别为序列4-1的同源序列，序列具体如下表4所示。
序列4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
以上所述的RNA片段包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列能够在目标受体中被表达，所述补偿序列在目标受体中不能被表达。RNA序列可以为siRNA序列、shRNA序列或miRNA序列，优选为siRNA序列。
一个RNA序列的长度为15-25个核苷酸(nt)，优选为18-22nt，比如18nt、19nt、20nt、21nt、22nt均可。此序列长度的范围并不是随意选择的，而是经过反复的试验后确定的。大量试验证明，在RNA序列的长度小于18nt，特别是小于15nt的情况下，该RNA序列大多无效，不会发挥作用，而在RNA序列的长度大于22nt，特别是大于25nt的情况下，则不仅线路的成本大大提高，而且效果也并未优于长度为18-22nt的RNA序列，经济效益差。因此，在RNA序列的长度为15-25nt，特别是18-22nt时，能够兼顾成本与作用的发挥，效果最好。
RNA序列长度分别为18、20、21时，所构建的递送系统，也具有体内富集、自组装和肥胖症的治疗效果，具体序列如表5所示。
21nt序列 CTAACTTCAGTGTCTGGACTC
20nt序列 CTAACTTCAGTGTCTGGACT
18nt序列 CTAACTTCAGTGTCTGGA
病毒载体系统中携带有多种不同RNA片段的情况下，具有体内富集、自组装及针对肥胖症治疗效果，如图6所示，其中RNA片段分组如下所示：
1、6种RNA单独使用：siRNA-1单独、siRNA-2单独、shRNA-1单独、shRNA-2单独、miRNA-1单独、miRNA-2单独；
2、6种RNA序列中的任意2种组合成RNA片段：siRNA-1+siRNA-2、shRNA-1+shRNA-2、miRNA-1+miRNA-2；
3、6种RNA序列中的任意3种组合成RNA片段：siRNA-1+siRNA-2+shRNA-1、siRNA-1+siRNA-2+shRNA-2、siRNA-1+siRNA-2+miRNA-1、siRNA-1+siRNA-2+miRNA-2。
RNA序列具体如下表1所示。
Figure PCTCN2022083876-appb-000021
Figure PCTCN2022083876-appb-000022
实施例14
根据实施例2描述的方法构建携带抑制mHTT表达的mHTT siRNA的质粒CMV-siR mHTT，以及携带靶向肽RVG和mHTT siRNA的质粒CMV-RVG-siR P。类似的，根据实施例5描述的方法构建携带抑制mHTT表达的mHTT siRNA的病毒AAV-CMV-siR mHTT和携带靶向肽RVG的病毒AAV-CMV-RVG-siR mHTT。其中，mHTT基因的siRNA具有以下核苷酸序列：UAUGUUUUCACAUAUUGUCAG(SEQ ID NO.15)。
在第一个试验中，设置2个试验组和2个对照组。试验组分别为CMV-siR mHTT组、CMV-RVG-siR mHTT组，对照组分别为PBS组、CMV-scrR组。
试验流程如图45A所示，分别对CMV-siR mHTT组、CMV-RVG-siR mHTT组、PBS组、CMV-scrR组患有亨廷顿病的小鼠尾静脉注射CMV-siR mHTT质粒、CMV-RVG-siR mHTT质粒、PBS溶液、CMV-scrR质粒后，分离血浆外泌体，用PKH26染料标记后与细胞进行共培，观察细胞对外泌体的吸收情况。
如图45B所示，该图为各组小鼠血浆外泌体中siRNA水平对比图，可以看出，2个试验组小鼠血浆的外泌体中siRNA水平较高。
对各组小鼠注射质粒/溶液后提取的血浆外泌体，采用PKH26标记、与细胞共培养并用共聚焦显微镜进行拍照。结果如图45C所示，显示包裹siRNA的外泌体进入细胞。
