WO2022206781A1

WO2022206781A1 - Rna delivery system for treating colitis

Info

Publication number: WO2022206781A1
Application number: PCT/CN2022/083810
Authority: WO
Inventors: 张辰宇; 陈熹; 付正; 李菁; 张翔; 周心妍; 张丽; 余梦超; 郭宏源
Original assignee: 南京大学
Priority date: 2021-03-30
Filing date: 2022-03-29
Publication date: 2022-10-06
Also published as: CN115137835A

Abstract

Provided is an RNA delivery system for treating colitis, characterized in that the system comprises a viral vector. The viral vector carries an RNA fragment capable of treating colitis. The viral vector can be enriched in organ tissues of a host. In the host organ tissues, the viral vector can endogenously and spontaneously form a composite structure containing the RNA fragment. The composite structure can feed the RNA fragment into an intestinal tract, so as to treat colitis. The safety and reliability of the RNA delivery system for treating colitis have been fully verified, and the system has good druggability and high universality.

Description

An RNA delivery system for the treatment of colitis

technical field

The present application relates to the field of biomedical technology, in particular to an RNA delivery system for treating colitis.

Background technique

Colitis refers to inflammatory lesions of the colon caused by various reasons, which can be caused by bacteria, fungi, viruses, parasites, protozoa and other organisms, and can also be caused by allergic reactions and physical and chemical factors. According to different etiologies, it can be divided into specific inflammatory lesions and non-specific inflammatory lesions. The former refers to infectious colitis, ischemic colitis and pseudomembranous colitis, and the latter includes ulcerative colitis and colonic Crohn's disease. . The main clinical manifestations are diarrhea, abdominal pain, mucus stool, pus and blood stool, tenesmus, even constipation, inability to pass stool for several days; The incidence of ulcerative colitis in my country is gradually increasing, the course of disease is long, and there is a risk of colon cancer, so it has received more and more attention.

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.

It is generally believed that 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.

At present, there are many patents related to siRNA, mainly focusing on the following aspects: 1. Designing siRNA with medical effects. 2. Chemical modification of siRNA to improve the stability of siRNA in vivo and increase the yield. 3. Improve the design of various artificial carriers (such as lipid nanoparticles, cationic polymers and viruses) to improve the efficiency of siRNA delivery in vivo. Among them, there are many patents in the third aspect. The fundamental reason is that researchers have realized that there is currently a lack of suitable siRNA delivery systems to safely, precisely and efficiently deliver siRNA to target tissues. This problem has become the core restricting RNAi therapy. question.

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 α-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. These patents are all inventions and creations in terms of siRNA vectors, but their technical solutions have a common feature, that is, the vectors and siRNA are pre-assembled in vitro and then introduced into the host. In fact, this is the case with most of the delivery technologies currently designed. However, this type of delivery system has a common problem, that is, these synthetic exogenous delivery systems are easily cleared by the host's circulatory system, may also cause immunogenic responses, and may even be toxic to specific cell types and tissues.

The research team of the present invention found that endogenous cells can selectively encapsulate miRNAs into exosomes, and 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. For example, 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 .

However, such techniques of in vitro isolation or preparation of exosomes often require obtaining a large amount of exosomes through cell culture, coupled with the step of siRNA encapsulation, which makes the clinical cost of large-scale application of this product very high. Patients cannot afford it; more importantly, the complex production/purification process of exosomes makes it almost impossible to comply with GMP standards.

So far, drugs with exosomes as active ingredients have never been approved by the CFDA. The core problem is that the consistency of exosome products cannot be guaranteed, and this problem directly leads to the inability of such products to obtain drug production licenses. If this problem can be solved, it will be of great significance to promote RNAi therapy for colitis.

Therefore, the development of a safe, precise and efficient siRNA delivery system is a crucial part of improving the effect of RNAi therapy and advancing RNAi therapy.

SUMMARY OF THE INVENTION

In view of this, the embodiments of the present application provide an RNA delivery system for treating colitis 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 colitis, the system comprising a viral vector carrying an RNA fragment capable of treating colitis, and the viral vector can be used in the organ tissue of a host. It is enriched in the host organ and tissue, and a composite structure containing the RNA fragment is endogenously and spontaneously formed in the host organ tissue, and the composite structure can send the RNA fragment into the intestinal tract to realize the treatment of colitis. After the RNA fragment is delivered to the target tissue intestine, it can inhibit the expression of the matching gene, thereby inhibiting the further development of colitis.

Optionally, 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 capable of inhibiting or hindering the development of colitis.

Optionally, the RNA sequence is 15-25 nucleotides in length. For example, the length of the RNA sequence can be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides. Preferably, the RNA sequence is 18-22 nucleotides in length.

Optionally, the RNA fragment is selected from any one or more of the following: the siRNA of the TNF-α gene, the siRNA of the integrin-α gene, the siRNA of the B7 gene, or an RNA sequence with more than 80% homology to the above-mentioned 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.

Optionally, the siRNA of the TNF-α gene includes AAAACAUAAUCAAAAGAAGGC, UAAAAAACAUAAUCAAAAGAA, AAUAAUAAAUAAUCACAAGUG, UUUUCACGGAAAACAUGUCUG, AAACAUAAUCAAAAGAAGGCA, other sequences that inhibit the expression of the TNF-α gene, and sequences that are more than 80% homologous to the above sequences;

siRNA of integrin-α gene includes AUAAUCAUCUCCAUUAAUGUC, AAACAAUUCCUUUUUUAUCUU, AUUAAAACAGGAAACUUUGAG, AUAAUGAAGGAUAUACAACAG, UUCUUUUAUUCAUAAAAGUCUC, other sequences that inhibit the expression of integrin-α gene and sequences with more than 80% homology to the above sequences;

siRNA of B7 gene, UUUUCUUUGGGUAAUCUUCAG, AGAAAAAUUCCACUUUUUCUU, AUUUCAAAGUCAGAUAUACUA, ACAAAAAUUCCAUUUACUGAG, AUUAUUGAGUUAAGUAUUCCU, other sequences that inhibit the expression of B7 gene and sequences with more than 80% homology to the above sequences.

