Treatment and prevention of microbial infections

Abstract

The present invention provides an agent for use in a method for the continuous treatment of bacterial infections causing sepsis in humans or animals. [Solution] The bacterial infection is caused by a first species of bacterium or strain, which is an Enterobacteriaceae selected from the group consisting of Escherichia, Salmonella, Klebsiella, Shigella, Enterobacter, and Citrobacter, and the agent comprises a programmable Cas nuclease that can be programmed to cleave a target site contained in an essential gene contained in the chromosome of the bacteria that infected the subject, and the method for treating the bacterial infection comprises bringing the subject into contact with the nuclease.

Description

[Technical Field]

[0001] The present invention relates to a method for treating or preventing microbial (e.g., bacterial) infections, and such To provide means for carrying out the method. In particular, for septicemia, sepsis, Rapid or continuous therapy to treat acute conditions such as SIRS or septic shock. It enables the treatment of necessary infectious diseases. Furthermore, the present invention can be used, for example, in the environment, in food, and is particularly useful for treating microorganisms for beverage use. The present invention is particularly useful in industrial or Microbial corrosion (MIC, microbiologically influenced corrosion) of household substrates or fluids This relates to methods for controlling biofouling.

[0002] The present invention relates to transplantation, or to the treatment of diseases such as cancer, viral infections, or autoimmune diseases. It is also useful in treating pathogenic bacterial infections in patients receiving treatment for a condition. [Background technology]

[0003] Sepsis is an acute and severe blood infection. Sepsis is also known as bacteremia or hematotoxicity. Sepsis occurs when a bacterial infection from another part of the body, such as the lungs or skin, enters the bloodstream. This is dangerous because bacteria and their toxins can be carried throughout the body via the bloodstream. Yes, sepsis can rapidly become a life-threatening condition. Sepsis requires rapid treatment at a hospital or other medical facility. It must be treated. Sepsis can progress to sepsis if left untreated. be.

[0004] Sepsis and sepsis are not the same thing. Sepsis is a serious complication of sepsis. Ossus is a condition in which inflammation occurs throughout the body. This inflammation can lead to blood clots. This can prevent oxygen from reaching vital organs, potentially leading to organ failure. According to the National Institutes of Health (NIH), more than one million Americans contract severe sepsis each year. It is estimated that 28-50 percent of these patients will die from this condition. Yes. When inflammation occurs and blood pressure drops very low, it is called septic shock. Shock is often fatal.

[0005] The average age of the population is increasing, more people have chronic diseases, and they are under immunosuppressant medication. The increase in the number of invasive procedures performed is linked to an increase in the sepsis rate. Elderly people, especially those with health problems, are more likely to contract sepsis than any other group. It is easy. According to a study published in 2006, people aged 65 and over make up about 12% of the US population. This accounts for 65% of sepsis cases in hospitals.

[0006] Sepsis is caused by an infection in a part of the body. This infection is typically It is acute. Many types of bacteria can lead to sepsis. The exact source of the infection is... It is often difficult to make a decision. The most common infections that lead to sepsis are as follows: It is. · Urinary tract infection • Pneumonia and other lung infections · Kidney infection • Infections of the abdominal region

[0007] These infectious bacteria enter the bloodstream, multiply rapidly, and cause acute infections and immediate symptoms. To wake up.

[0008] People who are already hospitalized for surgery or something else are at a higher risk of developing sepsis. While in the hospital, secondary infections can occur. Bacteria can already be resistant to antibiotics, so these infections are often more dangerous. The risk of developing sepsis is high when the subject is in the following situations. · Having severe wounds or burns · Being very young or very old · Having an immune system deficiency that can result from diseases such as HIV or leukemia · Having a urinary tract or intravenous catheter · Wearing a mechanical ventilator · Receiving medical treatments that weaken the immune system, such as chemotherapy or steroid injections.

[0009] Usually, the symptoms of sepsis start very rapidly. Even in the first stage of the illness, an individual may look very sick. The symptoms of sepsis can occur after another localized (e.g., limited to one location) infection such as an injury, surgery, or pneumonia. The most common <D initial symptoms are as follows. · Chills · High body temperature (fever) · Very rapid breathing · Tachycardia

[0010] <D More severe symptoms will begin to appear as sepsis progression if appropriate treatment is not performed. More severe symptoms include the following. · Confusion or inability to think clearly · Nausea and vomiting · Red patches that appear on the skin · Reduction in urine volume · Insufficient blood flow (shock)

[0011] Sepsis that has started to affect organ or tissue function is an acute medical emergency. Sepsis It must be treated promptly in a hospital. Many people with sepsis require treatment and recovery. They are admitted to the hospital's ICU for recovery. They are also being offered a "wait and see" approach, or are being asked to deal with the problem at home. It is not recommended to do so. If the subject shows signs of sepsis, go to the hospital immediately. It is important to rush to the scene. Sepsis has a significant number of serious complications. If left untreated, or if treatment is inadequate, If delayed for a long period, these complications can be fatal.

[0012] Septic shock One complication of sepsis is a severe drop in blood pressure, known as septic shock. Toxins released by bacteria in the bloodstream can cause very low blood flow, This may result in organ or tissue damage. Septic shock is an acute medical condition. This is an emergency. People with septic shock are usually treated in the intensive care unit (ICU) of a hospital. In cases of septic shock, it is necessary to put the patient on a ventilator, i.e., a respiratory device. It may be necessary.

[0013] Acute respiratory distress syndrome (ARDS) Another complication of sepsis is acute respiratory distress syndrome (ARDS). This occurs when the lungs do not receive enough oxygen. It is a life-threatening condition that prevents blood from reaching the bloodstream. (National Heart, Lung, and Blood Institute (NHLBI)) According to the study, approximately one-third of ARDS cases are fatal. ARDS involves a certain level of permanent lung damage. It often causes bleeding. Also, ARDS can damage the brain and may lead to memory problems. be.

[0014] Sepsis Sepsis occurs when the body exhibits a strong immune response to an infection. It can lead to widespread inflammation throughout the body. If it leads to organ failure, it can result in severe sepsis. This is called sepsis. People with HIV or chronic diseases such as cancer are at higher risk of sepsis. This is because such people have weakened immune systems and cannot fight off infections on their own. Sepsis causes millions of deaths worldwide every year and is the most common cause of hospitalization. It is a common cause of death. The global incidence of sepsis is estimated at 18 million cases per year. In the United States, sepsis affects approximately 3 out of every 1,000 people, and severe sepsis can last for one year. It contributes to more than 200,000 deaths per case. Sepsis occurs in 1-2% of all hospitalizations, and 25 It accounts for a significant percentage of ICU bed occupancy.

[0015] Early initiation of therapy is key to reducing mortality from severe sepsis, therefore, early diagnosis is necessary. It is necessary to decisively manage sepsis appropriately. Within the diagnostic study, unless the use of the diagnostic study is delayed by more than 45 minutes, the white blood cell count will be... To measure serum lactate and to obtain a suitable culture before starting antibiotics. It should be included.

[0016] The most common primary sources of infection leading to sepsis are the lungs, abdomen, and urinary tract. Typically 50% of all sepsis cases begin with a lung infection.

[0017] Speed ​​of treatment is essential. Do not delay the initiation of antibiotics and perform two sets of blood cultures (aerobic). A sample (both sexual and anaerobic) should be collected. If an infection originating from these sites is suspected, a respiratory sample should be taken. Other sites such as aspirated secretions, urine, wounds, cerebrospinal fluid, and catheter insertion sites (in situ for 48 hours) Cultures may be collected from a longer period of time than the specified period. In severe sepsis and septic shock, broad-spectrum cultures may be used. Antibiotics (usually two broadly covering β-lactam antibiotics or fluorochemical antibiotics) (Nolones, macrolides, or broad-spectrum carbapenems combined with aminoglycosides) , which has been used conventionally. However, the combination of antibiotics broadens the antibacterial activity. Unless used for this purpose, it is not recommended for treating sepsis in immunocompromised individuals who do not show shock. No. The administration of antibiotics is important in determining an individual's survival. 1 hour after diagnosis. Some recommend administering the antibiotic within a certain timeframe, as each hour of delay in antibiotic administration increases the mortality rate. This is said to be accompanied by a 6% increase.

[0018] Early Goal-Oriented Therapy (EGDT) is designed to manage severe sepsis within the first six hours after diagnosis. This is a method. It is a stepwise approach, and the physiological goal is cardiac preload and afterload. The objectives are to optimize contractile force and to administer antibiotics early. nothing.

[0019] Neonatal sepsis is difficult to diagnose because newborns may be asymptomatic. If signs and symptoms suggestive of the disease are present, antibiotics should be started immediately, and the antibiotics should be used for diagnosis. The test may be modified to target specific organisms identified, or the cause of infection of the symptoms. Either it is excluded and then canceled.

[0020] Approximately 20-35% of people with severe sepsis and 30-70% of people with septic shock. He dies. The Surviving Sepsis Campaign (SSC) This is a global initiative that brings together specialized organizations to reduce the mortality rate from Sepsis. Antibiotics are administered within 2 hours of admission / diagnosis. After the onset of septic shock, the patient For every hour of refusal of antibiotic therapy, the patient's chances of survival decrease by 7.9% (Survivesepsis). (org 2005).

[0021] Therefore, acute microbiota such as bacterial infections associated with sepsis, sepsis, or septic shock. Rapid treatment of biological infections is needed, as treatment can sometimes last for several hours. It would be beneficial. Furthermore, rapid and sustained treatment of microorganisms is beneficial for the microbial activity of substrates in industrial and household applications. It is desirable for controlling material corrosion (MIC) or biofouling, and is intended for that purpose. [Prior art documents] [Patent Documents]

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[0024] Acute bacterial infections can, in some circumstances, be health-related or even life-threatening. This may apply, for example, to cancer patients, organ transplant recipients, or other individuals. The need for treatment of infectious diseases can be urgent, and in fact, it is a matter of immediate medical concern. The focus is on the negative impact on the effectiveness of cancer therapy or other separate therapies, which the patient also needs to respond to. To avoid causing harm, it is necessary to provide methods for treating such pathogenic bacterial infections. It will be useful. [Means for solving the problem]

[0025] This invention utilizes the action of programmable nuclease cleavage of microbial genomes. This provides a solution using metabolic inhibitors, as well as beta-L for treating infections. Unlike other mechanisms of action used by kutam and other conventional antibiotics, targeted cleavage is different. , propose selective microbial killing or reduction of growth or proliferation for the treatment or prevention of infectious diseases. To provide. Furthermore, the inventors have surprisingly found that in some embodiments, substantial elimination (several l) is achieved. It can achieve the goal of (og) very quickly (for example, within 15 minutes) and deliver sustainable effects. We found that it is possible to achieve this (for example, for a longer period than one hour after the start of treatment). (and for approximately 3 hours). Therefore, the present invention provides the following configuration.

[0026] First composition Programmable nuclei for use in methods to treat target microbial infections. - In which a microbial infection is caused by a first species or strain of microorganism, nucleotide Aase programmed to cleave target sites contained in the genome of the microorganism infecting the target. Gram may be able to kill the first species or strain of microorganism, or the microbial growth may be lost. Growth or proliferation is reduced, and the treatment method involves bringing the target into contact with the nuclease, and the nuclease This includes a process programmed to cut the target site, thereby reducing the amount of material in the object. The genome of the microorganism being introduced is cut, and the target microbial infection is treated in a programmable manner. Nuclease.

[0027] Second composition Used in conjunction with programmable nucleases in methods for treating target microbial infections. Multiple viruses for use (e.g., phages or phages for producing phages) (Mid) and microbial infections are caused by a first species or strain of microorganism, nucleus Aase programmed to cleave target sites contained in the genome of the microorganism infecting the target. Gram is possible, and thereby the first species or strain of microorganism is killed or the microorganism is Growth or proliferation is reduced, and the treatment method involves exposing the target to the nuclease and the virus. The nuclease includes a step in which it is programmed to cleave the target site, thereby Depending on the target, the genome of the microorganisms included is cut, and the infection caused by the target microorganism is treated. Each virus contains a copy of nucleic acid that encodes RNA in order to express RNA in the target. RNA complexes with nucleases, programming the nucleases to be included in the target. The target site of the microorganism is cut, Viruses can infect microorganisms present in their target organisms and deliver nucleic acids to them. These are multiple viruses.

[0028] Third composition Programmable nucleases are programmed in methods to treat target microbial infections. A composition comprising multiple nucleic acids for laminating, wherein microbial infections are caused by a first species or strain Caused by microorganisms, the nuclease is contained in the genome of the microorganism that infected the target. It can be programmed to cut the target site, thereby removing the microscopic structure of the first species or strain. The organism dies, or the growth or proliferation of the microorganism is reduced, and the treatment method involves targeting the organism. When contacted with rease and nucleic acids, the nuclease is programmed to cleave the target site. This process includes a step in which the genome of the microorganisms contained in the target is cut, and the target microorganisms Biological infections are treated, Each nucleic acid codes for RNA that is expressed in the target, and the RNA is compounded with a nuclease. It embodys, programs nucleases, and cleaves target sites of microorganisms contained in the target. a composition.

