WO2021121038A1 - Exosome formulation for treating drug-resistant bacterial infections - Google Patents
Exosome formulation for treating drug-resistant bacterial infections Download PDFInfo
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- WO2021121038A1 WO2021121038A1 PCT/CN2020/133392 CN2020133392W WO2021121038A1 WO 2021121038 A1 WO2021121038 A1 WO 2021121038A1 CN 2020133392 W CN2020133392 W CN 2020133392W WO 2021121038 A1 WO2021121038 A1 WO 2021121038A1
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- 229940124597 therapeutic agent Drugs 0.000 description 1
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- 229960003087 tioguanine Drugs 0.000 description 1
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- 238000003151 transfection method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
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- 210000002700 urine Anatomy 0.000 description 1
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/425—Thiazoles
- A61K31/429—Thiazoles condensed with heterocyclic ring systems
- A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
Definitions
- the present invention relates to the field of biomedicine, in particular to an exosomal preparation used for the treatment of drug-resistant bacterial infections.
- Small RNAs are small RNAs with a length of about 20-30 nucleotides. They do not encode proteins, but they participate in various important physiological and pathological processes of the body. They can combine with their complementary gene sequences to degrade mRNA or inhibit mRNA translation, leading to the silencing of specific genes. Endogenous small RNAs are mainly divided into three categories: microRNA (microRNA, miRNA), small interfering RNA (short interfering RNA, siRNA) and RNA that interacts with piwi (piRNA). Studies have confirmed that gene silencing caused by small RNAs can not only serve as a defense mechanism to silence genes of foreign species, such as viruses and transposons, but also play an important role in controlling and fine-tuning gene expression and participating in numerous cellular processes. .
- microRNA microRNA
- miRNA small interfering RNA
- siRNA small interfering RNA
- piRNA RNA that interacts with piwi
- RNAs from different species can regulate the gene expression of human cells through similar mechanisms, demonstrating the important role of small RNAs in cross-border regulation between species. More importantly, when the human body fights against the invasion of foreign species (parasites, viruses, etc.), endogenous (produced by the human body) or exogenous (food or drug sources) small RNAs often play an important defensive role. For example, for the blood-borne protozoan Plasmodium P. falciparum, miR-451, which is enriched in sickle-type red blood cells in the human body, is transported into Plasmodium at high concentrations and targets its cAMP-dependent protein kinase PKA-R.
- honeysuckle-derived miRNA-2911 can directly target influenza A virus, which can prevent infection in mice and reduce the mortality rate after infection with H5N1 virus.
- the purpose of the present invention is to provide a new method for the treatment of drug-resistant bacterial infections.
- the first aspect of the present invention provides an inhibitor-loaded exosome, which includes a substance that inhibits the expression of drug-resistant genes and/or protein activity thereof.
- the drug resistance gene is derived from a drug resistance gene of bacteria selected from the group consisting of Staphylococcus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
- the drug resistance gene derived from Staphylococcus is selected from the group consisting of mecA, kpc, imp, vim, ndm, or a combination thereof.
- the drug resistance gene derived from Escherichia coli is selected from the group consisting of AcrA, AcrB, TolC, pbp2, pbp3, ampC, or a combination thereof.
- the drug resistance gene derived from Pseudomonas aeruginosa is selected from the group consisting of vim, OprN, ampC, imp, or a combination thereof.
- the substance that inhibits the expression of the drug-resistant gene and/or its protein activity is selected from the following group: siRNA, miRNA, or a combination thereof.
- the length (bp) of the siRNA is 15-33, preferably 19-22.
- the sense strand sequence of the siRNA is shown in SEQ ID NO.:1.
- the second aspect of the present invention provides a preparation comprising:
- the antibiotic is selected from the group consisting of methicillin, carbapenem antibiotics, or a combination thereof.
- the carbapenem antibiotic is selected from the group consisting of imipenem, meropenem, ertapenem, or a combination thereof.
- the preparation also includes other drugs for treating drug-resistant bacterial infections.
- the drug-resistant bacteria are selected from the group consisting of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
- the content of the exosomes in the preparation is 30-800 ⁇ g, preferably, 50-600 ⁇ g, more preferably, 80-500 ⁇ g, more preferably, 100-300 ⁇ g, according to The total weight of the formulation.
- the content of the antibiotic in the formulation is 10-300 ⁇ M, preferably, 25-200 ⁇ M, more preferably, 30-100 ⁇ M, more preferably, 40-60 ⁇ M, according to the formulation The total concentration meter.
- the formulation is a liquid formulation and/or a lyophilized powder formulation.
- the dosage form of the preparation includes an injection.
- the pharmaceutically acceptable carrier is selected from the following group: phosphate buffer, physiological saline, or a combination thereof.
- the third aspect of the present invention provides a medicine kit including:
- the kit further includes:
- the antibiotic is selected from the group consisting of methicillin, carbapenem antibiotics, or a combination thereof.
- the carbapenem antibiotic is selected from the group consisting of imipenem, meropenem, ertapenem, or a combination thereof.
- the drug-resistant bacteria are selected from the group consisting of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
- first container, the second container, and the third container are the same or different containers.
- the medicine in the first container is a unilateral preparation containing the exosomes of claim 1.
- the medicine in the second container is a unilateral preparation containing antibiotics.
- the medicine in the third container is a single preparation containing other medicines for preventing and/or treating drug-resistant bacterial infections.
- the dosage form of the drug is an oral dosage form or an injection dosage form.
- the kit also contains instructions.
- the description records one or more descriptions selected from the following group:
- the fourth aspect of the present invention provides a preparation according to the second aspect of the present invention, or the use of the kit according to the third aspect of the present invention, for preparing a medicine for the treatment of drug-resistant bacterial infections.
- the medicine is also used for one or more purposes selected from the following group:
- the drug-resistant bacteria are selected from the group consisting of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
- the fifth aspect of the present invention provides a method for treating drug-resistant bacterial infections, including:
- the preparation according to the second aspect of the present invention or the kit according to the third aspect of the present invention is administered to a subject in need.
- the subject includes humans or non-human mammals infected by drug-resistant bacteria.
- the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.
- the administration dose of the exosomes is 0.1-40 mg/kg body weight, preferably 1-20 mg/kg body weight, more preferably, 2-10 mg/kg body weight.
- the dosage of the antibiotic is 0.1-50 mg/kg body weight, preferably 1-20 mg/kg body weight, more preferably 2-6 mg/kg body weight.
- the frequency of administration of the exosomes is 1-10 times/week, more preferably, 3-9 times/week, more preferably 5-8 times/week.
- the frequency of administration of the antibiotic is 1-10 times/week, more preferably, 3-9 times/week, more preferably 5-8 times/week.
- the administration time of the exosomes is 2-20 days, preferably 3-15 days, more preferably 5-8 days.
- the administration time of the antibiotic is 2-20 days, preferably 3-15 days, more preferably 5-8 days.
- the exosomes and antibiotics are administered simultaneously or sequentially.
- the sixth aspect of the present invention provides a non-therapeutic in vitro method for improving the sensitivity of drug-resistant bacteria to antibiotics, which includes the steps of: culturing drug-resistant bacteria strains in the presence of the exosomes described in the first aspect of the present invention, Thereby increasing the sensitivity of drug-resistant bacterial strains to antibiotics.
- the expression of drug-resistant genes in drug-resistant bacteria strains is regulated And/or activity, thereby improving the sensitivity of drug-resistant bacteria to antibiotics.
- substances such as siRNA, miRNA
- the concentration of the exosomes is 30-800 ⁇ g/ml, preferably, 50-600 ⁇ g/ml, more preferably, 80-500 ⁇ g/ml, more preferably, 100-300 ⁇ g/ml ml.
- the drug-resistant bacteria are selected from the group consisting of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
- Figure 1 shows that compared with free siRNA, exosome can efficiently transport its encapsulated siRNA (si-ada) into E. coli, and exhibit a dose effect.
- Figure 2 shows the dose-dependent effect of exosome-encapsulated siRNA (si-ada) on the inhibition of the target protein Ada expression after entering E. coli.
- FIG. 3 shows the amount of siRNA (si-mecA) introduced into methicillin-resistant S. aureus (MRSA) using exosome package transportation.
- FIG 4 shows the inhibitory effect of siRNA (si-mecA) introduced into MRSA by exosome packaging and transportation on the expression of the target protein PBP2a.
- Figure 5 shows that si-mecA imported into MRSA by exosome package transportation can significantly improve the inhibitory effect of antibiotic methicillin on MRSA.
- Fig. 6 is a schematic diagram of "small RNA-containing exosomal vesicles secreted by human cells deliver small RNAs into bacteria, and reduce bacterial drug resistance by inhibiting the expression of drug-resistant genes".
- Figure 7 shows that tail vein injection of siRNA expression vector, combined with antibiotic methicillin, significantly improved the survival rate of mice infected with drug-resistant MRSA bacteria.
- Figure 8 shows the amount of siRNA (siMecA) introduced into P. aeruginosa by exosome package transportation.
- exosomes loaded with substances that inhibit the expression of drug-resistant genes and/or their protein activity can significantly increase the sensitivity of drug-resistant bacteria to antibiotics, and the above-mentioned exosome Combination of body and antibiotics can also treat infections caused by resistant bacteria.
- the present invention designs and synthesizes siRNA against drug-resistant bacteria, such as methicillin-resistant S. aureus (MRSA) drug-resistant gene mecA, and utilizes exosomes for packaging and transportation.
- drug-resistant bacteria such as methicillin-resistant S. aureus (MRSA) drug-resistant gene mecA
- MRSA methicillin-resistant S. aureus
- the present inventors also unexpectedly discovered that the combination of exosomes encapsulated with drug-resistant genes against drug-resistant bacteria and antibiotics can significantly treat infections of drug-resistant bacteria. On this basis, the inventor completed the present invention.
- the term "about” may refer to a value or composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
- the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
- the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
- the terms "host”, “subject”, and “desired subject” refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, such as cows, horses, dogs, cats, pigs, sheep, goats, camels, rats, mice, hares, and rabbits.
- the drug resistance gene refers to a gene in bacteria that encodes a protein related to antibiotic resistance.
- the drug resistance gene is derived from a bacteria selected from the group consisting of Staphylococcus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
- the drug resistance gene derived from Staphylococcus is selected from the group consisting of mecA, kpc, imp, vim, ndm, or a combination thereof.
- the drug resistance gene derived from Escherichia coli is selected from the group consisting of AcrA, AcrB, TolC, pbp2, pbp3, ampC, or a combination thereof.
- the drug resistance gene derived from Pseudomonas aeruginosa is selected from the group consisting of vim, OprN, ampC, imp, or a combination thereof.
- the drug-resistant bacteria include, but are not limited to, Staphylococcus, Escherichia coli, and Pseudomonas aeruginosa.
- the terms "protein of the present invention”, “protein”, and “mecA polypeptide” are used interchangeably, and all refer to a protein or polypeptide having a mecA amino acid sequence. They include mecA proteins with or without starting methionine. In addition, the term also includes full-length mecA and fragments thereof.
- the mecA protein referred to in the present invention includes its complete amino acid sequence, its secreted protein, its mutant and its functionally active fragments.
- the mecA gene is integrated in a chromosomal genetic element called the Staphylococcal Chromosome Box (SCC) mec.
- SCC Staphylococcal Chromosome Box
- the encoded protein is PBP2a, the full name is "penicillin binding protein 2a", with 668 amino acids.
- the mecA gene is widely distributed in Staphylococcus. At present, the mecA gene has not been detected in bacteria other than Staphylococcus.
- mecA gene and “mecA polynucleotide” are used interchangeably, and both refer to a nucleic acid sequence having a mecA nucleotide sequence.
- PBP2a is the protein encoded by the mecA gene.
