WO2022206781A1 - Système d'administration d'arn pour le traitement de la colite - Google Patents
Système d'administration d'arn pour le traitement de la colite Download PDFInfo
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- WO2022206781A1 WO2022206781A1 PCT/CN2022/083810 CN2022083810W WO2022206781A1 WO 2022206781 A1 WO2022206781 A1 WO 2022206781A1 CN 2022083810 W CN2022083810 W CN 2022083810W WO 2022206781 A1 WO2022206781 A1 WO 2022206781A1
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- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
Definitions
- the present application relates to the field of biomedical technology, in particular to an RNA delivery system for treating colitis.
- 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.
- RNA cannot exist stably outside the cell for a long time, because RNA will be degraded into fragments by RNases rich in extracellular, so it is necessary to find a method that can make RNA stable outside the cell and can enter specific tissues in a targeted manner. Highlight the effect of RNAi therapy.
- Virus (Biological virus) is a small individual, simple structure, containing only one nucleic acid (DNA or RNA), must be parasitic in living cells and replicated non-cellular organisms. Viral vectors can bring genetic material into cells. The principle is to use the molecular mechanism of viruses to transmit their genomes into other cells for infection. It can occur in a complete living body (in vivo) or cell culture (in vitro), mainly used in Basic research, gene therapy or vaccines. However, there are few related studies on the use of viruses as vectors to deliver RNA, especially siRNA, using a special self-assembly mechanism.
- the Chinese Patent Publication No. CN108624590A discloses a siRNA capable of inhibiting the expression of DDR2 gene; the Chinese Patent Publication No. CN108624591A discloses a siRNA capable of silencing the ARPC4 gene, and the siRNA is modified with ⁇ -phosphorus-selenium;
- the Chinese Patent Publication No. CN108546702A discloses a siRNA targeting long-chain non-coding RNA DDX11-AS1.
- the Chinese Patent Publication No. CN106177990A discloses a siRNA precursor that can be used for various tumor treatments. These patents design specific siRNAs to target certain diseases caused by genetic changes.
- Chinese Patent Publication No. CN108250267A discloses a polypeptide, polypeptide-siRNA induced co-assembly, using polypeptide as a carrier of siRNA.
- the Chinese Patent Publication No. CN108117585A discloses a polypeptide for promoting apoptosis of breast cancer cells through targeted introduction of siRNA, and the polypeptide is also used as the carrier of siRNA.
- the Chinese Patent Publication No. CN108096583A discloses a nanoparticle carrier, which can be loaded with siRNA with breast cancer curative effect while containing chemotherapeutic drugs.
- exosomes can deliver miRNAs to recipient cells, which secrete miRNAs at relatively low concentrations , which can effectively block the expression of target genes.
- Exosomes are biocompatible with the host immune system and possess the innate ability to protect and transport miRNAs across biological barriers in vivo, thus becoming a potential solution to overcome problems associated with siRNA delivery.
- the Chinese Patent Publication No. CN110699382A discloses a method for preparing siRNA-delivering exosomes, and discloses the technology of separating exosomes from plasma and encapsulating siRNA into exosomes by electroporation .
- the embodiments of the present application provide an RNA delivery system for treating 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.
- 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.
- the RNA sequence is 15-25 nucleotides in length.
- the length of the RNA sequence can be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides.
- the RNA sequence is 18-22 nucleotides in length.
- the RNA fragment is selected from any one or more of the following: the siRNA of the TNF- ⁇ gene, the siRNA of the integrin- ⁇ 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.
- 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 siRNA of the TNF- ⁇ gene includes AAAACAUAAUCAAAAGAAGGC, UAAAAAACAUAAUCAAAAGAA, AAUAAUAAAUAAUCACAAGUG, UUUUCACGGAAAACAUGUCUG, AAACAUAAUCAAAAGAAGGCA, other sequences that inhibit the expression of the TNF- ⁇ gene, and sequences that are more than 80% homologous 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 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 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.
- 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 loop sequence is gttttggccactgactgac or a sequence whose homology is greater than 80%;
- the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
- the purpose of deleting bases 1-5 of the reverse complement of the RNA is to make the sequence unexpressed.