[0029] The fourth structure A CRISPR / Cas system comprising a nuclease according to the present invention for use in a therapeutic method, The nuclease is a Cas nuclease (e.g., Cas3 or Cas9), and the system consists of one or more nucleases. It contains several guide RNAs or DNA encoding one or more guide RNAs, and each guide RNA is The Cas nuclease is programmed to cleave target sites contained in the genome of microorganisms. CRISPR / Cas-based systems that can be used for various purposes.

[0030] Fifth composition A method for treating a target microbial infection, wherein the microbial infection is a first species or bacterium. Caused by a strain of microorganism, the nuclease is produced by the genome of the microorganism that infected the target. It can be programmed to cut the included target site, thereby eliminating the first species or strain. The microorganisms are killed, or their growth or proliferation is reduced, and the treatment method targets When brought into contact with a nuclease, the nuclease is programmed to cleave the target site. The process includes cutting the genome of the microorganisms contained in the target microorganism. Methods of treating infectious diseases.

[0031] The sixth composition A method for treating a target microbial infection, wherein the microbial infection is a first species or bacterium. Caused by a strain of microorganism, the nuclease is produced by the genome of the microorganism that infected the target. It can be programmed to cut the included target site, thereby eliminating the first species or strain. The microorganisms are killed, or their growth or proliferation is reduced, and the treatment method targets When the nuclease is brought into contact with multiple viruses, the nuclease is designed to cleave the target site. This includes a programmed process that thereby cleaves the genome of the microorganisms contained in the target. The target microbial infection is treated, and each virus is subjected to R to express RNA in the target. The RNA contains a copy of the nucleic acid encoding NA, and the RNA complexes with a nuclease, and the nuclease The program cleaves the target site of microorganisms contained in the target, and the virus is affected by the target. A method that allows for the delivery of nucleic acids to microorganisms contained within a substance by infecting them.

[0032] The seventh component A method for treating a target microbial infection, wherein the microbial infection is a first species or bacterium. Caused by a strain of microorganism, the nuclease is produced by the genome of the microorganism that infected the target. It can be programmed to cut the included target site, thereby eliminating the first species or strain. The microorganisms are killed, or their growth or proliferation is reduced, and the treatment method targets When a nuclease is brought into contact with multiple nucleic acids, the nuclease is designed to cleave the target site. The process includes programming, which thereby cuts the genome of the microorganisms contained in the target. The target microbial infection is treated, and each virus produces RNA to express RNA in the target. The RNA, containing a copy of the coding nucleic acid, complexes with a nuclease, and the nuclease processes the protein. Gram is used to cleave the target site of the microorganisms contained in the target, and each nucleic acid is used to extract RNA from the target. It codes for the RNA to be expressed, the RNA complexes with a nuclease, and the nuclease promotes A method of graining and cleaving the target site of microorganisms contained in the target.

[0033] The eighth composition In the manufacture of a composition for carrying out the treatment method specified herein, the present invention The use of crease, multiple viruses, systems, guide RNA, DNA, or vectors, and the target The use refers to a living organism other than a human or animal.

[0034] The ninth composition Compositions for carrying out ex vivo or in vitro methods to treat microbial infections of substrates In the manufacturing process, the nuclease of the present invention, multiple viruses, systems, guide RNA, DNA, or vector The use of a microbial agent, in which microbial infection is caused by a first species or strain of microorganism, Clease works by cleaving target sites contained within the genome of the microorganism that has infected the target. It is programmable, thereby killing the first species or strain of microorganism, or the microorganism The growth or proliferation of is reduced, and the treatment method involves bringing the target into contact with the nuclease, - The process includes a step programmed to cut the target site, thereby affecting the target The genomes of the microorganisms contained within are cut, and the acute microbial infection of the substrate is treated.

[0035] The tenth composition In the production of compositions for carrying out ex vivo methods to treat microbial infections of substrates The use of a programmable nuclease, wherein microbial infection is caused by the first species or strain of microorganisms. Triggered by living organisms, the nuclease is contained within the genome of the microorganism that infected the target. It can be programmed to cut the target site, thereby removing the microorganisms of the first species or strain. The substance is killed, or the growth or proliferation of microorganisms is reduced, and the treatment method involves nucleating the target. The nuclease is brought into contact with the ase, and the nuclease is programmed to cleave the target site. It contains, and as a result, the genome of the microorganisms contained in the target is cut, causing acute microbial infection of the substrate. Use in which dyeing treatment is applied.

[0036] In any configuration, for example, the infection is an acute infection. For example, the infection is treated quickly. It is an acute infectious disease. For example, the infection is treated quickly, and for example, this method is for treatment Reduce infection rates by at least 1 / 100th within the first 30 minutes (for example, within the first 15 minutes). This includes the process of reducing infections. For example, the treatment is persistent, and for example, the reduction of infections is the initial stage of treatment. It continues for at least 30 minutes immediately after 30 minutes. Also, optionally, at least 1 / 100 or A reduction in infection rates to 1 / 1000th is achieved within at least 60 minutes after the start of treatment (e.g., at least 120 minutes). (For several minutes) This is maintained. Surprisingly, rapid death lasted for about 3 hours after the start of treatment, among other things. Case studies demonstrating the effectiveness are provided below. For example, this method improves the survival of the subject, or the survival of a human patient who has contracted an infection caused by the first species or strain of microorganism Improve the rate.

[0037] Furthermore, the present invention also applies to cancer or other separate therapies that must also be effective. This provides a solution to the need for effective treatment of the target pathogenic bacterial infection. The present invention further provides the following:

[0038] The 11th composition Pathogenicity of humans or animals caused by the first species or strain of bacteria (the first bacterium) A method for treating a bacterial infection, wherein a target site included in the genome of a first bacterium By cutting, the primary bacteria contained in the target are selectively killed, and the cutting is the target. Using a programmable nuclease programmed to cut the tissue The process involves the treatment of subjects who are suffering from further diseases or conditions other than pathogenic bacterial infections. The above method involves the process of administering therapy to a subject in order to treat or prevent further diseases or conditions. Nucleases, including those that treat infections, are used in the presence of programmed nucleases. A method that is effective in treating or preventing a disease or condition.

[0039] The 12th composition Pathogenic bacterial infections in cancer patients caused by the first species or strain of bacteria (first bacterium) A method for treating the disease, comprising cutting a target site contained in the genome of a first bacterium. This selectively kills the first bacterium present in the target, and the cleavage is performed by guide RNA. This is performed using a Cas nuclease programmed to cut the target site. The above method includes a step of applying immunotherapy to treat a patient's cancer. Nucleases treat infections, and immunotherapy involves the presence of programmed nucleases. Below are some methods that are effective in treating cancer.

[0040] The 13th composition A programmable nuclease for use in the method of the present invention.

[0041] The 14th composition CRIS Including a Nuclease in a Thirteenth Composition for Use in the Method of the Eleventh or Twelfth Composition It is a PR / Cas system, and the nuclease is a Cas nuclease (e.g., Cas3 or Cas9). The system encodes one or more guide RNAs (gRNAs) or one or more guide RNAs. Each guide RNA contains DNA and is designed to cleave a target site contained within the genome of the first bacterium. The CRISPR / Cas system allows for the programming of Cas nucleases.

[0042] The 15th composition Guide RNA or guide R for use in systems or methods for treating pathogenic bacterial infections DNA that codes for NA.

[0043] The 16th composition A nucleic acid vector containing guide RNA or DNA.

[0044] The 17th composition A first nucleic acid vector (or more thereof) encoding a nuclease, and a guide RNA encoding A pharmaceutical composition comprising a second nucleic acid vector (or a plurality thereof). [Brief explanation of the drawing]

[0045] [Figure 1] This figure shows the time-death curve of the Escherichia coli (EHEC) ATCC43888 strain containing the CGV line. (a) CRISPR induction killed 99.98% of the population in 30 minutes (black line). Growth in the absence of induction is shown by the dotted line. CRISPR was induced at time 0 and monitored up to 60 minutes. (b) Dilution series (101-106) of drop spots (5 μl) of E. coli ATCC43888 containing the CGV line on an LB agar plate. [Figure 2]This figure shows CRISPR-induced death of the target strain Escherichia coli (EHEC) ATCC43888 in Galleria mellonella larvae. G. mellonella larvae were delivered by injecting the bacteria behind the final left ventral leg. Approximately one hour after injection, a CRISPR inducer was administered behind the final right ventral leg. The larvae were incubated at 37°C for 2 hours and then sacrificed. In addition, a control group was injected with control bacteria carrying an off-target single-stranded guide RNA plasmid. [Figure 3] This figure shows the CRISPR-death curve of Escherichia coli (EHEC) ATCC43888 in Galleria mellonella. G. mellonella larvae were delivered by injecting the bacteria behind the final left ventral leg. Approximately one hour after injection, a CRISPR inducer was administered behind the final right ventral leg. Larvae were incubated at 37°C and sacrificed at 0, 1, and 2 hours after induction. [Figure 4] This figure shows the Kaplan-Meier survival curves of Galleria mellonella larvae infected with Escherichia coli (EHEC) ATCC43888. CRISPR induction significantly improved larval survival (black line) compared to off-target controls (dotted line) carrying off-target single-stranded guide RNA plasmids. [Figure 5] This figure shows the time-mortem curves of Escherichia coli Nissle 1917 containing a CGV system targeting pks. (a) CRISPR induction killed 99.98% of the population in 15 minutes (black line). Growth in the absence of induction is shown by the dotted line. CRISPR was induced at time 0 and monitored for 3 hours. (b) Dilution series (101-106) of drop spots (5 μl) of E. coli Nissle 1917 containing a CGV system on an LB agar plate. [Figure 6]This figure shows the time-death curves of Escherichia coli Nissle 1917 containing a CGV system targeting yapH. (a) CRISPR induction killed 99.98% of the population in 15 minutes (black line). Growth in the absence of induction is shown by the dotted line. CRISPR was induced at time 0 and monitored for 3 hours. (b) Dilution series (101-106) of drop spots (5 μl) on an LB agar plate of E. coli Nissle 1917 containing a CGV system 15 minutes after induction. [Figure 7] This figure shows the complete death of transzygous C. difficile cells. The figure demonstrates the complete and precise death of Clostridium difficile using a gRNA-encoded CRISPR array delivered from a probiotic carrier bacterial species via a conjugation plasmid as a vector. Carrier bacteria (E. coli donor strains containing the vector) mated with Clostridium difficile were killed upon delivery of the specified array. This utilized the endogenous Cas3 mechanism of Clostridium difficile. 100% death of Clostridium difficile cells was achieved, as shown in this figure. [Figure 8] Antibiotic treatment during ICI therapy is associated with fatal outcomes. Kaplan-Meier curves for overall survival in a validated cohort at Memorial Sloan Ketterin Cancer Center, including 239 patients with advanced NSCLC treated with anti-PD-L1 / anti-PD-1 mAbs and either receiving an antibiotic (ATB) two months prior to immune checkpoint blocker injections (ATB, n=68) or not (no ATB, n=171). Median overall survival in the absence of antibiotic treatment was 21.9 months, compared to 9.8 months with antibiotic treatment. Therefore, median overall survival in patients treated with classic antibiotics was <50% (or >12 months) shorter than median overall survival in patients not receiving antibiotic treatment. [Figure 9A]This figure shows that the gut microbiome modulates the effectiveness of anti-PD-1 inhibitors in melanoma patients (Gopalakrishnan et al., Science 2018, Vol. 359, pp. 97-103). [Figure 9B] This figure shows that the gut microbiome modulates the effectiveness of anti-PD-1 inhibitors in melanoma patients (Gopalakrishnan et al., Science 2018, Vol. 359, pp. 97-103). [Modes for carrying out the invention]

[0046] The method of the present invention differs from conventional antibiotic methods. In the present invention, the program Targeted cleavage of microbial genomes using modified nucleases is used, but conventional antibiotics Substances exert their activity through metabolic processes and cell replication cycles, as well as through their inhibition. It exists. The present invention, surprisingly, by focusing instead on nuclease cleavage, It achieves extremely rapid, efficient, and remarkably persistent microbial elimination. This has been demonstrated in the following experiments using different microorganisms, different nucleases, and different delivery methods. Surprisingly, 99-100% mortality was observed repeatedly, typically. Also, 3-4 log mortality was observed. However, this was achieved very rapidly and lasted for a long period.

[0047] The present invention relates to a method for treating or preventing microbial (e.g., bacterial) infections, and such The method provides means for carrying it out. In particular, it provides means for treating sepsis, sepsis, SIRS, or septic shock. Infections requiring rapid treatment, such as those causing acute conditions like flu, can be treated. As described herein, a rapid response is necessary in many settings to prevent microbial infections. Speed ​​is crucial for managing the situation. Speed ​​is essential in many infectious disease scenarios, including acute infections requiring hospitalization. The benefit of this invention is to reduce the spread, severity, or progression of the target infectious disease; infectious disease (e.g.) For example, reduction of the onset, severity, or progression of symptoms of sepsis or septic shock; in humans or animals This may involve one or more increases in the patient's chances of survival.