- the total length of the genome of the mecA gene is 2007 bp (accession number is Gene ID: 5767902).
- a nucleic acid sequence encoding it can be constructed based on it, and a specific probe can be designed based on the nucleotide sequence.
- the full-length nucleotide sequence or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
- primers can be designed according to the mecA nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
- the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
- the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
- artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
- the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
- the DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.
- the polynucleotide sequence of the present invention can be used to express or produce recombinant mecA polypeptide. Generally speaking, there are the following steps:
- the mecA polynucleotide sequence can be inserted into a recombinant expression vector.
- any plasmid and vector can be used as long as it can replicate and stabilize in the host.
- An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
- expression vectors containing mecA encoding DNA sequences and appropriate transcription/translation control signals can be used to construct expression vectors containing mecA encoding DNA sequences and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
- the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
- the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
- the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
- selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
- a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.
- the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
- a prokaryotic cell such as a bacterial cell
- a lower eukaryotic cell such as a yeast cell
- a higher eukaryotic cell such as a mammalian cell.
- Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells, etc.
- Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
- the host is a prokaryote such as Escherichia coli
- competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, the transformation can also be carried out by electroporation.
- the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
- the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
- the medium used in the culture can be selected from various conventional mediums.
- the culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
- the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
- exosomes are tiny vesicles secreted from cells, with a diameter of about 30-200nm and a density of 1.13-1.21g/ml. They have a cup-shaped morphology and a double-layer membrane structure. They naturally exist in In biological fluids such as blood, urine, saliva, breast milk, and cell culture media. Almost all types of cells (immune cells, nerve cells, stem cells), including tumor cells, can produce and release exosomes.
- exosomes include inhibitory resistance genes (such as resistance genes derived from Staphylococcus (such as mecA, kpc, imp, vim, ndm); resistance genes derived from Escherichia coli (such as AcrA, AcrB, TolC, pbp2, pbp3, ampC; substances derived from the expression of resistance genes of Pseudomonas aeruginosa (such as vim, OprN, ampC, imp, etc.) and/or their protein activities (such as siRNA, miRNA, etc.).
- inhibitory resistance genes such as resistance genes derived from Staphylococcus (such as mecA, kpc, imp, vim, ndm)
- Escherichia coli such as AcrA, AcrB, TolC, pbp2, pbp3, ampC
- substances derived from the expression of resistance genes of Pseudomonas aeruginosa such as vim, OprN,
- the exosome preparation of the present invention contains a safe and effective amount of exosomes and a pharmaceutically acceptable carrier or excipient.
- a pharmaceutically acceptable carrier or excipient include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powder, and combinations thereof.
- the pharmaceutical preparation should match the mode of administration.
- the preparation of the present invention may also contain a safe and effective amount of other drugs for preventing and/or treating drug-resistant bacterial infections.
- the pharmaceutical composition of the present invention can be made into a liquid preparation, and its preparation can be carried out by a conventional method, and the liquid preparation should be manufactured under aseptic conditions.
- the dosage of the active ingredient is a therapeutically effective amount, for example, about 1 ⁇ g/kg body weight-about 50 mg/kg body weight, about 5 ⁇ g/kg body weight-about 10 mg/kg body weight, and about 10 ⁇ g/kg body weight-about 5 per day. Mg/kg body weight.
- the formulations of the present invention can also be used together with other therapeutic agents.
- a safe and effective amount of the drug is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases, does not exceed about 50 mg/kg body weight, preferably The dosage is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
- the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
- the term “substance that suppresses gene expression” means a substance that suppresses the transcription of mRNA of a target gene, a substance that decomposes transcribed mRNA, or a substance that suppresses the translation of mRNA into protein.
- examples of the above-mentioned substances include nucleic acids, such as siRNA, miRNA, and the like. Among them, siRNA is preferred.
- a “substance that inhibits gene expression” in addition to the above, it also includes proteins, peptides, or other small molecules.
- the target gene is a drug resistance gene, such as mecA gene, kpc, imp, vim, ndm, AcrA, AcrB, TolC, pbp2, pbp3, ampC, OprN, imp, etc.
- siRNA refers to an RNA molecule having a double-stranded RNA portion composed of about 15-40 bases, which cleaves the mRNA of a target gene having a complementary sequence to the antisense strand of the above-mentioned siRNA, and has an inhibitory target The function of gene expression.
- the siRNA in the present invention is an RNA comprising a double-stranded RNA portion composed of a sense RNA strand and an anti-sense RNA strand.
- the sense RNA strand is composed of drug-resistant genes such as mecA, kpc, imp, vim , ndm; AcrA, AcrB, TolC; pbp2, pbp3; ampC, OprN, and imp mRNA consist of the same sequence of consecutive RNA sequences, and the antisense RNA strand is composed of a sequence complementary to the sense RNA sequence.
- drug-resistant genes such as mecA, kpc, imp, vim , ndm; AcrA, AcrB, TolC; pbp2, pbp3; ampC, OprN, and imp mRNA consist of the same sequence of consecutive RNA sequences
- the antisense RNA strand is composed of a sequence complementary to the sense RNA sequence.
- the design and manufacture of the siRNA and the variant siRNA described later are within the skill of those skilled in the art.
- miRNA refers to an RNA molecule having a double-stranded RNA portion composed of approximately 21 to 25 bases, which has the function of suppressing the expression of the target gene by binding to the mRNA of the target gene.
- the miRNA in the present invention is an RNA comprising a double-stranded RNA portion composed of a sense RNA strand and an antisense RNA strand.
- the sense RNA strand is composed of a drug resistance gene of the present invention, such as mecA gene, kpc , imp, vim, ndm; AcrA, AcrB, TolC; pbp2, pbp3; ampC, OprN, imp mRNA sequence composed of the same sequence of consecutive RNA sequences, the antisense RNA strand is complementary to the sense RNA sequence Sequence composition.
- a drug resistance gene of the present invention such as mecA gene, kpc , imp, vim, ndm; AcrA, AcrB, TolC; pbp2, pbp3; ampC, OprN, imp mRNA sequence composed of the same sequence of consecutive RNA sequences
- the antisense RNA strand is complementary to the sense RNA sequence Sequence composition.
- the design and manufacture of the miRNA and the variant siRNA described later are within the skills of those skilled in the art.
- the base is preferably about 15 to 40 bases, more preferably 15 to 30 bases, still more preferably 15 to 25 bases, and still more preferably 18 to 23 bases, More preferably, it is 19 to 21 bases.
- the length of the double-stranded RNA portion of the miRNA is preferably about 21 to 25 bases, and more preferably 22 to 24 bases as bases.
- the end structure of the sense strand or antisense strand of siRNA and miRNA is not particularly limited, and can be appropriately selected according to the purpose. For example, it may have a smooth end or an overhang (overhang), preferably a 3'end overhang. Types of.
- SiRNA and miRNA that have an overhang composed of multiple bases, preferably 1 to 3 bases, more preferably 2 bases, at the 3'end of the sense RNA strand and the antisense RNA strand, inhibit the expression of the target gene in most cases The effect is greater, and it is the preferred one.
- the type of base at the overhang is not particularly limited, and it may be a base constituting RNA or a base constituting DNA.
- siRNA and miRNA in which one to several nucleotides have been deleted, substituted, inserted, and/or added in one or both of the sense strand or antisense strand of the above-mentioned siRNA and miRNA can also be used for The exosomes of the present invention.
- 1 to several bases means, and it is not particularly limited, but it is preferably 1 to 4 bases, more preferably 1 to 3 bases, and even more preferably 1 to 2 bases.
- the number of bases in the overhang at the 3'end is 0 to 3, the base sequence of the overhang at the 3'end is changed to another base sequence, or
- the length of the sense RNA strand and the antisense RNA strand are different from 1 to 3 bases by the insertion, addition or deletion of bases, and the bases in the sense strand and/or the antisense strand are replaced by other bases, etc., but Not limited to these.
- it is necessary that the sense strand and the antisense strand can hybridize in these variant siRNA and miRNA, and it is necessary that these variant siRNA and miRNA have the same gene expression inhibitory energy as the non-variant siRNA and miRNA.
- siRNA and miRNA may be molecules with a closed-end structure, such as siRNA and miRNA (Short hairpin RNA; shRNA) having a hairpin structure.
- shRNA is a sense strand RNA containing a specific sequence of a target gene, an antisense strand RNA composed of a sequence complementary to the sense strand RNA, and an RNA that connects the two strands of the linker sequence, and the sense strand part and the antisense strand The parts hybridize to form a double-stranded RNA part.
- Off-target effect refers to the effect of inhibiting the expression of other genes that are partially homologous to the used siRNA and miRNA in addition to the target gene.
- DNA microarrays can be used in advance to confirm that there is no cross-reaction.
- NCBI National Center for Biotechnology Information
- RNA and miRNA of the present invention known methods such as a chemical synthesis method and a method using gene recombination technology can be suitably used.
- double-stranded RNA can be synthesized by conventional methods based on sequence information.
- an expression vector inserted with a sense strand sequence or an antisense strand sequence is constructed, and after the vector is introduced into mammalian cells cultured in vitro, the sense strand generated by transcription is obtained respectively.
- RNA or antisense strand RNA can also be produced from this.
- RNA containing a specific sequence of the target gene an antisense strand RNA composed of a sequence complementary to the sense strand RNA, and a linker sequence connecting the two strands, and forming a hairpin structure, is expressed by This can also produce the required double-stranded RNA.
- a part of the nucleic acid constituting the siRNA and miRNA may be DNA.
- all or part of the nucleic acids constituting the siRNA and miRNA may be modified nucleic acids.
- a modified nucleic acid refers to a nucleoside (base site, sugar site) and/or an internucleoside bond site modified to have a structure different from that of a natural nucleic acid.
- modified nucleoside constituting the modified nucleic acid include: abasic nucleosides; arabinonucleosides, 2'-deoxyuridines, and ⁇ -deoxyribonucleosides , ⁇ -L-deoxyribonucleosides, nucleosides with other sugar modifications; peptide nucleic acid (PNA), phosphate-bonded peptide nucleic acid (PHONA), locked nucleic acid (LNA), morpholino nucleic acid, etc.
- PNA peptide nucleic acid
- PONA phosphate-bonded peptide nucleic acid
- LNA locked nucleic acid
- morpholino nucleic acid etc.
- the nucleosides with the above sugar modification include: 2'-O-methylribose, 2'-deoxy-2'-fluororibose, 3'-O-methylribose and other substituted pentoses; 1', 2' -Deoxyribose; arabinose; substituted arabinose; hexose and sugar-modified nucleosides with ⁇ -terminal groups.
- These nucleosides may also be modified bases with modified base positions. Examples of such modified bases include pyrimidines such as 5-hydroxycytosine, 5-fluorouracil, and 4-thiouracil; purines such as 6-methyladenine and 6-thioguanine; and others The heterocyclic base and so on.
- modified internucleoside linkage constituting the modified nucleic acid
- examples of the "modified internucleoside linkage" constituting the modified nucleic acid include alkyl linkers, glycerol linkers, amino linkers, poly(ethylene glycol) linkages, methylphosphonate internucleoside linkages, Methyl thiophosphonate, phosphotriester, phosphorothioate triester, phosphorothioate, phosphorodithioate, triester prodrug, sulfone, sulfonamide, sulfamate, methylal, Non-natural internucleoside bonds such as N-methylhydroxylamine, carbonate, carbamate, morpholino, borophosphonate, phosphoramidate, etc.
- exosomes prepared by the invention can effectively improve the sensitivity of drug-resistant bacteria to antibiotics, and the exosomes preparation prepared by the invention can effectively treat the infection of drug-resistant bacteria.
- a method of using exosomes to introduce small RNAs into bacteria and regulating gene expression includes: obtaining exosomes encapsulating siRNA, co-cultivating the exosomes and bacteria (E.coli, S.aureus), and detecting The content of siRNA in exosomes and bacteria.