- the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
- the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
- the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
- adjacent lines are connected by a sequence composed of sequences 1-3 (sequence 1-sequence 2-sequence 3);
- sequence 1 is CAGATC
- sequence 2 is a sequence consisting of 5-80 bases
- sequence 3 is TGGATC
- adjacent lines are connected by sequence 4 or a sequence with a homology of more than 80% to sequence 4;
- sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
- the organ tissue is liver
- the composite structure is exosome
- the targeting tag is selected from targeting peptides or targeting proteins with targeting function;
- the targeting peptides include RVG targeting peptides, GE11 targeting peptides, PTP targeting peptides, TCP-1 targeting peptides, and MSP targeting peptides;
- the targeting proteins include RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein, and MSP-LAMP2B fusion protein.
- the targeting tag is a TCP-1 targeting peptide or a TCP-1-LAMP2B fusion protein.
- the viral vector is an adenovirus-associated virus
- the adeno-associated virus is adeno-associated virus type 5, adeno-associated virus type 8 or adenovirus-associated virus type 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) .
- 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.
- the medicine is a medicine for the treatment of colitis and its related diseases
- 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 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 RNA delivery system for the treatment of colitis 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 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.
- RNA delivery system for the treatment of colitis 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.
- Fig. 1 is the mouse colitis treatment situation and RNA expression level comparison diagram provided by an embodiment of the present application
- FIG. 2 is a comparison diagram of mouse cytokine concentration and colon HE staining provided by an embodiment of the present application
- FIG. 3 is a comparison diagram of the treatment of colitis in mice provided by an embodiment of the present application.
- FIG. 4 is a comparison diagram of the mouse disease activity index and various siRNA levels provided by an embodiment of the present application.
- Figure 5 is a comparison diagram of various siRNA and mRNA levels in mice provided by an embodiment of the present application.
- FIG. 6 is a comparison diagram of 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- ⁇ siRNA by injecting mice with TNF- ⁇ siRNA-lentivirus according to an example of the present application.
- A is the liver enrichment result
- B is the plasma enrichment result
- Figure 8 is a graph showing the results of injecting mice with TNF- ⁇ siRNA-lentivirus and detecting TNF- ⁇ siRNA in plasma exosomes to determine the spontaneous formation of complex structures provided in an example of the present application.
- Fig. 9 is the result graph of the specific curative effect after injecting mice with TNF- ⁇ 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.
- 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- ⁇ ), miR- 124-3p (target gene TNF- ⁇ ), B7-siRNA-1, B7-siRNA-2, integrin ⁇ 4 shRNA-1, integrin ⁇ 4 shRNA-2, A is the disease index score in the figure, and B is the detection result of inflammatory factors.
- RNA 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- ⁇ )+B7-siRNA-1, miR-124-3p (target gene TNF- ⁇ )+B7-siRNA-2, B7-siRNA-1+integrin ⁇ 4 shRNA-1, B7- siRNA-2+integrin ⁇ 4 shRNA-2, in the figure
- A is the expression result of small RNA (sequence 2) detected in liver
- B is the expression result of small RNA (sequence 2) detected in plasma
- 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- ⁇ )+B7-siRNA-1, miR-124-3p (target gene TNF- ⁇ )+B7-siRNA-2, B7-siRNA-1+integrin ⁇ 4 shRNA-1, B7-siRNA -2+integrin ⁇ 4 shRNA-2, A is the disease index score in the figure, and B is the detection result of inflammatory factors.
- RNA 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- ⁇ )+B7-siRNA-1+integrin ⁇ 4 shRNA-1, miR-124-3p (target gene TNF- ⁇ )+B7-siRNA-2+integrin ⁇ 4 shRNA-2, miR-19a (target gene TNF- ⁇ )+B7-siRNA-1+integrin ⁇ 4 shRNA-2, in the figure
- A is the expression result of small RNA (sequence 1) detected in liver
- B is the expression result of small RNA (sequence 1) detected in plasma
- C is the expression result of small RNA (sequence 1) detected in colon.
- 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- ⁇ )+B7-siRNA-1+integrin ⁇ 4 shRNA-1, miR-124-3p (target gene TNF- ⁇ )+B7-siRNA-2+integrin ⁇ 4 shRNA-2, miR- 19a (target gene TNF- ⁇ )+B7-siRNA-1+integrin ⁇ 4 shRNA-2, in the figure A is the expression result of small RNA (sequence 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.