[0048] In this invention, programmable nuclease cleavage of the microbial genome is used. Cutting kills a wider range of microorganisms, such as several different species, as seen with conventional antibiotics. In contrast, selective microbial killing or growth or proliferation for the treatment or prevention of infectious diseases It provides a reduction. Selective killing leaves beneficial microorganisms that are not the target of treatment intact. It is advantageous to do so, and it may be beneficial to the patient. Furthermore, the inventors have discovered that it is surprising In some embodiments, substantial (several log) elimination can be achieved very quickly. For example, within 15 minutes, sustainable effects can be achieved (for example, longer than 1 hour). (over time) they found that. As illustrated below, the inventors were surprised In particular, it was possible to achieve a remarkable, immediate, and sustained killing effect for approximately 2-3 hours. .

[0049] Therefore, the present invention provides the following embodiments.

[0050] For use in methods to treat target microbial infections (e.g., acute microbial infections). A programmable nuclease for microbial infections, which is a first species or strain of microorganism. This is caused by the nuclease being contained in the genome of the microorganism that infected the target. It can be programmed to cut the target area, and the first species or strain of microorganism will be killed. , or the growth or proliferation of microorganisms is reduced, and the treatment method involves bringing the target into contact with the nuclease. The nuclease then includes a process in which it is programmed to cleave the target site, The genome of the microorganisms included in the target is cut, and the infection caused by the target microorganism is treated. A programmable nuclease.

[0051] Another aspect involves its use in methods for rapidly treating acute microbial (e.g., bacterial) infections. A programmable nuclease for use in microbial infections, the first species or fungus Caused by a strain of microorganism, the nuclease is produced by the genome of the microorganism that infected the target. It can be programmed to cut the included target site, thereby eliminating the first species or strain. The microorganisms are killed, or their growth or proliferation is reduced, and the treatment method targets When brought into contact with a nuclease, the nuclease is programmed to cleave the target site. The process includes cutting the genome of microorganisms contained in the target, and the acute microorganism of the target We provide a programmable nuclease for the rapid treatment of biological infections.

[0052] Another embodiment is for use in methods for treating infections caused by the target microorganism (e.g., bacteria). A programmable nuclease for microbial infections, which is a first species or strain of microorganism. This is caused by the nuclease being contained in the genome of the microorganism that infected the target. It is programmable to cut the target area, thereby the first species or strain of microorganism The microbial growth or proliferation is reduced, and the treatment method targets the nuclea Nucleases, and nucleic acids that program nucleases to recognize and cleave target sites, are used in conjunction with the nuclease. This process involves contact with the target, which in turn causes the genome of the microorganisms contained within the target to be cut. It provides a programmable nuclease that treats microbial infections.

[0053] Another aspect involves its use in methods for rapidly treating acute microbial (e.g., bacterial) infections. A programmable nuclease for use in microbial infections, the first species or fungus Caused by a strain of microorganism, the nuclease is produced by the genome of the microorganism that infected the target. It can be programmed to cut the included target site, thereby eliminating the first species or strain. The microorganisms are killed, or their growth or proliferation is reduced, and the treatment method targets , nuclease, and programming the nuclease to recognize and cleave the target site The process includes contacting nucleic acids, thereby cleaving the genome of microorganisms contained in the target. It provides a programmable nuclease that rapidly treats the target acute microbial infection. do.

[0054] Another aspect involves its use in methods for the sustained treatment of infections caused by the target microorganism (e.g., bacteria). A programmable nuclease for the purpose of treating microbial infections by targeting the first species or strain Caused by microorganisms, the nuclease is contained in the genome of the microorganism that infected the target. It can be programmed to cut the target site, thereby the first species or strain The microorganisms are permanently killed, or their growth or proliferation is reduced, and the treatment method is: The target is brought into contact with the nuclease, which is programmed to cleave the target site. This process includes a step in which the genome of the microorganisms contained in the target is cut, and the target We provide a programmable nuclease for treating microbial infections.

[0055] Another aspect involves its use in methods for the sustained treatment of infections caused by the target microorganism (e.g., bacteria). A programmable nuclease for the purpose of treating microbial infections by targeting the first species or strain Caused by microorganisms, the nuclease is contained in the genome of the microorganism that infected the target. It can be programmed to cut the target site, thereby the first species or strain The microorganisms are permanently killed, or their growth or proliferation is reduced, and the treatment method is: The target is programmed to recognize and cleave the nuclease, as well as the target site. The process includes contacting the nucleic acid to be lambed, thereby affecting the genome of the microorganisms contained in the target. It is cleaved and provides a programmable nuclease that treats the target microbial infection. .

[0056] Another aspect is a method for the sustained treatment of an acute microbial (e.g., bacterial) infection of a target. A programmable nuclease for use, which is used to treat microbial infections by the first species or Caused by the microorganism of the bacterial strain, the nuclease is derived from the genome of the microorganism that infected the target. It is programmable to cut the target site contained within, and the microorganism of the first species or strain. The microorganisms are permanently killed, or their growth or proliferation is reduced, and the treatment method targets the target. , nuclease, and programming the nuclease to recognize and cleave the target site The process includes contacting nucleic acids, thereby cleaving the genome of microorganisms contained in the target. The invention provides a programmable nuclease that treats the target acute microbial infection.

[0057] Surprisingly, as illustrated below, using nucleases (conventional anti In contrast to conventional methods for killing living organisms, it has a sustained effect of several log (e.g., 3 or 4 log) This was observed approximately 3 hours after the first contact between the programmed nuclease and the bacteria. In this aspect of the present invention, programmed nucleases (that is, programmed nucleases) Rammed nucleases, programmable nucleases, and / or nucleases (Low-frequency administration of nucleic acids for programming) and drug regimens for lower-frequency contact This makes it possible to program Cas and gRNA (or gRNA) to program nucleases. The DNA being programmed is processed by a programmable nuclease at the first time point (T1) and the second time point (T2). For example, administered to the target along with Cas9 or Cas3, or gRNA (or DNA encoding gRNA) This process involves the endogenous Cas nuclease (e.g., Cas9 or Cas3) of the first species or strain of bacteria. Administered in T1 and T2 to gram, programmed endogenous Cas is used to modify the bacterial genome. Cutting the bacteria kills them or reduces their growth or proliferation, thus treating the infection. Such less frequent medication is convenient for healthcare workers and patients, and And economic therapy is provided. Therefore, optionally, nucleases and / or nucleic acids are T The drug is administered to the target in T1 and T2, and T2 is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 of T1. , 13, 14, or 24 hours later. For example, T2 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 of T1. It is 12, 13, 14, or 24 hours later. For example, T2 is 2 to 7 hours after T1. For example, T2 is For example, T2 is 1 hour after T1. For example, T2 is 2 hours after T1. For example, T2 is 3 hours after T1. For example, T2 is 4 hours after T1. For example, T2 is 5 hours after T1.

[0058] Optionally, a nuclease (e.g., a programmed nuclease) and / or a target region. Nucleic acids that program nucleases to recognize and cleave specific sites are introduced into the target at T1 and T2. Given, T2 is at least one hour after T1 (e.g., 1, 1.5, 2, 2.5, or 3 hours).

[0059] Another embodiment is for use in methods for treating infections caused by the target microorganism (e.g., bacteria). Cas nuclease, and microbial infections are caused by the first species or strain of microorganism. Nucleases then cleave target sites contained in the genome of the microorganism that has infected the target. It is programmable together with guide RNA (gRNA), thereby allowing for the microscopic development of the first species or strain. The organism dies, or the growth or proliferation of the microorganism is reduced, and the treatment method is applied to the subject as described above. The process includes the step of administering nucleic acids, the nucleic acids being gRNA or DNA encoding gRNA, thereby Depending on the target, the nuclease is programmed to recognize and cleave the target site of the microorganisms contained in the target. This causes the genome of the microorganism to be cut, thereby treating the target microbial infection, and the above method The method includes the step of administering nucleic acids to a subject at a first time point (T1) and a second time point (T2), thereby The subjects were T1 and T2, which came into contact with the programmed nuclease, and T2 was in contact with T1 for more than 1 hour. The Cas nuclease is provided later.

[0060] Optionally, T2 is 2 hours or more after T1; optionally, T2 is 3 hours or more after T1. ;Optionally, T2 is 4 hours or more after T1;Optionally, T2 is 5 hours or more after T1 ;Optionally, T2 is 6 hours or more after T1;Optionally, T2 is 7 hours or more after T1 ;Optionally, T2 is 8 hours or more after T1;Optionally, T2 is 9 hours or more after T1 Optionally, T2 is 10 hours or more after T1; Optionally, T2 is 11 hours or more after T1 Yes; optional, T2 is 12 hours or more after T1; optional, T2 is 13 hours or more after T1 And; optionally, T2 is 14 hours or more after T1; optionally, T2 is 24 hours or more after T1. It is later. In addition or instead, optionally, T2 is less than or equal to 7 hours after T1; Optionally, T2 is less than or equal to 12 hours after T1; optionally, T2 is less than or equal to 24 hours after T1. ;Optionally, T2 is 2 to 7 hours after T1; Optionally, T2 is 24 hours after T1; Optionally, T2 is 7 hours after T1; optionally, T2 is 6 hours after T1; optional Then, T2 is 5 hours after T1; optionally, T2 is 4 hours after T1; optionally, T2 is , 3 hours after T1; optionally, T2 is 2 hours after T1; optionally, T2 is 1 It is a certain amount of time. For example, T2 is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 of T1, or This is 24 hours later. For example, T2 is 1 to 7 hours after T1; or T2 is 2 to 7 hours after T1. or T2 is 3-7 hours after T1; or T2 is 4-7 hours after T1; or T2 is 5- It is 7 hours later; or T2 is 6-7 hours after T1.

[0061] Optionally, this method involves testing for symptoms within the first 30 minutes of treatment (for example, within the first 15 minutes). The method includes a step to reduce the infection to at least 1 / 100th. Optionally, this method is used as the first step in treatment. Reduce infection rates by at least 1 / 1000 within 30 minutes (for example, within the first 15 minutes). This includes the process. Optionally, this method may be performed within the first 30 minutes of treatment (for example, the first 15 minutes). The process includes reducing the infection rate to at least 1 / 10,000th of the original rate by [date / time].

[0062] Optionally, this method reduces infection immediately after the first 30 minutes of treatment and continues for 30 minutes. The method includes steps to reduce infection for sustained results. Optionally, the method includes at least 100 A 1 / 1 reduction in infections is maintained for 30 minutes immediately after the first 30 minutes of treatment. The process includes steps to reduce infectious diseases. Optionally, this method reduces infectious diseases by at least 1 / 1000. Reduce infection so that the reduction lasts for 30 minutes immediately after the first 30 minutes of treatment. The process includes the following steps. Optionally, this method reduces infection rates by at least 1 / 10,000, making it the most effective treatment. The process includes steps to reduce infection immediately after the first 30 minutes, and to continue for another 30 minutes.

[0063] Optionally, this method involves testing for symptoms within the first 30 minutes of treatment (for example, within the first 15 minutes). The process includes steps to reduce the infection by at least 1 / 100th, and the reduction of the infection by at least 1 / 100th is This lasts for 30 minutes immediately after the first 30 minutes of treatment. Optionally, this method is used for treatment. Within the first 30 minutes (for example, within the first 15 minutes), reduce the infection rate to at least 1 / 1000th. The process includes reducing the number of infections by at least 1 / 1000, and the reduction is immediate after the first 30 minutes of treatment. It lasts for 0 minutes. Optionally, this method can be used within the first 30 minutes of treatment (for example, (Within the first 15 minutes) the process includes reducing the infection rate to at least 1 / 10,000th of the original rate. A reduction of at least 1 / 10,000 is observed immediately after the first 30 minutes of treatment and lasts for 30 minutes.

[0064] Optionally, this method involves bringing the subject into contact with a programmed nuclease, followed by a small amount of... For at least 60 minutes (for example, at least 120, 145, or 180 minutes), the infection was less severe. Both include a process that maintains a 1 / 100 reduction. Optionally, at least 1 / 100 of infections Reduction is achieved by exposing the subject to the programmed nuclease for at least 60 minutes (e.g.) For example, it is maintained for at least 120, 145, or 180 minutes. Optionally, this method After bringing the subject into contact with the programmed nuclease, leave it for at least 60 minutes (for example, A reduction of at least 1 / 1000 in infections over a period of at least 120, 145, or 180 minutes. Includes maintenance processes. Optionally, a reduction of at least 1 / 1000th of infections is targeted to the program. After contact with the programmed nuclease, leave for at least 60 minutes (e.g., at least 120 minutes) It is maintained for 145 or 180 minutes.Optionally, this method is used to program the subjects. After contact with the nuclease, allow to breathe for at least 60 minutes (for example, at least 120, 145 minutes). The process includes maintaining a reduction of at least 1 / 10,000 of the infection rate over a period of 180 minutes or longer. . Optionally, a reduction of at least 1 / 10,000 in infections will target the programmed NUK After contact with rease, leave for at least 60 minutes (for example, at least 120, 145, or 180 minutes) It is maintained over the course of ( ).

