WO2022206809A1 - 一种用于治疗肺癌的rna递送系统 - Google Patents

一种用于治疗肺癌的rna递送系统 Download PDF

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WO2022206809A1
WO2022206809A1 PCT/CN2022/083951 CN2022083951W WO2022206809A1 WO 2022206809 A1 WO2022206809 A1 WO 2022206809A1 CN 2022083951 W CN2022083951 W CN 2022083951W WO 2022206809 A1 WO2022206809 A1 WO 2022206809A1
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sequence
rna
lung cancer
targeting
delivery system
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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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Definitions

  • the present application relates to the field of biomedical technology, in particular to an RNA delivery system for the treatment of lung cancer.
  • Lung cancer is one of the malignant tumors with the fastest growing morbidity and mortality, and the greatest threat to the health and life of the population. Lung cancer is currently mainly treated by the following means: 1.
  • Chemotherapy is the main treatment method for lung cancer, and more than 90% of lung cancers need to be treated with chemotherapy. 1% of early-stage small cell lung cancers are cured with chemotherapy. However, chemotherapy inhibits the bone marrow hematopoietic system, mainly the decline of white blood cells and platelets.
  • Radiotherapy The radiation field of lung cancer radiotherapy should include the primary tumor and the mediastinal area of lymph node metastasis, and should be supplemented by drug therapy.
  • Surgical treatment it can completely remove the primary tumor and metastatic lymph nodes of lung cancer to achieve clinical cure; or remove most of the tumor to create favorable conditions for other treatments, that is, cytoreduction surgery; however, surgical treatment has many limitations and cannot large-scale application.
  • RNA interference (RNAi) therapy has been considered a promising strategy for the treatment of human diseases since its invention, but many problems have been encountered during clinical practice, and the development of this therapy has lagged far behind expectations.
  • RNA cannot exist stably outside the cell for a long time, because RNA will be degraded into fragments by RNases rich in extracellular, so it is necessary to find a method that can make RNA stable outside the cell and can enter specific tissues in a targeted manner. Highlight the effect of RNAi therapy.
  • Virus (Biological virus) is a small individual, simple structure, containing only one nucleic acid (DNA or RNA), must be parasitic in living cells and replicated non-cellular organisms. Viral vectors can bring genetic material into cells. The principle is to use the molecular mechanism of viruses to transmit their genomes into other cells for infection. It can occur in a complete living body (in vivo) or cell culture (in vitro), mainly used in Basic research, gene therapy or vaccines. However, there are few related studies on the use of viruses as vectors to deliver RNA, especially siRNA, using a special self-assembly mechanism.
  • the Chinese Patent Publication No. CN108624590A discloses a siRNA capable of inhibiting the expression of DDR2 gene; the Chinese Patent Publication No. CN108624591A discloses a siRNA capable of silencing the ARPC4 gene, and the siRNA is modified with ⁇ -phosphorus-selenium;
  • the Chinese Patent Publication No. CN108546702A discloses a siRNA targeting long-chain non-coding RNA DDX11-AS1.
  • the Chinese Patent Publication No. CN106177990A discloses a siRNA precursor that can be used for various tumor treatments. These patents design specific siRNAs to target certain diseases caused by genetic changes.
  • Chinese Patent Publication No. CN108250267A discloses a polypeptide, polypeptide-siRNA induced co-assembly, using polypeptide as a carrier of siRNA.
  • the Chinese Patent Publication No. CN108117585A discloses a polypeptide for promoting apoptosis of breast cancer cells through targeted introduction of siRNA, and the polypeptide is also used as the carrier of siRNA.
  • the Chinese Patent Publication No. CN108096583A discloses a nanoparticle carrier, which can be loaded with siRNA with breast cancer curative effect while containing chemotherapeutic drugs.
  • exosomes can deliver miRNAs to recipient cells, which secrete miRNAs at relatively low concentrations , which can effectively block the expression of target genes.
  • Exosomes are biocompatible with the host immune system and possess the innate ability to protect and transport miRNAs across biological barriers in vivo, thus becoming a potential solution to overcome problems associated with siRNA delivery.
  • the Chinese Patent Publication No. CN110699382A discloses a method for preparing siRNA-delivering exosomes, and discloses the technology of separating exosomes from plasma and encapsulating siRNA into exosomes by electroporation .
  • the embodiments of the present application provide an RNA delivery system for treating lung cancer and its application, so as to solve the technical defects existing in the prior art.
  • One of the inventions of the present application is to provide an RNA delivery system for treating lung cancer.
  • the system includes a viral vector carrying an RNA fragment capable of treating lung cancer, and the viral vector can enrich the organ tissue of a host. and endogenously and spontaneously form a complex structure containing the RNA in the host organ tissue, and the complex structure can deliver the RNA fragments into the lungs to realize the treatment of lung cancer.
  • the viral vector is an adenovirus-associated virus.
  • adenovirus-associated virus is adenovirus-associated virus type 5, adenovirus-associated virus type 8 or adenovirus-associated virus type 9.
  • RNA fragment comprises one, two or more specific RNA sequences with medical significance, and the RNA sequences are siRNA, shRNA or miRNA with medical significance.
  • the viral vector includes a promoter sequence and a targeting tag
  • the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host
  • the targeting structure is located on the surface of the composite structure, so The complex structure can seek and bind to the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
  • the viral vector includes any one of the following circuits or a combination of several circuits: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each of the viral vectors including at least one RNA segment and one targeting tag, the RNA segment and targeting tag are located in the same circuit or are located in different circuits.
  • the viral vector also includes a flanking sequence, a compensation sequence and a loop sequence that can make the circuit fold into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence;
  • the viral vector includes any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-target To tag, 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensating sequence-3'flanking sequence.
  • the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence whose homology is greater than 80%;
  • the loop sequence is gttttggccactgactgac or a sequence whose homology is greater than 80%;
  • the 3' flanking sequence is accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence whose homology is greater than 80%;
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the purpose of deleting bases 1-5 of the reverse complement of the RNA is to make the sequence unexpressed.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
  • the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
  • adjacent lines are connected by a sequence composed of sequences 1-3 (sequence 1-sequence 2-sequence 3);
  • sequence 1 is CAGATC
  • sequence 2 is a sequence consisting of 5-80 bases
  • sequence 3 is TGGATC.
  • adjacent lines are connected by sequence 4 or a sequence with more than 80% homology to sequence 4;
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • organ tissue is liver
  • composite structure is exosome
  • the targeting tag is selected from targeting peptides or targeting proteins with targeting function.
  • the targeting peptides include RVG targeting peptides, GE11 targeting peptides, PTP targeting peptides, TCP-1 targeting peptides, and MSP targeting peptides;
  • the targeting proteins include RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein, and MSP-LAMP2B fusion protein.
  • the length of the RNA sequence is 15-25 nucleotides.
  • the length of the RNA sequence can be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides.
  • the RNA sequence is 18-22 nucleotides in length.
  • RNA sequence capable of treating lung cancer is selected from any one or more of the following RNAs: EGFR gene siRNA, KRAS gene siRNA or nucleic acid molecules encoding the above RNAs.
  • EGFR gene siRNA includes UGUUGCUUCUCUUAAUUCCU, AAAUGAUCUUCAAAAGUGGCC, UCUUUAAGAAGGAAAGAUCAU, AAUAUUCGUAGCAUUUAUGGA, UAAAAAUCCUCACAUAUACUU, other sequences that inhibit EGFR gene expression and sequences with more than 80% homology to the above sequences.
  • the siRNA of KRAS gene includes UGAUUUAGUAUUAUUUAUGGC, AAUUUGUUCUCUAUAAUGGUG, UAAUUUGUUCUCUAUAAUGGU, UUAUGUUUUCGAAUUUCUCGA, UGUAUUUUACAUAAUUACACAC, other sequences that inhibit KRAS gene expression, and sequences with more than 80% homology to the above sequences.
  • sequences with more than 80% homology may be 85%, 88%, 90%, 95%, 98%, etc. homology.