It should be noted that the above-mentioned "sequences with more than 80% homology" may be 85%, 88%, 90%, 95%, 98%, etc. homology.

Optionally, 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.

In the present invention, 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. In one of the embodiments of the present invention, 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. In one of the embodiments of the present invention, 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. Preferably, 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.

Optionally, the viral vector also includes a promoter and a targeting tag, the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host, and the targeting structure is located on the surface of the composite structure, The complex structure is capable of finding and binding to the target tissue through the targeting structure, and delivering the RNA fragment into the target tissue.

Optionally, the viral vector includes any one of the following circuits or a combination of several circuits: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each of the virus At least one RNA fragment and one targeting tag are included in the vector, and the RNA fragment and targeting tag are located in the same circuit or in different circuits.

Optionally, the viral vector further comprises a flanking sequence, a compensation sequence and a loop sequence that enable the circuit to be folded into a correct structure and expressed, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence;

The viral vector includes any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-target To tag, 5'-promoter-targeting tag-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence;

Preferably, 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.

Preferably, the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.

More preferably, the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.

Most preferably, the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.

Optionally, when there are at least two lines in the viral vector, adjacent lines are connected by a sequence composed of sequences 1-3 (sequence 1-sequence 2-sequence 3);

Among them, sequence 1 is CAGATC, sequence 2 is a sequence consisting of 5-80 bases, and sequence 3 is TGGATC;

Preferably, when there are at least two lines in the viral vector, adjacent lines are connected by sequence 4 or a sequence with a homology of more than 80% to sequence 4;

Wherein, sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.

Optionally, the organ tissue is liver, and the composite structure is exosome.

Optionally, the targeting tag is selected from targeting peptides or targeting proteins with targeting function;

Preferably, 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.

Preferably, the targeting tag is a TCP-1 targeting peptide or a TCP-1-LAMP2B fusion protein.

Optionally, the viral vector is an adenovirus-associated virus;

Preferably, the adeno-associated virus is adeno-associated virus type 5, adeno-associated virus type 8 or adenovirus-associated virus type 9.

In addition to adeno-associated

virus types

5, 8, and 9, adeno-associated

virus types

2 and 7 as vectors also have similar in vivo enrichment, self-assembly and colitis therapeutic effects (Figure 34-35) .

Optionally, the delivery system is a delivery system for use in mammals, including humans.

The present application also provides an application of the RNA delivery system for treating colitis in medicine.

Optionally, the medicine is a medicine for the treatment of colitis and its related diseases, and the related diseases here refer to the associated diseases or complications, sequelae, etc. that occur in the formation or development of the above-mentioned colitis, or have a relationship with colitis. other related diseases.

The drug includes the above-mentioned viral vector, specifically, the viral vector here refers to a viral vector carrying RNA fragments, or carrying RNA fragments and targeting tags, and can enter the host body, can be enriched in the liver, and self-assemble. A composite structure exosome is formed, which can deliver RNA fragments to the target tissue, so that the RNA fragments are expressed in the target tissue, thereby inhibiting the expression of matching genes, and achieving the purpose of treating diseases.

The 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 technical effects of this application are:

The RNA delivery system for the treatment of colitis provided in this application 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. The RNA delivery system for the treatment of colitis 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.

The application of the RNA delivery system for the treatment of colitis provided in this application to medicines provides a drug delivery platform, which can greatly improve the therapeutic effect of colitis, and can also form a research and development basis for more RNA-based medicines through this platform. It has greatly promoted the development and use of RNA drugs.

Description of drawings

Fig. 1 is the mouse colitis treatment situation and RNA expression level comparison diagram provided by an embodiment of the present application;

2 is a comparison diagram of mouse cytokine concentration and colon HE staining provided by an embodiment of the present application;

3 is a comparison diagram of the treatment of colitis in mice provided by an embodiment of the present application;

4 is a comparison diagram of the mouse disease activity index and various siRNA levels provided by an embodiment of the present application;

Figure 5 is a comparison diagram of various siRNA and mRNA levels in mice provided by an embodiment of the present application;

FIG. 6 is a comparison diagram of HE staining of mouse colon provided by an example of the present application.

Figure 7 is a graph showing the results of in vivo enrichment of TNF-α siRNA by injecting mice with TNF-α siRNA-lentivirus according to an example of the present application. In the figure, A is the liver enrichment result, B is the plasma enrichment result, and C Results for colon enrichment.

Figure 8 is a graph showing the results of injecting mice with TNF-α siRNA-lentivirus and detecting TNF-α siRNA in plasma exosomes to determine the spontaneous formation of complex structures provided in an example of the present application.

Fig. 9 is the result graph of the specific curative effect after injecting mice with TNF-α siRNA-lentivirus according to an embodiment of the present application, in the figure A is the disease index score, B is the detection result of inflammatory factors, and C is the detection of target gene mRNA result.

Figure 10 is a graph of the enrichment results in vivo provided by an example of the present application after injecting mice with viral vectors containing 6 different RNAs. The 6 RNAs are: miR-19a (target gene TNF-α), miR -124-3p (target gene TNF-α), B7-siRNA-1, B7-siRNA-2, integrin α4 shRNA-1, integrin α4 shRNA-2, A is the result of intrahepatic detection of small RNA expression, B is The results of small RNA expression in plasma, and C is the result of small RNA expression in colon.

FIG. 11 is a graph showing the specific therapeutic effects of mice injected with viral vectors containing 6 different RNAs provided in an example of the present application. The 6 RNAs are: miR-19a (target gene TNF-α), miR- 124-3p (target gene TNF-α), B7-siRNA-1, B7-siRNA-2, integrin α4 shRNA-1, integrin α4 shRNA-2, A is the disease index score in the figure, and B is the detection result of inflammatory factors.

12 is a graph of the enrichment results in vivo provided by an embodiment of the present application after injecting mice with viral vectors containing 4 groups of different RNA fragments. The 4 groups of RNA fragments respectively contain any two RNA sequences, specifically: miR -19a (target gene TNF-α)+B7-siRNA-1, miR-124-3p (target gene TNF-α)+B7-siRNA-2, B7-siRNA-1+integrin α4 shRNA-1, B7-siRNA -2+integrin α4 shRNA-2, in the figure A is the expression result of small RNA (sequence 1) detected in liver, B is the expression result of small RNA (sequence 1) detected in plasma, C is the result of small RNA detected in colon (sequence 1) Express the result.