[0065] Optionally, this method involves testing for symptoms within the first 30 minutes of treatment (for example, within the first 15 minutes). The process includes steps to reduce the infection by at least 1 / 100th, and the reduction of the infection by at least 1 / 100th is The subject was exposed to the programmed nuclease for at least 60 minutes (for example, a short period of time). It is maintained for at least 120, 145, or 180 minutes. Optionally, this method is used for treatment. Within the first 30 minutes (for example, within the first 15 minutes), reduce the infection rate to at least 1 / 1000th. The process includes reducing the number of infections by at least 1 / 1000, and the target is programmed. After contact with the nuclease, leave for at least 60 minutes (for example, at least 120, 145, or 180 minutes) It is maintained over the course of (for example, (Within the first 15 minutes) the infection rate is reduced to at least 1 / 10,000, including the reduction of infection rates. A reduction of at least 1 / 10,000 is achieved after the subject is brought into contact with the programmed nuclease. It is maintained for at least 60 minutes (for example, at least 120, 145, or 180 minutes).

[0066] Optionally, this method reduces the infection to at least 1 / 10,000 within the first 15 minutes of treatment. The process includes a reduction step, and the reduction of infections by at least 1 / 10,000 is programmed to target... This is maintained for at least 45 minutes after contact with the nuclease. Examples are shown.

[0067] In the example, the infection was treated continuously, and at least a 1 / 100 reduction in the infection occurred after treatment commencement. It is maintained for at least 60 minutes thereafter (for example, at least 120, 145, or 180 minutes). In the example, the infection was continuously treated, and the infection was reduced by at least 1 / 1000th, indicating that treatment was effective. It is maintained for at least 60 minutes after initiation (for example, at least 120, 145, or 180 minutes). In the example, the infection was continuously treated, and the infection was reduced by at least 1 / 10,000. Maintained for at least 60 minutes after initiation (for example, at least 120, 145, or 180 minutes) It can be done.

[0068] Optionally, infections are reduced to at least 1 in 100,000 within the first 30 or 45 minutes of treatment. It is reduced. Optionally, infections are reduced to at least 100,000 within the first 30 or 45 minutes of treatment. It is reduced to 1 / 0, and the reduction is maintained up to 60 minutes into treatment.

[0069] Optionally, infections are reduced to at least 1 in 1,000,000 within the first 30 or 45 minutes of treatment. It is reduced. Optionally, infections are reduced to at least 1000,000 within the first 30 or 45 minutes of treatment. The level is reduced to 1 / 00th, and this reduction is maintained for up to 60 minutes into the treatment.

[0070] Optionally, infections are reduced to at least 1 / 100th within the first 15 minutes of treatment. Optionally, infection rates are reduced to at least 1 / 1000th within the first 15 minutes of treatment. Optionally, infections should be reduced to at least 1 / 100th within the first 15 minutes of treatment. It will be reduced to at least 1 / 1000th within 30 minutes.

[0071] For example, the reduction is maintained for at least 15 minutes, and for example, the infection is treated. The reduction is reduced to at least 1 / 100 or at least 1 / 1000 within the first 15 minutes, and the reduction is This is maintained from 15-30 minutes into the treatment, or from 15-45 minutes into the treatment, or from 15-60 minutes into the treatment.

[0072] For example, an infection is treated within the first 15 minutes, and within the first 15 minutes of treatment, at least 1 / 100th or The dose is reduced to at least 1 / 1000 or at least 1 / 10000, and the reduction occurs 15-30 minutes into the treatment. Alternatively, it can be maintained from 15 to 45 minutes into the session.

[0073] Optionally, this method involves testing for symptoms within the first 30 minutes of treatment (for example, within the first 15 minutes). The process includes reducing the infection rate to at least 1 / 100th of the original level.

[0074] Optionally, this method involves testing for symptoms within the first 30 minutes of treatment (for example, within the first 15 minutes). The process includes reducing the infection rate to at least 1 / 1000th of the original level.

[0075] Optionally, this method involves testing for symptoms within the first 30 minutes of treatment (for example, within the first 15 minutes). The process includes reducing the infection rate to at least 1 / 10,000th of the original level.

[0076] Optionally, this method is used when the reduction of infection is immediately after the first 30 minutes of treatment and continues for 30 minutes. The process includes steps to reduce infection, for example, reduction is less after the first 30 minutes of treatment. Both effects can last for up to 60 minutes. If treatment is administered at time zero (T0), the reduction in infection is It may exist at 60 minutes after T0, and in fact, it may persist for 60 minutes after that. In Figures 1(a), 5(a), and 6(a), for example, the reduction is observed 60 to 180 minutes after T0. In the selection process, the reduction in infection persists for at least 30 minutes after the first 30 minutes of treatment.

[0077] In the example, the infectious disease is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% within, for example, the first 15 minutes of treatment. In the example, the infectious disease is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90 , 91, 92, 93, 94, 95, 96, 97, 98, or 99% within, for example, the first 30 minutes of treatment.

[0078] To determine the killing or reduction of growth or proliferation of the target microorganism, for example, (i) in a sample taken from the subject immediately before the start of treatment (e.g., a blood, intestine, or leaf sample), and (ii) the difference in the number of microorganisms of the first species or strain in a sample taken from the subject at the 30 minute time point of treatment (a sample of the same type as the sample in (i), e.g., a blood, intestine, or leaf sample respectively) can be determined. For example, if the microorganism is a bacterium, the sample can be evaluated in terms of the difference in colony forming units (CFU) / ml of the sample when, for example, the sample is plated on the agar of a corresponding Petri dish and incubated under the same conditions. In another example, microscopic counting of microorganisms in the sample or other routine methods known to those skilled in the art may be used. In the example, at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% killing of the microorganism is achieved by the first 30, 60, 90, or 120 minutes of treatment (e.g., by the first 30 minutes, or by the first 120 minutes). For example, if the subject is a human or an animal, the killing is determined by the spread of microorganisms (e.g., bacteria) in a blood sample taken immediately before the start of treatment (e.g., by standard colony counting method on an agar plate), compared to a blood sample taken 15 or 30 minutes after the start of treatment.

[0079] In the example, at least 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% killing of the microorganism is achieved by the first 30, 60, 90, or 120 minutes of treatment (e.g., by the first 30 minutes, or by the first 120 minutes). For example, if the subject is a human or an animal, the killing is determined by the spread of microorganisms (e.g., bacteria) in a blood sample taken immediately before the start of treatment (e.g., by standard colony counting method on an agar plate), compared to a blood sample taken 15 or 30 minutes after the start of treatment. Determined by comparison with the sample. In the example, at least 80, 90, 91, 92, 93, 94, 95 of the microorganisms. 96, 97, 98, or 99% elimination is achieved within the first 0.5, 1, or 2 hours of treatment. This means that at least 99% of the microorganisms are killed within the first 30 minutes of treatment. For example, At least 99% of the microorganisms are killed within the first two hours of treatment. In some cases, 100% are killed. Elimination is achieved. These are illustrated below. In one embodiment, less than 100% of the microorganisms are eliminated. They die.

[0080] Examples of bacterial elimination are shown below. Surprisingly, programmed nuclei... When using the enzyme to target selected bacteria, specific cleavage leads to the rapid death of the target bacteria. This resulted in at least 3 or 4 log deaths (i.e., 1,000 or 10,000 times the death rate), extremely We were able to observe this in a short period of time. Surprisingly, these were observed over at least one hour. This was maintained. Optionally, infections were reduced within the first 15, 30, or 45 minutes of treatment. Both are reduced to 1 / 1000th. Optionally, infection is treated during the first 15, 30, or 45 minutes of treatment. The reduction is reduced to at least 1 / 1000th by that point, and the reduction is maintained until 60, 120, or 180 minutes into treatment. It is done. Optionally, infections are treated with at least 10,000 smears within the first 15, 30, or 45 minutes of treatment. It is reduced to 1 / 00th. Optionally, infection is reduced within the first 15, 30, or 45 minutes of treatment. The reduction is at least 1 / 10,000th, and the reduction is maintained for 60, 120, or 180 minutes of treatment. For example, please refer to the case example in Figure 5a.

[0081] In this example, 100% elimination is achieved within 24 hours of the start of treatment.

[0082] In this example, the infection was less severe for 2 hours or longer (e.g., 2-3 hours). Both are reduced to 1 / 1000th. Additionally, optionally, infections are reduced during the first 15 or 13 minutes of treatment. It will be reduced to at least 1 / 1000th by then.

[0083] In this example, the infection was less severe for 2 hours or longer (e.g., 2-3 hours). Both are reduced to 1 / 10,000th. Additionally, optionally, infections are treated within the first 15 or 13 minutes of treatment. It will be reduced to at least 1 / 10,000th by then.

[0084] In the example, the infection lasted for one hour, or for one hour or longer. or for a period of 2 hours or longer (for example, 2-3 hours), at least 90, 9 The infection rate is reduced by 1, 92, 93, 94, 95, 96, 97, 98, or 99%. Optionally, infection rates are reduced by 1 hour. For a period of time, or for a period of time of one hour or longer, or for two hours or longer Also, over a long period (e.g., 2-3 hours), and optionally within the first 30 minutes of treatment (e.g.) For example, within the first 15 minutes, it is reduced by at least 90%. Optionally, infection can be treated for 1 hour. Or for a longer period of time, and within the first 30 minutes of treatment (for example, the first 15 minutes) (By the time of treatment) the infection is reduced by at least 90%. Optionally, infection is treated within the first 15 or 13 minutes of treatment. By the time of the event, it will be reduced by at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%. Optional In the selection, the infection should be at least 90, 91, 92, 93, 94 within the first 15 or 13 minutes of treatment. Reduced by 95, 96, 97, 98, or 99%, and the reduction lasts for one hour or longer (e.g.) For example, for 2 hours or longer, or for 3 hours or longer or for about 2 hours, or for 2 hours, or for about 3 hours, or for 3 hours). Exemplary conditions are provided below, and the bacteria is E. coli .

[0085] Optionally, the subject is a human or an animal, the microorganism is a bacterium (e.g., E. coli or C. difficile), and the bloodstream infection of the subject by the bacterium is reduced by at least 1 / 100 (e.g., by at least 1 / 1000) by the first 30 minutes of treatment (e.g., by the first 15 minutes of treatment). Optionally, the subject is a human or an animal, the microorganism is a bacterium (e.g., E. coli or C. difficile), and the bloodstream infection of the subject by the bacterium is reduced by at least 1 / 1000 by the first 30 minutes of treatment (e.g., by the first 15 minutes of treatment). Optionally, the subject is a human or an animal, the microorganism is a bacterium (e.g., E. coli or C. difficile), and the bloodstream infection of the subject by the bacterium is reduced by at least 1 / 10,000 by the first 30 minutes of treatment (by the first 15 minutes of treatment). Optionally, the E. coli is EHEC E. coli.

[0086] Optionally, the programmed nuclease (e.g., Cas9 or Cas3) is capable of cleaving the target site contained by E. coli (EHEC) ATCC 43888. Optionally, the programmed nuclease (e.g., Cas9 or Cas3) is capable of cleaving the target site contained by E. coli Nissle. Optionally, the present invention is a substrate-expanded beta-lactamase Kill E. coli and / or Klebsiella (e.g., K. pneumoniae) that produce extended-spectrum beta-lactamase (ESBL). Therefore, the present invention may be for treating or preventing ESBL bacterial infections in a subject, such as a human. For example, the infection may be a urinary tract infection (UTI) or septicaemia. In the example, the bacteria are resistant to one or all of cephalosporins, penicillins, fluoroquinolones, trimethoprim, and tetracyclines. In the example, the bacteria are carbapenem-resistant E. coli or Klebsiella.

[0087] Optionally, the subject's blood is infected with bacteria at 10 ~10 12 CFU / ml (e.g., 10 7 ~10 11 10 7 ~10 <00所求00994> 10 10 7 ~10 9 10 7 ~10 8 CFU / ml) just prior to treatment.

[0088] The following examples show improved survival when using the method of the present invention in an in vivo model. Therefore, in the example, the method of the present invention is for improving the survival of a subject by treating the subject's acute microbial infection. In the example, the programmed nuclease herein can effect the killing of bacteria of a first species or strain in a Galleria mellonella larva in vivo infection model.

[0089] The nuclease may be, for example, a DNase (e.g., Cpf1, Cas9, or Cas3) or an RNase (for example For example, Cas13b) may also be used. In this example, the nuclease is a class 1 nuclease. So, the nuclease is a class 2 nuclease. In the example, the nuclease is a type I nuclease. Reases, for example, IA, IB, IC, ID, IE, IF, or IU type nucleases. In the example, nu Clease is a type II nuclease, for example, IIA, IB, or an IC type nuclease, for example, C These are as9, spCas9, or saCas9. In the example, the nuclease is a type III nuclease, for example. These are type IIIA, IIIB, IIIC, or IIID nucleases. In this example, the nuclease is type IV nuclease. The nuclease is, for example, type IVA or type IVB nuclease. In this example, the nuclease is type V. A nuclease, for example, cpf1. In this example, the nuclease is a type VI nuclease, for example Cas13, for example, Cas13a, Cas13b, Cas13c, or Cas13d.

[0090] For example, the nuclease is type IA Cas3.

[0091] For example, nucleases include IB-type Cas3, such as Clostridium (e.g., C dificile)Cas3. That is the case.

