WO2022206819A1 - 一种用于治疗亨廷顿病的rna递送系统 - Google Patents

一种用于治疗亨廷顿病的rna递送系统 Download PDF

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WO2022206819A1
WO2022206819A1 PCT/CN2022/083992 CN2022083992W WO2022206819A1 WO 2022206819 A1 WO2022206819 A1 WO 2022206819A1 CN 2022083992 W CN2022083992 W CN 2022083992W WO 2022206819 A1 WO2022206819 A1 WO 2022206819A1
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sequence
rna
targeting
huntington
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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Priority to JP2023559683A priority Critical patent/JP2024511207A/ja
Priority to EP22779001.1A priority patent/EP4317434A1/en
Publication of WO2022206819A1 publication Critical patent/WO2022206819A1/zh
Priority to US18/477,107 priority patent/US20240141347A1/en

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    • 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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Definitions

  • the present application relates to the field of biomedical technology, in particular to an RNA delivery system for the treatment of Huntington's disease.
  • Huntington's disease also known as major chorea or Huntington's chorea
  • Huntington's chorea is an autosomal dominant neurodegenerative disease.
  • the main cause is the mutation of the Huntington gene on the patient's fourth chromosome, which produces a mutated protein that gradually aggregates in cells to form large molecular clusters that accumulate in the brain and affect the function of nerve cells.
  • patients have onset in middle age, showing dance-like movements. As the disease progresses, they gradually lose the ability to speak, move, think, and swallow. The disease will continue to develop for about 10 to 20 years, and eventually lead to the death of the patient.
  • RNA interference (RNAi) therapy has been considered a promising strategy for the treatment of human diseases since its invention, but many problems have been encountered during clinical practice, and the development of this therapy has lagged far behind expectations.
  • RNA cannot exist stably outside the cell for a long time, because RNA will be degraded into fragments by RNases rich in extracellular, so it is necessary to find a method that can make RNA stable outside the cell and can enter specific tissues in a targeted manner. Highlight the effect of RNAi therapy.
  • Virus (Biological virus) is a small individual, simple structure, containing only one nucleic acid (DNA or RNA), must be parasitic in living cells and replicated non-cellular organisms. Viral vectors can bring genetic material into cells. The principle is to use the molecular mechanism of viruses to transmit their genomes into other cells for infection. It can occur in a complete living body (in vivo) or cell culture (in vitro), mainly used in Basic research, gene therapy or vaccines. However, there are few related studies on the use of viruses as vectors to deliver RNA, especially siRNA, using a special self-assembly mechanism.
  • the Chinese Patent Publication No. CN108624590A discloses a siRNA capable of inhibiting the expression of DDR2 gene; the Chinese Patent Publication No. CN108624591A discloses a siRNA capable of silencing the ARPC4 gene, and the siRNA is modified with ⁇ -phosphorus-selenium;
  • the Chinese Patent Publication No. CN108546702A discloses a siRNA targeting long-chain non-coding RNA DDX11-AS1.
  • the Chinese Patent Publication No. CN106177990A discloses a siRNA precursor that can be used for various tumor treatments. These patents design specific siRNAs to target certain diseases caused by genetic changes.
  • Chinese Patent Publication No. CN108250267A discloses a polypeptide, polypeptide-siRNA induced co-assembly, using polypeptide as a carrier of siRNA.
  • the Chinese Patent Publication No. CN108117585A discloses a polypeptide for promoting apoptosis of breast cancer cells through targeted introduction of siRNA, and the polypeptide is also used as the carrier of siRNA.
  • the Chinese Patent Publication No. CN108096583A discloses a nanoparticle carrier, which can be loaded with siRNA with breast cancer curative effect while containing chemotherapeutic drugs.
  • exosomes can deliver miRNAs to recipient cells, which secrete miRNAs at relatively low concentrations , which can effectively block the expression of target genes.
  • Exosomes are biocompatible with the host immune system and possess the innate ability to protect and transport miRNAs across biological barriers in vivo, thus becoming a potential solution to overcome problems associated with siRNA delivery.
  • the Chinese Patent Publication No. CN110699382A discloses a method for preparing siRNA-delivering exosomes, and discloses the technology of separating exosomes from plasma and encapsulating siRNA into exosomes by electroporation .
  • the embodiments of the present application provide an RNA delivery system for the treatment of Huntington's disease and its application, so as to solve the technical defects existing in the prior art.
  • An invention of the present application is to provide an RNA delivery system for the treatment of Huntington's disease, the system comprising a viral vector carrying an RNA fragment capable of treating Huntington's disease, and the viral vector is capable of delivering in the organs and tissues of a host. enriched in the host organ and tissue, and endogenously and spontaneously form a composite structure containing the RNA fragment capable of treating Huntington's disease, and the composite structure can deliver the RNA fragment into the target tissue to achieve the effect of Huntington's disease. disease treatment. After the RNA fragment is delivered to the target tissue's brain, it can inhibit the expression of the matching gene, thereby inhibiting the development of disease in the target tissue.
  • the viral vector is an adenovirus-associated virus.
  • adenovirus-associated virus is adenovirus-associated virus type 5, adenovirus-associated virus type 8 or adenovirus-associated virus type 9.
  • RNA fragment comprises one, two or more specific RNA sequences with medical significance, and the RNA sequences are siRNA, shRNA or miRNA with medical significance.
  • the viral vector comprises a promoter and a targeting tag
  • the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host
  • the targeting structure is located on the surface of the composite structure
  • the The complex structure is capable of finding and binding to the target tissue through the targeting structure, delivering the RNA fragment into the target tissue.