- A is the expression result of small RNA (sequence 3) detected in liver
- B is the expression
- 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- ⁇ )+B7-siRNA-1+integrin ⁇ 4 shRNA-1, miR-124-3p (target gene TNF- ⁇ )+B7-siRNA-2+integrin ⁇ 4 shRNA-2, miR-19a (target gene TNF- ⁇ )+B7-siRNA-1+integrin ⁇ 4 shRNA-2, A is the disease index score in the figure, and B is the detection result of inflammatory factors.
- 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- ⁇ -siRNA-1 (siRNA length 18bp), TNF- ⁇ -siRNA-1 (siRNA length 18bp), - ⁇ -siRNA-2 (siRNA length 20bp), TNF- ⁇ -siRNA-3 (siRNA length 22bp), in the figure
- A is the result of intrahepatic detection of siRNA expression
- B is the result of siRNA expression detected in plasma
- 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- ⁇ -siRNA-1 (siRNA length 18bp), TNF- ⁇ -siRNA-1 (siRNA length 18bp), - ⁇ -siRNA-2 (siRNA length 20bp), TNF- ⁇ -siRNA-3 (siRNA length 22bp),
- A is the disease index score in the figure
- B is the detection result of inflammatory factors
- C is the detection result of target gene mRNA.
- FIG 21 is a graph showing the results of in vivo enrichment of viral vectors containing 3 homologous TNF- ⁇ -siRNA sequences provided in an example of the present application after injection into mice.
- the homologous TNF- ⁇ -siRNA sequences are: TNF- ⁇ -siRNA-4, TNF- ⁇ -siRNA-5, TNF- ⁇ -siRNA-6, in the figure A is the expression result of TNF- ⁇ -siRNA detected in liver, B is the expression result of TNF- ⁇ -siRNA detected in plasma, C The expression results of TNF- ⁇ -siRNA were detected in colon, and D was the expression results of TNF- ⁇ -siRNA detected in plasma exosomes.
- Figure 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 ⁇ 4 siRNA sequences respectively provided in another embodiment of the present application after injection into mice.
- the homologous integrin ⁇ 4 siRNA sequences are: integrin ⁇ 4 siRNA-1, integrin ⁇ 4 siRNA-2, integrin ⁇ 4 siRNA-3, in the figure A is the result of detecting integrin ⁇ 4 siRNA expression in liver, B is the result of detecting integrin ⁇ 4 siRNA expression in plasma, C is the result of detecting integrin ⁇ 4 siRNA expression in colon, D is the result of plasma detection Detection of integrin ⁇ 4 siRNA expression results in exosomes.
- Figure 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- ⁇ -siRNA-4, TNF- ⁇ - siRNA-5, TNF- ⁇ -siRNA-6, B7-siRNA-1, B7-siRNA-2, B7-siRNA-3, integrin ⁇ 4 siRNA-1, integrin ⁇ 4 siRNA-2, integrin ⁇ 4 siRNA-3, 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- ⁇ -siRNA Expression results
- B is the expression result of TNF- ⁇ -siRNA in plasma
- C is the expression result of TNF- ⁇ -siRNA in colon
- D is the expression result of TNF- ⁇ -siRNA in plasma exosome.
- Figure 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 is the disease index score
- B is the detection of inflammatory factors
- 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- ⁇ -siRNA detected in the liver Results, B is the result of detecting the expression of TNF- ⁇ -siRNA in plasma, and C is the result of detecting the expression of TNF- ⁇ -siRNA in colon.
- Figure 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 ⁇ 4-siRNA detected in the liver Results, B is the result of detecting the expression of integrin ⁇ 4-siRNA in plasma, and C is the result of detecting the expression of integrin ⁇ 4-siRNA in colon.
- Figure 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- ⁇ -siRNA expression in the liver, B is the result of detecting the expression of TNF- ⁇ -siRNA in the plasma, and C is the result of detecting the expression of TNF- ⁇ -siRNA in the colon.
- Figure 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
- 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 ⁇ 4-siRNA in the liver
- B is the result of detecting the expression of integrin ⁇ 4-siRNA in the plasma
- C is the result of detecting the expression of integrin ⁇ 4-siRNA in the colon.
- 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- ⁇ -siRNA loaded in mice when adenovirus-associated virus types 2, 7 and 8 are used as viral vectors provided in an example of the present application, and A in the figure is the liver
- the expression results of TNF- ⁇ -siRNA were detected in B, the expression results of TNF- ⁇ -siRNA in plasma were detected in B, and the expression results of TNF- ⁇ -siRNA in colon were detected in C.
- HE staining Hematoxylin-eosin staining, referred to as HE staining.