[0092] For example, the nuclease is an IC-type Cas3.

[0093] For example, the nuclease is an ID-type Cas3.

[0094] For example, nucleases include IE-type Cas3, e.g., E coli or Pseudomonas (e.g., Paerugi) (nosa)Cas3.

[0095] For example, nucleases include IF-type Cas3, such as Pseudomonas (e.g., P aeruginosa) Cas3. That is the case.

[0096] For example, the nuclease is IU-type Cas3.

[0097] In this example, the nuclease is Cas6, for example, Cas6f.

[0098] In the example, the nuclease is an isolated or recombinant nuclease. For example, nuclease This is a synthetic or non-natural nuclease. In this example, the nuclease is nicasse.

[0099] In the example, the nuclease is ex vivo, for example, in vitro. In the example, the nucleic acid is ex vivo In this example, the guide RNA or the DNA encoding the guide RNA is ex vivo, for example. It is in vitro.

[0100] Optionally, the nuclease is a Cas nuclease (e.g., Cpf1, CasX, CasY, Cas13b, Cas3 or Cas9), meganuclease, TALEN (transcription activator-like effector nuclease) Cas is a zinc finger nuclease. For example, Cas is Streptococcus (for example). ,pyogenes or aureus)Cas9, Clostridium (e.g., dificile), Salmonella (e.g., typ It is himurium, or E coli Cas3. For example, Cas is spCas. In the example, Cas9 is Ca It is combined with tracrRNA or DNA encoding tracrRNA that can work with s. For example, tr acrRNA is of the same species as Cas, for example, S pyogenes tracrRNA or the DNA that encodes it. .

[0101] In the example, the nuclease is a nucleic acid containing SEQ ID NO: 9, or at least 80, 85, 90, 9 Cas3 is encoded by sequences that are 5, 96, 97, 98, or 99% identical. Also, optional. So, is the bacterium Clostridium (e.g., C dificile) bacterium, or is it listed in Table 1 (Tables 1-29)? Any Clostridium shown is an example. This is illustrated below.

[0102] In the example, the nuclease is a nucleic acid containing SEQ ID NO: 10, or at least 80, 85, 90, Cas9 encoded by sequences that are 95, 96, 97, 98, or 99% identical. In the example, Rease is a nucleic acid containing SEQ ID NO: 11, or at least 80, 85, 90, 95, 96, 97, 98 , or Cas9 encoded by a sequence that is 99% identical. Also, optionally, bacteria are C It is a lostridium (e.g., C dificile) bacterium, or one of the bacteria shown in Table 1 (Tables 1-29). Any Clostridium species is used. Additionally, the bacterium is optionally E coli (e.g., EHEC). This is illustrated below.

[0103] Optionally, this method uses RNA or nucleic acids that encode RNA to express RNA in the target. For example, the process includes administering a substance (such as DNA) to a target, and the RNA complexes with a nuclease, and the nuclease The enzyme is programmed to cleave the target site of the microorganisms contained in the target material.

[0104] Optionally, nucleases are administered to the target simultaneously with or sequentially with RNA or nucleic acids.

[0105] Optionally, the target may include a nuclease before administering RNA or nucleic acid to the target. For example. The nuclease is the endogenous Cas nuclease of the bacterial cell of the first species or strain contained in the subject. It is a Cas nuclease. Therefore, in this example, it is administered to RNA or nucleic acids. Furthermore, endogenous Cas contained in bacteria can be introduced into bacteria to program them. This leads to the formation of programmed Cas nucleases that cleave target sites in the bacterial genome. This results in the death of bacteria or a reduction in bacterial growth or proliferation, and therefore The infection is treated or prevented.

[0106] Multiple viruses (e.g., phages or phagemids) are administered to the target subject at their discretion. Each virus contains copies of nucleic acid (e.g., one or more copies), Russ infects microorganisms present in the target and delivers nucleic acids to them. For example, this specification The virus in this book infects (or is capable of infecting) the first species or strain of bacteria. It is a diphagemide.

[0107] The ratio of viruses to microorganisms administered is optional and varies from 10 to 150 depending on the target. For example, microorganisms are bacteria, and the ratio is 10 to 100, that is, 1 to 100, for example, 10 to 100. This is the MOI (memory of interest) (for example, whether a virus is replicable, for example, whether it is a phage). (If not a phagemid). The ratio is derived, for example, from a human or animal subject immediately before treatment. Using a sample (e.g., blood sample or intestinal sample), microorganisms (e.g., per 1 ml of blood sample or intestinal sample) The number of bacteria can be determined. Then, the virus to be administered can be determined. The amount can be calculated according to the determination made using the sample.

[0108] By choice, microorganisms are bacteria. Alternatively, microorganisms are archaea. Instead Instead, microorganisms are viruses. Instead, microorganisms are fungi. Instead, microorganisms The substance is algae. Instead, the microorganism is a protozoan.

[0109] In the example, the subject is a human, and the infection is a hospital-acquired infection. In the example, the subject is a plant, and the subject is a yeast. They are protists or amoebas.

[0110] The subjects are selected at will, and include humans (e.g., adults, children, newborns, infants, teenagers). (male or female), or animals (e.g., dogs, cats, horses, cows, sheep, goats, salmon, (Chickens, turkeys, pigs, companion animals, or domesticated animals).

[0111] In the example, the subjects are humans or animals, and selection is optional. The infectious disease affects the lungs, abdomen, or urinary tract. It is an infectious disease. For example, the subjects include urinary tract infections, lung infections such as pneumonia, kidney infections, or abdominal infections. The subject is suffering from an infectious disease. In this example, the subject is a surgical patient. In this example, the subject is a burn patient. In this example, the subject has an infected wound (e.g., a wound infected with bacteria). In this example, the patient has AIDS. They have a disease or are infected with HIV. For example, the subject is a blood cancer, such as leukemia. For example, they have cancer such as AML, CML, CLL, or lymphoma. This refers to patients who have received tissue or organ transplants, such as hematopoietic stem cell transplants or bone marrow transplants. In the example, the subject has a urinary catheter or intravenous catheter. In the example, the subject has, The patient is on a mechanical ventilator. In this example, the patient is receiving an immunosuppressant. It is being received. In the example, the subject has pneumonia. In the example, the subject is in the intensive care unit (I In this example, the subject is a patient with acute respiratory distress syndrome (ARDS). This includes meningitis, pregnancy infections, and gallbladder rupture (gallbladder rupture is a medical condition in which the gallbladder leaks or ruptures). This is the condition. Rupture is generally caused by inflammation of the gallbladder, abortion, septic shock ( Abortion-induced septic shock is an acute, life-threatening illness), endometritis (endometritis is a common illness). (Usually an inflammation of the endometrium due to infection), acute respiratory distress syndrome (acute respiratory distress syndrome) This is a lung condition (which occurs when the alveoli become filled with fluid), or when suffering from cellulitis. ru.

[0112] The average age of the population is increasing, more people have chronic diseases, and they are under immunosuppressants. The increase in the number of invasive procedures performed is linked to an increase in the sepsis rate. The subjects are those who have undergone surgery, are on immunosuppressant therapy, and / or I have a chronic illness.

[0113] Participants are selected on an optional basis, and the subjects are individuals older than 60, 65, 70, 75, or 80 years of age, or children. The patient is a medical patient. In alternative forms, the target is pediatric patients (e.g., human infants or children) or adolescents. The subject is a young person. In this example, this method treats or prevents neonatal sepsis. The subjects are immunocompromised individuals suffering from acute viral infections such as HIV infection, or Is it an animal, or does the subject have cancer, such as blood cancer like leukemia, or The patient is, for example, a transplant patient who has received an organ transplant, tissue transplant, or bone marrow transplant. For example, the subjects are humans who are positive for Gram-negative bacterial lipopolysaccharide or lipid A. It is an animal. In the example, the subject is a Gram-positive bacterial cell wall that is positive for lipoteichoic acid. It is either a to or an animal.

[0114] Optionally, this method is used to treat sepsis and / or sepsis (e.g., septic shock) To treat or prevent.

[0115] The SIRS (Systemic Inflammatory Response Syndrome) criteria are used to define sepsis. SIRS can manifest in two or more ways: abnormal body temperature, heart rate, respiratory rate, or This includes blood gases and white blood cell count. Sepsis is, for example, SIRS in response to an infection process. Severe sepsis is, for example, sepsis-induced organ dysfunction or sepsis with reduced tissue blood flow. Septic shock is a condition that manifests as low blood pressure, elevated lactate levels, or decreased urine output. Furthermore, despite severe sepsis and intravenous fluid administration, the patient's blood pressure remains persistently low.

[0116] In this embodiment, the method is used to treat end-organ dysfunction in the subject (when the subject is a human or animal). To prevent or delay the progression of the disease.

[0117] Examples of end-organ dysfunction include the following: ·Lungs: Acute respiratory distress syndrome (ARDS) (PaO2 / FiO2<300) • Brain: Encephalopathy symptoms including excitement, confusion, and coma; causes include ischemia, hemorrhage, and blood clots in the microvessels. These include formation, microabscesses, and multiple necrotizing leukoencephalopathy. • Liver: Destruction of protein synthesis function leads to progressive breakdown of blood coagulation due to inability to synthesize coagulation factors. It manifests acutely as a breakdown, and the disruption of metabolic function is linked to bilirubin metabolism disorders, and unconjugated bilirubin This results in an increase in serum bilirubin levels. • Kidneys: Low urine output and anuria, electrolyte abnormalities, or volume overload • Heart: Systolic and diastolic heart problems likely caused by chemical signals that impair muscle cell function. Failure and cell damage manifest as troponin leakage (though not necessarily ischemic in nature).

[0118] A more specific definition exists for end-organ dysfunction in relation to SIRS in children. • Cardiovascular dysfunction (after fluid resuscitation with at least 40 ml / kg of crystalloid fluid) • Blood pressure at the <5th percentile for each age group, or a contraction of <2 standard deviations below the normal range for each age group. Hypotension showing a circumstance, or • The need for a vasopressor, or The following two criteria: • Metabolic acidosis of unknown cause with a base deficiency > 5 mEq / l • Lactic acidosis: Serum lactate levels twice the upper limit of normal. • Oliguria (urine volume <0.5 ml / kg / h) • Long-term capillary refill time > 5 seconds • Core-to-periphery temperature difference > 3°C • Respiratory dysfunction (in the absence of cyanotic heart disease or known chronic lung disease) • The ratio of arterial oxygen partial pressure to the oxygen fraction in the inhaled gas (PaO2 / FiO2) is <300 (indicating acute lung injury). Definition), or • Arterial carbon dioxide partial pressure (PaCO2) relative to baseline PaCO2 is >65 torr (20 mmHg) (hypercapnic respiration) Evidence of failure), or • The need for oxygen supplementation greater than FiO20.5 to maintain oxygen saturation ≥92% • Neurological dysfunction • Glasgow Coma Score (GCS) ≤ 11, or • A GCS score of 3 points or more in individuals with developmental delay / intellectual disability. The following changes in mental state • Hematological dysfunction ·Platelet count<80,000 / mm 3 Or a 50% reduction from the maximum value of chronic thrombocytopenia. • International Normalized Ratio (INR) > 2 ·Disseminated intravascular coagulation • Renal dysfunction • Serum creatinine levels are ≥2 times the upper limit of normal for their age, or they have chronic kidney disease. A twofold increase in baseline creatinine in people • Liver dysfunction (applicable only to infants > 1 month old) • Total serum bilirubin ≥ 4 mg / dl, or • Alanine aminotransferase (ALT) levels are ≥2 times the upper limit of normal.

[0119] Table 2 (Table 30) shows the criteria for a positive diagnosis of sepsis.

[0120] Optionally, this method is used to detect fever, hypothermia, rapid breathing, increased heart rate, confusion, and metabolic abnormalities. Sidosis, respiratory alkalinity, hypotension, blood coagulation dysfunction (in one or more organs or To reduce one or more symptoms in the patient, selected from subcutaneous bleeding (blood clotting, etc.) and edema. Optionally, this method reduces septic shock. Optionally, sepsis is used for severe sepsis. It is a psis.

[0121] Optionally, at the start of treatment, the subject (e.g., human) must have a body temperature of <36°C or >38°C and >90°C / min. Heart rate >20 breaths / min or PaCO2 <4.3kPa, and <4000 / mm 3 or >12,000 / mm 3 White It has a blood cell count.

[0122] Optionally, at the start of treatment, the subject (e.g., human) may have abnormal body temperature, abnormal heart rate, or other abnormalities. It shows the presence of two or more abnormalities: normal respiratory rate, abnormal blood gases, and abnormal white blood cell count. vinegar.

[0123] The subject is optional, and it must be a plant. In this example, the subject is a protist, such as an amoeba. Optionally, in this example, microorganisms are viruses (e.g., large viruses or giant viruses). Viruses (e.g., Mimivirus). Nucleases are, for example, Cas, and viral microorganisms. It is programmable using guide RNA delivered by virophages that infect objects. ru.

[0124] In this example, the microorganism is yeast, such as Candida.