  • the RNA fragment includes an RNA sequence ontology and a modified RNA sequence obtained by modifying the RNA sequence ontology with ribose sugar. That is, the RNA fragment can be composed of only at least one RNA sequence ontology, or only at least one modified RNA sequence, and can also be composed of RNA sequence ontology and modified RNA sequence.
  • the isolated nucleic acid also includes its variants and derivatives.
  • the nucleic acid can be modified by one of ordinary skill in the art using general methods. Modification methods include (but are not limited to): methylation modification, hydrocarbyl modification, glycosylation modification (such as 2-methoxy-glycosyl modification, hydrocarbyl-glycosyl modification, sugar ring modification, etc.), nucleic acid modification, peptide modification Segment modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification) and the like.
  • the modification is an internucleotide linkage, for example selected from: phosphorothioate, 2'-O methoxyethyl (MOE), 2'-fluoro, phosphine Acid alkyl esters, phosphorodithioates, alkyl phosphorothioates, phosphoramidates, carbamates, carbonates, phosphoric triesters, acetamidates, carboxymethyl esters, and combinations thereof.
  • phosphorothioate 2'-O methoxyethyl (MOE), 2'-fluoro
  • phosphine Acid alkyl esters phosphorodithioates, alkyl phosphorothioates, phosphoramidates, carbamates, carbonates, phosphoric triesters, acetamidates, carboxymethyl esters, and combinations thereof.
  • the modification is a modification of nucleotides, such as selected from: peptide nucleic acid (PNA), locked nucleic acid (LNA), arabinose-nucleic acid (FANA), analogs, derivatives objects and their combinations.
  • the modification is a 2' fluoropyrimidine modification.
  • 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on RNA with 2'-F.
  • 2'-F can make RNA not easily recognized by RNase in vivo, thereby increasing the stability of RNA fragment transmission in vivo. sex.
  • the delivery system is a delivery system for use in mammals including humans.
  • Another inventive point of the present application is to provide an application of the above-mentioned RNA delivery system for treating lung cancer in medicine.
  • administration modes of the drug include oral, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection.
  • the dosage forms of the drug can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes and the like.
  • the RNA delivery system for the treatment of lung cancer uses a virus as a vector, and the virus vector is used as a mature injection, and its safety and reliability have been fully verified, and the drugability is very good.
  • the final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of viral vectors is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • RNA delivery system for the treatment of lung cancer provided in this application can be tightly combined with AGO 2 and enriched into a composite structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation, but also maintain its stability in circulation It is also beneficial to receptor cell uptake, intracytoplasmic release and lysosomal escape, and the required dose is low.
  • RNA delivery system for the treatment of lung cancer provided in this application to medicines provides a drug delivery platform, which can greatly improve the therapeutic effect of lung cancer, and can also form the basis for the research and development of more RNA-based drugs through this platform. Drug research and development and use have a great impetus.
  • FIG. 1 is a diagram of the treatment situation of mouse lung cancer based on KRAS siRNA provided by an embodiment of the present application
  • FIG. 2 is a diagram of the treatment situation of mouse lung cancer based on EGFR siRNA provided by an embodiment of the present application
  • FIG. 3 is a comparison diagram of the content of various enzymes in mice provided in an example of the present application.
  • Figure 4 shows the enrichment effect of the lentiviral vector in liver, lung, plasma, and exosomes and the detection of EGFR gene expression provided by an embodiment of the present application.
  • 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 5 shows the enrichment effect of adenovirus vector in liver, lung, plasma, and exosomes and the detection of EGFR gene expression provided by an embodiment 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 embodiment 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
  • Fig. 6 is the fluorescence signal statistics 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 fluorescence signal statistics after siRNA is constructed into adeno-associated virus vectors to treat lung cancer
  • B is the fluorescence signal statistics after siRNA was constructed into adeno-associated virus vector for treatment of lung cancer
  • C is the fluorescence signal statistics of miR-7 constructed into adeno-associated virus vector for lung cancer treatment
  • D is the fluorescence signal statistics after shRE was constructed into adeno-associated virus vector for lung cancer treatment
  • the fluorescence signal statistics of E is the fluorescence signal statistics of shRT 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.
  • Fig. 7 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 siR E +shR in the figure
  • 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. 8 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 in the figure is siR E +shR 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 9 shows the enrichment results of siRNA in liver, lung, plasma, and exosome species and the detection results of EGFR protein and mRNA expression after intravenous injection provided by an embodiment of the present application
  • a in the figure is AAV-siR E and AAV -
  • the enrichment results of GE11-siRE E in liver and lung B is the enrichment results of AAV-siRE E and AAV-GE11-siRE E in plasma and exosomes
  • C is the enrichment results of AAV-siRE E and AAV-GE11 - EGFR protein expression of siR E
  • D is the EGFR mRNA expression of AAV-siR E and AAV-GE11-siR E.
  • Figure 10 shows the enrichment effect and therapeutic effect in the lungs after 2 sequences with more than 80% homology to the 5' flanking sequence provided by an embodiment of the present application are constructed into the 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 11 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 in an example 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 12 shows the enrichment effect and therapeutic effect in the lungs 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 13 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 AAV vectors, and EGFR siRNA in lung tissue after intravenous injection for 9 hours 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 14 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.
  • A is the result of EGFR protein content
  • B is the result of EGFR mRNA content.
  • Western Blot (Western Blot) is to transfer the protein to the membrane, and then use the antibody for detection.
  • the corresponding antibody can be used as the primary antibody for detection, and the expression product of the new gene can be detected by the fusion part of the antibody. .
  • Western Blot uses polyacrylamide gel electrophoresis, the detected object is protein, the "probe” is an antibody, and the "color development” is a labeled secondary antibody.
  • the protein sample separated by PAGE is transferred to a solid phase carrier (such as nitrocellulose membrane), and the solid phase carrier adsorbs proteins in the form of non-covalent bonds, and can keep the types of polypeptides separated by electrophoresis and their biological activities unchanged.
  • the protein or polypeptide on the solid phase carrier is used as an antigen, which reacts with the corresponding antibody, and then reacts with the enzyme or isotope-labeled secondary antibody to detect the specific target gene separated by electrophoresis through substrate color development or autoradiography.
  • expressed protein components The steps mainly include: protein extraction, protein quantification, gel preparation and electrophoresis, membrane transfer, immunolabeling and development.
  • the detection of the siRNA level, the protein content and the mRNA content involved in the present invention is to establish the mouse stem cell in vitro model by injecting the RNA delivery system into the mouse.
  • the expression levels of mRNA and siRNA in cells and tissues were detected by qRT-PCR. Absolute quantification of siRNA was determined by plotting a standard curve using the standards.
  • the internal reference gene is U6snRNA (in tissue) or miR-16 (in serum, exosomes)
  • the gene is GAPDH or 18s RNA.
  • Western blotting was used to detect protein expression levels in cells and tissues, and ImageJ software was used for protein quantitative analysis.
  • This embodiment provides an RNA delivery system for treating lung cancer.
  • the system includes a viral vector carrying an RNA fragment capable of treating lung cancer.
  • the viral vector can be enriched in the organ tissue of a host and can A composite structure containing the RNA is formed endogenously and spontaneously in the host organ tissue, and the composite structure can deliver the RNA fragments into the lungs to achieve the treatment of lung cancer.
  • Figure 4 shows the enrichment effect of lentiviral vectors in liver, lung, plasma, and exosomes and the detection of EGFR gene expression.
  • Figure 5 shows adeno-associated virus in liver, lung, plasma, and exosomes. The enrichment effect and EGFR gene expression detection.
  • the viral vector also includes a promoter and a targeting tag.
  • the viral vector includes any one of the following circuits or a combination of several circuits: promoter-RNA sequence, promoter-targeting tag, promoter-RNA sequence-targeting tag, and each of the viral vectors includes at least one RNA fragments and a targeting tag, either in the same circuit or in different circuits.