13 is a graph of the enrichment results in vivo provided by another embodiment of the present application after injecting mice with viral vectors containing 4 groups of different RNA fragments, respectively. The 4 groups of RNA fragments respectively contain any 2 kinds of RNA sequences, specifically: miR-19a (target gene TNF-α)+B7-siRNA-1, miR-124-3p (target gene TNF-α)+B7-siRNA-2, B7-siRNA-1+integrin α4 shRNA-1, B7- siRNA-2+integrin α4 shRNA-2, in the figure A is the expression result of small RNA (sequence 2) detected in liver, B is the expression result of small RNA (sequence 2) detected in plasma, and C is the result of small RNA detected in colon (sequence 2). ) to express the result.

Figure 14 is a graph showing the results of specific therapeutic effects after injecting mice with viral vectors containing 4 groups of different RNA fragments provided in an embodiment of the present application. The 4 groups of RNA fragments respectively contain any two RNA sequences, specifically: miR -19a (target gene TNF-α)+B7-siRNA-1, miR-124-3p (target gene TNF-α)+B7-siRNA-2, B7-siRNA-1+integrin α4 shRNA-1, B7-siRNA -2+integrin α4 shRNA-2, A is the disease index score in the figure, and B is the detection result of inflammatory factors.

15 is a graph of the enrichment results in vivo provided by an embodiment of the present application after injecting mice with viral vectors containing 3 groups of different RNA fragments. The 3 groups of RNA fragments respectively contain any 3 kinds of RNA sequences, specifically: miR -19a (target gene TNF-α)+B7-siRNA-1+integrin α4 shRNA-1, miR-124-3p (target gene TNF-α)+B7-siRNA-2+integrin α4 shRNA-2, miR-19a (target gene TNF-α)+B7-siRNA-1+integrin α4 shRNA-2, in the figure A is the expression result of small RNA (sequence 1) detected in liver, B is the expression result of small RNA (sequence 1) detected in plasma, C is the expression result of small RNA (sequence 1) detected in colon.

Figure 16 is a graph of the in vivo enrichment results provided by another embodiment of the present application after injecting mice with viral vectors containing 3 groups of different RNA fragments respectively. The 3 groups of RNA fragments respectively contain any 3 kinds of RNA sequences, specifically: miR-19a (target gene TNF-α)+B7-siRNA-1+integrin α4 shRNA-1, miR-124-3p (target gene TNF-α)+B7-siRNA-2+integrin α4 shRNA-2, miR- 19a (target gene TNF-α)+B7-siRNA-1+integrin α4 shRNA-2, in the figure A is the expression result of small RNA (sequence 2) detected in liver, and B is the expression result of small RNA (sequence 2) detected in plasma , C is the expression result of small RNA (sequence 2) detected in colon.

Figure 17 is a graph of the enrichment results in vivo after injecting mice with viral vectors containing 3 groups of different RNA fragments, respectively, provided in another embodiment of the present application. The 3 groups of RNA fragments respectively contain any 3 kinds of RNA sequences, specifically: miR-19a (target gene TNF-α)+B7-siRNA-1+integrin α4 shRNA-1, miR-124-3p (target gene TNF-α)+B7-siRNA-2+integrin α4 shRNA-2, miR- 19a (target gene TNF-α)+B7-siRNA-1+integrin α4 shRNA-2, in the figure A is the expression result of small RNA (sequence 3) detected in liver, and B is the expression result of small RNA (sequence 3) detected in plasma , C is the expression result of small RNA (SEQ ID NO: 3) detected in colon.

Figure 18 is a graph showing the results of specific curative effects after injecting mice with viral vectors containing 3 groups of different RNA fragments, respectively, provided in an embodiment of the present application. The 3 groups of RNA fragments respectively contain any 3 kinds of RNA sequences, specifically: miR -19a (target gene TNF-α)+B7-siRNA-1+integrin α4 shRNA-1, miR-124-3p (target gene TNF-α)+B7-siRNA-2+integrin α4 shRNA-2, miR-19a (target gene TNF-α)+B7-siRNA-1+integrin α4 shRNA-2, A is the disease index score in the figure, and B is the detection result of inflammatory factors.

Fig. 19 is a graph showing the results of in vivo enrichment of viral vectors containing RNA sequences of different lengths provided in an example of the present application after injection into mice. The RNA sequences of different lengths are: TNF-α-siRNA-1 (siRNA length 18bp), TNF-α-siRNA-1 (siRNA length 18bp), -α-siRNA-2 (siRNA length 20bp), TNF-α-siRNA-3 (siRNA length 22bp), in the figure A is the result of intrahepatic detection of siRNA expression, B is the result of siRNA expression detected in plasma, and C is the detection of siRNA in colon siRNA expression results.

Figure 20 is a graph showing the specific curative effect results after injecting mice with viral vectors containing RNA sequences of different lengths provided by another embodiment of the present application. The RNA sequences of different lengths are: TNF-α-siRNA-1 (siRNA length 18bp), TNF-α-siRNA-1 (siRNA length 18bp), -α-siRNA-2 (siRNA length 20bp), TNF-α-siRNA-3 (siRNA length 22bp), A is the disease index score in the figure, B is the detection result of inflammatory factors, and C is the detection result of target gene mRNA.

Figure 21 is a graph showing the results of in vivo enrichment of viral vectors containing 3 homologous TNF-α-siRNA sequences provided in an example of the present application after injection into mice. The homologous TNF-α-siRNA sequences are: TNF-α -siRNA-4, TNF-α-siRNA-5, TNF-α-siRNA-6, in the figure A is the expression result of TNF-α-siRNA detected in liver, B is the expression result of TNF-α-siRNA detected in plasma, C The expression results of TNF-α-siRNA were detected in colon, and D was the expression results of TNF-α-siRNA detected in plasma exosomes.