  • the viral vector includes any one of the following circuits or a combination of several circuits: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each of the viral vectors including at least one RNA segment and one targeting tag, the RNA segment and targeting tag are located in the same circuit or are located in different circuits.
  • the viral vector also includes a flanking sequence, a compensation sequence and a loop sequence that can make the circuit fold into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence;
  • the viral vector includes any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-target To tag, 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensating sequence-3'flanking sequence.
  • the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence whose homology is greater than 80%;
  • the loop sequence is gttttggccactgactgac or a sequence whose homology is greater than 80%;
  • the 3' flanking sequence is accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence whose homology is greater than 80%;
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the purpose of deleting bases 1-5 of the reverse complement of the RNA is to make the sequence unexpressed.
  • siRNA and miRNA precursors due to deletion of the 9th and 10th bases of the reverse complementary strand of the active strand (such as the reverse complementary strand of siRNA), the active strand of siRNA and miRNA will form a bulge. structure, which facilitates more efficient silencing of gene expression. To sum up, it is an accepted conclusion that the silencing efficiency can be improved by deleting the reverse complementary sequence of the 9th and 10th bases.
  • 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 targeting tag is preferably an RVG targeting peptide or RVG-LAMP2B fusion protein that can precisely target brain tissue.
  • the length of the RNA sequence is 15-25 nucleotides.
  • the length of the RNA sequence can be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides.
  • the RNA sequence is 18-22 nucleotides in length.
  • RNA capable of treating Huntington's disease is selected from siRNA of mHTT gene, or an RNA sequence with more than 80% homology to the above sequence, or a nucleic acid molecule encoding the above RNA. It should be noted that the RNA sequences in the "nucleic acid molecules encoding the above RNA sequences" here also include RNA sequences with a homology of more than 80% of each RNA.
  • the mHTT gene siRNA includes UAUGUUUUCACAUAUUGUCAG, AUUUAGUAGCCAACUAUAGAA, AUGUUUUUCAAUAAAUGUGCC, UAUGAAUAGCAUUCUUAUCUG, UAUUUGUUCCUCUUAAUACAA, other sequences that inhibit the expression of the mHTT gene, and sequences with more than 80% homology to the above sequences.
  • 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 invention of the present application is to provide the application of the RNA delivery system for the treatment of Huntington's disease in medicine.
  • the drug is a drug for the treatment of Huntington's disease and its related diseases, and the related diseases here refer to the related diseases or complications, sequelae, etc. that occur in the formation or development of the above-mentioned Huntington's disease, or have a certain correlation with Huntington's disease. other illnesses.
  • the medicine includes the above-mentioned viral vector, specifically, the viral vector here means a viral vector carrying an RNA fragment, or carrying an RNA fragment and a targeting tag, and can enter the host and can be enriched in the liver. , self-assembled to form a composite structure exosome, the composite structure can deliver RNA fragments to the target tissue, so that the RNA fragments are expressed in the target tissue, and then inhibit the expression of matching genes to achieve the purpose of treating diseases.
  • the viral vector here means a viral vector carrying an RNA fragment, or carrying an RNA fragment and a targeting tag, and can enter the host and can be enriched in the liver. , self-assembled to form a composite structure exosome, the composite structure can deliver RNA fragments to the target tissue, so that the RNA fragments are expressed in the target tissue, and then inhibit the expression of matching genes to achieve the purpose of treating diseases.
  • the dosage forms of the drug can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes and the like.
  • administration modes of the drug include oral, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection.
  • the RNA delivery system for the treatment of Huntington's disease 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 Huntington's disease 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 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 Huntington's disease provided in this application is applied to medicines, that is, a drug delivery platform is provided, which can greatly improve the therapeutic effect of Huntington's disease, and can also form a research and development basis for more RNA-based drugs through this platform. It has greatly promoted the development and use of RNA drugs.
  • Fig. 1 is the comparison diagram of mouse Huntington's disease treatment provided by an embodiment of the present application.
  • FIG. 2 is a comparison diagram of the descending latency of mice provided in 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.
  • FIG. 9 is a graph showing the effect of in vivo enrichment of the adeno-associated virus type 8 vector loaded with 6 individual RNA sequences provided in an example 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.
  • 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 body.
  • 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 Huntington's disease, the system comprising a viral vector carrying an RNA fragment capable of treating Huntington's disease, the viral vector being capable of enriching in the organ tissue of a host, And a composite structure containing the RNA fragments capable of treating Huntington's disease is endogenously and spontaneously formed in the host organ tissue, and the composite structure can send the RNA fragments into target tissues to achieve the treatment of Huntington's disease.
  • lentivirus In addition to adenovirus, other viral vectors also have in vivo enrichment, self-assembly and therapeutic effects on Huntington's disease, such as lentivirus.
  • CTX stands for cortex
  • STR stands for Striatum (Striatum).
  • the viral vector is preferably adeno-associated virus, more preferably adeno-associated virus type 5, adenovirus-associated virus type 8 or adenovirus-associated virus type 9.
  • adeno-associated virus types 5, 8, and 9 other viral vectors also have in vivo enrichment, self-assembly, and therapeutic effects on Huntington's disease, such as adenovirus types 1, 2, 4, and 7. Data such as shown in Figure 6-8.
  • 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.
  • 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 viral vector contains 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensation sequence-3'flanking sequence
  • its RNA delivery system has in vivo enrichment and Huntington's disease treatment
  • the effect is shown in Figure 16-17, the targeting tag is RVG, and the RNA fragment is siRNA with therapeutic effect.
  • 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 2 is specifically shown in Table 1 below.