  • the viral vector may only include a promoter-RNA sequence-targeting tag, or may include a combination of a promoter-RNA sequence, a promoter-targeting tag, or a promoter-targeting tag, a promoter A combination of RNA-seq-targeting tags.
  • 5'-promoter-5' flanking sequence-RNA sequence-loop sequence-compensation sequence-3' flanking sequence corresponds to the delivery system name AAV-siR E , 5'-promoter-targeting tag-5 'Flanking sequence-RNA sequence-loop sequence-compensation sequence-3' flanking
  • the corresponding delivery system name is AAV-GE11- siRE
  • Figure 9 shows the enrichment results of siRNA in liver, lung, plasma and exosomes after intravenous injection and EGFR protein and mRNA expression detection results.
  • 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.
  • flanking sequence CTGGAGGCTTGCTGAAGGCTGTATGCTGAATTCG 5' flanking sequence-2 CTGGAGGGCTTGCTGAAGGCTGGCAGCTGAATTCG loop-1 GTTTTGGCCACTGACTGAC loop-2 GTTGGTAACTGACTGAC 3' flanking sequence -1 CACCGGTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC 3' flanking sequence-2 CACCGGTCTGAACACAAGGCCTGTTACTAGCACGTCCATGGAACAAATGGCC
  • 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.
  • Figure 10 shows the enrichment effect and therapeutic effect of the 5' flanking sequence with more than 80% homology into the AAV vector
  • Figure 11 shows the construction of the loop sequence with more than 80% homology
  • the enrichment effect and therapeutic effect of the imported AAV vector in the lungs shows the enrichment effect and therapeutic effect of the imported AAV vector in the lung of the sequence with the 3' flanking sequence homology greater than 80%.
  • 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 vector, and the detection result of EGFR siRNA content in lung tissue 9 hours after intravenous injection .
  • 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 functional structural region of the viral vector can be expressed as: (promoter-siRNA1)-connector sequence-(promoter-siRNA2)-connector sequence- (promoter-targeting tag), or (promoter-targeting tag-siRNA1)-linker-(promoter-targeting tag-siRNA2), or (promoter-siRNA1)-linker-(promoter- Targeting tag-siRNA2) etc.
  • RNA sequence length is respectively 18,20,22 corresponds to AAV- siRE (18), AAV- siRE (20), AAV- siRE (22) respectively, and Fig. 14 is above-mentioned 3 kinds Detection of EGFR expression after gene loop intravenous injection.
  • RNAs comprise one, two or more specific RNA sequences of medical significance, the RNA sequences can be expressed in the target receptor, and the compensatory sequence cannot be expressed in the target receptor.
  • RNA capable of treating lung cancer is selected from any one or more of the following RNAs: EGFR gene siRNA, KRAS gene siRNA or nucleic acid molecules encoding the above RNAs.
  • RNA effective sequences capable of treating lung cancer is one, two or more.
  • EGFR gene siRNA and KRAS gene siRNA can be used in combination on the same viral vector, or EGFR gene siRNA or KRAS gene siRNA can be used alone.
  • the functional structural region of the viral vector can be expressed as: (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-connector sequence-(5'-promoter - 5' flanking sequence - siRNA2-loop sequence - compensation sequence - 3' flanking sequence) - linking sequence - (5'-promoter-targeting tag), or (5'-promoter-targeting tag-5' flanking sequence-siRNA1-loop sequence-compensation sequence-3' flanking sequence)-linker sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'flanking sequence), or (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-linking sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'flanking
  • the above RNA can also be obtained by ribose modification of the RNA sequence (siRNA, shRNA or miRNA) therein, preferably 2' fluoropyrimidine modification.
  • 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on siRNA, shRNA or miRNA with 2'-F.
  • 2'-F can make it difficult for RNase in the human body to recognize siRNA, shRNA or miRNA, so it can Increases the stability of RNA transport in vivo.
  • the liver will phagocytose exogenous viruses, and up to 99% of the exogenous viruses will enter the liver. Therefore, when viruses are used as vectors, they can be enriched in liver tissue without specific design. After being opened, RNA molecules (siRNA, shRNA, or miRNA) are released, and liver tissue spontaneously wraps the above RNA molecules into exosomes, and these exosomes become RNA delivery mechanisms.
  • RNA molecules siRNA, shRNA, or miRNA
  • RNA delivery mechanism in order to make the RNA delivery mechanism (exosome) have the ability of "precision guidance”, we design a targeting tag in the virus injected into the body, and the targeting tag will also be assembled into exosomes by liver tissue , especially when certain specific targeting tags are selected, the targeting tags can be inserted into the surface of exosomes to become targeting structures that can guide exosomes, which greatly improves the RNA delivery mechanism of the present invention
  • the amount of viral vector that needs to be introduced can be greatly reduced, and on the other hand, the efficiency of potential drug delivery can be greatly improved.
  • the targeting tag is selected from one of the peptides, proteins or antibodies with targeting function.
  • the selection of the targeting tag is a process that requires creative work. On the one hand, it is necessary to select the available targeting tags according to the target tissue. It is ensured that the targeting label can stably appear on the surface of exosomes, so as to achieve the targeting function. Targeting peptides, targeting proteins and antibodies that have been screened.
  • targeting peptides include but are not limited to RVG targeting peptide (nucleotide sequence shown in SEQ ID No: 1), GE11 targeting peptide (nucleotide sequence shown in SEQ ID No: 2), PTP targeting peptide Peptide (nucleotide sequence shown in SEQ ID No: 3), TCP-1 targeting peptide (nucleotide sequence shown in SEQ ID No: 4), MSP targeting peptide (nucleotide sequence shown in SEQ ID No: 4) : 5); targeting proteins include but are not limited to RVG-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6), GE11-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 7) shown), PTP-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 8), TCP-1-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No:
  • the viral vector can also be composed of multiple viruses with different structures, one of which contains a promoter promoters and targeting tags, other viruses contain promoters and RNA segments. Loading the targeting tag and RNA fragment into different viral vectors, and injecting the two viral vectors into the body, the targeting effect is no worse than the targeting effect produced by loading the same targeting tag and RNA fragment into one viral vector .
  • the viral vector containing the RNA sequence can be injected first, and then the viral vector containing the targeting tag can be injected after 1-2 hours, so that a better target can be achieved. to the effect.
  • the delivery systems described above can all be used in mammals, including humans.
  • the RNA delivery system for the treatment of lung cancer uses a virus as a vector, and the virus vector is used as a mature injection. Its safety and reliability have been fully verified, and the drugability is very good.
  • the final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of viral vectors is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • RNA delivery system for the treatment of lung cancer provided in this example can be tightly combined with AGO 2 and enriched into a composite structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation, but also maintain its circulation in the circulation. It is stable, and facilitates uptake by recipient cells, intracytoplasmic release and lysosomal escape, and requires a low dose.
  • RNA delivery system for the treatment of lung cancer, the system comprising a viral vector carrying an RNA fragment capable of treating lung cancer, the viral vector being capable of enriching in the organ tissue of a host, and expressing in the organ of the host
  • a complex structure containing the RNA is formed endogenously and spontaneously in the tissue, and the complex structure can deliver the RNA fragments into the lungs to achieve the treatment of lung cancer.
  • RNA fragment comprises one, two or more specific RNA sequences with medical significance, and the RNA sequences are siRNA, shRNA or miRNA with medical significance.
  • Fig. 6 is the fluorescence signal statistics after 6 kinds of different RNAs provided are built into adeno-associated virus vector to treat lung cancer, 6 kinds of RNAs are respectively: siRNA (target gene is EGFR), siRNA ( target base) Because TNC), shRE (target gene is EGFR), shRT (target gene is TNC), miR-7 (target gene is EGFR), miR-133b (target gene is EGFR);
  • Figure 7 is the 6 kinds provided above Fluorescence signal statistics of 4 groups consisting of any 2 RNA sequences in the RNA after treatment of lung cancer;
  • Figure 8 is the fluorescence signal statistics of 3 groups consisting of any 3 RNA sequences of the 6 RNAs provided above after treatment of lung cancer.