Figure 22 is a graph showing the results of in vivo enrichment of viral vectors containing 3 homologous B7-siRNA sequences provided in another embodiment of the present application after injection into mice. The homologous B7-siRNA sequences are: B7-siRNA-1, B7-siRNA-2, B7-siRNA-3, in the figure A is the expression result of B7-siRNA detected in liver, B is the expression result of B7-siRNA detected in plasma, C is the expression result of B7-siRNA detected in colon, D is the expression result of plasma B7-siRNA expression results were detected in exosomes.

Figure 23 is a graph showing the results of in vivo enrichment of viral vectors containing 3 homologous integrin α4 siRNA sequences respectively provided in another embodiment of the present application after injection into mice. The homologous integrin α4 siRNA sequences are: integrin α4 siRNA-1, integrin α4 siRNA-2, integrin α4 siRNA-3, in the figure A is the result of detecting integrin α4 siRNA expression in liver, B is the result of detecting integrin α4 siRNA expression in plasma, C is the result of detecting integrin α4 siRNA expression in colon, D is the result of plasma detection Detection of integrin α4 siRNA expression results in exosomes.

Figure 24 is a graph showing the specific curative effect results of virus vectors containing 9 homologous siRNA sequences provided in an example of the present application after injection into mice. The homologous siRNA sequences are: TNF-α-siRNA-4, TNF-α- siRNA-5, TNF-α-siRNA-6, B7-siRNA-1, B7-siRNA-2, B7-siRNA-3, integrin α4 siRNA-1, integrin α4 siRNA-2, integrin α4 siRNA-3, in the figure A is the disease index score, and B is the detection result of inflammatory factors.

Figure 25 is a graph showing the results of in vivo enrichment of mice injected with viral vectors containing RNA fragments with different flanking sequences, loop sequences and reverse complementary sequences provided in an embodiment of the present application. Definite 5' flanking sequences with more than 80% homology, 2 unambiguous sequences with more than 80% homology to the identified loop sequence, and 2 clear sequences with more than 80% homology to the identified 3' flanking sequences A clear sequence, a reverse complementary sequence of a normal sequence, and a clear reverse complementary sequence of a clear sequence whose 5' flanking sequence homology is greater than 80%, A in the figure is the intrahepatic detection of TNF-α-siRNA Expression results, B is the expression result of TNF-α-siRNA in plasma, C is the expression result of TNF-α-siRNA in colon, D is the expression result of TNF-α-siRNA in plasma exosome.

Figure 26 is a graph of the specific curative effect results after injecting mice with viral vectors containing RNA fragments with different flanking sequences, loop sequences and reverse complementary sequences provided by an embodiment of the present application. The 5' flanking sequence homology is greater than 80% of the clear sequence, 2 clear sequences with the identified loop sequence homology greater than 80%, 2 clear sequences with the identified 3' flanking sequence homology greater than 80% A clear sequence, a reverse complementary sequence of a normal sequence, and a clear reverse complementary sequence of a clear sequence whose 5' flanking sequence homology is greater than 80%. In the figure, A is the disease index score, and B is the detection of inflammatory factors As a result, C is the detection result of target gene mRNA.

Figure 27 shows that when the adenovirus vector provided in an embodiment of the present application carries multiple lines, adjacent lines are connected by sequence 1-sequence 2-sequence 3, and wherein sequence 2 is 5 bases, 10 bases, In the case of 20 bases, 30 bases, 40 bases, 50 bases, and 80 bases, the enrichment results of mice in vivo, the figure A is the expression of TNF-α-siRNA detected in the liver Results, B is the result of detecting the expression of TNF-α-siRNA in plasma, and C is the result of detecting the expression of TNF-α-siRNA in colon.

Figure 28 shows that when the adenovirus vector provided by another embodiment of the present application carries multiple lines, adjacent lines are connected by sequence 1-sequence 2-sequence 3, wherein sequence 2 is 5 bases and 10 bases respectively , 20 bases, 30 bases, 40 bases, 50 bases, 80 bases, the in vivo enrichment results of mice, the figure A is the intrahepatic detection of B7-1-siRNA Expression results, B is the expression result of B7-1-siRNA detected in plasma, C is the expression result of B7-1-siRNA detected in colon.

Figure 29 shows that when the adenovirus vector provided by another embodiment of the present application carries multiple lines, adjacent lines are connected by sequence 1-sequence 2-sequence 3, wherein sequence 2 is 5 bases and 10 bases respectively , 20 bases, 30 bases, 40 bases, 50 bases, and 80 bases, the enrichment results of mice in vivo, in the figure A is the expression of integrin α4-siRNA detected in the liver Results, B is the result of detecting the expression of integrin α4-siRNA in plasma, and C is the result of detecting the expression of integrin α4-siRNA in colon.

Figure 30 is a graph showing the enrichment results in mice in vivo when the adenovirus vector provided in an embodiment of the present application carries multiple lines and the connecting sequence is sequence 4 and two sequences with a homology of more than 80% to sequence 4 , in the figure, A is the result of detecting TNF-α-siRNA expression in the liver, B is the result of detecting the expression of TNF-α-siRNA in the plasma, and C is the result of detecting the expression of TNF-α-siRNA in the colon.

Figure 31 shows the in vivo enrichment results of mice when the adenovirus vector provided in another embodiment of the present application carries multiple lines, and the connecting sequence is sequence 4 and two sequences with a homology of more than 80% to sequence 4 Figure A is the result of B7-1-siRNA expression detected in liver, B is the result of B7-1-siRNA expression detected in plasma, and C is the result of B7-1-siRNA expression detected in colon.

Figure 32 shows the enrichment results in vivo in mice when the adenovirus vector provided by another embodiment of the present application carries multiple lines and the connecting sequence is sequence 4 and two sequences with a homology of more than 80% to sequence 4 Figure A is the result of detecting the expression of integrin α4-siRNA in the liver, B is the result of detecting the expression of integrin α4-siRNA in the plasma, and C is the result of detecting the expression of integrin α4-siRNA in the colon.

Figure 33 shows that when the adenoviral vector provided by an embodiment of the present application carries multiple lines, and the connecting sequence is sequence 4 and two sequences with more than 80% homology to sequence 4, the small The specific curative effect results of mice, A is the disease index score, B is the target gene mRNA detection result.