- HE staining is one of the most basic and widely used technical methods in histology and pathology teaching and research.
- the hematoxylin staining solution is alkaline and can stain the basophilic structure of the tissue (such as ribosome, nucleus and ribonucleic acid in the cytoplasm) into blue-violet; eosin is an acid dye, which can stain the eosinophilic structure of the tissue ( Such as intracellular and intercellular proteins, including Lewy bodies, alcohol bodies, and most of the cytoplasm) stained pink, making the morphology of the entire cell organization clearly visible.
- the basophilic structure of the tissue such as ribosome, nucleus and ribonucleic acid in the cytoplasm
- eosin is an acid dye, which can stain the eosinophilic structure of the tissue ( Such as intracellular and intercellular proteins, including Lewy bodies, alcohol bodies, and most of the cytoplasm) stained pink, making the morphology of the entire cell organization clearly visible.
- 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.
- RNA fragments 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:
- adeno-associated virus types 2 and 7 as vectors also have similar in vivo enrichment, self-assembly and colitis therapeutic effects (Figure 34-35) .
- the viral vector may also include a flanking sequence, a compensation sequence and a loop sequence that can make the circuit fold into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence; the viral vector Including any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag, 5' - Promoter - Targeting Tag - 5' Flanking Sequence - RNA Fragment - Loop Sequence - Compensation Sequence - 3' Flanking Sequence.
- the 5' flanking sequence is preferably ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with ggatcctggaggcttgctgaaggctgtatgctgaattc, etc.
- the loop sequence is preferably gttttggccactgactgac or a sequence with more than 80% homology thereto, including sequences with 85%, 90%, 92%, 95%, 98%, 99% homology with gttttggccactgactgac, and the like.
- the 3' flanking sequence is preferably accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag, etc.
- the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
- the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-5 bases therein.
- the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
- the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-3 bases therein.
- the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
- the compensation sequence may be the reverse complementary sequence of the RNA sequence by deleting any 1-3 consecutively arranged bases.
- the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
- the compensation sequence may be the reverse complementary sequence of the 9th position and/or the 10th position in the deletion of the RNA sequence. Deleting bases 9 and 10 works best.
- flanking sequences are not randomly selected, but are determined based on a large number of theoretical studies and experiments. increase the expression rate of RNA fragments.
- 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).
- sequence 1 is preferably CAGATC
- sequence 2 can be composed of 5-80 bases
- Sequence of bases such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases
- sequence of 10-50 bases is preferable, and the sequence of 20-40 bases is more preferable.
- Sequence 3 is preferably TGGATC.
- the 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).
- sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
- the vector system 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).
- 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.
- RNA sequences 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).
- RNA capable of treating colitis is selected from any one or more of the following RNAs: siRNA of TNF- ⁇ gene, siRNA of integrin- ⁇ gene, siRNA of B7 gene or nucleic acid molecules encoding the above RNAs.
- the number of RNA sequences capable of treating colitis is one, two or more.
- three siRNAs including TNF- ⁇ gene siRNA, integrin- ⁇ gene siRNA and B7 gene siRNA can be used at the same time, or TNF- ⁇ gene siRNA and integrin- ⁇ gene siRNA can be used at the same time.
- TNF- ⁇ gene siRNA and integrin- ⁇ gene siRNA can be used at the same time.
- Any two kinds of siRNA among siRNA and siRNA of B7 gene, siRNA of TNF- ⁇ gene, siRNA of integrin- ⁇ gene or siRNA of B7 gene can also be used alone.
- 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-siRNA 2-loop sequence - Compensation sequence-3' flanking
- the above RNA can also be obtained by ribose modification of the RNA sequence (siRNA, shRNA or miRNA) therein, preferably 2' fluoropyrimidine modification.
- 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on siRNA, shRNA or miRNA with 2'-F.
- 2'-F can make it difficult for RNase in the human body to recognize siRNA, shRNA or miRNA, so it can Increases the stability of RNA transport in vivo.
- the liver will phagocytose exogenous viruses, and up to 99% of the exogenous viruses will enter the liver. Therefore, when viruses are used as vectors, they can be enriched in liver tissue without specific design. After being opened, RNA molecules (siRNA, shRNA, or miRNA) are released, and liver tissue spontaneously wraps the above RNA molecules into exosomes, and these exosomes become RNA delivery mechanisms.