  • the viral vector includes a promoter sequence and a targeting tag
  • the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host
  • the targeting structure is located on the surface of the composite structure, so The complex structure can seek and bind to the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
  • the drug can be administered orally, inhaled, subcutaneously injected, intramuscularly injected or intravenously injected into the human body, it can be delivered to the target tissue lung cancer through the RNA delivery system described in Example 1 to play a therapeutic role.
  • the drug can also be used in combination with other lung cancer drugs to enhance the effect of treatment.
  • other lung cancer drugs such as gefitinib, erlotinib, icotinib, afatinib, etc.
  • the medicine of this embodiment may also include a pharmaceutically acceptable carrier, which includes but is not limited to diluents, buffers, emulsions, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorption Agents, wetting agents, disintegrating agents, absorption enhancers, surfactants, colorants, flavoring agents, adjuvants, desiccants, adsorption carriers, etc.
  • a pharmaceutically acceptable carrier includes but is not limited to diluents, buffers, emulsions, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorption Agents, wetting agents, disintegrating agents, absorption enhancers, surfactants, colorants, flavoring agents, adjuvants, desiccants, adsorption carriers, etc.
  • the dosage forms of the medicine provided in this embodiment can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes, and the like.
  • the medicine provided in this example uses the virus as the carrier and the virus carrier as the mature injection, and its safety and reliability have been fully verified, and the druggability is very good.
  • the final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the drug can deliver various kinds of small molecule RNAs and has strong versatility. And the preparation of viral vectors is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • the drug provided in this application can be closely combined with AGO 2 and enriched into a composite structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation and maintain its stability in circulation, but also benefit the receptor.
  • Cellular uptake, intracytoplasmic release and lysosomal escape require low doses.
  • this embodiment provides an application of an RNA delivery system for treating lung cancer in medicine. Here, it will be specifically explained by the following test.
  • liver high-affinity AAV-5 adeno-associated virus-encapsulated EGFR siRNA system AAV-CMV-EGFR siRNA
  • KRAS siRNA system AAV-CMV-KRAS siRNA
  • mice mouse lung cancer cells
  • CT scanning technology was used to observe the progress of mouse model construction.
  • the mice in the successfully constructed mice were administered once every two days, that is, the mice in the PBS group/AAV-CMV-scrR group/AAV-CMV-KRAS siRNA group were injected with PBS buffer/AAV-CMV once every two days -scrR/AAV-CMV-KRAS siRNA treatment, survival analysis and tumor assessment were performed in mice, respectively, and the treatment was stopped after 7 doses.
  • FIG. 1B “PBS pre” indicates the PBS group before administration, “PBS post” indicates the PBS group after administration; “AAV-CMV-scrR pre” indicates the AAV-CMV-scrR group before administration, “AAV-CMV-scrR post” indicates the AAV-CMV-scrR group after administration; “AAV-CMV-KRAS-siRNA pre” indicates the AAV-CMV-KRAS siRNA group before administration, “AAV-CMV-KRAS-siRNA pre” post” indicates the AAV-CMV-KRAS siRNA group after administration.
  • the tumor volume of the mice in the AAV-CMV-KRAS siRNA group decreased significantly after administration, while the tumor volume of the mice in the PBS group and AAV-CMV-scrR group not only did not decrease after administration, but also showed increased to varying degrees.
  • 1 experimental group and 2 control groups were set, wherein the experimental group was the AAV-CMV-EGFR siRNA group, and the control groups were the PBS group and the AAV-CMV-scrR group.
  • mice were administered to the successfully constructed mice, once every two days, that is, to the PBS group/AAV-CMV-scrR group/