  • adjacent lines are connected by sequence 4 or a sequence with more than 80% homology to sequence 4; wherein, sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGA TC.
  • sequence 4 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-1 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-3 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 account, and the effect is the best.
  • nt nucleotides
  • the RNA capable of treating Huntington's disease is selected from siRNA of mHTT gene or nucleic acid molecules encoding the above RNA.
  • RNA effective sequences capable of treating Huntington's disease is one, two or more.
  • the functional structural region of the viral vector can be expressed as: (promoter-siRNA1)-connector sequence-(promoter-siRNA2)-connector sequence- (promoter-targeting tag), or (promoter-targeting tag-siRNA1)-linker-(promoter-targeting tag-siRNA2), or (promoter-siRNA1)-linker-(promoter- Targeting tag-siRNA2) etc.
  • the functional structural region of the viral vector can be expressed as: (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-connector sequence-(5'-promoter - 5' flanking sequence - siRNA2-loop sequence - compensation sequence - 3' flanking sequence) - linking sequence - (5'-promoter-targeting tag), or (5'-promoter-targeting tag-5' flanking sequence-siRNA1-loop sequence-compensation sequence-3'flanking sequence)-linker sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'flanking sequence), or (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-linking sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'flan
  • the above RNA can also be obtained by ribose modification of the RNA sequence (siRNA, shRNA or miRNA) therein, preferably 2' fluoropyrimidine modification.
  • 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on siRNA, shRNA or miRNA with 2'-F.
  • 2'-F can make it difficult for RNase in the human body to recognize siRNA, shRNA or miRNA, so it can Increases the stability of RNA transport in vivo.
  • the liver will phagocytose exogenous viruses, and up to 99% of the exogenous viruses will enter the liver. Therefore, when viruses are used as vectors, they can be enriched in liver tissue without specific design. After being opened, RNA molecules (siRNA, shRNA, or miRNA) are released, and liver tissue spontaneously wraps the above RNA molecules into exosomes, and these exosomes become RNA delivery mechanisms.
  • RNA molecules siRNA, shRNA, or miRNA
  • RNA delivery mechanism in order to make the RNA delivery mechanism (exosome) have the ability of "precision guidance”, we design a targeting tag in the viral vector injected into the body, and the targeting tag will also be assembled into exosomes by liver tissue
  • the targeting tags can be inserted into the surface of exosomes to become targeting structures that can guide exosomes, which greatly improves the RNA delivery of the present invention.
  • the accuracy of the mechanism on the one hand, can greatly reduce the amount of viral vector that needs to be introduced, and on the other hand, greatly improves the efficiency of potential drug delivery.
  • the targeting tag is selected from one of the peptides, proteins or antibodies with targeting function.
  • the selection of the targeting tag is a process that requires creative work. On the one hand, it is necessary to select the available targeting tags according to the target tissue. It is ensured that the targeting label can stably appear on the surface of exosomes, so as to achieve the targeting function.
  • Targeting tags that have been screened include: targeting peptides, targeting proteins, and antibodies.
  • targeting peptides include but are not limited to RVG targeting peptide (nucleotide sequence shown in SEQ ID No: 1), GE11 targeting peptide (nucleotide sequence shown in SEQ ID No: 2), PTP targeting peptide Peptide (nucleotide sequence shown in SEQ ID No: 3), TCP-1 targeting peptide (nucleotide sequence shown in SEQ ID No: 4), MSP targeting peptide (nucleotide sequence shown in SEQ ID No: 4) : 5); targeting proteins include but are not limited to RVG-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6), GE11-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 7) shown), PTP-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 8), TCP-1-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No:
  • RVG-LAMP2B fusion protein on adenovirus vector and the therapeutic effect of Huntington's disease are shown in Figure 18, and its therapeutic effect is reflected by the level of HTT mRNA.
  • 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 Huntington's disease 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 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 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 Huntington's disease 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 beneficial to receptor cell uptake, intracytoplasmic release and lysosomal escape, and the required dose is low.
  • this embodiment provides a medicine.
  • the medicament comprises a viral vector carrying an RNA segment capable of treating Huntington's disease, the viral vector being capable of enriching in the organ tissue of a host, and endogenously and spontaneously forming in the organ tissue of the host an RNA fragment capable of treating Huntington's disease
  • the composite structure of the RNA fragment for treating Huntington's disease, the composite structure can deliver the RNA fragment into a target tissue to achieve the treatment of Huntington's disease.
  • the RNA fragment comprises one, two or more specific RNA sequences with medical significance, and the RNA sequences are siRNA, shRNA or miRNA with medical significance.
  • Adenovirus-associated virus types 8 and 9 are used as viral vectors, which have in vivo enrichment, self-assembly and Huntington's disease treatment effects when carrying one or more RNA fragments.
  • Figure 9-11 shows that the vectors carry
  • Figures 12-13 show data presented for RNA fragments with 6 individual RNA sequences;
  • Figures 12-13 show data presented for vectors carrying 4 sets of RNA fragments containing any two RNA sequences;
  • Figures 14-15 show vectors carrying There are 3 sets of therapeutic data results presented for RNA fragments containing any of the 3 RNA sequences.
  • the viral vector includes a promoter and a targeting tag
  • the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host
  • the targeting structure is located on the surface of the composite structure, so The complex structure can seek and bind to the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
  • the drug can be administered orally, inhaled, subcutaneously injected, intramuscularly injected or intravenously injected into the human body, it can be delivered to the target tissue through the RNA delivery system described in Example 1 to exert a therapeutic effect.