[0125] Preferably, the microorganism is a bacterium. Optionally, the bacterium is a Gram-positive bacterium. Optional The selected bacteria are Staphylococcus, Streptococcus, Enterococcus, Legionella, and Heamoph. ilus, Ghonnorhea, Acinetobacter, Escherichia, Klebsiella, Pseudomonas, or Steno Trophomonas bacteria (e.g., E. coli (e.g., EHEC E. coli), C. dificile, V. cholera, Stap) Hylococcus (e.g., S aureus or MRSA), Streptococcus pyogenes, Acinetobacter baum These are bacteria (Annii, Legionella, Pseudomonas aeruginosa, Klebsiella pneumoniae).

[0126] The first species is optionally selected from the species listed in Table 1 (Tables 1 to 29).

[0127] Optionally, the first species is enterohemorrhagic E. coli (EHEC), E. coli serotype O157:H7, or Shiga toxin. This is a seroproducible E. coli (STEC). In the example, the bacteria are selected from the following: • Shiga toxin-producing E. coli (STEC) (STEC is also sometimes called verotoxin-producing E. coli (VTEC)) (There is), • Enterohemorrhagic E. coli (EHEC) (This pathogenic variant is the most frequently reported in news related to foodborne outbreaks) (This is a commonly seen and heard pathogenic form.) ·Toxigenic E. coli (ETEC), ·Enteropathogenic E. coli (EPEC), ·Enteroaggregative E. coli (EAEC), • Invasive E. coli (EIEC), and • Diffuse adhesion E. coli (DAEC).

[0128] Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 causes bloody diarrhea and hemolytic uremic symptoms. It is a human pathogen that causes the global epidemic of herd ulcerative encephalopathy (HUS). Conventional antibacterial agents are used against EHEC infection. EHEC promotes the release of potent Shiga toxin, which is responsible for many of the associated morbidity and mortality rates. It triggers the S response. Cattle are the natural host of EHEC, and approximately 75% of EHEC outbreaks are caused by contamination. It is linked to the consumption of bovine-derived products. EHEC causes disease in humans, but adult recurrent infections It is asymptomatic in stunt animals. A characteristic symptom of E. coli serotype O157:H7 (EHEC) infection is abdominal colic. Other complications include bloody diarrhea and hemolytic uremic syndrome (HUS), a life-threatening complication. Currently, there is a need for treatment of EHEC infection (Goldwater and Bettelheim, 2012). The use of conventional antibiotics exacerbates Shiga toxin-mediated cytotoxicity. The Centers for Disease Control and Prevention (CDC) In the epidemiological study conducted, patients treated for EHEC enteritis with antibiotics developed HUS. The risk was higher (Slutsker et al., 1998). Additional studies have investigated the effects of antibiotics on EHEC infections. Children who support the contraindications and are on antibiotic therapy for hemorrhagic colitis associated with EHEC may develop HUS. This showed an increase in opportunities (Wong et al., 2000; Zimmerhackl, 2000; Safdar et al., 2002; Tarr (2005). Conventional antibiotics are located within the lambda-shaped prophage genome integrated into the chromosome. This promotes Shiga toxin production by enhancing the replication and expression of the stx gene encoded in this gene. The inventive method relies on nuclease cleavage. Furthermore, Stx induction leads to the release of Shiga toxin into the environment. Promotes phage-mediated lysis of EHEC cell epithelium, enabling dispersal and dispersion (Karch et al., 1999) (Year; Matsushiro et al., 1999; Wagner et al., 2002). Therefore, advantageously, the present invention relates to human It also provides an alternative means for treating EHEC in animals. This involves nuclease activity. Regarding the rate and duration of the anti-EHEC effect produced (in contrast to conventional antibiotic action), The surprising results are illustrated below.

[0129] In the example, the subject (e.g., a human) is suffering from hemolytic uremic syndrome (HUS) or has There is a risk, for example, the subjects have E coli infections such as EHEC E coli infection.

[0130] Aspects of the present invention include a plurality of U for use together with the nuclease of the present invention in a therapeutic method. Viruses (for example, phages or phagemids for producing phages), each virus Rus includes copies of nucleic acids as described herein, and viruses are microorganisms contained by the subject. It provides multiple viruses that can infect and deliver nucleic acids to the host.

[0131] Aspects of the present invention relate to a method for treating a target microbial infection, which is programmable Multiple viruses (e.g., phages or phages) for use with nucleases Phagemids for production, and microbial infections are caused by a first species or strain of microorganism. This is triggered, and the nuclease is drawn to the target region contained in the genome of the microorganism that infected the target. It is programmable to sever the position, thereby killing the first species or strain of microorganism. The treatment method involves eliminating the microbial growth or proliferation, or reducing the growth or proliferation of the microbial cells, and targeting the nuclease The process involves bringing the nuclease into contact with the target site and programming it to cleave the target site. As a result, the genome of the microorganisms contained in the target is cut, and the infection caused by the target microorganism is treated. And so, Each virus contains a copy of nucleic acid that encodes RNA in order to express RNA in the target. RNA complexes with nucleases, programming the nucleases to be included in the target. The target site of the microorganism is cut, Viruses can infect microorganisms, depending on the target, and deliver nucleic acids to them. It offers several viruses.

[0132] Optionally, the above method is for continuous treatment, such as as described herein. And / or optionally, the infection is an acute infectious disease.

[0133] Optionally, the above method is for rapid treatment as described herein. , and / or optionally, the infectious disease is an acute infectious disease.

[0134] Optionally, the nuclease may be any nuclease of the present invention as described herein. Yes. Optionally, the nucleic acid may be any nucleic acid of the present invention as described herein.

[0135] Optionally, the nuclease may be any nuclease of the present invention as described herein. Yes. Optionally, the nucleic acid may be any nucleic acid of the present invention as described herein.

[0136] In an alternative form, if the microorganism is a virus, then multiple viruses contain the microorganism. It is a phage capable of infecting chief cells, and its nucleic acid is used in the host cell to express RNA there. It is introduced. The RNA complexes with a nuclease in the host cell, and guides the nuclease to micro It cleaves the target site of an organism (i.e., cleaves viral RNA or DNA), thereby causing virality To inactivate microorganisms. For example, microorganisms are viruses (e.g., in amoebas, or The viruses of the aforementioned plurality of viruses (in human, animal, or plant cells) are microorganisms It is possible to target them, thereby promoting the nuclease to cleave the microorganisms. Gram is obtained (for example, in an amoeba or in the aforementioned cell).

[0137] Aspects of the present invention include a plurality of methods for programming the nuclease of the present invention in a therapeutic method. A composition comprising nucleic acids, wherein each nucleic acid is a nucleic acid as defined herein. provide.

[0138] Aspects of the present invention relate to a method for treating a target microbial infection, which is programmable A composition comprising multiple nucleic acids for programming nucleases, for microbial infections This is caused by a first species or strain of microorganism, and the nuclease is released from the infected microorganism. It can be programmed to cleave target sites contained within the genome of an organism, and thereby The first species or strain of microorganism is killed, or its growth or proliferation is reduced. The treatment method involves contacting the target with a nuclease and nucleic acid, where the nuclease cleaves the target site. The process includes a programmed step to remove the genotypes of microorganisms contained in the subject. The molecule is cleaved, the target microbial infection is treated, and each nucleic acid expresses RNA in the target. It codes for RNA, the RNA complexes with a nuclease, and programs the nuclease. The present invention provides a composition that cleaves target sites of microorganisms contained in a given substance.

[0139] Optionally, the above method may be used for continuous treatment, such as as described herein. Yes, and / or optionally, the infection is an acute infection.

[0140] Optionally, the above method is for rapid treatment, such as as described herein. And / or optionally, the infectious disease is an acute infectious disease.

[0141] Optionally, the nuclease may be any nuclease of the present invention as described herein. Yes. Optionally, each nucleic acid is any nucleic acid of the present invention as described herein.

[0142] Optionally, the composition comprises nucleic acids and pharmaceutically acceptable diluents, carriers, or excipients. This is a pharmaceutical composition. Optionally, the composition may be administered orally or intravenously to humans or animals. pulmonary administration, rectal administration, local administration, buccal administration, ocular administration, intranasal administration, or subcutaneous administration It is for use. Optionally, the composition may be a herbicide or insecticide or insecticide or nematod It is a septic (or aracnicide). Optionally, the composition is toxic to yeast. The composition is optionally toxic to giant viruses.

[0143] One aspect of the present invention is a CRISPR comprising a nuclease according to the present invention for use in a therapeutic method. / Cas system, and the nuclease is a Cas nuclease (for example, Cas3 or Cas9 or Honmei Any other Cas mentioned in the details, and the system is one or more guide RNAs or 1 It contains DNA encoding one or more guide RNAs, each guide RNA being part of the microbial genome. It is possible to program Cas nuclease to cleave more of the target site. It provides a CRISPR / Cas system.

[0144] In the example, each guide RNA referred to herein is a single-stranded guide RNA (i.e., a chimeric one). In another example, each guide RNA is a crRNA that hybridizes with tracrRNA. Includes.

[0145] For example, the target sites referred to herein include essential bacterial genes, pathogenicity genes, and These are included by antibiotic resistance genes (comprsed). For example, the marks mentioned herein The target site is a multicopy sequence (i.e., one or more copies in each bacterial genome). Distributions present in (for example, 2, 3, 4, 5, 6, 7, 8, or 9, or more) copies The target site is included (comprsed) by the ribosomal RNA gene. For example, the target site is included by the ribosomal RNA gene. For example, the target sites referred to herein are ribosomal RNA genes (e.g., 23S Ribosomal RNA gene), yapH gene, or pks gene, or their homologs or ol It is included (comprsed) from the Solog.

[0146] Optionally, each guide RNA described herein may be high in the protospacer sequence containing the target site. It is possible to breed, and the protospacer sequence is 15-45 nucleotides long, for example. For example, the length is 15-25, 18-21, 20, or about 20 nucleotides. Optionally, each of the specified terms Guide RNA is 15-45 nucleotides long, for example, 15-25, 18-21, 20, or about 20 nucleos. It includes a spacer array with a tide length.

[0147] Optionally, each guide RNA specified herein may be, for example, if the bacterium is E. coli, then 5'-NGG It is the same species as the rotospacer adjacent motif (PAM). Optionally, each guide RNA described herein is For example, if the bacteria is C-difficult, the 5'-CCA or 5'-CCT protospacer adjacent motif It is the same type as PAM.

[0148] Aspects of the present invention are for treating target acute microbial infections, such as sepsis or sepsis. Guide RNA or coding for guide RNA for use in the system of the present invention for use in the method of the present invention Provides DNA.

[0149] One aspect of the present invention provides a nucleic acid vector comprising guide RNA or DNA.

[0150] The vectors are optional and include phages, phagemids, and viriophages. These are viruses, plasmids (e.g., conjugative plasmids), or transposons. See below. An example is achieving near-complete death by using a conjugating plasmid as a vector. This demonstrates that it is possible. Therefore, in the embodiment, each vector is a carrier bacterium, for example, Conjugated probiotics delivered from probiotic carrier bacteria for administration to humans or animals It is a smid. In the example, the carrier bacteria are Lactobacillus (e.g., L. reuteri) or E. coli. This is illustrated below, and complete (100%) extermination is achieved.

[0151] Aspects of the present invention are for administration to humans or animals to treat sepsis or septicemia. An anti-sepsis or anti-septic composition comprising a plurality of vectors, each vector being the present invention A composition containing a vector is provided.

[0152] Aspects of the present invention relate to the treatment of a target acute microbial infection (for example, rapidly and / or A method for continuous treatment, as specified herein. To provide.

[0153] Aspects of the present invention relate to the treatment of a target acute microbial infection (for example, rapidly and / or A method for continuous treatment of a microbial infection, in which the microbial infection is caused by a first species or strain of microorganism. This is triggered, and the nuclease is drawn to the target region contained in the genome of the microorganism that infected the target. It is programmable to sever the position, thereby killing the first species or strain of microorganism. Alternatively, the growth or proliferation of microorganisms is reduced, and the treatment method involves targeting the nuclease and The process involves contacting the nuclease, which is programmed to cleave the target site, and As a result, the genome of the microorganisms contained in the target is cut, and the acute microbial infection of the target is cured. To provide a method of treatment (for example, rapid and / or continuous treatment).

[0154] Aspects of the present invention relate to the treatment of a target acute microbial infection (for example, rapidly and / or A method for continuous treatment of a microbial infection, in which the microbial infection is caused by a first species or strain of microorganism. This is triggered, and the nuclease is drawn to the target region contained in the genome of the microorganism that infected the target. It is programmable to sever the position, thereby killing the first species or strain of microorganism. The growth or proliferation of microorganisms is reduced, and the treatment method involves targeting the target with nuclease and By exposing it to multiple viruses, the nuclease is programmed to cleave the target site. This process includes a step in which the genome of the microorganisms contained in the target is cut, and the target Sexually transmitted microbial infections are treated (e.g., rapidly and / or continuously), and each virus is The target contains a copy of nucleic acid encoding RNA to express RNA, and the RNA is a nuclea It complexes with the nuclease and programs the nuclease to target the microbial region contained in the target. The virus cleaves its stem and infects microorganisms contained within the target, delivering nucleic acids to them. This provides a method that makes this possible.