  • the mice in the AAV-CMV-EGFR siRNA group were injected with PBS buffer/AAV-CMV-scrR/AAV-CMV-EGFR siRNA once for treatment, and the mice were subjected to survival analysis and tumor assessment respectively, and the treatment was stopped after 7 administrations.
  • the tumor volume of the mice in the AAV-CMV-EGFR siRNA group decreased significantly after administration, while the tumor volume of the mice in the PBS group and AAV-CMV-scrR group not only did not decrease after administration, but also showed increased to varying degrees.
  • the experimental group was AAV-CMV-KRAS siRNA group, AAV-CMV-EGFR siRNA group, and the control group was PBS group and AAV-CMV-scrR Group.
  • mice were administered to the successfully constructed mice, once every two days, that is, to the PBS group/AAV-CMV-scrR group/
  • the mice in the AAV-CMV-EGFR siRNA group/AAV-CMV-KRAS siRNA group were injected once with PBS buffer/AAV-CMV-scrR/AAV-CMV-EGFR siRNA/AAV-CMV-KRAS siRNA for treatment.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • TBIL total bilirubin
  • ALP serum alkaline phosphatase
  • CREA creatinine
  • BUN blood urea nitrogen

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Abstract

提供了一种用于治疗肺癌的RNA递送系统。提供的用于治疗肺癌的RNA递送系统包括病毒载体，所述病毒载体携带有能够治疗肺癌的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述RNA的复合结构，所述复合结构能够将所述RNA片段送入肺部，实现肺癌的治疗。

Description

一种用于治疗肺癌的RNA递送系统 技术领域
本申请涉及生物医学技术领域，特别涉及一种用于治疗肺癌的RNA递送系统。
背景技术
肺癌是发病率和死亡率增长最快，对人群健康和生命威胁最大的恶性肿瘤之一。肺癌目前主要采用以下手段进行治疗：1、化学治疗：化疗是肺癌的主要治疗方法，90％以上的肺癌需要接受化疗治疗，化疗对小细胞肺癌的疗效无论早期或晚期均较肯定，甚至有约1％的早期小细胞肺癌通过化疗治愈。但化疗会抑制骨髓造血系统，主要是白细胞和血小板的下降。2、放射治疗：肺癌放疗照射野应包括原发灶、淋巴结转移的纵隔区，同时要辅以药物治疗。但放疗并发症较多，比如放射性肺炎、放射性食管炎、放射性肺纤维化和放射性脊髓炎等。3、外科治疗：其可以完全切除肺癌原发病灶及转移淋巴结，达到临床治愈；或切除肿瘤的绝大部分，为其他治疗创造有利条件，即减瘤手术；但外科治疗限制条件较多，无法大规模应用。
RNA干扰(RNAi)疗法自从被发明以来，一直被认为是治疗人类疾病的一种很有前途的策略，但在临床实践过程中遇到了许多问题，该疗法的发展进度远远落后于预期。
一般认为RNA无法在细胞外长期稳定存在，因为RNA会被细胞外富含的RNase降解成碎片，因此必须找到能够使RNA稳定存在于细胞外，并且能够靶向性地进入特定组织的方法，才能将RNAi疗法的效果凸显出来。
目前与siRNA相关的专利很多，主要聚焦在以下几个方面：1、设计具有医学效果的siRNA。2、对siRNA进行化学修饰，提高siRNA在生物体内的稳定性，提高产率。3、提高设计各种人工载体(如脂质纳米粒子、阳离子聚合物和病毒)，以提高siRNA在体内传递的效率。其中第3方面的专利很多，其根本原因是研究人员们已经意识到目前缺乏合适的siRNA传递系统，将siRNA安全地、精确地、高效地输送到目标组织，该问题已经成为制约RNAi疗法的核心问题。
病毒(Biological virus)是一种个体微小，结构简单，只含一种核酸(DNA或RNA)，必须在活细胞内寄生并以复制方式增殖的非细胞型生物。病毒载体可将遗传物质带入细胞，原理是利用病毒具有传送其基因组进入其他细胞，进行感染的分子机制，可发生于完整活体(in vivo)或是细胞培养(in vitro)中，主要应用于基础研究、基因疗法或疫苗。但是目前很少有针对将病毒作为载体利用特殊的自组装机制递送RNA，特别是siRNA的相关研究。
公开号为CN108624590A的中国专利公开了一种能够抑制DDR2基因表达的siRNA；公开号为CN108624591A的中国专利公开了一种能够沉默ARPC4基因的siRNA，并且对该siRNA进行了α-磷-硒修饰；公开号为CN108546702A的中国专利公开了一种靶向长链非编码RNA DDX11-AS1的siRNA。公开号为CN106177990A的中国专利公开了一种可以用于多种肿瘤治疗的siRNA前体。这些专利均设计了特定的siRNA并且来针对某些由基因变化引起的疾病。
公开号为CN108250267A的中国专利公开了一种多肽、多肽-siRNA诱导共组装体，使用多肽作为siRNA的载体。公开号为CN108117585A的中国专利公开了一种靶向导入siRNA促进乳腺癌细胞凋亡的多肽，同样使用多肽作为siRNA的载体。公开号为CN108096583A的中国专利公开了一种纳米粒子载体，该载体在包含化疗药物的同时还可以装载具有乳腺癌疗效的siRNA。这些专利均为在siRNA载体方面的发明创造，但是其技术方案具有一个共同特征，那就是载体和siRNA均在体外预先组装，然后再引入宿主体内。事实上，目前绝大部分设计的传递技术均是如此。然而这类传递体系具有共同的问题，那就是这 些人工合成的外源性传递体系很容易被宿主的循环系统清除，也有可能引起免疫原性反应，甚至可能对特定的细胞类型和组织有毒。
本发明的研究团队发现内源性细胞可以选择性地将miRNAs封装到外泌体(exosome)中，外泌体可以将miRNA传递到受体细胞中，其分泌的miRNA在相对较低的浓度下，即可有力阻断靶基因的表达。外泌体与宿主免疫系统生物相容，并具有在体内保护和运输miRNA跨越生物屏障的先天能力，因此成为克服与siRNA传递相关的问题的潜在解决方案。例如，公开号为CN110699382A的中国专利就公开了一种递送siRNA的外泌体的制备方法，公开了从血浆中分离外泌体，并将siRNA通过电穿孔的方式封装到外泌体中的技术。
但是这类在体外分离或制备外泌体的技术，往往需要通过细胞培养获取大量的外泌体，再加上siRNA封装的步骤，这使得大规模应用该产品的临床费用变得非常高，一般患者无法负担；更重要的是，外泌体复杂的生产/纯化过程，使其几乎不可能符合GMP标准。
到目前为止，以外泌体为有效成分的药物从未获得CFDA批准，其核心问题就是无法保证外泌体产品的一致性，而这一问题直接导致此类产品无法获得药品生产许可证。如果能解决这一问题，则对推动RNAi疗法治疗肺癌意义非凡。
因此，开发一个安全、精确和高效的siRNA传递系统是对提高RNAi治疗效果，推进RNAi疗法至关重要的一环。
发明内容
有鉴于此，本申请实施例提供了一种用于治疗肺癌的RNA递送系统及其应用，以解决现有技术中存在的技术缺陷。
本申请的一个发明点为提供一种用于治疗肺癌的RNA递送系统，该系统包括病毒载体，所述病毒载体携带有能够治疗肺癌的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述RNA的复合结构，所述复合结构能够将所述RNA片段送入肺部，实现肺癌的治疗。
进一步地，所述病毒载体为腺病毒相关病毒。
进一步地，所述腺病毒相关病毒为腺病毒相关病毒5型、腺病毒相关病毒8型或腺病毒相关病毒9型。
进一步地，所述RNA片段包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列是具有医学意义的siRNA、shRNA或miRNA。
进一步地，所述病毒载体包括启动子序列和靶向标签，所述靶向标签能够在宿主的器官组织中形成所述复合结构的靶向结构，所述靶向结构位于复合结构的表面，所述复合结构能够通过所述靶向结构寻找并结合目标组织，将所述RNA片段递送进入目标组织。
进一步地，所述病毒载体中包括以下任意一种线路或几种线路的组合：启动子-RNA片段、启动子-靶向标签、启动子-RNA片段-靶向标签；每一个所述病毒载体中至少包括一个RNA片段和一个靶向标签，所述RNA片段和靶向标签位于相同的线路中或位于不同的线路中。
进一步地，所述病毒载体还包括能够使所述线路折叠成正确结构并表达的侧翼序列、补偿序列和loop序列，所述侧翼序列包括5’侧翼序列和3’侧翼序列；
所述病毒载体中包括以下任意一种线路或几种线路的组合：5'-启动子-5'侧翼序列-RNA片段-loop序 列-补偿序列-3'侧翼序列、5'-启动子-靶向标签、5'-启动子-靶向标签-5'侧翼序列-RNA片段-loop序列-补偿序列-3'侧翼序列。