- RNA molecules siRNA, shRNA, or miRNA
- RNA delivery mechanism in order to make the RNA delivery mechanism (exosome) have the ability of "precision guidance”, we design a targeting tag in the 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 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 (nucleot
- 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 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.
- 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 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.
- 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 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 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.
- this embodiment provides an application of an RNA delivery system in medicine, and the medicine is a medicine for treating colitis.
- 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.
- the three experimental groups were the AAV-CMV-siR TNF- ⁇ (low) group and the AAV-CMV-siR TNF- ⁇ (medium) group.
- AAV-CMV-siR TNF- ⁇ (high) group; control group were Normal group and AAV-CMV-scrR group.
- the experimental process is shown in Figure 1A.
- the AAV-CMV-siR TNF- ⁇ (low) group, the AAV-CMV-siR TNF- ⁇ (medium) group, and the AAV-CMV-siR TNF- ⁇ (high) group were treated with high hepatic Affinity AAV-5 adeno-associated virus-encapsulated TNF- ⁇ siRNA system (AAV-CMV-siR TNF- ⁇ ), AAV solution with a titer of 10 12 Vg/ml was injected through the tail vein, 25 ⁇ L, 50 ⁇ L, 100 ⁇ L 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- ⁇ (high) group has an average The Average Radiance reached 8.42*105 (p/sec/cm 2 /sr), and the expression site was in the liver, which indicated that the expression of the AAV system had a dose-dependent effect.
- the disease index of the mice in each group was scored and counted, and the results are shown in Figure 1E. It can be seen that the disease index of the mice in the AAV-CMV-siR TNF- ⁇ (high) group was lower than that of the AAV-CMV-siR TNF- ⁇ (low) group, AAV-CMV-siR TNF- ⁇ (medium) group and AAV-CMV-scrR group.
- TNF- ⁇ siRNA The levels of TNF- ⁇ siRNA in the mice of each group were detected respectively. The results are shown in Figure 1F. It can be seen that the levels of TNF- ⁇ siRNA in the three experimental groups were higher, while the AAV-CMV-scrR group in the control group was smaller. There is almost no expression of TNF- ⁇ siRNA in mice, which indicates that the above-mentioned AAV system can produce a certain amount of TNF- ⁇ siRNA.
- liver-friendly AAV to encapsulate the CMV-siR TNF- ⁇ circuit can achieve long-term TNF- ⁇ siRNA expression and long-term TNF- ⁇ silencing, and can relieve colitis to a certain extent.
- Drug potential and clinical research value show that the use of liver-friendly AAV to encapsulate the CMV-siR TNF- ⁇ circuit can achieve long-term TNF- ⁇ siRNA expression and long-term TNF- ⁇ silencing, and can relieve colitis to a certain extent. Drug potential and clinical research value.
- the experimental groups were AAV-CMV-siR T+B+I (low) group, AAV-CMV-siR T+B+I (medium) group, AAV-CMV-siR T+B+I (high) group ;
- the control groups were Normal group and AAV-CMV-scrR group.
- the in vivo expression of the AAV system was monitored by small animals. The results are shown in Figure 3A. After 3 weeks, it can be seen that the AAV system is stably expressed in vivo, especially in the liver, and the expression of the AAV system has a dose-dependent effect.
- the disease index of mice in each group was scored and counted. The results are shown in Figure 4A. It can be seen that the disease index of mice in the AAV-CMV-siR T+B+I (high) group was lower than that of AAV-CMV-siR T +B+I (low) group, AAV-CMV-siR T+B+I (medium) group and AAV-CMV-scrR group.
- TNF- ⁇ siRNA, B7 siRNA and integrin ⁇ 4 siRNA in 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.
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Abstract
L'invention concerne un système d'administration d'ARN pour le traitement de la colite, caractérisé en ce que le système comprend un vecteur viral. Le vecteur viral porte un fragment d'ARN capable de traiter la colite. Le vecteur viral peut être enrichi en tissus d'organe d'un hôte. Dans les tissus d'organe d'un hôte, le vecteur viral peut former de manière endogène et spontanée une structure composite contenant le fragment d'ARN. La structure composite peut alimenter le fragment d'ARN dans un tractus intestinal, de façon à traiter la colite. La sécurité et la fiabilité du système de distribution d'ARN pour traiter la colite ont été complètement vérifiées, et le système a une bonne aptitude à la pharmacopotentialité et une universalité élevée.
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