Figure 34 is a graph showing the enrichment results of TNF-α-siRNA loaded in mice when adenovirus-associated

virus types

2, 7 and 8 are used as viral vectors provided in an example of the present application, and A in the figure is the liver The expression results of TNF-α-siRNA were detected in B, the expression results of TNF-α-siRNA in plasma were detected in B, and the expression results of TNF-α-siRNA in colon were detected in C.

Figure 35 is a graph showing the specific efficacy results of adenovirus-associated virus type 2, type 7 and type 8 as the viral vector provided in an example of the present application, after the TNF-α-siRNA loaded thereon is expressed in mice. A is the disease index score, B is the detection result of inflammatory factors, and C is the detection result of target gene mRNA.

Detailed ways

The specific embodiments of the present application will be described below with reference to the accompanying drawings.

First, technical terms, test methods, etc. related to the present invention are explained.

Hematoxylin-eosin staining, referred to as HE staining. HE staining is one of the most basic and widely used technical methods in histology and pathology teaching and research.

The hematoxylin staining solution is alkaline and can stain the basophilic structure of the tissue (such as ribosome, nucleus and ribonucleic acid in the cytoplasm) into blue-violet; eosin is an acid dye, which can stain the eosinophilic structure of the tissue ( Such as intracellular and intercellular proteins, including Lewy bodies, alcohol bodies, and most of the cytoplasm) stained pink, making the morphology of the entire cell organization clearly visible.

The specific steps of HE staining include: sample tissue fixation and sectioning; tissue sample dewaxing; tissue sample hydration; tissue section hematoxylin staining, differentiation and anti-blue; tissue section eosin staining and dehydration; tissue sample section air-drying and sealing; Observe and photograph under the microscope.

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 expression level of each siRNA or mRNA relative to the internal control can be represented by 2-ΔCT, where ΔCT=C sample-C internal control. When amplifying siRNA, the internal reference gene is U6snRNA (in tissue) or miR-16 (in serum, exosomes), and when amplifying mRNA, 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.

In the diagram provided by the present invention, "*" means P<0.05, "**" means P<0.01, and "***" means P<0.005.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the reagents, materials and operation steps used herein are the reagents, materials and conventional steps widely used in the corresponding fields.

Example 1

This embodiment provides an RNA delivery system for treating colitis, the system comprising a viral vector carrying an RNA fragment capable of treating colitis, and the viral vector can be enriched in the organ tissue of a host, And endogenously and spontaneously form a composite structure containing the RNA fragment in the host organ tissue, and the composite structure can send the RNA fragment into the intestinal tract to realize the treatment of colitis.

Figures 7-9 show that in addition to adenovirus, lentiviral vectors also have in vivo enrichment, spontaneous formation of complex structures, and therapeutic effects on colitis.

In the case of carrying different RNA fragments, the viral vector delivery system also has in vivo enrichment, spontaneous formation of composite structures and the treatment effect of colitis. The grouping of RNA fragments is as follows:

1), siRNA1 alone, siRNA2 alone, shRNA1 alone, shRNA2 alone, miRNA1 alone, miRNA2 alone (Figure 10-11);

2) In the above 1), there are 4 groups of RNA fragments of any 2 different RNA sequences (Fig. 12-14);

3) In the above 1), there are 3 sets of RNA fragments comprising any 3 kinds of RNA sequences (Figs. 15-18).

In addition to adeno-associated

virus types

5, 8, and 9, adeno-associated

virus types

In this embodiment, 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. In other words, 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.

Further, 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.

Wherein, 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. When the RNA fragment contains only one RNA sequence, the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-5 bases therein.

Preferably, the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted. When the RNA fragment contains only one RNA sequence, the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-3 bases therein.

More preferably, the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted. When the RNA fragment contains only one RNA sequence, the compensation sequence may be the reverse complementary sequence of the RNA sequence by deleting any 1-3 consecutively arranged bases.

Most preferably, the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted. When the RNA fragment contains only one RNA sequence, 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.

It should be noted that the above-mentioned flanking sequences, compensation sequences and loop 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.

When the vector system contains different 5' flanking sequences, loop sequences, 3' flanking sequences, reverse complementary sequences, and clear sequences with more than 80% homology to the above sequences, it also has in vivo enrichment and self-assembly. and the treatment effect of colitis (Figure 25-26).

In the case that the viral vector carries two or more lines, the adjacent lines can be connected by sequence 1-sequence 2-sequence 3; wherein, sequence 1 is preferably CAGATC, and 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 The sequence of 10-50 bases is preferable, and the sequence of 20-40 bases is more preferable. Sequence 3 is preferably TGGATC.

The vector system carries multiple lines, and adjacent lines are connected by sequence 1-sequence 2-sequence 3, wherein sequence 2 is respectively 5 bases, 10 bases, 20 bases, 30 bases, 40 bases In the case of bases, 50 bases, and 80 bases, it also has in vivo enrichment, self-assembly and colitis treatment effects (Figures 27-29).

More preferably, when the viral vector carries two or more lines, adjacent lines are connected by sequence 4 or a sequence with more than 80% homology to sequence 4; wherein, sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.

When the connecting sequence is the above-mentioned sequence 4 or a sequence with more than 80% homology to the sequence 4, the vector system also has the effect of in vivo enrichment, self-assembly and treatment of colitis (Figures 30-33).

The above-mentioned 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.

When the lengths of RNA sequences are different (18, 22, 20, respectively), they can also be enriched in vivo through the carrier and have corresponding therapeutic effects (Figures 19-20).

The RNA capable of treating colitis is selected from any one or more of the following RNAs: siRNA of TNF-α gene, siRNA of integrin-α gene, siRNA of B7 gene or nucleic acid molecules encoding the above RNAs.

The gene circuit also has in vivo enrichment, self-assembly and therapeutic effects on enteritis when it contains the listed RNA sequences or sequences with more than 80% homology (Figures 21-24).

The number of RNA sequences capable of treating colitis is one, two or more. For example, in the treatment of enteritis, three siRNAs including TNF-α gene siRNA, integrin-α gene siRNA and B7 gene siRNA can be used at the same time, or TNF-α gene siRNA and integrin-α gene siRNA can be used at the same time. Any two kinds of siRNA among siRNA and siRNA of B7 gene, siRNA of TNF-α gene, siRNA of integrin-α gene or siRNA of B7 gene can also be used alone.