进一步地，所述5’侧翼序列为ggatcctggaggcttgctgaaggctgtatgctgaattc或与其同源性大于80％的序列；
所述loop序列为gttttggccactgactgac或与其同源性大于80％的序列；
所述3’侧翼序列为accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag或与其同源性大于80％的序列；
所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-5位碱基。删除RNA反向互补序列的1-5位碱基的目的是使该序列不表达。
优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-3位碱基。
更为优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-3位连续排列的碱基。
最为优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中的第9位和/或第10位碱基。
进一步地，在病毒载体中存在至少两种线路的情况下，相邻的线路之间通过序列1-3(序列1-序列2-序列3)组成的序列相连；
其中，序列1为CAGATC，序列2是由5-80个碱基组成的序列，序列3为TGGATC。
进一步地，在病毒载体中存在至少两种线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；
其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
进一步地，所述器官组织为肝脏，所述复合结构为外泌体。
进一步地，所述靶向标签选自具有靶向功能的靶向肽或靶向蛋白。
进一步地，所述靶向肽包括RVG靶向肽、GE11靶向肽、PTP靶向肽、TCP-1靶向肽、MSP靶向肽；
所述靶向蛋白包括RVG-LAMP2B融合蛋白、GE11-LAMP2B融合蛋白、PTP-LAMP2B融合蛋白、TCP-1-LAMP2B融合蛋白、MSP-LAMP2B融合蛋白。
进一步地，所述RNA序列的长度为15-25个核苷酸。比如，所述RNA序列的长度可以为16、17、18、19、20、21、22、23、24、25个核苷酸。优选地，所述RNA序列的长度为18-22个核苷酸。
进一步地，所述能够治疗肺癌的RNA序列选自以下RNA中的任意一种或几种：EGFR基因的siRNA、KRAS基因的siRNA或编码上述RNA的核酸分子。
EGFR基因的siRNA包括UGUUGCUUCUCUUAAUUCCU、AAAUGAUCUUCAAAAGUGCCC、UCUUUAAGAAGGAAAGAUCAU、AAUAUUCGUAGCAUUUAUGGA、UAAAAAUCCUCACAUAUACUU、其他具有抑制EGFR基因表达的序列以及与上述序列同源性大于80％的序列。
KRAS基因的siRNA包括UGAUUUAGUAUUAUUUAUGGC、AAUUUGUUCUCUAUAAUGGUG、UAAUUUGUUCUCUAUAAUGGU、UUAUGUUUUCGAAUUUCUCGA、UGUAUUUACAUAAUUACACAC、其他具有抑制KRAS基因表达的序列以及与上述序列同源性大于80％的序列。
需要说明的是，以上所述的“同源性大于80％的序列”可以为同源性为85％、88％、90％、95％、98％等。
可选地，所述RNA片段包括RNA序列本体和对RNA序列本体进行核糖修饰得到的修饰RNA序列。即RNA片段既可以仅由至少一个RNA序列本体组成，也可以仅由至少一个修饰RNA序列组成，还可以由RNA序列本体与修饰RNA序列组成。
在本发明中，所述分离的核酸还包括其变体和衍生物。本领域的普通技术人员可以使用通用的方法对所述核酸进行修饰。修饰方式包括(但不限于)：甲基化修饰、烃基修饰、糖基化修饰(如2-甲氧基-糖基修饰、烃基-糖基修饰、糖环修饰等)、核酸化修饰、肽段修饰、脂类修饰、卤素修饰、核酸修饰(如“TT”修饰)等。在本发明的其中一种实施方式中，所述修饰为核苷酸间键合，例如选自：硫代磷酸酯、2'-O甲氧基乙基(MOE)、2'-氟、膦酸烷基酯、二硫代磷酸酯、烷基硫代膦酸酯、氨基磷酸酯、氨基甲酸酯、碳酸酯、磷酸三酯、乙酰胺酯、羧甲基酯及其组合。在本发明的其中一种实施方式中，所述修饰为对核苷酸的修饰，例如选自：肽核酸(PNA)、锁核酸(LNA)、阿拉伯糖-核酸(FANA)、类似物、衍生物及其组合。优选的，所述修饰为2’氟嘧啶修饰。2’氟嘧啶修饰是将RNA上嘧啶核苷酸的2’-OH用2’-F替代，2’-F能够使RNA不易被体内的RNA酶识别，由此增加RNA片段在体内传输的稳定性。
进一步地，所述递送系统为用于包括人在内的哺乳动物中的递送系统。
本申请的另一个发明点为提供一种如上所述的用于治疗肺癌的RNA递送系统在药物中的应用。
进一步地，所述药物的给药方式包括口服、吸入、皮下注射、肌肉注射、静脉注射。
所述药物的剂型可以为片剂、胶囊剂、粉剂、颗粒剂、丸剂、栓剂、软膏剂、溶液剂、混悬剂、洗剂、凝胶剂、糊剂等。
本申请的技术效果为：
本申请提供的用于治疗肺癌的RNA递送系统以病毒作为载体，病毒载体作为成熟的注入物，其安全性和可靠性已被充分验证，成药性非常好。最终发挥效果的RNA序列由内源性外泌体包裹输送，不存在任何免疫反应，无需验证该外泌体的安全性。该递送系统可以递送各类小分子RNA，通用性强。并且病毒载体的制备要比外泌体或是蛋白质、多肽等物质的制备便宜地多，经济性好。本申请提供的用于治疗肺癌的RNA递送系统在体内自组装后能够与AGO 2紧密结合并富集为复合结构(外泌体)，不仅能防止其过早降解，维持其在循环中的稳定性，而且有利于受体细胞吸收、胞浆内释放和溶酶体逃逸，所需剂量低。
本申请提供的用于治疗肺癌的RNA递送系统应用于药物中，即提供了一个药物递送平台，可以大大提高肺癌治疗效果，还可以通过该平台形成更多RNA类药物的研发基础，对RNA类药物研发和使用具有极大的推动作用。
附图说明
图1是本申请一实施例提供的基于KRAS siRNA的小鼠肺癌治疗情况图；
图2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3是本申请一实施例提供的小鼠多种酶含量对比图。
图4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5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6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7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8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9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EGFR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10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11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12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13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14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。
具体实施方式
下面结合附图对本申请的具体实施方式进行描述。
首先，对本发明涉及到的专业名词、试验方法等进行解释说明。
Western免疫印迹(Western Blot)是将蛋白质转移到膜上,然后利用抗体进行检测.对已知表达蛋白,可用相应抗体作为一抗进行检测,对新基因的表达产物,可通过融合部分的抗体检测。
Western Blot采用的是聚丙烯酰胺凝胶电泳，被检测物是蛋白质，“探针”是抗体，“显色”用标记的二抗。经过PAGE分离的蛋白质样品，转移到固相载体(例如硝酸纤维素薄膜)上，固相载体以非共价键形式吸附蛋白质,且能保持电泳分离的多肽类型及其生物学活性不变，以固相载体上的蛋白质或多肽作为抗原，与对应的抗体起免疫反应，再与酶或同位素标记的第二抗体起反应，经过底物显色或放射自显影以检测电泳分离的特异性目的基因表达的蛋白成分。其步骤主要包括：提取蛋白、蛋白定量、制胶和电泳、转膜、免疫标记及显影。
本发明中涉及到的siRNA水平、蛋白含量和mRNA含量的检测，均是通过向小鼠体内注射RNA递送系统，建立了小鼠干细胞体外模型。利用qRT-PCR检测细胞、组织中mRNA和siRNA表达水平。对于siRNA的绝对定量利用标准品绘制标准曲线的方式进行确定。每个siRNA或mRNA相对于内参的表达量可以用2-ΔCT表示，其中ΔCT＝C样品-C内参。扩增siRNA时内参基因为U6snRNA(组织中)或miR-16(血清、外泌体中)分子，扩增mRNA时基因为GAPDH或18s RNA。利用Western blotting实验检测细胞、组织中蛋白质的表达水平，用ImageJ软件进行蛋白定量分析。
在本发明所提供的图示中，“*”表示P<0.05，“**”表示P<0.01,“***”P表示<0.005。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的试剂、材料和操作步骤均为相应领域内广泛使用的试剂、材料和常规步骤。
实施例1