  • the drug can also be used in combination with other Huntington's disease drugs to enhance the treatment effect, such as bufenazine, olanzapine, risperidone, tiapride, baclofen, benzodiazepines, selective 5 - Serotonin reuptake inhibitors, mirtazapine, amantadine, sodium valproate, botulinum toxin, etc.
  • other Huntington's disease drugs such as bufenazine, olanzapine, risperidone, tiapride, baclofen, benzodiazepines, selective 5 - Serotonin reuptake inhibitors, mirtazapine, amantadine, sodium valproate, botulinum toxin, 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 in a medicine, and the medicine is a medicine for treating Huntington's disease.
  • This example specifically describes the application of the RNA delivery system in the treatment of Huntington's disease in conjunction with the following experiments.
  • the HTT siRNA system (AAV-CMV-siR mHTT /AAV-CMV-RVG-siR mHTT ) was encapsulated by the liver high-affinity AAV-5 adeno-associated virus, and the titer was 10 12 Vg in 100 ⁇ L of tail vein injection. /ml of AAV solution into mice. The in vivo expression of the AAV system was monitored by small animals. After 3 weeks, it was found that the AAV system was stably expressed in vivo, especially in the liver.
  • mice were then selected for modeling. After modeling, mice were injected with PBS buffer/AAV-CMV-scrR/AAV-CMV-siR mHTT /AAV-CMV-RVG-siR mHTT to form a PBS group/AAV-CMV- scrR group/AAV-CMV-siR mHTT group/AAV-CMV-RVG-siR mHTT group.
  • plasma exosomes were isolated, labeled with PKH26 dye, and co-cultured with cells to observe the absorption of exosomes by cells. The results are as follows.
  • FIG. 1A the figure shows the comparison of siRNA levels in the plasma exosomes of the mice in each group. siRNA levels were higher in vivo.
  • the figure is a comparison of the relative mHTT mRNA levels of mice in each group after co-culture of mouse plasma exosomes with cells. It can be seen that the AAV-CMV-siR mHTT group and AAV-CMV-RVG The relative mHTT mRNA level of mice in the -siR mHTT group was lower, which indicated that AAV-CMV-siRmHTT and AAV-CMV-RVG-siRmHTT could reduce the level of HTT mRNA, that is, siRNA assembled into exosomes could still exert gene silencing function.
  • this figure is a comparison chart of the descending latency of mice in wild-type mice (WT), AAV-CMV-scrR group, and AAV-CMV-RVG-siR mHTT group. It can be seen that at week 0, the three The descending latencies of the mice in the groups were relatively consistent. At the 4th and 8th weeks, the mice in the CMV-scrR group had the shortest descending latencies.
  • the figure is a comparison of the relative mHTT mRNA levels in the cortex and striatum of mice in the AAV-CMV-scrR group and 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.
  • mice 8-week-old N17182Q mice were selected for modeling. After modeling, mice were injected with AAV-CMV-scrR/AAV-CMV-RVG-siR m HTT to form AAV-CMV-scrR group/AAV-CMV-RVG- siR mHTT group. Spin tests were performed on days 0 and 14, and mice were sacrificed for analysis after 14 days.