- exosomes loaded with substances that inhibit the expression of drug-resistant genes and/or their protein activity can significantly increase the sensitivity of drug-resistant bacteria to antibiotics, and the above-mentioned exosomes are combined with antibiotics, It can also treat infections caused by resistant bacteria.
- siRNA targeting drug-resistant bacteria such as methicillin-resistant S. aureus (MRSA) drug-resistant gene mecA mRNA
- MRSA methicillin-resistant S. aureus
- the packaging and transportation functions of siRNA can be introduced into the cells of drug-resistant bacteria.
- exosomes are used to introduce small RNAs into bacteria and regulate gene expression. Taking Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as examples, the exocytosis is reported for the first time.
- E. coli Escherichia coli
- S. aureus Staphylococcus aureus
- the cross-border regulation of bacterial genes by body-mediated small RNAs, innovative content, and exploration of the regulation strategies and mechanisms of heterologous small RNAs on bacterial intracellular gene expression levels have important biological significance.
- the present invention further expands the application range of exosomes as transport carriers in vivo. It can not only transmit small RNA molecular signals between mammalian tissues, but also mediate the cross-border of mammals and bacteria at the small RNA level. communicate with.
- the present invention proposes to use the packaging and transport functions of exosomes to introduce small RNAs into the cells of drug-resistant bacteria. By reducing the expression of drug-resistant genes and the production of drug-resistant-related proteins, the drug resistance is relieved, and the drug is regained. Conventional antibiotic treatment is sensitive. This strategy will be extended to a series of gene regulation related to the important life activities of bacteria, virulence factors and anti-immune factors, so as to develop new therapeutic strategies against bacterial infections. In addition, through intravenous injection of siRNA expression vectors, siRNA targeting drug-resistant genes is expressed in vivo, and combined with antibiotics, to treat drug-resistant bacterial infections and improve survival rates.
- siRNA-exosome Place overnight at 4°C; the next day after centrifugation at 10000g x 1h at 4°C, discard the supernatant, and resuspend the pellet in PBS. This method obtains siRNA-exosome (siRNA-exosome).
- the BCA protein quantification method was used to obtain the concentration of exosomes ( ⁇ g/ ⁇ l).
- the sense strand sequence of siRNA is shown in SEQ ID NO.:1:
- the antisense strand sequence of siRNA is shown in SEQ ID NO.: 2:
- mice were infected by intravenous injection of 5 ⁇ 10 7 CFU MRSA bacteria into the orbit to establish a bacteremia model.
- Infected mice received 3 mg/kg of antibiotic methicillin via intraperitoneal injection and 5 mg/kg of siRNA-containing exosomes via tail vein injection. The injection interval was 24 hours and the treatment was 7 days.
- the combination of methicillin and exosomes containing siRNA effectively protected mice from lethal doses of MRSA bacterial infection (Figure 7).
- the survival rate of the mice was significantly improved, and they remained healthy during the 14-day observation period after stopping the medication, indicating that the medication had no serious side effects.
- Figure 6 shows a mechanism diagram of the exosomes of the present invention in treating drug-resistant bacterial infections.
- the bacteremia model mice were constructed, and siRNA expression vector was injected through the tail vein to express the siRNA targeting the drug-resistant gene mecA in vivo, and the antibiotic methicillin was administered at the same time to observe the survival curve of the mice.
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Abstract
Provided is an exosome formulation for treating drug-resistant bacterial infections. Specifically, provided is an inhibitor-loaded exosome, comprising a substance that inhibits the expression of drug-resistant genes and/or the protein activity thereof. Also provided is an exosome formulation, which can significantly improve the sensitivity of drug-resistant bacteria to antibiotics. The exosome formulation can significantly treat infections caused by drug-resistant bacteria.
Description
本发明涉及生物医药领域，具体地，涉及一种用于治疗耐药菌感染的外泌体制剂。The present invention relates to the field of biomedicine, in particular to an exosomal preparation used for the treatment of drug-resistant bacterial infections.
小RNA是一类长约20-30个核苷酸的小分子RNA，它们不编码蛋白质，但却参与机体的各种重要的生理和病理过程。它们能够与其互补的基因序列结合，使mRNA降解或抑制mRNA翻译，从而导致特定基因的沉默。内源小RNA主要分为3类：微小RNA(microRNA，miRNA)，小干扰RNA(short interfering RNA，siRNA)和与piwi相互作用的RNA(piRNA)。研究证实，由小RNA引发的基因沉默现象不仅可以作为一种防御机制来沉默外来物种的基因，比如病毒和转座子，而且在控制和微调基因表达、参与众多的细胞过程中发挥着重要作用。Small RNAs are small RNAs with a length of about 20-30 nucleotides. They do not encode proteins, but they participate in various important physiological and pathological processes of the body. They can combine with their complementary gene sequences to degrade mRNA or inhibit mRNA translation, leading to the silencing of specific genes. Endogenous small RNAs are mainly divided into three categories: microRNA (microRNA, miRNA), small interfering RNA (short interfering RNA, siRNA) and RNA that interacts with piwi (piRNA). Studies have confirmed that gene silencing caused by small RNAs can not only serve as a defense mechanism to silence genes of foreign species, such as viruses and transposons, but also play an important role in controlling and fine-tuning gene expression and participating in numerous cellular processes. .
近年的研究进展表明，来自不同物种的小RNA进入人体后，均可通过相似机制对人体细胞的基因表达进行调控，展现了小RNA在物种间跨界调控的重要角色。更重要地，在人体对抗外来物种(寄生虫，病毒等)侵扰时，内源性(人体自身产生)或外源性(食物或药物来源)的小RNA常常扮演重要的防御作用。例如，针对通过血液传播的原生动物疟原虫P.falciparum，人体内镰刀型红细胞中富集存在的miR-451，以高浓度被转运进疟原虫，并靶向其cAMP依赖性蛋白激酶PKA-R，抑制蛋白翻译，使得疟原虫生长受抑制，缓解疟疾症状。再如，金银花来源的miRNA-2911能够直接靶向甲型流感病毒，可预防小鼠感染，并降低感染H5N1病毒后的死亡率。Research progress in recent years has shown that after small RNAs from different species enter the human body, they can regulate the gene expression of human cells through similar mechanisms, demonstrating the important role of small RNAs in cross-border regulation between species. More importantly, when the human body fights against the invasion of foreign species (parasites, viruses, etc.), endogenous (produced by the human body) or exogenous (food or drug sources) small RNAs often play an important defensive role. For example, for the blood-borne protozoan Plasmodium P. falciparum, miR-451, which is enriched in sickle-type red blood cells in the human body, is transported into Plasmodium at high concentrations and targets its cAMP-dependent protein kinase PKA-R. , Inhibits protein translation, inhibits the growth of malaria parasites and relieves the symptoms of malaria. For another example, honeysuckle-derived miRNA-2911 can directly target influenza A virus, which can prevent infection in mice and reduce the mortality rate after infection with H5N1 virus.
尽管围绕小RNA在不同物种中的生物学功能及医学应用已经进行了大量的研究，小RNA对细菌的基因表达调控仍属于起步阶段，知之甚少。而该领域对人类健康及医治由细菌感染引发的疾病，具有非常重要的意义。一方面，在人体内共生的细菌，尤其是肠道菌群，与人体健康密切相关；而人类肠道环境中含有大量miRNA，极有可能对肠道菌群的平衡及代谢状态具有重要调控作用。另一方面，在抗生素大量使用(甚至滥用)的背景下，由耐药性细菌引发的感染疾病正日益成为棘手的公众健康问题，缺少有效的应对手段。Although a large number of studies have been conducted on the biological functions and medical applications of small RNAs in different species, the regulation of bacterial gene expression by small RNAs is still in its infancy, and little is known. This field is of great significance to human health and the treatment of diseases caused by bacterial infections. On the one hand, the bacteria that symbiotically in the human body, especially the intestinal flora, are closely related to human health; and the human intestinal environment contains a large number of miRNAs, which is very likely to have an important regulatory effect on the balance and metabolic state of the intestinal flora . On the other hand, in the context of the massive use (or even abuse) of antibiotics, infectious diseases caused by drug-resistant bacteria are increasingly becoming a thorny public health problem, and there is a lack of effective countermeasures.
因此本领域迫切需要开发一种治疗耐药菌感染的新方法。Therefore, there is an urgent need in this field to develop a new method for the treatment of drug-resistant bacterial infection.
发明内容Summary of the invention
本发明的目的在于提供一种治疗耐药菌感染的新方法。The purpose of the present invention is to provide a new method for the treatment of drug-resistant bacterial infections.
本发明第一方面提供了一种负载抑制剂的外泌体，所述外泌体包括抑制耐药基因表达和/或其蛋白活性的物质。The first aspect of the present invention provides an inhibitor-loaded exosome, which includes a substance that inhibits the expression of drug-resistant genes and/or protein activity thereof.
在另一优选例中，所述耐药基因来源于选自下组的菌的耐药基因：葡萄球菌、大肠杆菌、铜绿假单胞菌、或其组合。In another preferred example, the drug resistance gene is derived from a drug resistance gene of bacteria selected from the group consisting of Staphylococcus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
在另一优选例中，所述来源于葡萄球菌的耐药基因选自下组：mecA、kpc、imp、vim、ndm、或其组合。In another preferred embodiment, the drug resistance gene derived from Staphylococcus is selected from the group consisting of mecA, kpc, imp, vim, ndm, or a combination thereof.
在另一优选例中，所述来源于大肠杆菌的耐药基因选自下组：AcrA、AcrB、TolC、pbp2、pbp3、ampC、或其组合。In another preferred example, the drug resistance gene derived from Escherichia coli is selected from the group consisting of AcrA, AcrB, TolC, pbp2, pbp3, ampC, or a combination thereof.
在另一优选例中，所述来源于铜绿假单胞菌的耐药基因选自下组：vim、OprN、ampC、imp、或其组合。In another preferred embodiment, the drug resistance gene derived from Pseudomonas aeruginosa is selected from the group consisting of vim, OprN, ampC, imp, or a combination thereof.
在另一优选例中，所述抑制耐药基因表达和/或其蛋白活性的物质选自下组：siRNA、miRNA、或其组合。In another preferred embodiment, the substance that inhibits the expression of the drug-resistant gene and/or its protein activity is selected from the following group: siRNA, miRNA, or a combination thereof.
在另一优选例中，所述siRNA的长度(bp)为15－33，较佳地，19－22。In another preferred example, the length (bp) of the siRNA is 15-33, preferably 19-22.
在另一优选例中，所述siRNA的正义链序列如SEQ ID NO.:1所示。In another preferred embodiment, the sense strand sequence of the siRNA is shown in SEQ ID NO.:1.
本发明第二方面提供了一种制剂，包括：The second aspect of the present invention provides a preparation comprising:
(a)本发明第一方面所述的外泌体；(a) The exosomes of the first aspect of the present invention;
(b)抗生素；和(b) Antibiotics; and
(c)药学上可接受的载体。(c) A pharmaceutically acceptable carrier.
在另一优选例中，所述抗生素选自下组：甲氧西林、碳青霉烯类抗生素、或其组合。In another preferred embodiment, the antibiotic is selected from the group consisting of methicillin, carbapenem antibiotics, or a combination thereof.
在另一优选例中，所述碳青霉烯类抗生素选自下组：亚胺培南、美罗培南、厄他培南、或其组合。In another preferred embodiment, the carbapenem antibiotic is selected from the group consisting of imipenem, meropenem, ertapenem, or a combination thereof.
在另一优选例中，所述制剂还包括其他治疗耐药菌感染的药物。In another preferred embodiment, the preparation also includes other drugs for treating drug-resistant bacterial infections.