Taking the combined use of "siRNA1" and "siRNA2" on the same viral vector as an example, 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.

More specifically, 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-siRNA 2-loop sequence - Compensation sequence-3' flanking sequence), (5'-promoter-targeting tag-5' flanking sequence-siRNA1-siRNA2-loop sequence-compensating sequence-3' flanking sequence), etc. Other situations can be deduced by analogy, and details are not repeated here. The above connecting sequence can be "sequence 1-sequence 2-sequence 3" or "sequence 4", and a bracket indicates a complete circuit.

Preferably, 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.

Specifically, 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.

Preferably, 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 In particular, 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 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 peptides that have been screened so far include but are not limited to RVG targeting peptide (nucleotide sequence shown in SEQ ID No: 1), GE11 targeting peptide (nucleotide sequence shown in SEQ ID No: 2), PTP targeting peptide (nucleotide sequence shown in SEQ ID No: 3), TCP-1 targeting peptide (nucleotide sequence shown in SEQ ID No: 4), MSP targeting peptide (nucleotide sequence shown in SEQ ID No: 4) SEQ ID No: 5); targeting proteins include but are not limited to RVG-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6), GE11-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6) : 7), PTP-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 8), TCP-1-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 9), MSP- LAMP2B fusion protein (nucleotide sequence is shown in SEQ ID No: 10). In this embodiment, TCP-1 targeting peptide and TCP-1-LAMP2B fusion protein that can precisely target colon are preferably used as targeting tags.

In addition, in order to achieve the purpose of precise delivery, we have experimented with a variety of viral vector loading schemes, and came up with another optimized scheme: 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 .

More preferably, when two different viral vectors are injected into the host, 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 colitis provided in this example uses a virus as a vector, and the virus vector is used as a mature injectable substance. 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. The RNA delivery system for the treatment of colitis 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 is beneficial to receptor cell uptake, intracytoplasmic release and lysosomal escape, and the required dose is low.

The sequences involved above are specifically shown in Tables 1-10 below.

Table 1

Table 2

table 3

Table 4

table 5

Table 6

Table 7

Table 8

Table 9

Table 10

Example 2

On the basis of Embodiment 1, this embodiment provides a medicine. The medicament comprises a viral vector carrying an RNA fragment capable of treating colitis, the viral vector being capable of being enriched in the organ tissue of a host, and endogenously forming spontaneously in the organ tissue of the host containing all the The composite structure of the RNA fragment, the composite structure can send the RNA fragment into the intestinal tract to realize the treatment of colitis. After the RNA fragment is delivered to the target tissue intestine, it can inhibit the expression of the matching gene, thereby inhibiting the further development of colitis.

For explanations about the above-mentioned viral vectors, RNA fragments, targeting tags, etc. in this embodiment, reference may be made to Embodiment 1, which will not be repeated here.

After 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.

This drug can also be used in combination with other drugs for colitis to enhance the effect of treatment. Such as norfloxacin, entericine, mesalazine, sulfasalazine and so on.

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.

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 as the mature injectable substance, and its safety and reliability have been fully verified, and the drugability is very good. The final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes. The drug can deliver various kinds of small molecule RNAs and has strong versatility. And the preparation of viruses is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances. The drug provided in this application can be closely combined with AGO ₂ and enriched into a composite structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation and maintain its stability in circulation, but also benefit the receptor. Cellular uptake, intracytoplasmic release and lysosomal escape require low doses.

Example 3

On the basis of

embodiment

1 or 2, this embodiment provides an application of an RNA delivery system in medicine, and the medicine is a medicine for treating colitis. In this embodiment, the following experiments will be conducted to investigate the application of RNA delivery system in the treatment of colitis. The effect is described in detail.

In the first experiment, we set up three experimental groups and two control groups, the three experimental groups were the AAV-CMV-siR ^TNF-α (low) group and the AAV-CMV-siR ^TNF-α (medium) group. , AAV-CMV-siR ^TNF-α (high) group; control group were Normal group and AAV-CMV-scrR group.

The experimental process is shown in Figure 1A. The AAV-CMV-siR ^TNF-α (low) group, the AAV-CMV-siR ^TNF-α (medium) group, and the AAV-CMV-siR ^TNF-α (high) group were treated with high hepatic Affinity AAV-5 adeno-associated virus-encapsulated TNF-α siRNA system (AAV-CMV-siR ^TNF-α ), AAV solution with a titer of 10 ¹² Vg/ml was injected through the tail vein, 25 μL, 50 μL, 100 μL to small in mice.

The in vivo expression of the AAV system was monitored by small animals. The results are shown in Figure 1B. After 3 weeks, it can be seen that the AAV system is stably expressed in vivo, especially in the liver. The AAV-CMV-siR ^TNF-α (high) group has an average The Average Radiance reached 8.42*105 (p/sec/cm ² /sr), and the expression site was in the liver, which indicated that the expression of the AAV system had a dose-dependent effect.

Immediately, the DSS-induced chronic colitis model was constructed, and the weighing recording was performed every two days. The results are shown in Figure 1C. It can be seen that the AAV-encapsulated CMV-siR TNF-α system can reduce the weight loss of chronic colitis mice. In addition, the mice in the three experimental groups recovered significantly faster than the AAV-CMV-scrR group during the period of inflammation remission.

At the end of the tenth week of model construction, the in vivo expression of the AAV system was monitored by small animals, and then the mice were sacrificed to observe the colon. The results are shown in Figure 1D. It can be seen that the AAV-CMV-siR ^TNF-α (low) group, In the AAV-CMV-siR ^TNF-α (medium) group and the AAV-CMV-siR ^TNF-α (high) group, the redness and swelling of the colon of the mice in the AAV-CMV-siR TNF-α (high) group were alleviated to varying degrees, and the shortening of the colon length caused by chronic inflammation was also improved in varying degrees. Among them, the AAV-CMV-siR ^TNF-α (high) group had the most significant improvement in inflammation.