  • the figure is a comparison chart of the descending latency of mice in wild-type mice, AAV-CMV-scrR group, and AAV-CMV-RVG-siR mHTT group. It can be seen that at day 0, AAV-CMV-scrR The descending latency of mice in the AAV-CMV-RVG-siR mHTT group was relatively consistent, and on the 14th day, the descending latency of the mice in the AAV-CMV-scrR group was significantly reduced.

Abstract

一种用于治疗亨廷顿病的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亨廷顿病的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有能够治疗亨廷顿病的所述RNA片段的复合结构，所述复合结构能够将所述RNA片段送入目标组织，实现对亨廷顿病的治疗。所述用于治疗亨廷顿病的RNA递送系统安全性和可靠性已被充分验证，成药性好、通用性强，具有经济效益和应用前景。

Description

一种用于治疗亨廷顿病的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亨廷顿病的RNA递送系统。
背景技术
亨廷顿病(Huntington's disease，缩写HD)，又称大舞蹈病或亨廷顿舞蹈症(Huntington's chorea)，是一种常染色体显性遗传性神经退行性疾病。主要病因是患者第四号染色体上的Huntington基因发生变异，产生了变异的蛋白质，该蛋白质在细胞内逐渐聚集在一起，形成大的分子团，在脑中积聚，影响神经细胞的功能。一般患者在中年发病，表现为舞蹈样动作，随着病情进展逐渐丧失说话、行动、思考和吞咽的能力，病情大约会持续发展10年到20年，并最终导致患者死亡。
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疗法治疗亨廷顿病意义非凡。
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亨廷顿病的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亨廷顿病的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亨廷顿病的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有能够治疗亨廷顿病的所述RNA片段的复合结构，所述复合结构能够将所述RNA片段送入目标组织，实现对亨廷顿病的治疗。RNA片段送入目标组织大脑后，能够抑制与其相匹配的基因的表达，进而抑制目标组织中疾病的发展。
进一步地，所述病毒载体为腺病毒相关病毒。
进一步地，所述腺病毒相关病毒为腺病毒相关病毒5型、腺病毒相关病毒8型或腺病毒相关病毒9型。
进一步地，所述RNA片段包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列是具有医学意义的siRNA、shRNA或miRNA。
进一步地，所述病毒载体包括启动子和靶向标签，所述靶向标签能够在宿主的器官组织中形成所述复合结构的靶向结构，所述靶向结构位于复合结构的表面，所述复合结构能够通过所述靶向结构寻找并结合目标组织，将所述RNA片段递送进入目标组织。
进一步地，所述病毒载体中包括以下任意一种线路或几种线路的组合：启动子-RNA片段、启动子-靶向标签、启动子-RNA片段-靶向标签；每一个所述病毒载体中至少包括一个RNA片段和一个靶向标签，所述RNA片段和靶向标签位于相同的线路中或位于不同的线路中。
进一步地，所述病毒载体还包括能够使所述线路折叠成正确结构并表达的侧翼序列、补偿序列和loop序列，所述侧翼序列包括5’侧翼序列和3’侧翼序列；
所述病毒载体中包括以下任意一种线路或几种线路的组合：5'-启动子-5'侧翼序列-RNA片段-loop序列-补偿序列-3'侧翼序列、5'-启动子-靶向标签、5'-启动子-靶向标签-5'侧翼序列-RNA片段-loop序列-补偿序列-3'侧翼序列。
进一步地，所述5’侧翼序列为ggatcctggaggcttgctgaaggctgtatgctgaattc或与其同源性大于80％的序列；
所述loop序列为gttttggccactgactgac或与其同源性大于80％的序列；
所述3’侧翼序列为accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag或与其同源性大于80％的序列；
所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-5位碱基。删除RNA反向互补序列的1-5位碱基的目的是使该序列不表达。
与shRNA不同，在siRNA和miRNA前体中，由于删除作用链的反向互补链(例如siRNA的反向互补链)的第9，10位碱基，导致siRNA和miRNA的作用链会形成凸起结构，以上结构有利于更有效的沉默基因的表达。综上，通过删除第9，10位碱基的反向互补序列来提高沉默效率是目前已公认的结论。
优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-3位碱基。
更为优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中任意1-3位连续排列的碱基。
最为优选地，所述补偿序列为所述RNA片段的反向互补序列，并删除其中的第9位和/或第10位碱基。
进一步地，在病毒载体中存在至少两种线路的情况下，相邻的线路之间通过序列1-3组成的序列(序列1-序列2-序列3)相连；
其中，序列1为CAGATC，序列2是由5-80个碱基组成的序列，序列3为TGGATC。
进一步地，在病毒载体中存在至少两种线路的情况下，相邻的线路之间通过序列4或与序列4同源性大于80％的序列相连；
其中，序列4为CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC。
进一步地，所述器官组织为肝脏，所述复合结构为外泌体。
进一步地，所述靶向标签选自具有靶向功能的靶向肽或靶向蛋白。
进一步地，所述靶向肽包括RVG靶向肽、GE11靶向肽、PTP靶向肽、TCP-1靶向肽、MSP靶向肽；
所述靶向蛋白包括RVG-LAMP2B融合蛋白、GE11-LAMP2B融合蛋白、PTP-LAMP2B融合蛋白、TCP-1-LAMP2B融合蛋白、MSP-LAMP2B融合蛋白。
所述靶向标签优选为能够精准靶向脑组织的RVG靶向肽或RVG-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，所述能够治疗亨廷顿病的RNA选自mHTT基因的siRNA，或与上述序列同源性大于80％的RNA序列，或编码上述RNA的核酸分子。需要说明的是，此处“编码上述RNA序列的核酸分子”中的RNA序列也同时包括每种RNA的同源性大于80％的RNA序列。