在另一优选例中，所述耐药菌选自下组：金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌、或其组合。In another preferred embodiment, the drug-resistant bacteria are selected from the group consisting of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
在另一优选例中，所述制剂中，所述外泌体的含量为30－800μg，较佳地， 50－600μg，更佳地，80-500μg，更佳地，100-300μg，按所述制剂的总重量计。In another preferred example, the content of the exosomes in the preparation is 30-800 μg, preferably, 50-600 μg, more preferably, 80-500 μg, more preferably, 100-300 μg, according to The total weight of the formulation.
在另一优选例中，所述制剂中，所述抗生素的含量为10－300μM，较佳地，25－200μM，更佳地，30-100μM，更佳地，40－60μM，按所述制剂的总浓度计。In another preferred example, the content of the antibiotic in the formulation is 10-300μM, preferably, 25-200μM, more preferably, 30-100μM, more preferably, 40-60μM, according to the formulation The total concentration meter.
在另一优选例中，所述制剂为液态制剂、和/或冻干粉制剂。In another preferred embodiment, the formulation is a liquid formulation and/or a lyophilized powder formulation.
在另一优选例中，所述制剂的剂型包括注射剂。In another preferred embodiment, the dosage form of the preparation includes an injection.
在另一优选例中，所述药学上可接受的载体选自下组：磷酸盐缓冲液、生理盐水、或其组合。In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the following group: phosphate buffer, physiological saline, or a combination thereof.
本发明第三方面提供了一种药盒，包括：The third aspect of the present invention provides a medicine kit including:
(a1)第一容器，以及位于所述第一容器中的本发明第一方面所述的外泌体，或含有本发明第一方面所述的外泌体的药物；(a1) A first container, and the exosomes according to the first aspect of the present invention located in the first container, or a drug containing the exosomes according to the first aspect of the present invention;
(b1)任选的第二容器，以及位于所述第二容器中的抗生素，或含有抗生素的药物。(b1) Optional second container, and antibiotics in the second container, or drugs containing antibiotics.
在另一优选例中，所述药盒还包括：In another preferred embodiment, the kit further includes:
(c1)第三容器，以及位于所述第三容器中的其他预防和/或治疗耐药菌感染的药物，或含有其他预防和/或治疗耐药菌感染的药物的药物。(c1) The third container, and other drugs for preventing and/or treating drug-resistant bacterial infections, or drugs containing other drugs for preventing and/or treating drug-resistant bacterial infections.
在另一优选例中，所述抗生素选自下组：甲氧西林、碳青霉烯类抗生素、或其组合。In another preferred embodiment, the antibiotic is selected from the group consisting of methicillin, carbapenem antibiotics, or a combination thereof.
在另一优选例中，所述碳青霉烯类抗生素选自下组：亚胺培南、美罗培南、厄他培南、或其组合。In another preferred embodiment, the carbapenem antibiotic is selected from the group consisting of imipenem, meropenem, ertapenem, or a combination thereof.
在另一优选例中，所述耐药菌选自下组：金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌、或其组合。In another preferred embodiment, the drug-resistant bacteria are selected from the group consisting of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
在另一优选例中，所述的第一容器和第二容器、第三容器、是相同或不同的容器。In another preferred embodiment, the first container, the second container, and the third container are the same or different containers.
在另一优选例中，所述的第一容器的药物是含权利要求1所述的外泌体的单方制剂。In another preferred embodiment, the medicine in the first container is a unilateral preparation containing the exosomes of claim 1.
在另一优选例中，所述的第二容器的药物是含抗生素的单方制剂。In another preferred embodiment, the medicine in the second container is a unilateral preparation containing antibiotics.
在另一优选例中，所述的第三容器的药物是含其他预防和/或治疗耐药菌感染的药物的单方制剂。In another preferred embodiment, the medicine in the third container is a single preparation containing other medicines for preventing and/or treating drug-resistant bacterial infections.
在另一优选例中，所述药物的剂型为口服剂型或注射剂型。In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injection dosage form.
在另一优选例中，所述的试剂盒还含有说明书。In another preferred embodiment, the kit also contains instructions.
在另一优选例中，所述说明书记载了选自下组的一个或多个说明：In another preferred embodiment, the description records one or more descriptions selected from the following group:
(a)将本发明第一方面所述的外泌体用于提高耐药菌(比如MRSA)对抗生素(比如methicillin)敏感性的方法；(a) A method for using the exosomes described in the first aspect of the present invention to increase the sensitivity of drug-resistant bacteria (such as MRSA) to antibiotics (such as methicillin);
(b)将本发明第一方面所述的外泌体与抗生素、和/或任选的其他预防和/或治疗耐药菌感染的药物联用来治疗耐药菌感染的方法。(b) A method of combining the exosomes described in the first aspect of the present invention with antibiotics, and/or optionally other drugs for preventing and/or treating drug-resistant bacterial infections.
本发明第四方面提供了一种本发明第二方面所述的制剂、或本发明第三方面所述的药盒的用途，用于制备治疗耐药菌感染的药物。The fourth aspect of the present invention provides a preparation according to the second aspect of the present invention, or the use of the kit according to the third aspect of the present invention, for preparing a medicine for the treatment of drug-resistant bacterial infections.
在另一优选例中，所述药物还用于选自下组的一种或多种用途：In another preferred embodiment, the medicine is also used for one or more purposes selected from the following group:
(a)提高耐药菌对抗生素的敏感性；(a) Improve the sensitivity of drug-resistant bacteria to antibiotics;
(b)在转录后水平调节细菌基因表达。(b) Regulate bacterial gene expression at the post-transcriptional level.
在另一优选例中，所述耐药菌选自下组：金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌、或其组合。In another preferred embodiment, the drug-resistant bacteria are selected from the group consisting of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
本发明第五方面提供了一种治疗耐药菌感染的方法，包括：The fifth aspect of the present invention provides a method for treating drug-resistant bacterial infections, including:
给需要的对象施用本发明第二方面所述的制剂、或本发明第三方面所述的药盒。The preparation according to the second aspect of the present invention or the kit according to the third aspect of the present invention is administered to a subject in need.
在另一优选例中，所述对象包括受耐药菌感染的人或非人哺乳动物。In another preferred embodiment, the subject includes humans or non-human mammals infected by drug-resistant bacteria.
在另一优选例中，所述非人哺乳动物包括啮齿动物和灵长目动物，优选小鼠、大鼠、兔、猴。In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.
在另一优选例中，所述外泌体的施用剂量0.1-40－mg/kg体重，较佳地为1-20mg/kg体重，更佳地，2-10mg/kg体重。In another preferred example, the administration dose of the exosomes is 0.1-40 mg/kg body weight, preferably 1-20 mg/kg body weight, more preferably, 2-10 mg/kg body weight.
在另一优选例中，所述抗生素的施用剂量为0.1－50mg/kg体重，较佳地为1－20mg/kg体重，更佳地为2-6mg/kg体重。In another preferred example, the dosage of the antibiotic is 0.1-50 mg/kg body weight, preferably 1-20 mg/kg body weight, more preferably 2-6 mg/kg body weight.
在另一优选例中，所述外泌体的施用频率为1-10次/周，更佳地，3－9次/周，更佳地为5-8次/周。In another preferred example, the frequency of administration of the exosomes is 1-10 times/week, more preferably, 3-9 times/week, more preferably 5-8 times/week.
在另一优选例中，所述抗生素的施用频率为1-10次/周，更佳地，3－9次/周，更佳地为5-8次/周。In another preferred example, the frequency of administration of the antibiotic is 1-10 times/week, more preferably, 3-9 times/week, more preferably 5-8 times/week.
在另一优选例中，所述外泌体的施用时间为2-20天，较佳地为3-15天，更佳地，5-8天。In another preferred example, the administration time of the exosomes is 2-20 days, preferably 3-15 days, more preferably 5-8 days.
在另一优选例中，所述抗生素的施用时间为2-20天，较佳地为3-15天，更佳地，5-8天。In another preferred embodiment, the administration time of the antibiotic is 2-20 days, preferably 3-15 days, more preferably 5-8 days.
在另一优选例中，所述外泌体与抗生素同时或先后施用。In another preferred embodiment, the exosomes and antibiotics are administered simultaneously or sequentially.
本发明第六方面提供了一种体外非治疗性的提高耐药菌对抗生素敏感性的方法，包括步骤：在本发明第一方面所述的外泌体的存在下，培养耐药菌菌株，从而提高耐药菌菌株对抗生素的敏感性。The sixth aspect of the present invention provides a non-therapeutic in vitro method for improving the sensitivity of drug-resistant bacteria to antibiotics, which includes the steps of: culturing drug-resistant bacteria strains in the presence of the exosomes described in the first aspect of the present invention, Thereby increasing the sensitivity of drug-resistant bacterial strains to antibiotics.
在另一优选例中，通过将外泌体中的抑制耐药基因表达和/或其蛋白活性的物质(比如siRNA、miRNA)导入耐药菌菌株，调控耐药菌菌株中的耐药基因表达和/或活性，从而提高耐药菌对抗生素敏感性的方法。In another preferred example, by introducing substances (such as siRNA, miRNA) in exosomes that inhibit the expression of drug-resistant genes and/or their protein activity into drug-resistant bacteria strains, the expression of drug-resistant genes in drug-resistant bacteria strains is regulated And/or activity, thereby improving the sensitivity of drug-resistant bacteria to antibiotics.
在另一优选例中，所述外泌体的作用浓度为30－800μg/ml，较佳地，50－600μg/ml，更佳地，80-500μg/ml，更佳地，100-300μg/ml。In another preferred example, the concentration of the exosomes is 30-800 μg/ml, preferably, 50-600 μg/ml, more preferably, 80-500 μg/ml, more preferably, 100-300 μg/ml ml.
在另一优选例中，所述耐药菌选自下组：金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌、或其组合。In another preferred embodiment, the drug-resistant bacteria are selected from the group consisting of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them one by one here.
图1显示和游离状态的siRNA相比，exosome能够高效将其包裹的siRNA(si-ada)运输进E.coli，并且呈现剂量效应。Figure 1 shows that compared with free siRNA, exosome can efficiently transport its encapsulated siRNA (si-ada) into E. coli, and exhibit a dose effect.
图2显示exosome包裹的siRNA(si-ada)进入E.coli后对靶蛋白Ada表达抑制的剂量依赖效应。Figure 2 shows the dose-dependent effect of exosome-encapsulated siRNA (si-ada) on the inhibition of the target protein Ada expression after entering E. coli.
图3显示利用exosome包裹运输导入methicillin-resistant S.aureus(MRSA)的siRNA(si-mecA)量。Figure 3 shows the amount of siRNA (si-mecA) introduced into methicillin-resistant S. aureus (MRSA) using exosome package transportation.
图4显示利用exosome包裹运输导入MRSA的siRNA(si-mecA)对靶蛋白PBP2a表达的抑制效果。Figure 4 shows the inhibitory effect of siRNA (si-mecA) introduced into MRSA by exosome packaging and transportation on the expression of the target protein PBP2a.
图5显示利用exosome包裹运输导入MRSA的si-mecA能显著提高抗生素methicillin对MRSA的抑制效果。Figure 5 shows that si-mecA imported into MRSA by exosome package transportation can significantly improve the inhibitory effect of antibiotic methicillin on MRSA.
图6是示意图“通过人体细胞分泌出的含有小RNA的外泌体囊泡将小RNA传递入细菌内，并通过抑制耐药基因的表达降低细菌耐药性”。Fig. 6 is a schematic diagram of "small RNA-containing exosomal vesicles secreted by human cells deliver small RNAs into bacteria, and reduce bacterial drug resistance by inhibiting the expression of drug-resistant genes".
图7显示尾静脉注射siRNA表达载体，联合抗生素甲氧西林，显著改善耐药菌MRSA感染小鼠的生存率。Figure 7 shows that tail vein injection of siRNA expression vector, combined with antibiotic methicillin, significantly improved the survival rate of mice infected with drug-resistant MRSA bacteria.