[0155] Optionally, the nuclease may be any nuclease of the present invention as described herein. Yes. Optionally, the nucleic acid may be any nucleic acid of the present invention as described herein.

[0156] Aspects of the present invention relate to the treatment of a target acute microbial infection (for example, rapidly and / or A method for continuous treatment of a microbial infection, in which the microbial infection is caused by a first species or strain of microorganism. This is triggered, and the nuclease is drawn to the target region contained in the genome of the microorganism that infected the target. It is programmable to sever the position, thereby killing the first species or strain of microorganism. The growth or proliferation of microorganisms is reduced, and the treatment method involves targeting the target with nuclease and By contacting multiple nucleic acids, the nuclease is programmed to cleave the target site. The process includes cutting the genome of microorganisms contained in the target, and the acute microorganism of the target When a biological infection is treated (e.g., rapidly and / or continuously), each virus is treated against In elephants, it contains a copy of the nucleic acid encoding RNA for RNA expression, and the RNA is a nuclease. It complexes with and programs the nuclease to target the microbial sites contained in the target. When cleaved, each nucleic acid codes for RNA to be expressed in the target, and the RNA is a nuclease. It complexes with and programs the nuclease to target the microbial sites contained in the target. A method for cutting is provided.

[0157] Optionally, the nuclease may be any nuclease of the present invention as described herein. Yes. Optionally, each nucleic acid is any nucleic acid of the present invention as described herein.

[0158] For example, the present invention is for medical use, or dental use, or ophthalmic (opthalmic) use. For example, to treat or prevent infectious diseases in organisms, or to limit the spread of infectious diseases in organisms. (It is intended for this purpose.)

[0159] For example, the present invention is for cosmetic use (e.g., use in cosmetic products, e.g., makeup), or For hygienic use (for example, use with hygienic products, such as soap).

[0160] In the example, the vector and / or nuclease administered to the subject is one of the following: It is contained in a composition in which the host is a first species or strain of microorganism (referring to). For example, compositions include medical compositions, ophthalmic (opthalmic) compositions, dental compositions, and This is a pharmaceutical composition (for example, contained in an anti-host vaccine). In this example, the composition is an anti-virus. A bacterial composition, for example, an antibiotic or antiviral agent, for example, a pharmaceutical, a disinfectant, or a mouthwash. Yes. In an example, the composition is a cosmetic composition (for example, a facial or body makeup composition). In the example, the composition is a herbicide. In the example, the composition is an insecticide (for example, when the host is B (If the host is Acillus (e.g., thuringiensis)). In the example, the composition is a beverage (e.g., biscuits). It is an additive (for alcohol, wine, or alcoholic beverages). For example, the composition is a food additive. (For example, if the host is E. coli, Salmonella, Listeria, or Clostridium (for example, botulinum) (um) If it is a host). In the example, the composition is a water additive. In the example, the composition is aquatic (acqua tic) Additives for animal environments (e.g., fish tanks). For example, the composition is oil or It is a petrochemical industrial composition, or is contained in such a composition (for example, the host is sulfur (If the host is an acid-reducing bacterium, e.g., Desulfovibrio). For example, the composition is oil or petrochemical. It is a chemical additive. In the example, the composition is a chemical additive. In the example, the composition is a disinfectant. (For example, for use in humans or animals, for example, for use in surgery or medicine) (For use in or for sterile equipment for infant feeding). For example, the composition is used in humans or animals. This is a sanitary composition for individual use. For example, the composition is for environmental use, such as soil treatment or environmental Decontamination (for example, if the host is a sulfate-reducing bacterium, e.g., Desulfovibrio, e.g., A composition for use from sewage, or from oil, petrochemicals, or chemicals. In the example, the composition is a plant growth stimulant. In the example, the composition is an oil extract, petrochemical. This is a composition for use in extraction, metal extraction, or mineral extraction. For example, the composition is used in fabric. It is a treatment agent or additive. For example, the composition is a treatment agent or additive for animal hides, leather, or suede. It is an agent. In the example, the composition is a dye additive. In the example, the composition is a beverage (e.g., a beverage). It is a brewing or fermentation additive (for example, when the host is Lactobacillus) (Combined). In the example, the composition is a paper additive. In the example, the composition is an ink additive. Example The composition is an adhesive additive. For example, the composition is anti-human, anti-animal, or anti-plant parasite. It is a chemical composition. For example, the composition is an air additive (for example, when the host is a Legionella host). In that case, for example, in or resulting from an air conditioning device (For air). For example, the composition is an antifreeze additive (for example, if the host is a Legionella host). (If it is the primary component). For example, the composition is an eye wash or ophthalmic (opthalmic) composition (e.g., co It is an anticonduct solution. For example, the composition is contained in dairy food (for example, the host is For example, if the host is Lactobacillus, Streptococcus, Lactococcus, or Listeria. (The composition is in or is in milk or dairy products.) The composition is contained in household or industrial cleaning products, or household or or industrial cleaning products (for example, host organisms such as E. coli, Salmonella, Listeria) or if the host is Clostridium (e.g., botulinum). In the example, the composition is fuel It is included. In the example, the composition is included by a solvent (e.g., water). In the example, the composition The substance is a baking additive (e.g., a food baking additive). In the example, the composition is actual Laboratory reagents (for example, for use in biotechnology or recombinant DNA or RNA technology) In this example, the composition is composed of a fiber-impregnating agent. In this example, the composition is composed of vitamin compounds. It is intended for use in the manufacturing process. For example, the composition is an antigravity or anti-grain or anti-plant decay composition. (For example, if the host is a saprophytic bacterium). In an example, the composition prevents, for example, metal corrosion. To stop or reduce (for example, when the host is a sulfate-reducing bacterium, e.g., Desulfovibrio host) For example, oil extraction, processing, or containment equipment; metal extraction, processing, or containment For use in corrosion reduction or prevention in equipment; or in mineral extraction, processing, or containment equipment. It is a corrosion-preventive composition. For example, the composition may be an agricultural composition or a farm composition. or contained in such a composition. In an example, the composition is a silage additive. The present invention, for example, when the host cell is a bacterial cell or an archaeal cell, is described in this paragraph. For use in any of the compositions described herein, or in any application described in this paragraph For use, the CRISPR arrays, gRNA-coding nucleotide sequences, and vectors described herein are available. - or provides multiple vectors. The present invention provides any application described in this paragraph A method for the purpose of using a CRISPR array, a gRNA coding nucleotide sequence, a vector, or multiple A method comprising the step of combining the present invention with a host cell (e.g., a bacterial cell or an archaeal cell). Provided. In embodiments, the host cell is also human (or human embryo) or animal, and human (or human It does not exist in embryos or animals.

[0161] Any aspect of the present invention may, for example, be for industrial use or household use, or It is used in a manner for such use. For example, any aspect of the present invention is used in agriculture, oil and the petroleum industry, food or beverage industry, apparel industry, packaging industry, electronics industry, Computer industry, environmental industry, chemical industry, aerospace industry, automotive industry, biotech Technology industry, medical industry, healthcare industry, dental industry, energy industry, consumer product industry Industry, pharmaceutical industry, mining, cleaning industry, forestry, fisheries, leisure industry, recycling industry, cosmetics Plastics industry, pulp or paper industry, textile industry, apparel industry, leather or leather For use in the ade or animal hide industry, the tobacco industry, or the iron and steel industry, or related to them It will be used.

[0162] In this specification, host cells refer to microorganisms of the first species or strain. Optionally, the host cells of this specification may be used as described herein. The host cell is a bacterial or archaeal cell. In this example, the cell is in a quiescent state. In one example, the cell is in the exponential growth phase. In another example, the cell is in the retardation phase. In yet another example, the cell is in the exponential growth phase. These are wild-type cells or naturally occurring cells, for example, from humans, animals, plants, soil, water, It is contained in the naturally occurring microbiome of the ocean, waterways, or environment. In this example, the cells have been genetically modified.

[0163] In the example, multiple vectors of the present invention are introduced into multiple host cells, and the host cells are, for example For example, this includes bacterial populations ex vivo, in vivo, or in vitro. In this example, the host cell is: A community of organisms or the environment (for example, aquatic microbiota, aquatic microbiota, humans or This includes animal intestinal microbiota, human or animal oral microbiota, human or animal vaginal microbiota, and (Contains microbiome from human or animal skin or hair, or from the axillary microbiome of humans or animals) The population consists of a first bacterium that is symbiotic or mutually beneficial with an organism or the environment, and the host cell. It contains a second bacterium, and the host cell is harmful to the organism or the environment (e.g., pathogenic). In embodiments, the population is ex vivo. In the example, the second bacterial subpopulation of the first bacterial subpopulation. The ratio to is increased. For example, the first bacterium is Bacteroides (e.g., B fragalis and / or B (thetaiotamicron) is a bacterium. Optionally, Bacteroides include caccae, capillosus, and cel. lulosilyticus, coprocola, coprophilus, coprosuis, distasonis, dorei, eggerthii, faecis, finegoldii, fluxus, fragalis, intestinalis, melaninogenicus, nordii, ole iciplenus, oralis, ovatus, pectinophilus, plebeius, stercoris, thetaiotaomicron One, two, three, or more selected from uniformis, vulgatus, and xylanisolvens. It includes many Bacteroides species. For example, Bacteroides is B thetaiotaomicron. or containing B thetaiotaomicron. For example, Bacteroides may be B fragalis or B fra Includes galis.

[0164] In the example, the host, the first or second cell, is specified in U.S. Patent Application Publication No. 20160333348, UK. Patent No. 1609811.3, PCT / EP2017 / 063593 brochure, and all U.S. equivalent applications. Any bacterial species disclosed in such disclosures (especially in PCT / EP2017 / 063593) The entirety of the frets (including Table 1) is incorporated herein, and one or more of the specifications herein Potentially included in one or more disclosures of this specification as in the claims.

[0165] In the example, host cells or bacterial populations are found in drinking water or water intended for human consumption (e.g., canal water or drinking water). It is contained within water. For example, the host cell or population is contained within its gut microbiota or oral microbiota. Compositions for administration to humans or non-human animals to plant and rebalance flocks (e.g.) For example, it is contained in medicines (e.g., bacterial enterotransplants), beverages, mouthwashes, or foods (e.g., (The aforementioned use of the pharmaceutical product is for the treatment or prevention of a disease or condition in a human or animal.) So, are the host cells or the population located on a solid surface or on a biofilm (for example, an intestinal biofilm)? This includes (biofilms on industrial equipment). Industrial or medical fluids, solid surfaces, equipment or This refers to containers (for example, for food, consumer products, cosmetics, personal healthcare products, petroleum or oil production). For in vitro treatment of (of), or for treating waterways, water, beverages, food products, or cosmetics. In an example of the present invention, the host cell is a fluid, surface, device, container, waterway, water, beverage, food. It may be included in or on products or cosmetics.

[0166] For example, the present invention relates to a container for medical or nutritional use. The present invention provides a container containing a vector for use in this method. For example, the container is a sterile container. For example, it may be an inhaler, or it may be connected to a syringe or IV needle.

[0167] Examples include vectors or compositions, mucosal administration, intestinal administration, oral administration, intranasal administration, and rectal administration. For administration to humans or non-human animals by oral administration, vaginal administration, ocular administration, or buccal administration. It is (or is administered).

[0168] Optionally, each host cell is a strain or species found in the human microbiome. The host cells are, at will, mixed with cells from different strains or species, and the different cells are, Humans and probiotics, symbiotic or mutually beneficial gut (for example, in the human gut) It is a bacteriaceae or bacterium. In examples, the host cell is an E coli or Salmonella cell.

[0169] The present invention optionally changes the relative ratio of the first and second bacterial subpopulations in a mixed bacterial population. This is for the purpose of, for example, Bacteroidetes in the human microbiome (e.g., Bacteroides For example, fragalis, and / or thetaiotamicron), Firmicutes, and / or Gram-positive cells. To change the proportion of bacteria or Gram-negative bacteria, etc., the human or animal microbiome It is for the purpose of making changes.

[0170] For example, the vector or composition of the present invention is used to lyse host cells in the host cells. It contains a nucleotide sequence for expressing drisin, and optionally, endolysin is f Phage Phi11, Phage Twort, Phage P68, Phage PhiWMY, or Phage K Endorsil (For example, MV-L endolysin or P-27 / HP endolysin).

[0171] In the example, the target site is contained within the chromosome of each microbial host cell, for example, the sequence is These are included by the host cell's antibiotic resistance gene, pathogenicity gene, or essential gene. Examples include The vector of the present invention, combined with an antibiotic (for example, a beta-lactam antibiotic) Provides, for example, a vector that can be used in the host cell genome or episome (for example, by the host cell) Protospacers included by antibiotic resistance genes (containing plasmids) - Targets sequences. For example, episomes target plasmids, transposons, and mobile genetic elements. It is a raw element or a viral sequence (e.g., a phage or prophage sequence).

[0172] In this example, the targets are chromosome sequences, endogenous host cell sequences, wild-type host cell sequences, and nonviral sequences. It is a chromosomal host cell sequence, but not an exogenous sequence and / or a non-phage sequence (that is, this (One or all of these), for example, sequences containing antibiotic resistance, are included by the host cell chromosome. This refers to wild-type host chromosome cell sequences, such as gene sequences or essential gene sequences. In the example, the sequence is This includes host cell plasmid sequences, such as antibiotic resistance gene sequences.