mHTT基因的siRNA包括UAUGUUUUCACAUAUUGUCAG、AUUUAGUAGCCAACUAUAGAA、AUGUUUUUCAAUAAAUGUGCC、UAUGAAUAGCAUUCUUAUCUG、UAUUUGUUCCUCUUAAUACAA、其他具有抑制mHTT基因表达的序列以及与上述序列同源性大于80％的序列。
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％、9 8％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如上所述的用于治疗亨廷顿病的RNA递送系统在药物中的应用。
所述药物为治疗亨廷顿病及其相关疾病的药物，这里的相关疾病指的是上述亨廷顿病的形成或发展过程中出现的关联疾病或并发症、后遗症等，或与亨廷顿病具有一定相关性的其他疾病。
进一步地，所述药物包括上述病毒载体，具体而言，此处的病毒载体表示携带有RNA片段、或携带有RNA片段及靶向标签的病毒载体，并且能够进入宿主体内能够在肝脏部位富集，自组装形成复合结构外泌体，该复合结构能够将RNA片段递送至目标组织，使RNA片段在目标组织中表达，进而抑制与其匹配的基因的表达，实现治疗疾病的目的。
所述药物的剂型可以为片剂、胶囊剂、粉剂、颗粒剂、丸剂、栓剂、软膏剂、溶液剂、混悬剂、洗剂、凝胶剂、糊剂等。
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。
本申请的技术效果为：
本申请提供的用于治疗亨廷顿病的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亨廷顿病的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亨廷顿病的RNA递送系统应用于药物中，即提供了一个药物递送平台，可以大大提高亨廷顿病的治疗效果，还可以通过该平台形成更多RNA类药物的研发基础，对RNA类药物研发和使用具有极大的推动作用。
附图说明
图1是本申请一实施例提供的小鼠亨廷顿病治疗情况对比图；
图2是本申请一实施例提供的小鼠下降潜伏期对比图。
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。
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。
本发明中涉及到的siRNA水平、蛋白含量和mRNA含量的检测，均是通过向小鼠体内注射RNA递 送系统，建立了小鼠干细胞体外模型。利用qRT-PCR检测细胞、组织中mRNA和siRNA表达水平。对于siRNA的绝对定量利用标准品绘制标准曲线的方式进行确定。每个siRNA或mRNA相对于内参的表达量可以用2-ΔCT表示，其中ΔCT＝C样品-C内参。扩增siRNA时内参基因为U6snRNA(组织中)或miR-16(血清、外泌体中)分子，扩增mRNA时基因为GAPDH或18s RNA。利用Western blotting实验检测细胞、组织中蛋白质的表达水平，用ImageJ软件进行蛋白定量分析。
在本发明所提供的图示中，“*”表示P<0.05，“**”表示P<0.01,“***”表示P<0.005。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的试剂、材料和操作步骤均为相应领域内广泛使用的试剂、材料和常规步骤。
实施例1
本实施例提供一种用于治疗亨廷顿病的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亨廷顿病的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有能够治疗亨廷顿病的所述RNA片段的复合结构，所述复合结构能够将所述RNA片段送入目标组织，实现对亨廷顿病的治疗。
除了腺病毒外，其它病毒载体也具有体内富集、自组装和亨廷顿病的治疗效果，如慢病毒，慢病毒的数据如图3-5所示，其中，CTX代表皮层(cortex)，STR代表纹状体(Striatum)。
所述病毒载体优选为腺病毒相关病毒，更优选为腺病毒相关病毒5型、腺病毒相关病毒8型或腺病毒相关病毒9型。
除了腺病毒相关病毒5型、8型和9型以外，其它病毒载体也具有体内富集、自组装及亨廷顿病治疗效果，如腺病毒1型、2型、4型和7型，其数据如图6-8所示。
在本实施例中，病毒载体还包括启动子和靶向标签。所述病毒载体包括以下任意一种线路或几种线路的组合：启动子-RNA序列、启动子-靶向标签、启动子-RNA序列-靶向标签，每一个所述病毒载体中至少包括一个RNA片段和一个靶向标签，所述RNA片段和靶向标签位于相同的线路中或位于不同的线路中。换而言之，病毒载体中可以仅包括启动子-RNA序列-靶向标签，也可以包括启动子-RNA序列、启动子-靶向标签的组合，或是启动子-靶向标签、启动子-RNA序列-靶向标签的组合。
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。
病毒载体中包含有5'-启动子--靶向标签-5'侧翼序列-RNA片段-loop序列-补偿序列-3'侧翼序列时，其RNA递送系统所具有的体内富集和亨廷顿病治疗效果如图16-17所示，靶向标签为RVG，RNA片段为具有治疗作用的siRNA。
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1所示。
Figure PCTCN2022083992-appb-000001
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CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGA TC。
序列具体如下表2所示。
序列4 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-1 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC
序列4-2 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC
序列4-3 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。
所述能够治疗亨廷顿病的RNA选自mHTT基因的siRNA或编码上述RNA的核酸分子。
以AUUUAGUAGCCAACUAUAGAA为例，设计了AUUUAGUAGCCAACUAUAUGA(85％)，AUUUAGUAGCCAACUAUAACC(90％)和AUUUAGUAGCCAACUAUAGAC(98％)，均有良好的基因沉默作用，分别负载有siRNA三个同源序列的递送系统，其所具有的体内富集和亨廷顿病治疗效果如图19-20所示。
能够治疗亨廷顿病的RNA有效序列的数量为1条、2条或多条。
以在同一个病毒载体上联合使用“siRNA1”和“siRNA2”为例，该病毒载体的功能结构区可以表示为：(启动子-siRNA1)-连接序列-(启动子-siRNA2)-连接序列-(启动子-靶向标签)，或(启动子-靶向标签-siRNA1)-连接序列-(启动子-靶向标签-siRNA2)，或(启动子-siRNA1)-连接序列-(启动子-靶向标签-siRNA2)等。