The disease index of the mice in each group was scored and counted, and the results are shown in Figure 1E. It can be seen that the disease index of the mice in the AAV-CMV-siR ^TNF-α (high) group was lower than that of the AAV-CMV-siR ^TNF-α (low) group, AAV-CMV-siR ^TNF-α (medium) group and AAV-CMV-scrR group.

The levels of TNF-α siRNA in the mice of each group were detected respectively. The results are shown in Figure 1F. It can be seen that the levels of TNF-α siRNA in the three experimental groups were higher, while the AAV-CMV-scrR group in the control group was smaller. There is almost no expression of TNF-α siRNA in mice, which indicates that the above-mentioned AAV system can produce a certain amount of TNF-α siRNA.

The levels of TNF-α mRNA in the mice in each group were detected respectively, and the results are shown in Figure 1G. It can be seen that the levels of TNF-α mRNA in the mice in the Normal group and the three experimental groups were relatively low, while the AAV-CMV-scrR group was lower. The level of TNF-αmRNA in mice was higher, which indicated that AAV system could reduce the expression and secretion of colonic TNF-α.

The pro-inflammatory cytokines IL-6, IL-12, and ^IL -23 in the colon of mice were detected, and the results were shown in Figure 2A. The secretion of inflammatory cytokines was the least, and the secretion of pro-inflammatory cytokines was the most in the AAV-CMV-scrR group.

HE staining and pathological score statistics were performed on the mouse colon sections. The results are shown in Figure 2B and Figure 2C. It can be seen that the experimental group, especially the AAV-CMV-siR ^TNF-α (high) group, had better colonic mucosa integrity. The degree of infiltration of immune cells was more shallow, and the colonic crypt abscess and the congestion and hemorrhage of the colon were also significantly lighter than those in the AAV-CMV-scrR group.

The above experiments show that the use of liver-friendly AAV to encapsulate the CMV-siR ^TNF-α circuit can achieve long-term TNF-α siRNA expression and long-term TNF-α silencing, and can relieve colitis to a certain extent. Drug potential and clinical research value.

In the second experiment, we set up three experimental groups and two control groups. Among them, the experimental groups were AAV-CMV-siR ^T+B+I (low) group, AAV-CMV-siR ^T+B+I (medium) group, AAV-CMV-siR ^T+B+I (high) group ; The control groups were Normal group and AAV-CMV-scrR group.

AAV-CMV-siR ^T+B+I (low) group, AAV-CMV-siR ^T+B+I (medium) group, and AAV-CMV-siR ^T+B+I (high) group used liver high affinity The AAV-5 adeno-associated virus encapsulated TNF-α siRNA, B7-siRNA and Integrin α4 siRNA element tandem drug delivery system (AAV-CMV-siR ^T+B+I ), the titer was 10 ¹² Vg/ by tail vein injection ml of AAV solution 25μL, 50μL, 100μL into mice.

The in vivo expression of the AAV system was monitored by small animals. The results are shown in Figure 3A. After 3 weeks, it can be seen that the AAV system is stably expressed in vivo, especially in the liver, and the expression of the AAV system has a dose-dependent effect.

Immediately, the DSS-induced chronic colitis model was constructed, during which weighing and recording were performed every two days. The results are shown in Figure 3B. It can be seen that the AAV-encapsulated CMV-siRT+B+I system can reduce the chronic colitis in mice. weight loss, and the mice in the three experimental groups regained body weight significantly faster than the AAV-CMV-scrR group during the period of inflammation remission.

After the tenth week of model construction, the in vivo expression of the AAV system was monitored by small animals, and then the mice were sacrificed to observe the colon. The results are shown in Figure 3C, it can be seen that AAV-CMV-siR ^T+B+I (low) Group, AAV-CMV-siR ^T+B+I (medium) group, AAV-CMV-siR ^T+B+I (high) group, the colonic redness and swelling of mice were alleviated to varying degrees, and the length of colon was shortened by chronic inflammation. There were also different degrees of improvement, among which the AAV-CMV-siR ^T+B+I (high) group had the most significant improvement in inflammation.

The disease index of mice in each group was scored and counted. The results are shown in Figure 4A. It can be seen that the disease index of mice in the AAV-CMV-siR ^T+B+I (high) group was lower than that of AAV-CMV-siR ^{T +B+I} (low) group, AAV-CMV-siR ^T+B+I (medium) group and AAV-CMV-scrR group.

TNF-α siRNA, B7 siRNA and integrin α4 siRNA in mouse plasma were detected. The results are shown in Figure 4B, Figure 4C, and Figure 4D. It can be seen that AAV-encapsulated CMV-siR ^T+B+I system in mouse plasma A certain amount of stably expressed siRNA was generated and showed a dose-dependent effect.

The TNF-α siRNA, B7 siRNA and integrin α4 siRNA in the mouse liver were detected. The results are shown in Figure 4E, Figure 4F, and Figure 4G. It can be seen that the AAV-encapsulated CMV-siR ^T+B+I system in the mouse liver A certain amount of stably expressed siRNA was generated and showed a dose-dependent effect.

The detection of TNF-α siRNA, B7 siRNA and integrin α4 siRNA in the mouse colon, the results are shown in Figure 5A, Figure 5B, Figure 5C, it can be seen that the AAV-encapsulated CMV-siR ^T+B+I system in the mouse colon A certain amount of stably expressed siRNA was generated and showed a dose-dependent effect.

The TNF-α mRNA, B7 mRNA and ^integriα4 mRNA in the mouse colon were detected, and the results were shown in Figure 5D, Figure 5E, and Figure 5F. TNF-α, B7 and integrin α4 mRNA expression.

HE staining and pathological score statistics were performed on mouse colon sections, and the results are shown in Figure 6A and Figure 6B. It can be seen that in the experimental group, especially in the AAV-CMV-siR ^T+B+I (high) group, the colonic mucosa has higher integrity, and the infiltration of immune cells is shallower. Significantly lighter than the AAV-CMV-scrR group.

The above experiments show that the use of liver-friendly AAV to encapsulate the CMV-siR ^T+B+I circuit can achieve long-term TNF-α siRNA, B7siRNA and integrin α4 siRNA expression and multiple target gene silencing, and significantly alleviate the degree of colonic inflammation. It has great drug potential and clinical research value.