提取各组小鼠血浆外泌体与细胞共培后，检测各组小鼠的HTT蛋白水平以及mRNA水平的变化，结果如图45D-图45F所示，表明CMV-siR mHTT与CMV-RVG-siR mHTT可以降低HTT蛋白水平，说明组装进入外泌体的siRNA仍然可以发挥基因沉默功能。
将提取的小鼠血浆外泌体与细胞共培后，观察并统计各组小鼠HTT蛋白的聚集情况，结果如图45G-图45H所示，表明CMV-siR mHTT与CMV-RVG-siR mHTT可以降低亨廷顿HTT聚集细胞模型中病理HTT蛋白的聚集，说明组装进入外泌体的siRNA仍然可以发挥基因沉默功能，并可以有效降低突变蛋白聚集。
分别检测统计各组小鼠肝脏、血浆、皮质、纹状体中绝对siRNA的表达水平。如图46A所示，该图为各组小鼠肝脏siRNA绝对水平对比图，可以看出，CMV-siR mHTT组、CMV-RVG-siR mHTT组的小鼠绝对siRNA水平较高；如图46B所示，该图为各组小鼠血浆中绝对siRNA水平对比图，可以看出，CMV-siR mHTT组、CMV-RVG-siR mHTT组的小鼠绝对siRNA水平较高；如图46C所示，该图为各组小鼠皮层和纹状体绝对siRNA水平对比图，可以看出，注射CMV-RVG-siR mHTT的小鼠绝对 siRNA水平较高。
如图46D所示，该图为各组小鼠肝、皮层、纹状体组织原位杂交图，可以看出，CMV-siR mHTT组、CMV-RVG-siR mHTT组小鼠肝脏组织切片有明显的荧光，CMV-RVG-siR mHTT组小鼠皮层、纹状体组织切片有明显的荧光。这说明RVG可以引导外泌体siRNA进入通过血脑屏障并发挥功能。
在第二个试验中，设置2个试验组和2个对照组。试验组分别为CMV-siR GFP组、CMV-RVG-siR GFP组，对照组分别为PBS组、CMV-scrR组。分别对CMV-siRGFP组、CMV-RVG-siRGFP组、PBS组、CMV-scrR组GFP转基因小鼠的小鼠尾静脉注射CMV-siR GFP质粒、CMV-RVG-siR GFP质粒、PBS溶液、CMV-scrR质粒。
如图46E、图46F所示，该图为各组小鼠肝、皮层、纹状体组织切片图，
可以看出，肝脏中注射CMV-siR GFP/CMV-RVG-siR GFP的GFP转基因小鼠GFP荧光水平降低，皮层纹状体中注射CMV-RVG-siR GFP的GFP荧光水平降低。说明RVG可以引导外泌体siRNA进入通过血脑屏障并发挥功能。
在第三个试验中，设置两个试验组和一个对照组，试验组分别为CMV-siR mHTT组、CMV-RVG-siR mHTT组，对照组为CMV-scrR组。
试验过程如图47A所示，选取8周龄的N17182Q小鼠，分别为CMV-siR mHTT组、CMV-RVG-siR mHTT组、CMV-scrR组患有亨廷顿病的小鼠尾静脉注射CMV-siR mHTT质粒、CMV-RVG-siR mHTT质粒、CMV-scrR质粒，在第0天和第14天进行旋转试验，14天后处死小鼠进行分析。
如图47B所示，该图为野生型小鼠、CMV-scrR组、CMV-RVG-siR mHTT组的小鼠下降潜伏期对比图，可以看出在0天时，CMV-scrR组、CMV-RVG-siR mHTT组的小鼠的下降潜伏期比较一致，在第14天时，CMV-scrR组的小鼠下降潜伏期最短。
如图47C和图47D所示，图47C为CMV-scrR组、CMV-RVG-siR mHTT组小鼠纹状体的western bolt图，图47D为CMV-scrR组、CMV-RVG-siR mHTT组小鼠纹状体的相对mHTT mRNA水平对比图，可以看出，CMV-scrR组小鼠纹状体中N171-mHTT蛋白含量较高、相对mHTT mRNA水平同样较高。
在第四个试验中，设置一个试验组和一个对照组，试验组为CMV-RVG-siR mHTT组，对照组为CMV-scrR组。
试验过程如图47E所示，选取3月龄的BACHD小鼠，分别为CMV-RVGsiR mHTT组、CMV-scrR组患有亨廷顿病的小鼠尾静脉注射CMV-RVG-siR mHTT质粒、CMV-scrR质粒，14天后处死小鼠进行分析。