图8显示了利用exosome包裹运输导入铜绿假单胞菌P.aeruginosa的siRNA(siMecA)量。Figure 8 shows the amount of siRNA (siMecA) introduced into P. aeruginosa by exosome package transportation.
本发明人经过广泛而深入的研究，意外地发现，负载有抑制耐药基因表达和/或其蛋白活性的物质的外泌体可显著提高耐药菌对抗生素的敏感性，并且将上述外泌体与抗生素联用，还可治疗耐药菌的感染。After extensive and in-depth research, the inventors unexpectedly discovered that exosomes loaded with substances that inhibit the expression of drug-resistant genes and/or their protein activity can significantly increase the sensitivity of drug-resistant bacteria to antibiotics, and the above-mentioned exosome Combination of body and antibiotics can also treat infections caused by resistant bacteria.
具体地，本发明通过设计合成针对耐药菌，比如耐甲氧西林金黄色葡萄球菌(methicillin-resistant S.aureus,MRSA)耐药基因mecA的siRNA，利用外泌体(exosome)的包裹及运输功能，将siRNA导入耐药菌细胞内，通过降低其耐药性基因的表达及耐药相关蛋白的生产，解除其耐药性，使其重新对常规的抗生素治疗敏感。此外，本发明人还意外的发现，将包裹有针对耐药菌的耐药基因的siRNA的外泌体与抗生素联用，可显著治疗耐药菌的感染。在此基础上，本发明人完成了本发明。Specifically, the present invention designs and synthesizes siRNA against drug-resistant bacteria, such as methicillin-resistant S. aureus (MRSA) drug-resistant gene mecA, and utilizes exosomes for packaging and transportation. Function to introduce siRNA into the cells of drug-resistant bacteria, by reducing the expression of drug-resistant genes and the production of drug-resistant related proteins, the drug resistance is relieved, and it is resusceptible to conventional antibiotic treatment. In addition, the present inventors also unexpectedly discovered that the combination of exosomes encapsulated with drug-resistant genes against drug-resistant bacteria and antibiotics can significantly treat infections of drug-resistant bacteria. On this basis, the inventor completed the present invention.
术语the term
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。In order to make the present disclosure easier to understand, first define certain terms. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning given below. Other definitions are stated throughout the application.
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。The term "about" may refer to a value or composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。As used herein, the term "containing" or "including (including)" can be open, semi-closed, and closed. In other words, the term also includes "substantially consisting of" or "consisting of".
如本文使用的，术语“宿主”、“受试者”、“所需对象”指任何哺乳动物或非哺乳动物。哺乳动物包括但不限于人类、脊椎动物诸如啮齿类、非人类灵长类，如牛、马、狗、猫、猪、绵羊、山羊、骆驼、大鼠、小鼠、野兔和家兔。As used herein, the terms "host", "subject", and "desired subject" refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, such as cows, horses, dogs, cats, pigs, sheep, goats, camels, rats, mice, hares, and rabbits.
耐药基因Resistance gene
在本发明中，耐药基因指细菌中编码与抗生素耐药相关蛋白的基因。In the present invention, the drug resistance gene refers to a gene in bacteria that encodes a protein related to antibiotic resistance.
在另一优选例中，所述耐药基因来源于选自下组的菌：葡萄球菌、大肠杆菌、铜绿假单胞菌、或其组合。In another preferred embodiment, the drug resistance gene is derived from a bacteria selected from the group consisting of Staphylococcus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
在另一优选例中，所述来源于葡萄球菌的耐药基因选自下组：mecA、kpc、imp、vim、ndm、或其组合。In another preferred embodiment, the drug resistance gene derived from Staphylococcus is selected from the group consisting of mecA, kpc, imp, vim, ndm, or a combination thereof.
在另一优选例中，所述来源于大肠杆菌的耐药基因选自下组：AcrA、AcrB、TolC、pbp2、pbp3、ampC、或其组合。In another preferred example, the drug resistance gene derived from Escherichia coli is selected from the group consisting of AcrA, AcrB, TolC, pbp2, pbp3, ampC, or a combination thereof.
在另一优选例中，所述来源于铜绿假单胞菌的耐药基因选自下组：vim、OprN、ampC、imp、或其组合。In another preferred embodiment, the drug resistance gene derived from Pseudomonas aeruginosa is selected from the group consisting of vim, OprN, ampC, imp, or a combination thereof.
在一优选实施方式中，所述耐药菌包括，但并不限于，葡萄球菌、大肠杆菌、铜绿假单胞菌。In a preferred embodiment, the drug-resistant bacteria include, but are not limited to, Staphylococcus, Escherichia coli, and Pseudomonas aeruginosa.
mecA蛋白和多核苷酸mecA protein and polynucleotide
在本发明中，术语“本发明蛋白”、“蛋白”、“mecA多肽”可互换使用，都指具有mecA氨基酸序列的蛋白或多肽。它们包括含有或不含起始甲硫氨酸的mecA蛋白。此外，该术语还包括全长的mecA及其片段。本发明所指的mecA蛋白包括其完整的氨基酸序列、其分泌蛋白、其突变体以及其功能上活性的片段。In the present invention, the terms "protein of the present invention", "protein", and "mecA polypeptide" are used interchangeably, and all refer to a protein or polypeptide having a mecA amino acid sequence. They include mecA proteins with or without starting methionine. In addition, the term also includes full-length mecA and fragments thereof. The mecA protein referred to in the present invention includes its complete amino acid sequence, its secreted protein, its mutant and its functionally active fragments.
mecA基因整合在称为葡萄球菌染色体盒(SCC)mec的染色体遗传元件中，编码蛋白为PBP2a，全称“青霉素结合蛋白2a”，668个氨基酸。mecA基因在葡萄球菌属中分布广泛。目前葡萄球菌属以外的细菌中尚未检出mecA基因。The mecA gene is integrated in a chromosomal genetic element called the Staphylococcal Chromosome Box (SCC) mec. The encoded protein is PBP2a, the full name is "penicillin binding protein 2a", with 668 amino acids. The mecA gene is widely distributed in Staphylococcus. At present, the mecA gene has not been detected in bacteria other than Staphylococcus.
在本发明中，术语“mecA基因”、“mecA多核苷酸”可互换使用，都指具有mecA核苷酸序列的核酸序列。PBP2a是mecA基因的编码蛋白。In the present invention, the terms "mecA gene" and "mecA polynucleotide" are used interchangeably, and both refer to a nucleic acid sequence having a mecA nucleotide sequence. PBP2a is the protein encoded by the mecA gene.
在本发明中，mecA基因的基因组全长为2007bp(登录号为Gene ID:5776902)。在得到了mecA的氨基酸片段的情况下，可根据其构建出编码它的核酸序列，并且根据核苷酸序列来设计特异性探针。核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的mecA核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。In the present invention, the total length of the genome of the mecA gene is 2007 bp (accession number is Gene ID: 5767902). When an amino acid fragment of mecA is obtained, a nucleic acid sequence encoding it can be constructed based on it, and a specific probe can be designed based on the nucleotide sequence. The full-length nucleotide sequence or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the mecA nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通 常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(如载体)和细胞中。At present, the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.
通过常规的重组DNA技术，可利用本发明的多核苷酸序列可用来表达或生产重组的mecA多肽。一般来说有以下步骤：Through conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant mecA polypeptide. Generally speaking, there are the following steps:
(1).用本发明的编码mecA多肽的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；(1) Use the polynucleotide (or variant) encoding the mecA polypeptide of the present invention, or use a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell;
(2).在合适的培养基中培养的宿主细胞；(2). Host cells cultured in a suitable medium;
(3).从培养基或细胞中分离、纯化蛋白质。(3). Separate and purify protein from culture medium or cells.
本发明中，mecA多核苷酸序列可***到重组表达载体中。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。In the present invention, the mecA polynucleotide sequence can be inserted into a recombinant expression vector. In short, any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
本领域的技术人员熟知的方法能用于构建含mecA编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。Methods well known to those skilled in the art can be used to construct expression vectors containing mecA encoding DNA sequences and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属的细菌细胞；真菌细胞如酵母；植物细胞；昆虫细胞；动物细胞等。The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells, etc.
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获， 用CaCl
2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl
2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Another method is to use MgCl 2 . If necessary, the transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used in the culture can be selected from various conventional mediums. The culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。The recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
外泌体Exosomes
如本文所用，外泌体(Exosome)是由细胞分泌而来的微小囊泡，直径约为30-200nm，密度在1.13-1.21g/ml，具有杯状形态、双层膜结构，天然存在于血液、尿液、唾液、母乳和细胞培养基等生物体液中。包括肿瘤细胞在内几乎所有类型的细胞(免疫细胞、神经细胞、干细胞)，都可以产生并释放exosome。As used herein, exosomes are tiny vesicles secreted from cells, with a diameter of about 30-200nm and a density of 1.13-1.21g/ml. They have a cup-shaped morphology and a double-layer membrane structure. They naturally exist in In biological fluids such as blood, urine, saliva, breast milk, and cell culture media. Almost all types of cells (immune cells, nerve cells, stem cells), including tumor cells, can produce and release exosomes.
在本发明中，外泌体包括抑制耐药基因(比如来源于葡萄球菌的耐药基因(如mecA、kpc,imp,vim,ndm)；来源于大肠杆菌的耐药基因(如AcrA,AcrB,TolC，pbp2,pbp3，ampC；来源于铜绿假单胞菌的耐药基因(如vim,OprN，ampC,imp等)的表达和/或其蛋白活性的物质(比如siRNA、miRNA等)。In the present invention, exosomes include inhibitory resistance genes (such as resistance genes derived from Staphylococcus (such as mecA, kpc, imp, vim, ndm); resistance genes derived from Escherichia coli (such as AcrA, AcrB, TolC, pbp2, pbp3, ampC; substances derived from the expression of resistance genes of Pseudomonas aeruginosa (such as vim, OprN, ampC, imp, etc.) and/or their protein activities (such as siRNA, miRNA, etc.).
外泌体制剂Exosomal preparations
本发明的胞外泌体制剂，含有安全有效量的外泌体以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、粉剂、及其组合。药物制剂应与给药方式相匹配。The exosome preparation of the present invention contains a safe and effective amount of exosomes and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powder, and combinations thereof. The pharmaceutical preparation should match the mode of administration.
在一优选实施方式中，本发明的制剂还可含有安全有效量的其他预防和/或治疗耐药菌感染的药物。In a preferred embodiment, the preparation of the present invention may also contain a safe and effective amount of other drugs for preventing and/or treating drug-resistant bacterial infections.
本发明的药物组合物可以被制成液态制剂，其制备可通过常规方法进行,液态制剂应在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约50毫克/千克体重，约5微克/千克体重-约10毫克/千克体重，约10微克/千克体重-约5毫克/千克体重。此外，本发明制剂还可与其他治疗剂一起使用。The pharmaceutical composition of the present invention can be made into a liquid preparation, and its preparation can be carried out by a conventional method, and the liquid preparation should be manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg/kg body weight-about 50 mg/kg body weight, about 5 μg/kg body weight-about 10 mg/kg body weight, and about 10 μg/kg body weight-about 5 per day. Mg/kg body weight. In addition, the formulations of the present invention can also be used together with other therapeutic agents.