In this document, "upper", "lower", "front", "rear", "left", "right", etc. are only used to indicate the relative positional relationship between related parts, rather than limit the absolute positions of these related parts .

In this document, "first", "second", etc. are only used to distinguish each other, but do not indicate the degree of importance and order, and the premise of mutual existence.

In this paper, "equal", "same" and the like are not limitations in strict mathematical and/or geometric senses, and also include errors that can be understood by those skilled in the art and allowed by manufacturing or use.

Unless otherwise indicated, numerical ranges herein include not only the entire range between its two endpoints, but also several subranges subsumed therein.

The preferred specific embodiments and embodiments of the present application have been described in detail above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned embodiments and embodiments. Various changes are made under the premise of the application concept.

Claims

An RNA delivery system for the treatment of colitis, characterized in that the system comprises a viral vector, the viral vector carries an RNA fragment capable of treating colitis, and the viral vector can be enriched in the organ tissue of a host, And endogenously and spontaneously form a composite structure containing the RNA fragment in the host organ tissue, and the composite structure can send the RNA fragment into the intestinal tract to realize the treatment of colitis.
The RNA delivery system for treating colitis of claim 1, wherein the RNA fragment comprises one, two or more specific RNA sequences with medical significance, and the RNA sequences are medically significant siRNA, shRNA or miRNA capable of inhibiting or hindering the development of colitis.
The RNA delivery system for treating colitis of claim 2, wherein the RNA sequence is 15-25 nucleotides in length.
The RNA delivery system for treating colitis according to claim 3, wherein the RNA fragment is selected from any one or more of the following: siRNA of TNF-α gene, siRNA of integrin-α gene, B7 siRNA of a gene, or an RNA sequence with more than 80% homology to the above sequence, or a nucleic acid molecule encoding the above RNA.
The RNA delivery system for treating colitis according to claim 4, wherein the siRNA of the TNF-α gene comprises AAAACAUAAUCAAAAGAAGGC, UAAAAAACAUAAUCAAAAGAA, AAUAAUAAAUAAUCACAAGUG, UUUUCACGGAAAACAUGUCUG, AAACAUAAUCAAAAGAAGGCA, other sequences that inhibit the expression of the TNF-α gene, and other sequences that inhibit the expression of the TNF-α gene. Sequences with more than 80% homology of the above sequences;

The siRNA of integrin-α gene includes AUAAUCAUCUCCAUUAAUGUC, AAACAAUUCCUUUUUUAUCUU, AUUAAAACAGGAAACUUUGAG, AUAAUGAA GGAUAUACAACAG, UUCUUUUAUUCAUAAAAGUCUC, other sequences that inhibit the expression of integrin-α gene and sequences with more than 80% homology to the above sequences;

siRNA of B7 gene, UUUUCUUUGGGUAAUCUUCAG, AGAAAAAUUCCACUUUUUCUU, AUUUCAAAGUCAGAUAUACUA, ACAAAAAUUCCAUUUACUGAG, AUUAUUGAGUUAAGUAUUCCU, other sequences that inhibit the expression of B7 gene and sequences with more than 80% homology to the above sequences.
The RNA delivery system for treating colitis according to claim 1, wherein the viral vector further comprises a promoter and a targeting tag, and the targeting tag can form the complex in the organ tissue of the host The targeting structure of the structure, the targeting structure is located on the surface of the composite structure, and the composite structure can find and bind the target tissue through the targeting structure, and deliver the RNA fragment into the target tissue.
The RNA delivery system for treating colitis according to claim 6, wherein the viral vector comprises any one of the following lines or a combination of several lines: promoter-RNA fragment, promoter-targeting Tag, promoter-RNA segment-targeting tag; each of the viral vectors includes at least one RNA segment and one targeting tag, and the RNA segment and targeting tag are located in the same line or in different lines.
The RNA delivery system for treating colitis according to claim 7, wherein the viral vector further comprises a flanking sequence, a compensation sequence and a loop sequence that can fold the circuit into a correct structure and express it, and the Flanking sequences include 5' flanking sequences and 3' flanking sequences;

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 RNA delivery system for treating colitis of claim 8, wherein

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 RNA delivery system for treating colitis according to claim 7, characterized in that, when there are at least two lines in the viral vector, adjacent lines are connected by a sequence consisting of sequences 1-3;

Wherein, sequence 1 is CAGATC, sequence 2 is a sequence consisting of 5-80 bases, and sequence 3 is TGGATC.
The RNA delivery system for treating colitis according to claim 10, characterized in that, when there are at least two lines in the viral vector, adjacent lines pass through sequence 4 or have homology to sequence 4. More than 80% of the sequences are connected;

Wherein, sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
The RNA delivery system for treating colitis according to claim 1, wherein the organ tissue is liver, and the composite structure is exosome.
The RNA delivery system for treating colitis according to claim 6, wherein the targeting tag is selected from targeting peptides or targeting proteins with targeting function.
The RNA delivery system for treating colitis according to claim 13, wherein the targeting peptide comprises RVG targeting peptide, GE11 targeting peptide, PTP targeting peptide, TCP-1 targeting peptide, MSP targeting peptide targeting peptide;

The targeting proteins include RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein, and MSP-LAMP2B fusion protein.
The RNA delivery system for treating colitis according to claim 14, wherein the targeting tag is a TCP-1 targeting peptide or a TCP-1-LAMP2B fusion protein.
The RNA delivery system for treating colitis according to claim 1, wherein the viral vector is an adeno-associated virus.
The RNA delivery system for treating colitis according to claim 16, wherein the adeno-associated virus is adeno-associated virus type 5, adenovirus-associated virus type 8 or adeno-associated virus type 9.
The RNA delivery system for treating colitis of claim 1, wherein the delivery system is a delivery system for use in mammals including humans.
A use of the RNA delivery system for treating colitis according to any one of claims 1-18 in medicine.
The application according to claim 19, wherein the medicine is a medicine for the treatment of Parkinson's and related diseases, and the administration mode of the medicine comprises oral administration, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection.