WO2022206779A1 - Système d'administration d'arn pour le traitement de l'obésité - Google Patents

Système d'administration d'arn pour le traitement de l'obésité Download PDF

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WO2022206779A1
WO2022206779A1 PCT/CN2022/083800 CN2022083800W WO2022206779A1 WO 2022206779 A1 WO2022206779 A1 WO 2022206779A1 CN 2022083800 W CN2022083800 W CN 2022083800W WO 2022206779 A1 WO2022206779 A1 WO 2022206779A1
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sequence
rna
targeting
delivery system
treating obesity
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Chinese (zh)
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张辰宇
陈熹
付正
李菁
张翔
周心妍
张丽
余梦超
郭宏源
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南京大学
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present application relates to the field of biomedical technology, in particular to an RNA delivery system for treating obesity.
  • Obesity is a group of common metabolic disorders. When the human body eats more calories than it consumes, the excess calories are stored in the body in the form of fat, and the amount exceeds the normal physiological requirement, and when it reaches a certain value, it evolves into obesity. Normal male adult adipose tissue weight accounts for 15% to 18% of body weight, and female adipose tissue accounts for 20% to 25%. With age, the percentage of body fat increases accordingly. If there is no obvious cause, it is called simple obesity, and if there is a clear cause, it is called secondary obesity.
  • 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 being 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 obesity 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 obesity, the system comprising a viral vector carrying an RNA fragment capable of treating obesity, and the viral vector can be used in the organ tissue of a host. It is enriched in the host organ tissue and endogenously and spontaneously forms a composite structure containing fragments of the RNA capable of treating obesity, and the composite structure can deliver the RNA fragments into target tissues to achieve obesity. disease treatment.
  • the target tissue is the brain.
  • the viral vector is an adenovirus-associated virus.
  • adenovirus-associated virus is adenovirus-associated virus type 5, adenovirus-associated virus type 8 or adenovirus-associated virus type 9.
  • RNA fragment comprises one, two or more specific RNA sequences with medical significance, and the RNA sequences are siRNA, shRNA or miRNA with medical significance.
  • the viral vector comprises a promoter and a targeting tag
  • the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host
  • the targeting structure is located on the surface of the composite structure
  • the The complex structure is capable of finding and binding to the target tissue through the targeting structure, delivering the RNA fragment into the target tissue.
  • the viral vector includes any one of the following circuits or a combination of several circuits: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each of the viral vectors including at least one RNA segment and one targeting tag, the RNA segment and targeting tag are located in the same circuit or are located in different circuits.
  • the viral vector also includes a flanking sequence, a compensation sequence and a loop sequence that can make the circuit fold into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence;
  • the viral vector includes any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-target To tag, 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensating sequence-3'flanking sequence.
  • the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence whose homology is greater than 80%;
  • the loop sequence is gttttggccactgactgac or a sequence whose homology is greater than 80%;
  • the 3' flanking sequence is accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence whose homology is greater than 80%;
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the purpose of deleting bases 1-5 of the reverse complement of the RNA is to make the sequence unexpressed.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
  • the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
  • adjacent lines are connected by a sequence composed of sequences 1-3 (sequence 1-sequence 2-sequence 3);
  • sequence 1 is CAGATC
  • sequence 2 is a sequence consisting of 5-80 bases
  • sequence 3 is TGGATC.
  • adjacent lines are connected by sequence 4 or a sequence with more than 80% homology to sequence 4;
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • organ tissue is liver
  • composite structure is exosome
  • the targeting tag is selected from targeting peptides or targeting proteins with targeting function.
  • the targeting peptides include RVG targeting peptides, GE11 targeting peptides, PTP targeting peptides, TCP-1 targeting peptides, and MSP targeting peptides;
  • the targeting proteins include RVG-LAMP2B fusion protein, GE11-LAMP2B fusion protein, PTP-LAMP2B fusion protein, TCP-1-LAMP2B fusion protein, and MSP-LAMP2B fusion protein.
  • the length of the RNA sequence is 15-25 nucleotides.
  • the length of the RNA sequence can be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides.
  • the RNA sequence is 18-22 nucleotides in length.
  • RNA capable of treating obesity is selected from siRNA of the PTP1B gene, or an RNA sequence with a homology of more than 80% to the above sequence, or a nucleic acid molecule encoding the above RNA. It should be noted that the RNA sequences in the "nucleic acid molecules encoding the above RNA sequences" here also include RNA sequences with a homology of more than 80% of each RNA.
  • the siRNA of PTP1B gene includes UGAUAUAGUCAUUAUCUUCUU, UCCAUUUUUAUCAAACUAGCG, AUUGUUUAAAUAAAUAUGGAG, AAUUUUAAUACAUUAUUGGUU, UUUAUUAUUGUACUUUUUGAU, other sequences that inhibit the expression of PTP1B gene, and sequences with more than 80% homology to the above sequences.
  • sequences with more than 80% homology may be 85%, 88%, 90%, 95%, 98%, etc. homology.
  • the RNA fragment includes an RNA sequence ontology and a modified RNA sequence obtained by modifying the RNA sequence ontology with ribose sugar. That is, the RNA fragment can be composed of only at least one RNA sequence ontology, or only at least one modified RNA sequence, and can also be composed of RNA sequence ontology and modified RNA sequence.
  • the isolated nucleic acid also includes its variants and derivatives.
  • the nucleic acid can be modified by one of ordinary skill in the art using general methods. Modification methods include (but are not limited to): methylation modification, hydrocarbyl modification, glycosylation modification (such as 2-methoxy-glycosyl modification, hydrocarbyl-glycosyl modification, sugar ring modification, etc.), nucleic acid modification, peptide modification Segment modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification) and the like.
  • the modification is an internucleotide linkage, for example selected from: phosphorothioate, 2'-O methoxyethyl (MOE), 2'-fluoro, phosphine Acid alkyl esters, phosphorodithioates, alkyl phosphorothioates, phosphoramidates, carbamates, carbonates, phosphoric triesters, acetamidates, carboxymethyl esters, and combinations thereof.
  • phosphorothioate 2'-O methoxyethyl (MOE), 2'-fluoro
  • phosphine Acid alkyl esters phosphorodithioates, alkyl phosphorothioates, phosphoramidates, carbamates, carbonates, phosphoric triesters, acetamidates, carboxymethyl esters, and combinations thereof.
  • the modification is a modification of nucleotides, such as selected from: peptide nucleic acid (PNA), locked nucleic acid (LNA), arabinose-nucleic acid (FANA), analogs, derivatives objects and their combinations.
  • the modification is a 2' fluoropyrimidine modification.
  • 2'Fluoropyrimidine modification is to replace the 2'-OH of pyrimidine nucleotides on RNA with 2'-F.
  • 2'-F can make RNA not easily recognized by RNase in vivo, thereby increasing the stability of RNA fragment transmission in vivo. sex.
  • the delivery system is a delivery system for use in mammals including humans.
  • Another inventive point of the present application is to provide the application of the above-mentioned RNA delivery system for treating obesity in medicine.
  • administration modes of the drug include oral, inhalation, subcutaneous injection, intramuscular injection, and intravenous injection. Intravenous injection is preferred.
  • the medicine includes the above-mentioned viral vector, specifically, the viral vector here means a viral vector carrying an RNA fragment, or carrying an RNA fragment and a targeting tag, and can enter the host and can be enriched in the liver. , self-assembled to form a composite structure exosome, the composite structure can deliver RNA fragments to the target tissue, so that the RNA fragments are expressed in the target tissue, and then inhibit the expression of matching genes to achieve the purpose of treating diseases.
  • the viral vector here means a viral vector carrying an RNA fragment, or carrying an RNA fragment and a targeting tag, and can enter the host and can be enriched in the liver. , self-assembled to form a composite structure exosome, the composite structure can deliver RNA fragments to the target tissue, so that the RNA fragments are expressed in the target tissue, and then inhibit the expression of matching genes to achieve the purpose of treating diseases.
  • the dosage forms of the drug can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes and the like.
  • the RNA delivery system for the treatment of obesity 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 obesity provided by this application can be tightly combined with AGO 2 and enriched into a complex 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 obesity provides a drug delivery platform, which can greatly improve the therapeutic effect of obesity, and can also form the basis for the research and development of more RNA drugs through this platform. It has greatly promoted the development and use of RNA drugs.
  • Fig. 1 is the comparison diagram of the mouse obesity treatment situation provided by an embodiment of the present application.
  • FIG. 2 is a comparison diagram of various obesity indicators in mice provided in an embodiment of the present application.
  • Fig. 3 is an RNA delivery system constructed with adenovirus and lentivirus as viral vectors provided in an embodiment of the present application, and has a detection diagram of in vivo enrichment effect.
  • A is the detection of siRNA content in blood after injection of the delivery system
  • B is the detection result of the siRNA content in the hypothalamus after injection of the delivery system.
  • FIG. 4 is a detection diagram of an RNA delivery system constructed with adenovirus and lentivirus as viral vectors provided by another embodiment of the present application, and has an enrichment effect in vivo. The figure shows that after injection of the delivery system, blood exosomes Detection results of siRNA content.
  • Fig. 5 is an RNA delivery system constructed with adenovirus and lentivirus as viral vectors provided by an embodiment of the present application, and has a detection diagram of in vivo self-assembly effect and obesity treatment effect, and A in the figure is the detection result of the mRNA content of PTP1B , B is the detection result of protein content of PTP1B, C is the change value of body weight with the increase of days.
  • Fig. 6 is the detection result of in vivo enrichment, self-assembly and treatment effect for obesity when the viral vector system provided by an embodiment of the present application carries a variety of different RNA fragments
  • a in the figure is the relative mRNA of PTP1B Quantitative detection results
  • B is the detection results of the relative amount of PTP1B protein.
  • FIG. 7 is a graph showing the detection results of in vivo enrichment, self-assembly and obesity treatment effects when the adenoviral vector delivery system provided by an embodiment of the present application contains multiple RNA fragments and multiple targeting tags, wherein targeting The label is RVG, and the RNA fragments are siRNA-1, siRNA-2, siRNA-1+siRNA-2.
  • A is the detection result of the relative amount of mRNA of PTP1B
  • B is the detection result of the relative amount of protein of PTP1B
  • C is the number of days Change in weight gain.
  • FIG. 8 is a graph showing the detection results of in vivo enrichment when three 5' flanking sequences/loop sequences/3' flanking sequences with homology greater than 80% are included in the adenovirus vector delivery system provided in an example of the present application (shown as siRNA content in blood).
  • Fig. 9 is a structure constructed when the adenovirus vector provided in an embodiment of the present application carries multiple lines, and adjacent lines are connected by sequence 1-sequence 2-sequence 3, wherein sequence 2 contains multiple bases.
  • the delivery system has a graph of in vivo enrichment assays (shown as siRNA levels in blood).
  • Fig. 10 is a graph showing the detection results of in vivo enrichment of the delivery system constructed by the linker sequence provided by an embodiment of the present application as sequence 4 and a sequence with more than 80% homology to sequence 4 (based on the siRNA content in blood show).
  • Figure 11 is a graph of the detection results of the delivery system constructed when the lengths of RNA sequences provided in an embodiment of the present application are respectively 18, 20, and 21, and has the effect of treating obesity.
  • A is the relative PTP1B mRNA of RNA sequences of different lengths.
  • Quantitative detection results B is the relative detection results of PTP1B protein of different length RNA sequences.
  • the detection of the siRNA level, the protein content and the mRNA content involved in the present invention is to establish the mouse stem cell in vitro model by injecting the RNA delivery system into the mouse.
  • the expression levels of mRNA and siRNA in cells and tissues were detected by qRT-PCR. Absolute quantification of siRNA was determined by plotting a standard curve using the standards.
  • the internal reference gene is U6snRNA (in tissue) or miR-16 (in serum, exosomes)
  • the gene is GAPDH or 18s RNA.
  • Western blotting was used to detect protein expression levels in cells and tissues, and ImageJ software was used for protein quantitative analysis.
  • This embodiment provides an RNA delivery system for treating obesity, the system comprising a viral vector carrying an RNA fragment capable of treating obesity, and the viral vector can be enriched in the organ tissue of a host, And endogenously and spontaneously form a composite structure containing fragments of the RNA capable of treating obesity in the host organ tissue, and the composite structure sends the RNA fragments into the target tissue (brain) to achieve obesity. treat.
  • Both adenovirus and lentivirus can be used as viral vectors, and the RNA delivery systems constructed by them have in vivo enrichment, self-assembly and therapeutic effects on obesity, as shown in Figure 3-5.
  • the viral vector also includes a promoter and a targeting tag.
  • the viral vector includes any one of the following circuits or a combination of several circuits: promoter-RNA sequence, promoter-targeting tag, promoter-RNA sequence-targeting tag, and each of the viral vectors includes at least one RNA fragments and a targeting tag, either in the same circuit or in different circuits.
  • the viral vector may only include a promoter-RNA sequence-targeting tag, or may include a combination of a promoter-RNA sequence, a promoter-targeting tag, or a promoter-targeting tag, a promoter A combination of RNA-seq-targeting tags.
  • the adenovirus vector delivery system contains multiple RNA fragments and multiple targeting tags, it has in vivo enrichment, self-assembly and obesity treatment effects, as shown in Figure 7, where the targeting tag is RVG, and the RNA fragment is siRNA-1, siRNA-2, siRNA-1+siRNA-2.
  • the viral vector may also include a flanking sequence, a compensation sequence and a loop sequence that can make the circuit fold into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence; the viral vector Including any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-targeting tag, 5' - Promoter - Targeting Tag - 5' Flanking Sequence - RNA Fragment - Loop Sequence - Compensation Sequence - 3' Flanking Sequence.
  • the 5' flanking sequence is preferably ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with ggatcctggaggcttgctgaaggctgtatgctgaattc, etc.
  • the loop sequence is preferably gttttggccactgactgac or a sequence with more than 80% homology thereto, including sequences with 85%, 90%, 92%, 95%, 98%, 99% homology with gttttggccactgactgac, and the like.
  • the 3' flanking sequence is preferably accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence with a homology greater than 80%, including a sequence with 85%, 90%, 92%, 95%, 98%, 99% homology with accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag, etc.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-5 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 bases are deleted.
  • the compensation sequence can be the reverse complementary sequence of the RNA sequence by deleting any 1-3 bases therein.
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-3 consecutive bases are deleted.
  • the compensation sequence may be the reverse complementary sequence of the RNA sequence by deleting any 1-3 consecutively arranged bases.
  • the compensation sequence is the reverse complement of the RNA fragment, and the 9th and/or 10th bases are deleted.
  • the compensation sequence may be the reverse complementary sequence of the 9th position and/or the 10th position in the deletion of the RNA sequence. Deleting bases 9 and 10 works best.
  • flanking sequences are not randomly selected, but are determined based on a large number of theoretical studies and experiments. increase the expression rate of RNA fragments.
  • adenovirus vectors containing 3 homologous sequences they also have in vivo enrichment, self-assembly and obesity treatment effects, as shown in Figure 8, the sequences are grouped as follows:
  • sequence 1 is preferably CAGATC
  • sequence 2 can be composed of 5-80 bases
  • Sequence of bases such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 bases
  • sequence of 10-50 bases is preferable, and the sequence of 20-40 bases is more preferable.
  • Sequence 3 is preferably TGGATC.
  • the adenovirus vector When the adenovirus vector carries multiple lines, the adjacent lines are connected by sequence 1-sequence 2-sequence 3, wherein sequence 2 contains multiple bases, and the constructed delivery system also has in vivo enrichment, self-assembly and obesity.
  • the treatment effect of the disease is shown in Figure 9.
  • Sequence 2 is specifically shown in Table 3 below.
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • the connecting sequence is sequence 4 and a sequence with more than 80% homology to sequence 4, the constructed delivery system also has in vivo enrichment, self-assembly and obesity treatment effects, as shown in Figure 10, sequence 4-1 in Figure 10 That is, the sequence 4, the sequence 4-2/4-3/4-4 is the homologous sequence of the sequence 4-1, and the sequence is specifically shown in Table 4 below.
  • Sequence 4-1 CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-2 CAGATCTGGCCGCACTCGTAGAGGTGAGTCGACCAGTGGATC Sequence 4-3 CAGATCTGGCACCCGTCGAGGTAGTGAGTCGACCAGTGGATC Sequence 4-4 CAGATCTGGCCGCACAGGTCGTAGTGAGTCGACCAGTGGATC
  • RNA fragments comprise one, two or more specific RNA sequences of medical significance, the RNA sequences can be expressed in the target receptor, and the compensatory sequence cannot be expressed in the target receptor.
  • the RNA sequence can be an siRNA sequence, a shRNA sequence or a miRNA sequence, preferably an siRNA sequence.
  • the length of an RNA sequence is 15-25 nucleotides (nt), preferably 18-22nt, such as 18nt, 19nt, 20nt, 21nt, and 22nt. This range of sequence lengths was not chosen arbitrarily, but was determined through trial and error. A large number of experiments have proved that when the length of the RNA sequence is less than 18nt, especially less than 15nt, the RNA sequence is mostly invalid and will not play a role. The cost of the line is greatly increased, and the effect is not better than the RNA sequence with a length of 18-22nt, and the economic benefit is poor. Therefore, when the length of the RNA sequence is 15-25nt, especially 18-22nt, the cost and the effect can be taken into account, and the effect is the best.
  • nt nucleotides
  • RNA sequence lengths were 18, 20, and 21, respectively, the constructed delivery system also had in vivo enrichment, self-assembly, and therapeutic effects on obesity, as shown in Figure 11 , and the specific sequences as shown in Table 5.
  • the RNA capable of treating obesity is selected from the siRNA of the PTP1B gene or the nucleic acid molecule encoding the above RNA.
  • the number of RNA effective sequences capable of treating obesity is one, two or more.
  • the functional structural region of the viral vector can be expressed as: (promoter-siRNA1)-connector sequence-(promoter-siRNA2)-connector sequence- (promoter-targeting tag), or (promoter-targeting tag-siRNA1)-linker-(promoter-targeting tag-siRNA2), or (promoter-siRNA1)-linker-(promoter- Targeting tag-siRNA2) etc.
  • the functional structural region of the viral vector can be expressed as: (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-connector sequence-(5'-promoter - 5' flanking sequence - siRNA2-loop sequence - compensation sequence - 3' flanking sequence) - linking sequence - (5'-promoter-targeting tag), or (5'-promoter-targeting tag-5' flanking sequence-siRNA1-loop sequence-compensation sequence-3' flanking sequence)-linker sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'flanking sequence), or (5'-promoter-5'flanking sequence-siRNA1-loop sequence-compensating sequence-3'flanking sequence)-linking sequence-(5'-promoter-targeting tag-5'flanking sequence-siRNA2-loop sequence-compensating sequence-3'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.
  • the available targeting tags need to be selected according to the target tissue, and on the other hand It is ensured that the targeting tag can stably appear on the surface of exosomes, so as to achieve the targeting function.
  • Targeting tags that have been screened include targeting peptides, targeting proteins, and antibodies.
  • targeting peptides include but are not limited to RVG targeting peptide (nucleotide sequence shown in SEQ ID No: 1), GE11 targeting peptide (nucleotide sequence shown in SEQ ID No: 2), PTP targeting peptide Peptide (nucleotide sequence shown in SEQ ID No: 3), TCP-1 targeting peptide (nucleotide sequence shown in SEQ ID No: 4), MSP targeting peptide (nucleotide sequence shown in SEQ ID No: 4) : 5); targeting proteins include but are not limited to RVG-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 6), GE11-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 7) shown), PTP-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No: 8), TCP-1-LAMP2B fusion protein (nucleotide sequence shown in SEQ ID No:
  • the viral vector can also be composed of multiple viruses with different structures, one of which contains a promoter promoters and targeting tags, other viruses contain promoters and RNA segments. Loading the targeting tag and RNA fragment into different viral vectors, and injecting the two viral vectors into the body, the targeting effect is no worse than the targeting effect produced by loading the same targeting tag and RNA fragment into one viral vector .
  • the viral vector containing the RNA sequence can be injected first, and then the viral vector containing the targeting tag can be injected after 1-2 hours, so that a better target can be achieved. to the effect.
  • the delivery systems described above can all be used in mammals, including humans.
  • the RNA delivery system for the treatment of obesity uses a virus as a vector, and the virus vector is used as a mature injection. Its safety and reliability have been fully verified, and the drugability is very good.
  • the final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the delivery system can deliver all kinds of small molecule RNAs, and has strong versatility. And the preparation of viral vectors is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • RNA delivery system for the treatment of obesity 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 medicament comprises a viral vector carrying an RNA fragment capable of treating obesity, the viral vector being capable of enriching in the organ tissue of a host, and endogenously and spontaneously forming in the organ tissue of the host an RNA fragment capable of treating obesity
  • a complex structure of fragments of the RNA for the treatment of obesity the complex structure being capable of entering and binding to a target tissue, delivering the RNA fragments into the target tissue.
  • RNA capable of treating obesity is one or more of siRNA, shRNA and miRNA having medical significance and capable of inhibiting or hindering the development of obesity.
  • RNA fragments When the viral vector system carries a variety of different RNA fragments, it has in vivo enrichment, self-assembly and therapeutic effects on obesity, as shown in Figure 6, where the RNA fragments are grouped as follows:
  • RNA-1 alone siRNA-2 alone, shRNA-1 alone, shRNA-2 alone, miRNA-1 alone, miRNA-2 alone;
  • RNA-1+siRNA-2 siRNA-1+siRNA-2, shRNA-1+shRNA-2, miRNA-1+miRNA-2;
  • RNA-1+siRNA-2+shRNA-1 siRNA-1+siRNA-2+shRNA-2
  • siRNA-1+siRNA-2+miRNA- 1 siRNA-1+siRNA-2+miRNA-2.
  • RNA sequences are specifically shown in Table 1 below.
  • the viral vector comprises a promoter and a targeting tag
  • the targeting tag can form the targeting structure of the composite structure in the organ tissue of the host
  • the targeting structure is located on the surface of the composite structure
  • the The complex structure is capable of finding and binding to the target tissue through the targeting structure, delivering the RNA fragment into the target tissue.
  • the viral vector includes any one of the following circuits or a combination of several circuits: promoter-RNA fragment, promoter-targeting tag, promoter-RNA fragment-targeting tag; each of the viral vectors including at least one RNA segment and one targeting tag, the RNA segment and targeting tag are located in the same circuit or are located in different circuits.
  • the viral vector further comprises a flanking sequence, a compensation sequence and a loop sequence that can fold the circuit into a correct structure and express, and the flanking sequence includes a 5' flanking sequence and a 3' flanking sequence;
  • the viral vector includes any one of the following lines or a combination of several lines: 5'-promoter-5' flanking sequence-RNA fragment-loop sequence-compensating sequence-3' flanking sequence, 5'-promoter-target To tag, 5'-promoter-targeting tag-5'flanking sequence-RNA fragment-loop sequence-compensating sequence-3'flanking sequence.
  • the 5' flanking sequence is ggatcctggaggcttgctgaaggctgtatgctgaattc or a sequence whose homology is greater than 80%;
  • the loop sequence is gttttggccactgactgac or a sequence whose homology is greater than 80%;
  • the 3' flanking sequence is accggtcaggacacaaggcctgttactagcactcacatggaacaaatggcccagatctggccgcactcgag or a sequence whose homology is greater than 80%;
  • the compensation sequence is the reverse complementary sequence of the RNA fragment, and any 1-5 bases are deleted.
  • adjacent lines are connected by a sequence consisting of sequences 1-3;
  • sequence 1 is CAGATC
  • sequence 2 is a sequence consisting of 5-80 bases
  • sequence 3 is TGGATC.
  • adjacent lines are connected by sequence 4 or a sequence with more than 80% homology to sequence 4;
  • sequence 4 is CAGATCTGGCCGCACTCGAGGTAGTGAGTCGACCAGTGGATC.
  • organ tissue is liver
  • composite structure is exosome
  • the targeting tag is selected from targeting peptides or targeting proteins with targeting function.
  • the targeting structure is located on the surface of the composite structure.
  • 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 RNA sequence is 15-25 nucleotides in length.
  • the targeting tag is RVG-LAMP2B fusion protein, or GE11-LAMP2B fusion protein.
  • RNA effective sequences capable of treating obesity is one, two or more.
  • the medicament can be used in mammals including humans.
  • RNA capable of treating obesity is selected from the siRNA of the PTP1B gene or the nucleic acid molecule encoding the above RNA.
  • the drug can be administered orally, inhaled, subcutaneously injected, intramuscularly injected or intravenously injected into the human body, it can be delivered to the target tissue through the RNA delivery system described in Example 1 to exert a therapeutic effect.
  • the drug can also be used in combination with other obesity treatment drugs to enhance the treatment effect, such as orlistat, metformin, liraglutide, etc.
  • the medicine of this embodiment may also include a pharmaceutically acceptable carrier, which includes but is not limited to diluents, buffers, emulsions, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorption Agents, wetting agents, disintegrating agents, absorption enhancers, surfactants, coloring agents, 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, coloring agents, flavoring agents, adjuvants, desiccants, adsorption carriers, etc.
  • the dosage forms of the medicine provided in this embodiment can be tablets, capsules, powders, granules, pills, suppositories, ointments, solutions, suspensions, lotions, gels, pastes, and the like.
  • the medicine provided in this example uses the virus as the carrier and the virus carrier as the mature injection, and its safety and reliability have been fully verified, and the druggability is very good.
  • the final effective RNA sequence is packaged and delivered by endogenous exosomes, and there is no immune response, so there is no need to verify the safety of the exosomes.
  • the drug can deliver various kinds of small molecule RNAs and has strong versatility. And the preparation of viral vectors is much cheaper and more economical than the preparation of exosomes or proteins, polypeptides and other substances.
  • the drug provided in this application can be closely combined with AGO 2 and enriched into a composite structure (exosome) after self-assembly in vivo, which can not only prevent its premature degradation and maintain its stability in circulation, but also benefit the receptor.
  • Cellular uptake, intracytoplasmic release and lysosomal escape require low doses.
  • this embodiment provides an application of an RNA delivery system for treating obesity in medicine, and the medicine is a medicine for treating obesity.
  • This example specifically describes the application of the RNA delivery system in obesity treatment in conjunction with the following experiments.
  • the PTP1B siRNA system (AAV-CMV-siR P /AAV-CMV-RVG- siRP ) was encapsulated by the high-affinity AAV-5 adeno-associated virus in the liver, and 100 ⁇ L of AAV solution with a titer of 10 12 Vg/ml was injected into the tail vein. into mice.
  • the in vivo expression of the AAV system was monitored by small animals. After 3 weeks, it was found that the AAV system was stably expressed in vivo, especially in the liver.
  • mice C57BL/6 mice were selected and injected with PBS buffer/AAV-CMV-scrR/AAV-CMV- siRP /AAV-CMV-RVG- siRP after 12 weeks to form PBS group/AAV-CMV-scrR group/AAV -CMV- siRP group/AAV-CMV-RVG- siRP group and injected every two days for 24 days.
  • Changes in body weight, weight of covered fat pads, initial food intake, serum leptin content, blood glucose content, basal glucose content, serum total cholesterol (TC), triglyceride (TG), low-density lipid The detection and statistics of protein (LDL), body length and food intake, the results are as follows.
  • Figure 1A is a comparison chart of the body weight of mice in each group, it can be seen that the body weight of mice in the AAV-CMV-RVG- siRP group is the most stable.
  • FIG. 1B is a comparison chart of the weight of the epididymal fat pad of the mice in each group, it can be seen that the weight of the epididymal fat pad of the mice in the AAAV-CMV-RVG- siRP group is the lightest.
  • the figure is a comparison chart of the initial food intake curves of mice in each group. As can be seen, the mice in the AAV-CMV-RVG- siRP group had the least food intake.
  • FIG. 1D the figure is a comparison chart of serum leptin content of mice in each group. It can be seen that the serum leptin content of mice in the AAV-CMV-RVG- siRP group was the lowest.
  • FIG. 1E the figure is a comparison chart of blood glucose change curves of mice in each group. It can be seen that the blood glucose content of mice in the AAV-CMV-RVG- siRP group was the lowest.
  • the figure is a comparison chart of the basal glucose change curves of mice in each group. It can be seen that the mice in the AAV-CMV-RVG- siRP group had the lowest basal glucose content.
  • the three pictures are the comparison charts of serum total cholesterol (TC), triglyceride (TG), and low density lipoprotein (LDL) of mice in each group. It can be seen that AAV-CMV -RVG- siRP group had the lowest TC, TG and LDL.
  • Figure 2D is a comparison chart of the body lengths of the mice in each group, it can be seen that the body lengths of the four groups of mice are almost the same.

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Abstract

L'invention concerne un système d'administration d'ARN pour le traitement de l'obésité. Le système comprend un vecteur viral, le vecteur viral portant un fragment d'ARN permettant de traiter l'obésité et pouvant être enrichi en tissus d'organe d'un hôte, et des formes de manière endogène et spontanée, dans les tissus d'organe de l'hôte, d'une structure composite contenant le fragment d'ARN permettant le traitement de l'obésité. La structure composite peut administrer le fragment d'ARN dans un tissu cible pour réaliser le traitement de l'obésité.
PCT/CN2022/083800 2021-03-30 2022-03-29 Système d'administration d'arn pour le traitement de l'obésité WO2022206779A1 (fr)

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