如图47F所示，图47F为CMV-scrR组、CMV-RVG-siR mHTT组小鼠皮质和纹状体的western bolt图，可以看出CMV-RVG-siR mHTT组小鼠皮质和纹状体的突变体HTT(Mutant HTT)、内源HTT(Endogenous HTT)含量均较少。
如图47G所示，图47G为CMV-scrR组、CMV-RVG-siR mHTT组小鼠皮质和纹状体的相对mHTT蛋白水平对比图，可见，不论是小鼠皮质还是纹状体，CMV-RVG-siR mHTT组小鼠的相对mHTT蛋白水平均较低。
如图47H和图47I所示，图47H为CMV-scrR组、CMV-RVG-siR mHTT组小鼠的免疫荧光图，图47I为CMV-scrR组、CMV-RVG-siR mHTT组小鼠皮质和纹状体的相对mHTT mRNA水平对比图，可见，不论是小鼠皮质还是纹状体，CMV-RVG-siR mHTT组小鼠的相对mHTT mRNA水平均较低。
以上试验证明了静脉注射CMV-RVG-siR mHTT质粒有助于抑制纹状体和皮质的mHTT，从而改善HD小鼠的运动能力和减轻神经病理学。
在第五个试验中，设置一个试验组和一个对照组，试验组为CMV-RVG-siR mHTT组，对照组为CMV-scrR组。
试验过程如图48A所示，选取6周龄的YAC128小鼠，分别为CMV-RVG-siR mHTT组、CMV-scrR组患有亨廷顿病的小鼠尾静脉注射CMV-RVG-siR mHTT质粒、CMV-scrR质粒，在试验开始第0天、 第4周、第8周时分别进行旋转试验，而后处死小鼠进行分析。
如图48B所示，该图为野生型小鼠、CMV-RVG-siR mHTT组、CMV-scrR组小鼠下降潜伏期对比图，可以看出在0天时，CMV-RVG-siR mHTT组、CMV-scrR组的小鼠的下降潜伏期比较一致，在第四周和第八周时，CMV-scrR组的小鼠下降潜伏期最短。
如图48C所示，该图为CMV-RVG-siR mHTT组、CMV-scrR组小鼠皮质和纹状体的western bolt图，可以看出，CMV-RVG-siR mHTT组小鼠皮质突变体HTT和内源HTT含量均较低，其纹状体突变体HTT含量较低，纹状体内源HTT含量较高。
如图48D、图48E所示，这两张图分别为CMV-RVG-siR mHTT组、CMV-scrR组小鼠皮质和纹状体的相对mHTT mRNA水平对比图、相对mHTT蛋白水平对比图，可以看出，不论是皮质还是纹状体，CMV-RVG-siR mHTT组小鼠的相对mHTT mRNA水平、相对mHTT蛋白水平均相对更低。
如图48F所示，该图是CMV-RVG-siR mHTT组、CMV-scrR组小鼠皮质和纹状体的免疫荧光图，可以看出CMV-RVG-siRmHTT组小鼠的NeuN、EM48表达均低于CMV-scrR组。
以上试验可以说明，静脉注射MV-RVG-siR mHTT质粒有助于纹状体和皮层mHTT蛋白和毒性聚集体减少，从而改善行为缺陷和纹状体和皮层神经病理学。
另外对病毒载体做类似实验。在小鼠尾静脉注射100μL滴度为1012V.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49A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49B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-RVGsiR mHTT组小鼠相对mHTT mRNA水平较低，这说明AAV-CMV-siR mHTT与AAV-CMV-RVG-siR mHTT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49C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49D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49E所示，该图为野生型小鼠(WT)、AAV-CMV-siR mHTT组和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49F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3所示。