使用本发明的制剂时，是将安全有效量的药物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。When the preparation of the present invention is used, a safe and effective amount of the drug is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases, does not exceed about 50 mg/kg body weight, preferably The dosage is about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
抑制基因表达的物质Substances that inhibit gene expression
如本文所用，术语“抑制基因表达的物质”是指，只要是抑制靶基因的mRNA转录的物质、分解已转录的mRNA的物质、或抑制由mRNA翻译成蛋白质的物质即可没有特别限定。作为所述的物质，可举出核酸，可示例：siRNA、miRNA等。其中，优选siRNA。作为“抑制基因表达的物质”，除上述之外，还包含蛋白质或肽、或者其他的小分子。需说明的是，在本发明中，靶基因是耐药基因，如mecA基因、kpc、imp、vim、ndm、AcrA、AcrB、TolC、pbp2、pbp3、ampC、OprN、imp等。As used herein, the term "substance that suppresses gene expression" means a substance that suppresses the transcription of mRNA of a target gene, a substance that decomposes transcribed mRNA, or a substance that suppresses the translation of mRNA into protein. Examples of the above-mentioned substances include nucleic acids, such as siRNA, miRNA, and the like. Among them, siRNA is preferred. As a "substance that inhibits gene expression", in addition to the above, it also includes proteins, peptides, or other small molecules. It should be noted that, in the present invention, the target gene is a drug resistance gene, such as mecA gene, kpc, imp, vim, ndm, AcrA, AcrB, TolC, pbp2, pbp3, ampC, OprN, imp, etc.
在本发明中，“siRNA”是指，具有由约15～40碱基组成的双链RNA部分的RNA分子，切割与上述siRNA的反义链具有互补序列的靶基因的mRNA，并具有抑制靶基因表达的功能。详细而言，本发明中的siRNA是包含由有义RNA链和反义RNA链组成的双链RNA部分的RNA，所述有义RNA链由与耐药基因，如mecA、kpc,imp,vim,ndm；AcrA,AcrB,TolC；pbp2,pbp3；ampC、OprN，imp的mRNA中的连续的RNA序列相同的序列组成，所述反义RNA链由与该有义RNA序列互补的序列组成。所述siRNA和后述的变异体siRNA的设计和制造在本领域技术人员的技能范围内。In the present invention, "siRNA" refers to an RNA molecule having a double-stranded RNA portion composed of about 15-40 bases, which cleaves the mRNA of a target gene having a complementary sequence to the antisense strand of the above-mentioned siRNA, and has an inhibitory target The function of gene expression. In detail, the siRNA in the present invention is an RNA comprising a double-stranded RNA portion composed of a sense RNA strand and an anti-sense RNA strand. The sense RNA strand is composed of drug-resistant genes such as mecA, kpc, imp, vim , ndm; AcrA, AcrB, TolC; pbp2, pbp3; ampC, OprN, and imp mRNA consist of the same sequence of consecutive RNA sequences, and the antisense RNA strand is composed of a sequence complementary to the sense RNA sequence. The design and manufacture of the siRNA and the variant siRNA described later are within the skill of those skilled in the art.
在本发明中，“miRNA”是指，具有由约21～25碱基组成的双链RNA部分的RNA分子，通过与靶基因的mRNA结合而具有抑制靶基因表达的功能。详细而言，本发明中的miRNA是包含由有义RNA链和反义RNA链组成的双链RNA部 分的RNA，所述有义RNA链由与本发明的耐药基因，如mecA基因、kpc,imp,vim,ndm；AcrA,AcrB,TolC；pbp2,pbp3；ampC、OprN，imp的mRNA中的连续的RNA序列相同的序列组成，所述反义RNA链由与该有义RNA序列互补的序列组成。所述miRNA和后述的变异体siRNA的设计和制造在本领域技术人员的技能范围内。In the present invention, "miRNA" refers to an RNA molecule having a double-stranded RNA portion composed of approximately 21 to 25 bases, which has the function of suppressing the expression of the target gene by binding to the mRNA of the target gene. In detail, the miRNA in the present invention is an RNA comprising a double-stranded RNA portion composed of a sense RNA strand and an antisense RNA strand. The sense RNA strand is composed of a drug resistance gene of the present invention, such as mecA gene, kpc , imp, vim, ndm; AcrA, AcrB, TolC; pbp2, pbp3; ampC, OprN, imp mRNA sequence composed of the same sequence of consecutive RNA sequences, the antisense RNA strand is complementary to the sense RNA sequence Sequence composition. The design and manufacture of the miRNA and the variant siRNA described later are within the skills of those skilled in the art.
关于siRNA的双链RNA部分的长度，作为碱基，优选为约15～40碱基，更优选为15～30碱基，进一步优选为15～25碱基，进一步优选为18～23碱基，更进一步优选是19～21碱基。另外，关于miRNA的双链RNA部分的长度，作为碱基，优选为约21～25碱基，更优选为22～24碱基。作为siRNA和miRNA的有义链或反义链的末端结构，没有特别限定，可以根据目的适当选择，例如可以具有平滑末端，也可以具有突出末端(overhang，突出端)，优选3'末端突出的类型。在有义RNA链和反义RNA链的3'末端具有由多个碱基、优选1～3个碱基、更优选2个碱基组成的突出端的siRNA和miRNA，大多情形抑制靶基因表达的效果较大，是优选者。突出端的碱基的种类没有特别限定，可以是构成RNA的碱基或构成DNA的碱基。Regarding the length of the double-stranded RNA portion of the siRNA, the base is preferably about 15 to 40 bases, more preferably 15 to 30 bases, still more preferably 15 to 25 bases, and still more preferably 18 to 23 bases, More preferably, it is 19 to 21 bases. In addition, the length of the double-stranded RNA portion of the miRNA is preferably about 21 to 25 bases, and more preferably 22 to 24 bases as bases. The end structure of the sense strand or antisense strand of siRNA and miRNA is not particularly limited, and can be appropriately selected according to the purpose. For example, it may have a smooth end or an overhang (overhang), preferably a 3'end overhang. Types of. SiRNA and miRNA that have an overhang composed of multiple bases, preferably 1 to 3 bases, more preferably 2 bases, at the 3'end of the sense RNA strand and the antisense RNA strand, inhibit the expression of the target gene in most cases The effect is greater, and it is the preferred one. The type of base at the overhang is not particularly limited, and it may be a base constituting RNA or a base constituting DNA.
而且，在上述siRNA和miRNA的有义链或反义链的一方或两方中有1～数个为止的核苷酸被缺失、取代、***和/或添加的siRNA和miRNA，也可以用于本发明的外泌体。在此，1～数个碱基是指，没有特别限定但优选为1～4碱基、更优选为1～3碱基、进一步优选为1～2碱基。作为所述变异的具体例，可举出：3'末端的突出端部分的碱基数为0～3个，将3'末端的突出端部分的碱基序列变更成其他的碱基序列、或通过碱基的***、添加或缺失上述有义RNA链和反义RNA链的长度为1～3碱基不同，有义链和/或反义链中碱基被另外的碱基取代等，但不限于这些。然而，在这些的变异体siRNA和miRNA中有义链和反义链可以杂交、以及这些的变异体siRNA和miRNA与不具有变异的siRNA和miRNA具有同等的基因表达抑制能是必要的。In addition, siRNA and miRNA in which one to several nucleotides have been deleted, substituted, inserted, and/or added in one or both of the sense strand or antisense strand of the above-mentioned siRNA and miRNA can also be used for The exosomes of the present invention. Here, 1 to several bases means, and it is not particularly limited, but it is preferably 1 to 4 bases, more preferably 1 to 3 bases, and even more preferably 1 to 2 bases. As a specific example of the mutation, the number of bases in the overhang at the 3'end is 0 to 3, the base sequence of the overhang at the 3'end is changed to another base sequence, or The length of the sense RNA strand and the antisense RNA strand are different from 1 to 3 bases by the insertion, addition or deletion of bases, and the bases in the sense strand and/or the antisense strand are replaced by other bases, etc., but Not limited to these. However, it is necessary that the sense strand and the antisense strand can hybridize in these variant siRNA and miRNA, and it is necessary that these variant siRNA and miRNA have the same gene expression inhibitory energy as the non-variant siRNA and miRNA.
而且，该siRNA和miRNA可以是一方的端封闭的结构的分子、例如具有发夹结构的siRNA和miRNA(Short hairpin RNA；shRNA)。shRNA是包含靶基因的特定序列的有义链RNA、由与该有义链RNA互补的序列组成的反义链RNA和连接其两链的接头序列的RNA，并且有义链部分和反义链部分进行杂交，形成双链RNA部分。Furthermore, the siRNA and miRNA may be molecules with a closed-end structure, such as siRNA and miRNA (Short hairpin RNA; shRNA) having a hairpin structure. shRNA is a sense strand RNA containing a specific sequence of a target gene, an antisense strand RNA composed of a sequence complementary to the sense strand RNA, and an RNA that connects the two strands of the linker sequence, and the sense strand part and the antisense strand The parts hybridize to form a double-stranded RNA part.
希望siRNA和miRNA在临床应用时不显示所谓的脱靶(off-target)效应。 脱靶效应是指，除了靶基因以外，抑制与使用的siRNA和miRNA具有部分同源的其他基因表达的作用。为了避免脱靶效应，对于候选siRNA和miRNA，可以预先利用DNA微阵列等确认不存在交叉反应。另外，使用NCBI(National Center for Biotechnology Information)等提供的已知数据库，除了成为靶的基因之外，还确认是否存在包含与候选siRNA和miRNA序列同源性高的部分的基因，由此可以避免脱靶效应。It is hoped that siRNA and miRNA will not show so-called off-target effects in clinical applications. Off-target effect refers to the effect of inhibiting the expression of other genes that are partially homologous to the used siRNA and miRNA in addition to the target gene. In order to avoid off-target effects, for candidate siRNA and miRNA, DNA microarrays can be used in advance to confirm that there is no cross-reaction. In addition, using known databases provided by NCBI (National Center for Biotechnology Information), etc., in addition to target genes, it is also confirmed whether there are genes that contain high homology to candidate siRNA and miRNA sequences, which can avoid Off-target effect.
为了制作本发明的siRNA和miRNA，可以适当使用：化学合成的方法和使用基因重组技术的方法等已知的方法。在合成的方法中，可以根据序列信息、通过常规方法合成双链RNA。另外，在使用基因重组技术的方法中，构建***了有义链序列或反义链序列的表达载体，将该载体导入到体外培养的哺乳动物细胞后，分别获取通过转录而生成的有义链RNA或反义链RNA，由此也可以制作。另外，使包含靶基因的特定序列的有义链RNA、由与该有义链RNA互补的序列组成的反义链RNA和连接其两链的接头序列，且形成发夹结构的shRNA表达，由此也可以制作所需的双链RNA。In order to produce the siRNA and miRNA of the present invention, known methods such as a chemical synthesis method and a method using gene recombination technology can be suitably used. In the method of synthesis, double-stranded RNA can be synthesized by conventional methods based on sequence information. In addition, in the method using gene recombination technology, an expression vector inserted with a sense strand sequence or an antisense strand sequence is constructed, and after the vector is introduced into mammalian cells cultured in vitro, the sense strand generated by transcription is obtained respectively. RNA or antisense strand RNA can also be produced from this. In addition, expression of a sense strand RNA containing a specific sequence of the target gene, an antisense strand RNA composed of a sequence complementary to the sense strand RNA, and a linker sequence connecting the two strands, and forming a hairpin structure, is expressed by This can also produce the required double-stranded RNA.
siRNA和miRNA只要具有靶基因的表达抑制活性，则构成siRNA和miRNA的核酸的一部分也可以是DNA。另外，siRNA和miRNA只要具有靶基因的表达抑制活性，则构成siRNA和miRNA的核酸全部或其一部分也可以是经修饰的核酸。As long as the siRNA and miRNA have the activity of suppressing the expression of the target gene, a part of the nucleic acid constituting the siRNA and miRNA may be DNA. In addition, as long as siRNA and miRNA have a target gene expression inhibitory activity, all or part of the nucleic acids constituting the siRNA and miRNA may be modified nucleic acids.