本实施例提供一种用于治疗肺癌的RNA递送系统，该系统包括病毒载体，所述病毒载体携带有能够治疗肺癌的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述RNA的复合结构，所述复合结构能够将所述RNA片段送入肺部，实现肺癌的治疗。
见附图4-5，图4为慢病毒载体在肝、肺、血浆、外泌体的富集效果以及EGFR基因表达量检测，图5为腺相关病毒在肝、肺、血浆、外泌体的富集效果以及EGFR基因表达量检测。
在本实施例中，病毒载体还包括启动子和靶向标签。所述病毒载体包括以下任意一种线路或几种线路的组合：启动子-RNA序列、启动子-靶向标签、启动子-RNA序列-靶向标签，每一个所述病毒载体中至少包括一个RNA片段和一个靶向标签，所述RNA片段和靶向标签位于相同的线路中或位于不同的线路中。换而言之，病毒载体中可以仅包括启动子-RNA序列-靶向标签，也可以包括启动子-RNA序列、启动子-靶向标签的组合，或是启动子-靶向标签、启动子-RNA序列-靶向标签的组合。
见附图9，5’-启动子-5’侧翼序列-RNA序列-loop序列-补偿序列-3’侧翼序列对应递送系统名称为AAV-siR E，5’-启动子-靶向标签-5’侧翼序列-RNA序列-loop序列-补偿序列-3’侧翼对应递送系统名称为AAV-GE11-siR E，图9为静脉注射后siRNA在肝、肺、血浆、外泌体中的富集结果和EGFR蛋白及mRNA的表达量检测结果。
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。
序列具体如下表1所示。
名称 序列
5'侧翼序列-1 CTGGAGGCTTGCTGAAGGCTGTATGCTGAATTCG
5'侧翼序列-2 CTGGAGGCTTGCTGAAGGCTGGCAGCTGAATTCG
loop-1 GTTTTGGCCACTGACTGAC
loop-2 GTTTTGGTAACTGACTGAC
3'侧翼序列-1 CACCGGTCAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC
3'侧翼序列-2 CACCGGTCTGAACACAAGGCCTGTTACTAGCACGTCCATGGAACAAATGGCC
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。
见附图10-12，图10为5’侧翼序列同源性大于80％的序列构建进AAV载体在肺部富集效果以及治疗效果，图11为loop序列同源性大于80％的序列构建进AAV载体在肺部富集效果以及治疗效果，图12为3’侧翼序列同源性大于80％的序列构建进AAV载体在肺部富集效果以及治疗效果。
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。
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。
见附图13，图为序列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2所示。
Figure PCTCN2022083951-appb-000001
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。以在同一个病毒载体上联合使用“siRNA1”和“siRNA2”为例，该病毒载体的功能结构区可以表示为：(启动子-siRNA1)-连接序列-(启动子-siRNA2)-连接序列-(启动子-靶向标签)，或(启动子-靶向标签-siRNA1)-连接序列-(启动子-靶向标签-siRNA2)，或(启动子-siRNA1)-连接序列-(启动子-靶向标签-siRNA2)等。
见附图14，RNA序列长度分别为18、20、22的载体系统分别对应AAV-siR E(18)、AAV-siR E(20)、AAV-siR E(22)，图14为上述3种基因环路静脉注射后EGFR表达量检测。
具体序列如下表3所示。
名称 序列
siRE(18) ACCTATTCCGTTACACACT
siRE(20) ATACCTATTCCGTTACACAC
siRE(22) ATACCTATTCCGTTACACACTT
以上所述的RNA包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列能够在目标受体中被表达，所述补偿序列在目标受体中不能被表达。
所述能够治疗肺癌的RNA选自以下RNA中的任意一种或几种：EGFR基因的siRNA、KRAS基因的siRNA或编码上述RNA的核酸分子。
能够治疗肺癌的RNA有效序列的数量为1条、2条或多条。比如可以在同一个病毒载体上联合使用EGFR基因的siRNA和KRAS基因的siRNA，也可以单独使用EGFR基因的siRNA或KRAS基因的siRNA。
更加具体地，该病毒载体的功能结构区可以表示为：(5’-启动子-5’侧翼序列-siRNA1-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-5’侧翼序列-siRNA2-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-靶向标签)，或(5’-启动子-靶向标签-5’侧翼序列-siRNA1-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-靶向标签-5’侧翼序列-siRNA2-loop序列-补偿序列-3’侧翼序列)，或(5’-启动子-5’侧翼序列-siRNA1-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-靶向标签-5’侧翼序列-siRNA2-loop序列-补偿序列-3’侧翼序列)、(5’-启动子-靶向标签-5’侧翼序列-siRNA1-siRNA2-loop序列-补偿序列 -3’侧翼序列)等。其他情况均可以此类推，在此不再赘述。以上连接序列可以为“序列1-序列2-序列3”或“序列4”，一个括号表示一个完整的线路(circuit)。
优选地，上述RNA还可以通过对其中的RNA序列(siRNA、shRNA或miRNA)进行核糖修饰得到，优选2’氟嘧啶修饰。2’氟嘧啶修饰是将siRNA、shRNA或miRNA上嘧啶核苷酸的2’-OH用2’-F替代，2’-F能够使人体内的RNA酶不易识别siRNA、shRNA或miRNA，如此能够增加RNA在体内传输的稳定性。
具体地，肝脏会吞噬外源性的病毒，高达99％的外源性病毒会进入肝脏，因此当以病毒作为载体时并不需要做特异性设计即可在肝脏组织中富集，随后病毒载体被打开，释放出RNA分子(siRNA、shRNA或miRNA)，肝脏组织自发地将上述RNA分子包裹进外泌体内部，这些外泌体就变成了RNA输送机构。
优选地，为了使该RNA输送机构(外泌体)具有“精确制导”的能力，在注入体内的病毒中我们设计了靶向标签，该靶向标签也会被肝脏组织组装到外泌体中，尤其是当选择某些特定的靶向标签时，靶向标签能够被插入外泌体表面，从而成为能够引导外泌体的靶向结构，这就大大提高了本发明所述的RNA输送机构的精准性，一方面可以使所需引入的病毒载体的用量大大减少，另一方面还大大提高了潜在药物递送的效率。
靶向标签选自具有靶向功能的肽、蛋白质或抗体中的一种，靶向标签的选择是需要创造性劳动的过程，一方面需要根据目标组织选取可用的靶向标签，另一方面还需要保证该靶向标签能够在稳定地出现在外泌体的表面，从而达到靶向功能。目前已经筛选出的靶向肽、靶向蛋白、抗体。其中，靶向肽包括但不限于RVG靶向肽(核苷酸序列如SEQ ID No：1所示)、GE11靶向肽(核苷酸序列如SEQ ID No：2所示)、PTP靶向肽(核苷酸序列如SEQ ID No：3所示)、TCP-1靶向肽(核苷酸序列如SEQ ID No：4所示)、MSP靶向肽(核苷酸序列如SEQ ID No：5所示)；靶向蛋白包括但不限于RVG-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：6所示)、GE11-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：7所示)、PTP-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：8所示)、TCP-1-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：9所示)、MSP-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：10所示)。优选采用GE11靶向肽、GE11-LAMP2B融合蛋白。
此外，为了达到精准递送的目的，我们实验了多种病毒载体搭载的方案，得出另一优化的方案：所述病毒载体还可以由具有不同结构的多种病毒构成，其中一种病毒包含启动子和靶向标签，其他病毒包含启动子和RNA片段。即将靶向标签与RNA片段装载到不同的病毒载体中，将两种病毒载体注入体内，其靶向效果不差于将相同的靶向标签与RNA片段装载在一个病毒载体中产生的靶向效果。
更优选地，两种不同的病毒载体注入宿主体内时，可以先将装有RNA序列的病毒载体注入，在1-2小时后再注入含有靶向标签的病毒载体，如此能够达到更优的靶向效果。
以上所述的递送系统均可用于包括人在内的哺乳动物。
本实施例提供的用于治疗肺癌的RNA递送系统以病毒作为载体，病毒载体作为成熟的注入物，其安全性和可靠性已被充分验证，成药性非常好。最终发挥效果的RNA序列由内源性外泌体包裹输送，不存在任何免疫反应，无需验证该外泌体的安全性。该递送系统可以递送各类小分子RNA，通用性强。并且病毒载体的制备要比外泌体或是蛋白质、多肽等物质的制备便宜地多，经济性好。本实施例提供的用于治疗肺癌的RNA递送系统在体内自组装后能够与AGO 2紧密结合并富集为复合结构(外泌体)，不仅能防止其过早降解，维持其在循环中的稳定性，而且有利于受体细胞吸收、胞浆内释放和溶酶体逃逸，所需剂量低。
实施例2