Figure PCTCN2022083876-appb-000023
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。
序列具体如下表4所示。
序列4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
实施例15
根据实施例2描述的方法构建携带抑制LRRK2表达的LRRK2siRNA的质粒CMV-siR LRRK2，以及携带靶向肽RVG和LRRK2siRNA的质粒CMV-RVG-siR LRRK2。其中，LRRK2基因的siRNA为AUUAACAUGAAAAUAUCACUU(SEQ ID NO.16)。
研究所述载体系统在帕金森治疗方面的应用。
在此试验中，选择LRRK2R1441G转基因小鼠3月龄时进行试验，试验设置LPS干预组和LPS非干预组。LPS干预组LPS干预7天后进行CMV-scrR/CMV-RVG-siR LRRK2的治疗。
如图50A和图50B所示，图50A为注射CMV-scrR/CMV-RVG-siR LRRK2的LRRK2R1441G转 基因小鼠western bolt图，图50B注射CMV-scrR/CMV-RVG-siR LRRK2的LRRK2R1441G转基因小鼠蛋白灰度分析图，可以看出注射CMV-RV G-siR LRRK2的小鼠LRRK2蛋白和S935蛋白水平降低，说明CMV-RVG-siR LRRK2在肝脏释放siRNA组装进入外泌体后可以穿过血脑屏障降低脑深部蛋白的表达。
如图50C所示，该图为注射CMV-scrR/CMV-RVG-siR  LRRK2的LRRK2R1441G转基因小鼠黑质区TH+神经元免疫荧光图，结果表明注射CMV-RVG-siR LRRK2的小鼠挽救了TH神经元的丢失，说明CMV-RVG-siR LRRK2在肝脏释放siRNA组装进入外泌体后可以穿过血脑屏障进入脑深部发挥功能。
如图50D所示，该图为注射CMV-scrR/CMV-RVG-siR LRRK2的LRRK2R1441G转基因小鼠小胶质细胞激活水平免疫荧光图，结果表明注射CMV-RVG-siR LRRK2小鼠能够抑制小胶质的激活，说明CMV-RVG-siR LRRK2在肝脏释放siRNA组装进入外泌体后可以穿过血脑屏障进入脑深部发挥功能。
以上试验可以说明，静脉注射CMV-RVG-siR LRRK2质粒有助于抑制多巴胺能神经元中的LRRK2，从而减轻帕金森PD小鼠的神经病理发展。
在质粒携带两个或多个线路的情况下，相邻的线路之间可以通过序列1-序列2-序列3相连；其中，序列1优选为CAGATC，序列2可以为由5-80个碱基组成的序列，比如10个、15个、20个、25个、30个、35个、40个、45个、50个、55个、60个、65个、70个、75个碱基组成的序列均可，优选为10-50个碱基组成的序列，更优选为20-40个碱基组成的序列，序列3优选为TGGATC。
序列2具体如下表3所示。
Figure PCTCN2022083876-appb-000024
更为优选地，在质粒携带两个或多个线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
序列具体如下表4所示。
序列4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
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3所示。
Figure PCTCN2022083876-appb-000025
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。
序列具体如下表4所示。
序列4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