在本发明中，经修饰的核酸是指，对核苷(碱基位点，糖位点)和/或核苷间键位点实施修饰、与天然的核酸具有不同的结构。作为构成经修饰的核酸的“经修饰的核苷”，例如可举出：脱碱基(abasic无碱基)核苷；阿糖核苷、2'-脱氧尿苷、α-脱氧核糖核苷、β-L-脱氧核糖核苷、具有其他的糖修饰的核苷；肽核酸(PNA)、键合有磷酸基的肽核酸(PHONA)、锁定核酸(LNA)、吗啉代核酸等。具有上述糖修饰的核苷中，包含：2'-O-甲基核糖、2'-脱氧-2'-氟核糖、3'-O-甲基核糖等的取代戊糖；1',2'-脱氧核糖；***糖；取代***糖；己糖和具有α-端基的糖修饰的核苷。这些的核苷也可以是碱基位点被修饰的修饰碱基。这样的修饰碱基中，例如可举出：5-羟基胞嘧啶、5-氟尿嘧啶、4-硫尿嘧啶等的嘧啶；6-甲基腺嘌呤、6-硫代鸟嘌呤等的嘌呤；和其他的杂环碱基等。作为构成经修饰的核酸的“经修饰的核苷间键”，例如可举出：烷基接头、甘油接头、氨基接头、聚(乙二醇)键、甲基膦酸酯核苷间键、甲基硫代膦酸酯、磷酸三酯、硫代磷酸三酯、硫代磷酸酯、二硫代磷酸酯、三酯前 体药物、砜、氨磺酰、氨基磺酸酯、甲缩醛、N-甲基羟胺、碳酸酯、氨基甲酸酯、吗啉代、硼代膦酸酯、氨基磷酸酯等的非天然核苷间键。In the present invention, a modified nucleic acid refers to a nucleoside (base site, sugar site) and/or an internucleoside bond site modified to have a structure different from that of a natural nucleic acid. Examples of the "modified nucleoside" constituting the modified nucleic acid include: abasic nucleosides; arabinonucleosides, 2'-deoxyuridines, and α-deoxyribonucleosides , Β-L-deoxyribonucleosides, nucleosides with other sugar modifications; peptide nucleic acid (PNA), phosphate-bonded peptide nucleic acid (PHONA), locked nucleic acid (LNA), morpholino nucleic acid, etc. The nucleosides with the above sugar modification include: 2'-O-methylribose, 2'-deoxy-2'-fluororibose, 3'-O-methylribose and other substituted pentoses; 1', 2' -Deoxyribose; arabinose; substituted arabinose; hexose and sugar-modified nucleosides with α-terminal groups. These nucleosides may also be modified bases with modified base positions. Examples of such modified bases include pyrimidines such as 5-hydroxycytosine, 5-fluorouracil, and 4-thiouracil; purines such as 6-methyladenine and 6-thioguanine; and others The heterocyclic base and so on. Examples of the "modified internucleoside linkage" constituting the modified nucleic acid include alkyl linkers, glycerol linkers, amino linkers, poly(ethylene glycol) linkages, methylphosphonate internucleoside linkages, Methyl thiophosphonate, phosphotriester, phosphorothioate triester, phosphorothioate, phosphorodithioate, triester prodrug, sulfone, sulfonamide, sulfamate, methylal, Non-natural internucleoside bonds such as N-methylhydroxylamine, carbonate, carbamate, morpholino, borophosphonate, phosphoramidate, etc.
用途use
本发明所制备的外泌体，可有效提高耐药菌对抗生素的敏感性，并且本发明制备的外泌体制剂，可有效治疗耐药菌的感染。The exosomes prepared by the invention can effectively improve the sensitivity of drug-resistant bacteria to antibiotics, and the exosomes preparation prepared by the invention can effectively treat the infection of drug-resistant bacteria.
在一优选实施方式中，本发明用途可通过如下技术方案实现：In a preferred embodiment, the use of the present invention can be achieved through the following technical solutions:
1、一种利用外泌体将小RNA导入细菌并调控基因表达的方法，该方法包括：获取包裹siRNA的外泌体，外泌体和细菌(E.coli、S.aureus)共培养，检测外泌体和细菌内siRNA的含量。1. A method of using exosomes to introduce small RNAs into bacteria and regulating gene expression. The method includes: obtaining exosomes encapsulating siRNA, co-cultivating the exosomes and bacteria (E.coli, S.aureus), and detecting The content of siRNA in exosomes and bacteria.
2、一种靶向MRSA耐药基因mecA mRNA的siRNA序列。2. A siRNA sequence targeting the MRSA drug resistance gene mecA mRNA.
3、一种利用外泌体的包裹及运输功能，将小RNA导入耐药菌细胞内，通过降低其耐药相关蛋白的生产，提高其对抗生素治疗敏感性的方法。3. A method that utilizes the packaging and transportation functions of exosomes to introduce small RNAs into drug-resistant bacteria cells to reduce the production of drug-resistant-related proteins and improve their sensitivity to antibiotic treatment.
4、一种体内治疗耐药菌感染的方法。4. A method of treating drug-resistant bacteria infection in vivo.
本发明的主要优点包括：The main advantages of the present invention include:
(1)本发明首次发现，负载有抑制耐药基因表达和/或其蛋白活性的物质的外泌体可显著提高耐药菌对抗生素的敏感性，并且将上述外泌体与抗生素联用，还可治疗耐药菌的感染。(1) The present invention found for the first time that exosomes loaded with substances that inhibit the expression of drug-resistant genes and/or their protein activity can significantly increase the sensitivity of drug-resistant bacteria to antibiotics, and the above-mentioned exosomes are combined with antibiotics, It can also treat infections caused by resistant bacteria.
(2)本发明首次发现，通过设计合成针对耐药菌，比如耐甲氧西林金黄色葡萄球菌(methicillin-resistant S.aureus,MRSA)耐药基因mecA mRNA的siRNA，利用外泌体(exosome)的包裹及运输功能，将siRNA导入耐药菌细胞内，通过降低其耐药性基因的表达及耐药相关蛋白的生产，解除其耐药性，使其重新对常规的抗生素治疗敏感。(2) The present invention finds for the first time that siRNA targeting drug-resistant bacteria, such as methicillin-resistant S. aureus (MRSA) drug-resistant gene mecA mRNA, is designed and synthesized, and exosomes are used. The packaging and transportation functions of siRNA can be introduced into the cells of drug-resistant bacteria. By reducing the expression of drug-resistant genes and the production of drug-resistant-related proteins, the drug resistance is relieved and it is resusceptible to conventional antibiotic treatment.
(3)本发明首次发现，将包裹有针对耐药菌的耐药基因的siRNA的外泌体与抗生素联用，可显著治疗耐药菌的感染。(3) The present invention found for the first time that the combination of exosomes coated with drug-resistant genes against drug-resistant bacteria and antibiotics can significantly treat infections of drug-resistant bacteria.
(4)在本发明中，利用外泌体将小RNA导入细菌并调控基因表达的方法，以大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)为例，首次报道了外泌体介导的小RNA对细菌基因的跨界调控方式，内容创新，探索异源小RNA对细菌胞内基因表达水平的调控策略及机制，具有重要的生物学意义。(4) In the present invention, exosomes are used to introduce small RNAs into bacteria and regulate gene expression. Taking Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as examples, the exocytosis is reported for the first time. The cross-border regulation of bacterial genes by body-mediated small RNAs, innovative content, and exploration of the regulation strategies and mechanisms of heterologous small RNAs on bacterial intracellular gene expression levels have important biological significance.
(5)本发明进一步拓展了外泌体作为体内运输载体的应用范围，它不仅可 以在哺乳动物组织之间传递小RNA分子信号，还能介导哺乳动物与细菌在小RNA层面上的跨界交流。本发明提出利用外泌体的包裹及运输功能，将小RNA导入耐药菌细胞内，通过降低其耐药性基因的表达及耐药相关蛋白的生产，解除其耐药性，使其重新对常规的抗生素治疗敏感。此策略将推广到一系列与细菌重要生命活动，毒力因子和抗免疫因子产生相关的基因调控上，从而开发新的抗击细菌感染的治疗策略。并且，通过静脉注射siRNA表达载体，在体内表达靶向耐药基因的siRNA，和抗生素联用，治疗耐药菌感染，提高生存率。(5) The present invention further expands the application range of exosomes as transport carriers in vivo. It can not only transmit small RNA molecular signals between mammalian tissues, but also mediate the cross-border of mammals and bacteria at the small RNA level. communicate with. The present invention proposes to use the packaging and transport functions of exosomes to introduce small RNAs into the cells of drug-resistant bacteria. By reducing the expression of drug-resistant genes and the production of drug-resistant-related proteins, the drug resistance is relieved, and the drug is regained. Conventional antibiotic treatment is sensitive. This strategy will be extended to a series of gene regulation related to the important life activities of bacteria, virulence factors and anti-immune factors, so as to develop new therapeutic strategies against bacterial infections. In addition, through intravenous injection of siRNA expression vectors, siRNA targeting drug-resistant genes is expressed in vivo, and combined with antibiotics, to treat drug-resistant bacterial infections and improve survival rates.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing The conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.
除非特别说明，否则本发明说明书中所用的试剂和材料均为市售产品。Unless otherwise specified, the reagents and materials used in the specification of the present invention are all commercially available products.
实施例1 获取外泌体Example 1 Obtaining exosomes
利用lipofectamine 2000试剂(Invitrogen)将siRNA转染进体外培养的哺乳动物细胞例如293T细胞，然后使用低血清培液培养细胞24-48小时，收集培液，使用exosome isolation kit(from cell culture)(Invitrogen)提取293T细胞分泌的外泌体，具体操作如下：培液先经过300g x 5min，3000g x 25min预离心去除细胞碎片，取上清，然后向其中加入一半体积的exosome isolation kit，颠倒混匀后4℃放置过夜；第二天经10000g x 1h，4℃离心后，弃上清，沉淀以PBS重悬。该方法获得包裹有siRNA的外泌体(siRNA-exosome)。使用BCA蛋白定量法得到外泌体的浓度(μg/μl)。Use lipofectamine 2000 reagent (Invitrogen) to transfect siRNA into mammalian cells cultured in vitro, such as 293T cells, and then use low-serum culture medium to culture the cells for 24-48 hours. Collect the culture medium and use exosome isolation kit (from cell culture) (Invitrogen) ) To extract exosomes secreted by 293T cells, the specific operation is as follows: the culture solution is pre-centrifuged at 300g x 5min, 3000g x 25min to remove cell debris, take the supernatant, and then add half the volume of the exosome isolation kit to it, and mix it upside down. Place overnight at 4°C; the next day after centrifugation at 10000g x 1h at 4°C, discard the supernatant, and resuspend the pellet in PBS. This method obtains siRNA-exosome (siRNA-exosome). The BCA protein quantification method was used to obtain the concentration of exosomes (μg/μl).
其中siRNA的正义链序列如SEQ ID NO.:1所示：The sense strand sequence of siRNA is shown in SEQ ID NO.:1:
si-mecA sense GCAAUCGCUAAAGAACUAATT。si-mecA sense GCAAUCGCUAAAGAACUAATT.
siRNA的反义链序列如SEQ ID NO.:2所示：The antisense strand sequence of siRNA is shown in SEQ ID NO.: 2:
si-mecA antisense UUAGUUCUUUAGCGAUUGCTT。si-mecA antisense UUAGUUCUUUAGCGAUUGCTT.
实施例2 外泌体和细菌共培养Example 2 Co-cultivation of exosomes and bacteria
(1)外泌体和细菌共培养：取过夜培养的菌液，按1％的标准接种于LB培 养基中。向菌液中加入外泌体使其终浓度为200μg/ml，混合均匀，在37℃，220rpm的条件下培养若干小时后，收取菌液。(1) Co-cultivation of exosomes and bacteria: Take the overnight cultured bacteria solution and inoculate it in the LB medium according to the standard of 1%. Add exosomes to the bacterial solution to a final concentration of 200 μg/ml, mix well, and incubate for several hours at 37° C. and 220 rpm, and then collect the bacterial solution.