更加具体地，该病毒载体的功能结构区可以表示为：(5’-启动子-5’侧翼序列-siRNA1-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-5’侧翼序列-siRNA2-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-靶向标签)，或(5’-启动子-靶向标签-5’侧翼序列-siRNA1-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-靶向标签-5’侧翼序列-siRNA2-loop序列-补偿序列-3’侧翼序列)，或(5’-启动子-5’侧翼序列-siRNA1-loop序列-补偿序列-3’侧翼序列)-连接序列-(5’-启动子-靶向标签-5’侧翼序列-siRNA2-loop序列-补偿序列-3’侧翼序列)、(5’-启动子-靶向标签-5’侧翼序列-siRNA1-siRNA2-loop序列-补偿序列-3’侧翼序列)等。其他情况均可以此类推，在此不再赘述。以上连接序列可以为“序列1-序列2-序列3” 或“序列4”，一个括号表示一个完整的线路(circuit)。
优选地，上述RNA还可以通过对其中的RNA序列(siRNA、shRNA或miRNA)进行核糖修饰得到，优选2’氟嘧啶修饰。2’氟嘧啶修饰是将siRNA、shRNA或miRNA上嘧啶核苷酸的2’-OH用2’-F替代，2’-F能够使人体内的RNA酶不易识别siRNA、shRNA或miRNA，如此能够增加RNA在体内传输的稳定性。
具体地，肝脏会吞噬外源性的病毒，高达99％的外源性病毒会进入肝脏，因此当以病毒作为载体时并不需要做特异性设计即可在肝脏组织中富集，随后病毒载体被打开，释放出RNA分子(siRNA、shRNA或miRNA)，肝脏组织自发地将上述RNA分子包裹进外泌体内部，这些外泌体就变成了RNA输送机构。
优选地，为了使该RNA输送机构(外泌体)具有“精确制导”的能力，在注入体内的病毒载体中我们设计了靶向标签，该靶向标签也会被肝脏组织组装到外泌体中，尤其是当选择某些特定的靶向标签时，靶向标签能够被插入外泌体表面，从而成为能够引导外泌体的靶向结构，这就大大提高了本发明所述的RNA输送机构的精准性，一方面可以使所需引入的病毒载体的用量大大减少，另一方面还大大提高了潜在药物递送的效率。
靶向标签选自具有靶向功能的肽、蛋白质或抗体中的一种，靶向标签的选择是需要创造性劳动的过程，一方面需要根据目标组织选取可用的靶向标签，另一方面还需要保证该靶向标签能够在稳定地出现在外泌体的表面，从而达到靶向功能。目前已经筛选出的靶向标签包括：靶向肽、靶向蛋白、抗体。其中，靶向肽包括但不限于RVG靶向肽(核苷酸序列如SEQ ID No：1所示)、GE11靶向肽(核苷酸序列如SEQ ID No：2所示)、PTP靶向肽(核苷酸序列如SEQ ID No：3所示)、TCP-1靶向肽(核苷酸序列如SEQ ID No：4所示)、MSP靶向肽(核苷酸序列如SEQ ID No：5所示)；靶向蛋白包括但不限于RVG-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：6所示)、GE11-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：7所示)、PTP-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：8所示)、TCP-1-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：9所示)、MSP-LAMP2B融合蛋白(核苷酸序列如SEQ ID No：10所示)。在本实施例中，靶向标签优选为能够精准靶向脑组织的RVG靶向肽或RVG-LAMP2B融合蛋白。
RVG-LAMP2B融合蛋白在腺病毒载体上所具有的体内富集和亨廷顿病治疗效果如图18所示，其治疗效果通过HTT mRNA的水平来体现。
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亨廷顿病的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亨廷顿病的RNA递送系统在体内自组装后能够与AGO 2紧密结合并富集为复合结构(外泌体)，不仅能防止其过早降解，维持其在循环中的稳定性，而且有利于受体细胞吸收、胞浆内释放和溶酶体逃逸， 所需剂量低。
实施例2
在实施例1的基础上，本实施例提供一种药物。该药物包括病毒载体，所述病毒载体携带有能够治疗亨廷顿病的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有能够治疗亨廷顿病的所述RNA片段的复合结构，所述复合结构能够将所述RNA片段送入目标组织，实现对亨廷顿病的治疗。
可选地，所述RNA片段包含1个、两个或多个具有医疗意义的具体RNA序列，所述RNA序列是具有医学意义的siRNA、shRNA或miRNA。
以腺病毒相关病毒8型和9型为病毒载体，其在携带有1个或多个RNA片段的情况下，具有体内富集、自组装和亨廷顿病治疗效果，图9-11显示了载体携带有6种单独RNA序列的RNA片段所呈现的数据结果；图12-13显示了载体携带有4组包含有任意2种RNA序列的RNA片段所呈现的数据结果；图14-15显示了载体携带有3组包含有任意3种RNA序列的RNA片段所呈现的治疗数据结果。
序列具体如下表3所示。
Figure PCTCN2022083992-appb-000002
可选地，所述病毒载体包括启动子和靶向标签，所述靶向标签能够在宿主的器官组织中形成所述复合结构的靶向结构，所述靶向结构位于复合结构的表面，所述复合结构能够通过所述靶向结构寻找并结合目标组织，将所述RNA片段递送进入目标组织。
关于本实施例中上述病毒载体、RNA片段、靶向标签等的解释说明均可以参考实施例1，在此不再赘述。
该药物可以通过口服、吸入、皮下注射、肌肉注射或静脉注射的方式进入人体后，通过实施例1所述的RNA递送系统递送至目标组织，发挥治疗作用。
该药物还可以与其他治疗亨廷顿病的药物联合使用，以增强治疗效果，比如丁苯喹嗪、奥氮平、利培酮、硫必利、巴氯芬、苯二氮卓类、选择性5-羟色胺再摄取抑制剂、米氮平、金刚烷胺、丙戊酸钠、肉毒杆菌毒素等。
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RNA递送系统在药物中的应用，该药物为治疗亨廷顿病的药物。本实施例结合以下试验对RNA递送系统在亨廷顿病治疗方面的应用进行具体说明。
在此试验中，利用肝脏高亲和的AAV-5型腺相关病毒包裹HTT siRNA系统(AAV-CMV-siR mHTT/AAV-CMV-RVG-siR mHTT)，尾静脉注射100μL滴度为10 12V.g/ml的AAV溶液至小鼠体内。通过小动物活体监测AAV系统的体内表达情况，3周后可见AAV系统在体内尤其是肝脏，稳定表达。
随即选取小鼠进行造模，造模完成后向小鼠注射PBS缓冲液/AAV-CMV-scrR/AAV-CMV-siR mHTT/AAV-CMV-RVG-siR mHTT,形成PBS组/AAV-CMV-scrR组/AAV-CMV-siR mHTT组/AAV-CMV-RVG-siR mHTT组。尾静脉注射上述溶液后，分离血浆外泌体，用PKH26染料标记后与细胞进行共培，观察细胞对外泌体的吸收情况，结果如下。