实施例16安全性检测
在食蟹猕猴(Macaca fascicularis)对本发明提供的RNA递送载体系统进行了更详细的研究。食蟹猕猴是一种用于安全性评估研究的著名非人灵长类动物模型。
经伦理学批准，用4只成年猕猴静脉注射5mg/kg CMV-siR E质粒，在注射前或注射后不同时间点采集血样。一个月后，这些猕猴每天静脉注射5mg/kg CMV-siR E质粒，共注射5次，并在注射前或最后一次注射后的不同时间点采集血样。
如图51所示，图51A为单次注射的食蟹猕猴全血siRNA浓度变化图，图51B位多次注射的食蟹猕猴全血siRNA浓度变化图，可以看出单次注射的食蟹猕猴siRNA浓度在静脉注射6小时后达到峰值，随即降低，多次注射的食蟹猕猴siRNA浓度在静脉注射3小时后达到峰值，随即降低，多次注射的食蟹猕猴siRNA浓度的降低速度更为缓慢。
以上实验可以说明，CMV-siR E质粒能够对食蟹猕猴等灵长类动物安全且有效。
上面是对本发明进行的说明，不能将其看成是对本发明进行的限制。除非另外指出，本发明的实践将使用有机化学、聚合物化学、生物技术等的常规技术，显然除在上述说明和实施例中所特别描述之外，还可以别的方式实现本发明。其它在本发明范围内的方面与改进将对本发明所属领域的技术人员显而易见。根据本发明的教导，许多改变和变化是可行的，因此其在本发明的范围之内。

Claims (14)

  1. 一种分离的核酸，其包括编码能够抑制基因表达的RNA的核苷酸序列，其包含：
    (a)编码一个或多个抑制基因表达的RNA的核苷酸序列，所述RNA为miRNA、shRNA、siRNA、mRNA、ncRNA、sgRNA或任意这些RNA的组合；
    其中，所述核苷酸序列包括针对所述基因的RNA片段序列，以及包括侧翼序列(如5’侧翼序列和3’侧翼序列)、茎环序列和所述RNA序列的补偿序列中的一个或多个；
    优选的，所述编码一个或多个抑制基因表达的RNA的核苷酸依序为：5’侧翼序列、RNA片段序列、茎环序列、补偿序列和3’侧翼序列；
    任选的，(b)编码靶向蛋白的核苷酸序列。
  2. 权利要求1所述的核酸，其中(a)为编码一个所述抑制基因表达的RNA的核苷酸序列。
  3. 权利要求1所述的核酸，其中(a)为编码多个所述抑制基因表达的RNA的核苷酸序列，优选的，所述多个抑制基因表达的RNA为2-4个抑制基因表达的RNA。
  4. 权利要求1-3中任一项所述的核酸，其中所述RNA为siRNA。
  5. 权利要求1-3中任一项所述的核酸，其中编码一个或多个抑制基因表达的RNA片段序列的长度为15-29个核苷酸，优选为21-23个核苷酸。
  6. 权利要求1所述的核酸，其中所述靶向蛋白为靶组织特异性靶向肽。
  7. 权利要求1所述的核酸，其中所述靶向蛋白为靶组织特异性靶向肽和膜蛋白的融合蛋白。
  8. 权利要求6或7所述的核酸，其中所述靶组织特异性靶向肽选自：RVG靶向肽、GE11靶向肽、PTP靶向肽、TCP-1靶向肽、MSP靶向肽。
  9. 权利要求6或7所述的核酸，其中所述膜蛋白选自：受体蛋白(例如生长因子受体)、LAMP1或LAMP2(例如为LAMP2B)、抗体或其结合片段。
  10. 抑制基因表达的RNA的载体，其包含：
    (a)编码一个或多个抑制基因表达的RNA的核苷酸序列，所述RNA为miRNA、shRNA、siRNA、mRNA、ncRNA、sgRNA或任意这些RNA的组合；
    任选的，(b)编码靶向蛋白的核苷酸序列。
  11. 权利要求10所述的表达载体，其为质粒。
  12. 权利要求10所述的表达载体，其为病毒载体，例如杆状病毒表达载体，腺病毒载体，逆转录病毒载体，孢疹病毒载体或慢病毒载体，优选为腺病毒载体。
  13. 药物组合物，其含有权利要求1-9中任一项的核酸或权利要求10-12中任一项的载体。
  14. 治疗疾病的方法，其中包括向对象施用权利要求1-9中任一项的核酸或权利要求10-12中任一项的载体。
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