(2)洗涤处理菌体：将菌液以3000rpm离心5min后获得菌体沉淀，经PBS洗涤后，加入含0.1％Triton X-100的PBS重悬，室温放置5min后，加入RNase A(终浓度100μg/ml)，37℃温育30min。该步骤是为了去除细菌外的exosome对后续检测的干扰。之后，细菌经3000rpm离心5min后弃上清，沉淀用PBS洗涤，并分成两等份，一份加入Trizol试剂，用于抽提RNA；另一份加入2x SDS loading buffer，95℃，加热10min，用于western blot样品。(2) Washing cells: Centrifuge the bacterial solution at 3000 rpm for 5 minutes to obtain a bacterial pellet. After washing with PBS, add 0.1% Triton X-100 in PBS to resuspend, and after standing at room temperature for 5 minutes, add RNase A (final concentration) 100μg/ml), incubate at 37°C for 30min. This step is to remove the interference of exosomes other than bacteria on subsequent detection. After that, the bacteria were centrifuged at 3000 rpm for 5 minutes and the supernatant was discarded. The pellet was washed with PBS and divided into two equal parts. One part was added with Trizol reagent for RNA extraction; the other part was added with 2x SDS loading buffer at 95°C and heated for 10 minutes. Used for western blot samples.
(3)检测进入E.coli的siRNA：将从(2)抽提的RNA逆转录为cDNA，用实时荧光定量PCR方法(探针法)对blank组、ctrl exosome组和siRNA-exosome组进行检测，得出siRNA进入E.coli的量。该结果显示出外泌体作为运输载体能使siRNA高效进入E.coli。(图1)(3) Detection of siRNA entering E. coli: reverse transcription of the extracted RNA from (2) into cDNA, and use real-time fluorescent quantitative PCR method (probe method) to detect the blank group, ctrl exosome group and siRNA-exosome group , Get the amount of siRNA into E.coli. This result shows that exosomes can be used as transport carriers to enable siRNA to enter E.coli efficiently. (figure 1)
(4)检测E.coli中的蛋白表达：将从(2)得到的蛋白样品用于western blot，检测靶蛋白Ada和内参DnaK蛋白的表达水平，得出利用外泌体运送进E.coli的siRNA对靶蛋白表达的调控效率。结果显示外泌体导入E.coli的siRNA可有效调控靶蛋白表达，并且该调控具有剂量依赖效应。(图2)(4) Detection of protein expression in E. coli: Use the protein sample obtained from (2) in western blot to detect the expression level of the target protein Ada and the internal reference DnaK protein, and obtain the use of exosomes to transport into E. coli Control efficiency of siRNA on target protein expression. The results showed that siRNA introduced into E.coli from exosomes can effectively regulate the expression of target proteins, and this regulation has a dose-dependent effect. (figure 2)
(5)检测进入S.aureus的siRNA：检测方法同(3)。该结果显示外泌体可以将小RNA导入S.aureus。(图3)(5) Detection of siRNA entering S.aureus: The detection method is the same as (3). This result shows that exosomes can introduce small RNA into S. aureus. (image 3)
(6)检测S.aureus中的蛋白表达：检测方法同(4)，靶蛋白是PBP2a。结果显示利用外泌体导入S.aureus的siRNA可有效调控靶蛋白表达。(图4)(6) Detection of protein expression in S. aureus: The detection method is the same as (4), and the target protein is PBP2a. The results show that the use of exosomes to introduce siRNA into S. aureus can effectively regulate the expression of the target protein. (Figure 4)
(7)检测MRSA对甲氧西林抗生素的敏感性：取过夜培养的菌液，在LB培养基中按1％的标准接种于96孔细胞培养板。实验分为三组：Mock组不加exosome，ctrl exo组加入ctrl exosome，siRNA exo组加入siRNA-exosome(终浓度为200μg/ml)。并分别加入不同浓度的抗生素methicillin，使得终浓度为0、10、25、50μM。在37℃，220rpm的条件下培养6h后测定菌液的光密度值(OD600)。结果显示siRNA-exosome可以显著提高MRSA对methicillin的敏感性，在抗生素浓度为10、25、50μM时尤为明显。(图5)(7) To detect the sensitivity of MRSA to methicillin antibiotics: take the overnight cultured bacterial solution and inoculate it in a 96-well cell culture plate according to the standard of 1% in LB medium. The experiment was divided into three groups: Mock group without exosome, ctrl exo group with ctrl exosome, and siRNA exo group with siRNA-exosome (final concentration 200μg/ml). And add different concentrations of antibiotic methicillin, so that the final concentration is 0, 10, 25, 50μM. After incubating at 37°C and 220 rpm for 6 hours, the optical density value (OD600) of the bacterial solution was measured. The results show that siRNA-exosome can significantly increase the sensitivity of MRSA to metthicillin, especially when the antibiotic concentration is 10, 25, and 50 μM. (Figure 5)
(8)检测进入铜绿假单胞菌P.aeruginosa的siRNA：检测方法同(3)。该结果显示外泌体可以将小RNA导入P.aeruginosa。(图8)(8) Detection of siRNA into P. aeruginosa: the detection method is the same as (3). This result shows that exosomes can introduce small RNA into P.aeruginosa. (Picture 8)
实施例3 体内治疗耐药菌感染Example 3 Treatment of drug-resistant bacterial infections in vivo
通过眼眶静脉注射5×10
7CFU MRSA细菌的方法来感染BALB/c小鼠，建立菌血症模型。感染小鼠通过腹膜注射接受3mg/kg抗生素甲氧西林的治疗剂量，并通过尾静脉注射接受5mg/kg包含siRNA的外泌体，注射间隔为24小时，治疗7天。甲氧西林和包含siRNA的外泌体联合用药，有效保护了小鼠免于致死剂量的MRSA细菌感染(图7)。联合用药期间，小鼠存活率显著提高，并在停药后14天的观察期内保持健康，说明药物没有严重的副作用。
BALB/c mice were infected by intravenous injection of 5×10 7 CFU MRSA bacteria into the orbit to establish a bacteremia model. Infected mice received 3 mg/kg of antibiotic methicillin via intraperitoneal injection and 5 mg/kg of siRNA-containing exosomes via tail vein injection. The injection interval was 24 hours and the treatment was 7 days. The combination of methicillin and exosomes containing siRNA effectively protected mice from lethal doses of MRSA bacterial infection (Figure 7). During the combined medication, the survival rate of the mice was significantly improved, and they remained healthy during the 14-day observation period after stopping the medication, indicating that the medication had no serious side effects.
图6显示了本发明的外泌体治疗耐药菌感染的机理图。Figure 6 shows a mechanism diagram of the exosomes of the present invention in treating drug-resistant bacterial infections.
构建菌血症模型小鼠，通过尾静脉注射siRNA表达载体，使体内表达靶向耐药基因mecA的siRNA，同时给药抗生素甲氧西林，观察小鼠的生存曲线。The bacteremia model mice were constructed, and siRNA expression vector was injected through the tail vein to express the siRNA targeting the drug-resistant gene mecA in vivo, and the antibiotic methicillin was administered at the same time to observe the survival curve of the mice.
结果显示，与不治疗组、单给甲氧西林治疗组相比，联用甲氧西林+siRNA组的小鼠生存率显著提高。(图7)The results showed that the survival rate of mice in the combined methicillin + siRNA group was significantly improved compared with the untreated group and the methicillin treatment group alone. (Picture 7)
此外，发明人发现，甲氧西林和包含siRNA的外泌体联合用药，也可有效保护了小鼠免于致死剂量的铜绿假单胞菌的感染。In addition, the inventors found that the combination of methicillin and exosomes containing siRNA can also effectively protect mice from a lethal dose of Pseudomonas aeruginosa infection.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
Claims (10)
- 一种负载抑制剂的外泌体，其特征在于，所述外泌体包括抑制耐药基因表达和/或其蛋白活性的物质。An inhibitor-loaded exosomes, characterized in that the exosomes include substances that inhibit the expression of drug-resistant genes and/or their protein activities.
- 如权利要求1所述的外泌体，其特征在于，所述耐药基因来源于选自下组的菌的耐药基因：葡萄球菌、大肠杆菌、铜绿假单胞菌、或其组合。The exosomes of claim 1, wherein the drug resistance gene is derived from a drug resistance gene of a bacteria selected from the group consisting of Staphylococcus, Escherichia coli, Pseudomonas aeruginosa, or a combination thereof.
- 如权利要求2所述的外泌体，其特征在于，所述来源于葡萄球菌的耐药基因选自下组：mecA、kpc、imp、vim、ndm、或其组合。The exosomes of claim 2, wherein the drug resistance gene derived from Staphylococcus is selected from the group consisting of mecA, kpc, imp, vim, ndm, or a combination thereof.
- 如权利要求1所述的外泌体，其特征在于，所述抑制耐药基因表达和/或其蛋白活性的物质选自下组：siRNA、miRNA、或其组合。The exosomes according to claim 1, wherein the substance that inhibits the expression of the drug-resistant gene and/or its protein activity is selected from the group consisting of siRNA, miRNA, or a combination thereof.
- 如权利要求4所述的外泌体，其特征在于，所述siRNA的正义链序列如SEQ ID NO.:1所示。The exosomes of claim 4, wherein the sense strand sequence of the siRNA is shown in SEQ ID NO.:1.
- 一种制剂，其特征在于，包括：A preparation, characterized in that it comprises:(a)权利要求1所述的外泌体；(a) The exosomes of claim 1;(b)抗生素；和(b) Antibiotics; and(c)药学上可接受的载体。(c) A pharmaceutically acceptable carrier.
- 如权利要求6所述的制剂，其特征在于，所述抗生素选自下组：甲氧西林、碳青霉烯类抗生素、或其组合。The formulation of claim 6, wherein the antibiotic is selected from the group consisting of methicillin, carbapenem antibiotics, or a combination thereof.
- 一种药盒，其特征在于，包括：A medicine box is characterized in that it comprises:(a1)第一容器，以及位于所述第一容器中的权利要求1所述的外泌体，或含有权利要求1所述的外泌体的药物；(a1) A first container, and the exosomes of claim 1 in the first container, or a drug containing the exosomes of claim 1;(b1)任选的第二容器，以及位于所述第二容器中的抗生素，或含有抗生素的药物。(b1) Optional second container, and antibiotics in the second container, or drugs containing antibiotics.
- 一种权利要求6所述的制剂、或权利要求8所述的药盒的用途，其特征在于，用于制备治疗耐药菌感染的药物。A preparation according to claim 6 or use of the kit according to claim 8, characterized in that it is used to prepare a medicine for the treatment of drug-resistant bacterial infections.
- 一种体外非治疗性的提高耐药菌对抗生素敏感性的方法，其特征在于，包括步骤：在权利要求1所述的外泌体的存在下，培养耐药菌菌株，从而提高耐药菌菌株对抗生素的敏感性。An in vitro non-therapeutic method for improving the sensitivity of drug-resistant bacteria to antibiotics, which is characterized in that it comprises the step of: culturing drug-resistant bacteria strains in the presence of the exosomes of claim 1, thereby increasing the drug-resistant bacteria The sensitivity of the strain to antibiotics.
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| GENG XIANLONG: "Rapid detection of Methicillin-Resistant Staphylococcus Aureus (MRSA) and Methodological Study of Antibiotic Resistance Gene Silencing", WANFANG DATA KNOWLEDGE SERVICE PLATFORM, 3 December 2015 (2015-12-03) * |
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| Publication number | Publication date |
|---|---|
| CN112972702A (en) | 2021-06-18 |
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