如图1A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1B所示，该图为小鼠血浆外泌体与细胞共培后，各组小鼠的相对mHTT mRNA水平对比图，可以看出，AAV-CMV-siR mHTT组和AAV-CMV-RVG-siR mHTT组小鼠相对mHTT mRNA水平较低，这说明AAV-CMV-siRmHTT与AAV-CMV-RVG-siRmHTT可以降低HTT mRNA水平，即组装进入外泌体的siRNA仍然可以发挥基因沉默功能。
如图1C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1D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1E所示，该图为野生型小鼠(WT)、AAV-CMV-scrR组、AAV-CMV-RVG-siR mHTT组的小鼠下降潜伏期对比图，可以看出在0周时，三组小鼠的下降潜伏期比较一致，在第4周和第8周时，CMV-scrR组的小鼠下降潜伏期最短。
如图1F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。
选取8周龄的N17182Q小鼠进行造模，造模完成后向小鼠注射AAV-CMV-scrR/AAV-CMV-RVG-siR m HTT,形成AAV-CMV-scrR组/AAV-CMV-RVG-siR mHTT组。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2所示，该图为野生型小鼠、AAV-CMV-scrR组、AAV-CMV-RVG-siR mHTT组的小鼠下降潜伏期 对比图，可以看出在0天时，AAV-CMV-scrR组、AAV-CMV-RVG-siR mHTT组小鼠的下降潜伏期比较一致，在第14天时，AAV-CMV-scrR组的小鼠下降潜伏期显著缩减。
以上试验可以说明，静脉注射AAV-CMV-RVG-siR mHTT有助于纹状体和皮层mHTT蛋白和毒性聚集体减少，从而发挥对亨廷顿舞蹈症的治疗作用。
在本文中，“上”、“下”、“前”、“后”、“左”、“右”等仅用于表示相关部分之间的相对位置关系，而非限定这些相关部分的绝对位置。
在本文中，“第一”、“第二”等仅用于彼此的区分，而非表示重要程度及顺序、以及互为存在的前提等。
在本文中，“相等”、“相同”等并非严格的数学和/或几何学意义上的限制，还包含本领域技术人员可以理解的且制造或使用等允许的误差。
除非另有说明，本文中的数值范围不仅包括其两个端点内的整个范围，也包括含于其中的若干子范围。
上面结合附图对本申请优选的具体实施方式和实施例作了详细说明，但是本申请并不限于上述实施方式和实施例，在本领域技术人员所具备的知识范围内，还可以在不脱离本申请构思的前提下做出各种变化。

Claims (19)

  1. 一种用于治疗亨廷顿病的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亨廷顿病的RNA片段，所述病毒载体能够在宿主的器官组织中富集，并在所述宿主器官组织中内源性地自发形成含有能够治疗亨廷顿病的所述RNA片段的复合结构，所述复合结构能够将所述RNA片段送入目标组织，实现对亨廷顿病的治疗。
  2. 如权利要求1所述的用于治疗亨廷顿病的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所述的用于治疗亨廷顿病的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所述的用于治疗亨廷顿病的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所述的用于治疗亨廷顿病的RNA递送系统，其特征在于，所述病毒载体包括启动子和靶向标签，所述靶向标签能够在宿主的器官组织中形成所述复合结构的靶向结构，所述靶向结构位于复合结构的表面，所述复合结构能够通过所述靶向结构寻找并结合目标组织，将所述RNA片段递送进入目标组织。
  6. 如权利要求5所述的用于治疗亨廷顿病的RNA递送系统，其特征在于，所述病毒载体中包括以下任意一种线路或几种线路的组合：启动子-RNA片段、启动子-靶向标签、启动子-RNA片段-靶向标签；每一个所述病毒载体中至少包括一个RNA片段和一个靶向标签，所述RNA片段和靶向标签位于相同的线路 中或位于不同的线路中。
  7. 如权利要求6所述的用于治疗亨廷顿病的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所述的用于治疗亨廷顿病的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所述的用于治疗亨廷顿病的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所述的用于治疗亨廷顿病的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所述的用于治疗亨廷顿病的RNA递送系统，其特征在于，所述器官组织为肝脏，所述复合结构为外泌体。
  12. 如权利要求5所述的用于治疗亨廷顿病的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所述的用于治疗亨廷顿病的RNA递送系统，其特征在于，所述靶向肽包括RVG靶向肽、GE11靶向肽、PTP靶向肽、TCP-1靶向肽、MSP靶向肽；
    所述靶向蛋白包括RVG-LAMP2B融合蛋白、GE11-LAMP2B融合蛋白、PTP-LAMP2B融合蛋白、TCP-1-LAMP2B融合蛋白、MSP-LAMP2B融合蛋白。
  14. 如权利要求4所述的用于治疗亨廷顿病的RNA递送系统，其特征在于，所述RNA序列的长度为15-25个核苷酸。
  15. 如权利要求14所述的用于治疗亨廷顿病的RNA递送系统，其特征在于，所述能够治疗亨廷顿病的RNA选自mHTT基因的siRNA，或与上述序列同源性大于80％的RNA序列，或编码上述RNA的核酸分子。
  16. 如权利要求15所述的用于治疗亨廷顿病的RNA递送系统，其特征在于，mHTT基因的siRNA包括UAUGUUUUCACAUAUUGUCAG、AUUUAGUAGCCAACUAUAGAA、AUGUUUUUCAAUAAAUGUGCC、UAUGAAUAGCAUUCUUAUCUG、UAUUUGUUCCUCUUAAUACAA、其他具有抑制mHTT基因表达的序列以及与上述序列同源性大于80％的序列。
  17. 如权利要求1所述的用于治疗亨廷顿病的RNA递送系统，其特征在于，所述递送系统为用于包括人在内的哺乳动物中的递送系统。
  18. 一种权利要求1-17任意一项所述的用于治疗亨廷顿病的RNA递送系统在药物中的应用。
  19. 如权利要求18所述的应用，其特征在于，所述药物为治疗亨廷顿病及其相关疾病的药物，所述药物的给药方式包括口服、吸入、皮下注射、肌肉注射、静脉注射。
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