在实施例1的基础上，本实施例提供一种药物。一种用于治疗肺癌的RNA递送系统，该系统包括病毒载体，所述病毒载体携带有能够治疗肺癌的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有所述RNA的复合结构，所述复合结构能够将所述RNA片段送入肺部，实现肺癌的治疗。
进一步地，所述RNA片段包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列是具有医学意义的siRNA、shRNA或miRNA。
见附图6-8，图6是提供的6种不同RNA构建进腺相关病毒载体治疗肺癌后的荧光信号统计，6种RNA分别为：siR E(靶基因为EGFR)、siR T(靶基因为TNC)、shR E(靶基因为EGFR)、shR T(靶基因为TNC)、miR-7(靶基因为EGFR)、miR-133b(靶基因为EGFR)；图7是以上提供的6种RNA中任意2种RNA序列组成的4组治疗肺癌后的荧光信号统计；图8是以上提供的6种RNA中任意3种RNA序列组成的3组治疗肺癌后的荧光信号统计。
具体序列(前体)如下表4所示。
Figure PCTCN2022083951-appb-000002
进一步地，所述病毒载体包括启动子序列和靶向标签，所述靶向标签能够在宿主的器官组织中形成所述复合结构的靶向结构，所述靶向结构位于复合结构的表面，所述复合结构能够通过所述靶向结构寻找并结合目标组织，将所述RNA片段递送进入目标组织。
关于本实施例中上述病毒载体、RNA片段、靶向标签等的解释说明均可以参考实施例1，在此不再赘述。
该药物可以通过口服、吸入、皮下注射、肌肉注射或静脉注射的方式进入人体后，通过实施例1所述的RNA递送系统递送至目标组织肺癌，发挥治疗作用。
该药物还可以与其他治疗肺癌的药物联合使用，以增强治疗效果。比如吉非替尼、厄洛替尼、埃克替尼、阿法替尼等。
本实施例的药物还可以包括药学上可以接受的载体，该载体包括但不限于稀释剂、缓冲剂、乳剂、包囊剂、赋形剂、填充剂、粘合剂、喷雾剂、透皮吸收剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、着色剂、矫味剂、佐剂、干燥剂、吸附载体等。
本实施例提供的药物的剂型可以为片剂、胶囊剂、粉剂、颗粒剂、丸剂、栓剂、软膏剂、溶液剂、混悬剂、洗剂、凝胶剂、糊剂等。
本实施例提供的药物以病毒作为载体，病毒载体作为成熟的注入物，其安全性和可靠性已被充分验证，成药性非常好。最终发挥效果的RNA序列由内源性外泌体包裹输送，不存在任何免疫反应，无需验证该外泌体的安全性。该药物可以递送各类小分子RNA，通用性强。并且病毒载体的制备要比外泌体或是蛋 白质、多肽等物质的制备便宜地多，经济性好。本申请提供的药物在体内自组装后能够与AGO 2紧密结合并富集为复合结构(外泌体)，不仅能防止其过早降解，维持其在循环中的稳定性，而且有利于受体细胞吸收、胞浆内释放和溶酶体逃逸，所需剂量低。
实施例3
在实施例1或2的基础上，本实施例提供一种用于治疗肺癌的RNA递送系统在药物中的应用。在此通过以下试验进行具体说明。
在第一个试验中，我们利用肝脏高亲和的AAV-5型腺相关病毒包裹EGFR siRNA系统(AAV-CMV-EGFR siRNA)和KRAS siRNA系统(AAV-CMV-KRAS siRNA)，尾静脉注射100μL滴度为10 12V.g/ml的AAV溶液至小鼠体内。通过小动物活体监测AAV系统的体内表达情况，3周后可见AAV系统在体内尤其是肝脏，稳定表达。
在第二个试验中，设置1个试验组和2个对照组，其中，试验组为AAV-CMV-KRAS-siRNA组，对照组为PBS组和AAV-CMV-scrR组。
各组选取相同数量的小鼠，向小鼠体内注射小鼠肺癌细胞(LLC细胞),采用CT扫描技术观察小鼠模型构建进展。30天后对构建成功的小鼠进行给药，两天给药一次，即每两日向PBS组/AAV-CMV-scrR组/AAV-CMV-KRAS siRNA组小鼠注射一次PBS缓冲液/AAV-CMV-scrR/AAV-CMV-KRAS siRNA进行治疗，分别对小鼠进行生存分析和肿瘤评估，给药7次后停止治疗。
统计各组小鼠在治疗后的100天内的生存情况，结果如图1A所示，可以看出，PBS组和AAV-CMV-scrR组小鼠存活率相差无几，而AAV-CMV-KRAS siRNA组小鼠存活率最高。
给药前后分别对各组小鼠进行CT扫描，根据CT影像图对小鼠肺组织进行3D建模，并计算肿瘤体积大小，结果如图1B所示。在图1B中，“PBS pre”表示给药前的PBS组，“PBS post”表示给药后的PBS组；“AAV-CMV-scrR pre”表示给药前的AAV-CMV-scrR组，“AAV-CMV-scrR post”表示给药后的AAV-CMV-scrR组；“AAV-CMV-KRAS-siRNA pre”表示给药前的AAV-CMV-KRAS siRNA组，“AAV-CMV-KRAS-siRNA post”表示给药后的AAV-CMV-KRAS siRNA组。可以看出，AAV-CMV-KRAS siRNA组的小鼠在给药后肿瘤体积显著减小，而PBS组和AAV-CMV-scrR组的小鼠在给药后肿瘤体积不仅没有减小，还呈现不同程度的增加。
分别通过RT-qPCR和Western blotting检测各组小鼠肺部KRAS蛋白和mRNA表达水平，结果如图1C、图1D所示。结果显示AAV-CMV-KRAS siRNA组的小鼠肺部KRAS蛋白和mRNA表达量相对于对照组有所降低。
以上试验说明，AAV-CMV-KRAS siRNA对小鼠肺癌肿瘤具有显著的治疗效果。
在第三个试验中，设置1个试验组和2个对照组，其中，试验组为AAV-CMV-EGFR siRNA组，对照组为PBS组和AAV-CMV-scrR组。
构建EGFR-DEL19小鼠模型，饲喂强力霉素饲料诱导肿瘤产生，30天后对构建成功的小鼠进行给药，两天给药一次，即每两日向PBS组/AAV-CMV-scrR组/AAV-CMV-EGFR siRNA组小鼠注射一次PBS缓冲液/AAV-CMV-scrR/AAV-CMV-EGFR siRNA进行治疗，分别对小鼠进行生存分析和肿瘤评估，给药7次后停止治疗。
统计各组小鼠在治疗后的100天内的生存情况，结果如图2A所示，可以看出，PBS组和AAV-CMV-scrR组小鼠存活率相差无几，而AAV-CMV-EGFR siRNA组小鼠存活率最高。
给药前后分别对各组小鼠进行CT扫描，结果如图2E所示，根据图2E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2B所示。在图2B中，“PBS pre”表示给药前的PBS组，“PBS post”表示给药后的PBS组；“AAV-CMV-scrR pre”表示给药前的AAV-CMV-scrR组，“AAV-CMV-scrR post”表示给药后的AAV-CMV-scrR组；“AAV-CMV-EGFR siRNA pre”表示给药前的AAV-CMV-EGFR siRNA组，“AAV-CMV-EGFR siRNA post”表示给药后的AAV-CMV-EGFR siRNA组。可以看出，AAV-CMV-EGFR siRNA组的小鼠在给药后肿瘤体积显著减小，而PBS组和AAV-CMV-scrR组的小鼠在给药后肿瘤体积不仅没有减小，还呈现不同程度的增加。
分别通过RT-qPCR和Western blotting检测各组小鼠肺部EGFR蛋白和mRNA表达水平，结果如图2C、图2D所示。结果显示AAV-CMV-EGFR siRNA组的小鼠肺部EGFR蛋白和mRNA表达量相对于对照组有所降低。
以上试验说明，AAV-CMV-EGFR siRNA对EGFR突变型小鼠肺癌肿瘤具有显著的治疗效果。
在第四个试验中，设置2个试验组和2个对照组，其中，试验组为AAV-CMV-KRAS siRNA组、AAV-CMV-EGFR siRNA组，对照组为PBS组和AAV-CMV-scrR组。
构建EGFR-DEL19小鼠模型，饲喂强力霉素饲料诱导肿瘤产生，30天后对构建成功的小鼠进行给药，两天给药一次，即每两日向PBS组/AAV-CMV-scrR组/AAV-CMV-EGFR siRNA组/AAV-CMV-KRAS siRNA组小鼠注射一次PBS缓冲液/AAV-CMV-scrR/AAV-CMV-EGFR siRNA/AAV-CMV-KRAS siRNA进行治疗。
在治疗后分别检测各组小鼠中谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TBIL)、血清碱性磷酸酶(ALP)、肌酐(CREA)、血尿素氮(BUN)的含量，结果如图3A-图3F所示，可见，PBS组、AAV-CMV-scrR组、AAV-CMV-EGFR siRNA组、AAV-CMV-KRAS siRNA组小鼠中上述酶的含量均相差无几。
以上试验可以说明，用肝脏高亲和的AAV-5型腺相关病毒包裹EGFR siRNA系统(AAV-CMV-EGFR siRNA)和KRAS siRNA系统(AAV-CMV-KRAS siRNA)安全性好，可靠性高，不会产生负面作用，适于大规模推广和应用。
在本文中，“上”、“下”、“前”、“后”、“左”、“右”等仅用于表示相关部分之间的相对位置关系，而非限定这些相关部分的绝对位置。
在本文中，“第一”、“第二”等仅用于彼此的区分，而非表示重要程度及顺序、以及互为存在的前提等。
在本文中，“相等”、“相同”等并非严格的数学和/或几何学意义上的限制，还包含本领域技术人员可以理解的且制造或使用等允许的误差。
除非另有说明，本文中的数值范围不仅包括其两个端点内的整个范围，也包括含于其中的若干子范围。
上面结合附图对本申请优选的具体实施方式和实施例作了详细说明，但是本申请并不限于上述实施方式和实施例，在本领域技术人员所具备的知识范围内，还可以在不脱离本申请构思的前提下做出各种变化。

Claims (20)

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