[0173] Optionally, the nuclease is Cas, and the target sites are adjacent NGG, NAG, NGA, NGC, N GGNG, NNGRRT, or NNAGAAW Protospacer Adjacent Motif (PAM), e.g., AAAGAAA or T The protospacer sequence is AAGAAA PAM (these sequences are written from 5' to 3'). It is included more. In the embodiment, the PAM is directly adjacent to the 3' end of the protospacer array. In the example, Cas is S aureus Cas, S theromophilus Cas, or S pyogenes Cas. In the example, Cas is Cpf1 and / or PAM is TTN or CTA. Optionally, Cas is: Type I (e.g., IA, IB, IC, ID, IE, or IF type) CRISPR-based Cas. (Arbitrary) By default, Cas is a type II CRISPR system Cas. By arbitrary choice, Cas is a type III CRISPR system Cas. Optionally, Cas is a type IV CRISPR system Cas. Optionally, Cas is a type V CRISPR system Cas. Yes, it exists. Optionally, Cas is a type VI CRISPR system Cas.

[0174] Optionally, the nuclease is a CAS, and each vector is designed to target a target site. Includes a homogeneous CRISPR array containing multiple copies of the same spacer. Optionally, the vector of the present invention A vector or multiple vectors are provided, and the vectors target host cell protospacer sequences. The CRISPR array comprises multiple gRNA coding sequences for the target site, and the protospacer is a target site It includes. Optionally, each vector contains two nucleic acid sequences (e.g., gRNA) that encode crRNA. It contains three or more copies, and each copy targets a host cell target site (e.g., toxic residues). The same method targets sites included in gene sequences, resistance gene sequences, or essential gene sequences. Includes a spacer array.

[0175] In the example, at least two target sequences, for example, an antibiotic resistance gene and an essential gene, are C Modified by as. Multiple targeting using this method reduces the evolution of evasive mutant host cells. It may be useful for that purpose.

[0176] In the example, Cas is the wild-type endogenous host cell Cas nuclease. In the example, target site cleavage. This is carried out by a dsDNA Cas nuclease (e.g., Cas9, e.g., spCas9 or saCas9), Therefore, the repair of the break is by non-homologous end joining (NHEJ), and instead Cas is exonu It is either Crease or Cas3.

[0177] In the example, the array, gRNA coding sequence, or vector is a repeat of the array or gRNA coding sequence. The Cas endonuclease coding sequence, which is found naturally in cells along with the sequence, is combined with the Cas endonuclease coding sequence. do not have.

[0178] The tracrRNA sequence is, for example, in the case of Cas systems that do not use tracrRNA, the one described in this invention. (i) or may be omitted from the vector, or endogenous tracrRNA may be encoded by the vector. It may be used in conjunction with cRNA.

[0179] In the example, the host target site consists of at least 5, 6, 7, 8, 9, 10, 20, 30, or 40 consecutive nuclei. It is contained by creotides.

[0180] In the example, each vector is an exogenous progenitor functional for transcribing crRNA or gRNA in microorganisms. Includes motor.

[0181] Optionally, each vector can be plasmid, cosmid, virus, virion, phage, It is a phagemide or prophage. For example, the present invention is, for example, that the vector is the same In some cases, the present invention provides multiple bacteriophages containing multiple vectors. The vector is a viral vector. Viral vectors are used for exogenous DNA insertion. It has particularly limited capabilities, and therefore virus packaging capabilities must be considered. Essential viral functions, such as expressing coat proteins and polymerases, are controlled by the coating. There needs to be room for the sequence to be added. In the example, the vector is a phage vector or It is either an AAV or lentiviral vector. If the host is a bacterial cell, it is a phage vector. This is useful. For example, the vector is a virus capable of infecting archaeal host cells. be.

[0182] Optionally, the vector component can be transferred into and / or between host cells. It is contained by a transposon. Transposons are described in U.S. Patent Application Publication No. 2016033334. Tragedy as described in Specification No. 8, British Patent No. 1609811.3, and all equivalent US applications These disclosures may also be transposons, and these disclosures, including the disclosure of such specific transposons, The entirety thereof is incorporated herein, and this specification in one or more claims herein. Potentially included in one or more of the disclosures.

[0183] In the example, each vector is provided either by nanoparticles or in liposomes.

[0184] In this example, each host cell (or first and / or second bacterium) is a Gram-positive bacterial cell. In the example, each host cell contains Enterobacteriaceae, such as Salmonella, Yersinia pestis, and Klebsiell. a. Shigella, Proteus, Enterobacter, Serratia, or Citrobacter cells. (Optional selection) By choice, each cell is either E coli (e.g., E coli K12) or Salmonella (e.g., Senteric serotype). These are typhimurium cells. By arbitrary selection, each host cell (or first and / or second bacterium) These are Gram-negative bacterial cells.

[0185] Optionally, the host (or first and / or second bacterium) may be Mycoplasma, Chlamydiae, or SPI. It is either rochete or mycobacterium. Optionally, the host (or the first and / or second bacterium) ) is a Streptococcus (e.g., pyogenes or thermophilus) host. Optionally, the host (or the first and / or second bacterium) is a Staphylococcus (e.g., aureus, e.g., MRSA) host. The host (or first and / or second bacterium) is optionally E. coli (e.g., O157: H7) is the host. Optionally, the host (or the first and / or second bacterium) is Pseudomonas (e.g., For example, *aeruginosa* is the host. By choice, the host (or the first and / or second bacterium) is: Vibro (e.g., cholerae (e.g., O139) or vulnificus) is the host. Optionally, the host ( Or the first and / or second bacterium) is Neisseria (e.g., gonnorrhoeae or meningitidis) ) is the host. By choice, the host (or the first and / or second bacterium) is Bordetella (for example) If pertussis is the host, then, by choice, the host (or the first and / or second bacterium) is Hae The host is a mophilus (e.g., influenzae).Optionally, the host (or the first and / or second) The bacteria (of which Shigella (e.g., dysenteriae) is the host. Optionally, the host (or the first and / or a second bacterium) is a Brucella (e.g., abortus) host. Optionally, the host (or The first and / or second bacterium is the Francisella host. Optionally, the host (or the first) The host (and / or the second bacterium) is a Xanthomonas host. Optionally, the host (or the first and / or) is a Xanthomonas host. The host (or the second bacterium) is an Agrobacterium host. Optionally, the host (or the first and / or The host (or the second bacterium) is an Erwinia host. Optionally, the host (or the first and / or second) The bacteria Legionella (e.g., pneumophila) is the host. Optionally, the host (or the first and / or a second bacterium) is a Listeria (e.g., monocytogenes) host. Optionally, The primary (or first and / or second) bacterium is a Campylobacter (e.g., jejuni) host. In selective breeding, the host (or first and / or second bacterium) is a Yersinia (e.g., pestis) host. Yes. By choice, the host (or first and / or second bacterium) is Borelia (e.g., burgdor). Helicobacter feri) is the host. Optionally, the host (or the first and / or second bacterium) is Helicobacter. (For example, pylori) is the host. Optionally, the host (or the first and / or second bacterium) is C Lostridium (e.g., dificile or botulinum) is the host. Optionally, the host (or the first and The host (or second bacterium) is Erlichia (e.g., chaffeensis). Optionally, The dominant (or first and / or second bacterium) is Salmonella (e.g., typhi or enterica, e.g.) The host is serotype typhimurium, e.g., DT 104). Optionally, the host (or the first and / or) (or the second bacterium) is a Chlamydia (e.g., pneumoniae) host. Optionally, the host (or The first and / or second bacterium is a Parachlamydia host. Optionally, the host (or the first) (and / or a second bacterium) is a host for Corynebacterium (e.g., amycolatum). In the selection, the host (or first and / or second bacterium) is Klebsiella (e.g., pneumoniae). Therefore, by choice, the host (or the first and / or second bacterium) is Enterococcus (for example, The host is *faecalis* or *faecim* (e.g., linezolid-resistant). Optionally, the host (or the first host) is selected. (and / or a second bacterium) can infest Acinetobacter (e.g., baumannii, e.g., multidrug-resistant). He is the Lord.

[0186] Optionally, the present invention relates to the environmental microbiome (for example, the host cells and yeast cells). Soil containing, composition, human microbiome, animal microbiome, or plant microbiome Some devices are used to reduce the growth or proliferation of host cells in the microbiome. If a microbiome exists naturally, it is useful.

[0187] Optionally, the nuclease is Cas, and the target is the 3' immediate vicinity of the allogeneic PAM in the host cell genome. A protospacer sequence containing at least 5, 6, 7, 8, 9, or 10 consecutive nucleotides. It is included by and PAM is selected from AWG, AAG, AGG, GAG, and ATG.

[0188] Non-medical ex vivo and in vitro use, etc. In one configuration, rapid and sustained microbial killing and growth or The observations of this invention relating to growth inhibition may be applied to subjects other than humans and animals (for example, plants or yeast). Ex vi Applications for VO or in vitro treatment have been found. Therefore, the present invention is based on the following concepts. Furthermore, any other features, configurations, aspects, embodiments, etc., of the present invention as described herein are provided. The options, and the examples above and elsewhere in this specification, may be modified as necessary (as per the claims of this specification). It is combinable with such concepts (including to provide combinations of features).

[0189] The concept relates to the manufacture of a composition for carrying out the therapeutic method specified herein. The present invention utilizes the nuclease, multiple viruses, systems, guide RNA, DNA, or vectors. It provides a use for organisms other than humans or animals.

[0190] The concept is for performing ex vivo or in vitro methods to treat microbial infections of substrates. In the production of the composition, the nuclease of the present invention, multiple viruses, systems, guide RNA, DNA, or use of a vector, in which the microbial infection is caused by the first species or strain of microorganism The nuclease then cleaves the target site contained in the genome of the microorganism that has infected the substrate. It is programmable to do so, thereby killing the first species or strain of microorganism, or The growth or proliferation of microorganisms is reduced, and the treatment method involves contacting the target with a nuclease. The nuclease involves a process programmed to cleave the target site, thereby, Depending on the target, the genome of the microorganisms contained is cut, and the acute microbial infection of the substrate is treated. To provide.

[0191] In this specification, treatment for substrate infection is incorporated on the surface of the substrate and / or into the material of the substrate. This may mean treating a group of bacteria (e.g., one or more colonies). For example, the procedure involves industrial equipment or instruments (e.g., medical devices such as scalpels or tubes). This could also be a treatment to kill bacteria on the surface of the medical device. In another example, the substrate (subj A strate is a medical fluid or petroleum product in fluid form (e.g., oil or hydrocarbon fluid or liquid). These are fluids (e.g., liquids or gases).

[0192] The concept is to manufacture a composition for carrying out ex vivo methods to treat microbial infections of a substrate. The use of a programmable nuclease in which microbial infection is caused by a first species or Caused by the microorganism of the bacterial strain, the nuclease is derived from the genome of the microorganism that infected the substrate. It can be programmed to cut the target site contained within, thereby eliminating the first species or fungus. The microorganisms in the strain are killed, or their growth or proliferation is reduced, and the treatment method is targeted. The substance is brought into contact with a nuclease, which is programmed to cleave the target site. This process includes a step in which the genome of microorganisms contained in the target is cut, and the acute It provides a use for treating microbial infections.

[0193] Optionally, a nuclease (e.g., a programmed nuclease) and / or a target region. Nucleic acids that program nucleases to recognize and cleave at a specific position are used at time point 1 (T1) and The drug is administered to the subject or substrate at the second time point (T2), where T2 is at least one hour after T1. T2 may be as defined herein.

[0194] Optionally, infections are minimized within the first 30 minutes of treatment (for example, within the first 15 minutes). It is reduced to at least 1 / 100th. At will, the infection targets the programmed nuclea. After contact with the -se, leave it for at least 60 minutes (for example, at least 120 minutes) Both are maintained at 1 / 100th.

[0195] Optionally, infection reduction is maintained for at least 30 minutes immediately after the first 30 minutes of treatment. To be continued.

[0196] Optionally, the above method may be applied to RNA, or to the target or substrate, or on the target or substrate. The process includes administering a nucleic acid encoding RNA to express RNA to a target or substrate, RNA complexes with a nuclease, and programs the nuclease to react to the target or substrate. It cuts the target site of the microorganisms contained within.

[0197] Optionally, nucleases may be administered to the target or substrate simultaneously with or sequentially to RNA or nucleic acids. It will be done.

[0198] Optionally, the subject or substrate may contain a nuclease before administering RNA or nucleic acid.

[0199] Multiple viruses (e.g., phages) are administered to the target or substrate at the discretion of the user, and each virus A virus contains a copy of nucleic acid, and viruses infect microorganisms present in the target or substrate. Then, nucleic acids are delivered to it.

[0200] The virus-to-microorganism ratio administered is optional and ranges from 10 to 150.

[0201] Optionally, infection lasts for one hour or longer, optionally, during the treatment period. Within the first 30 minutes (for example, within the first 15 minutes), it will be reduced by at least 90%.

[0202] Optionally, infection can be minimized within the first 30 minutes of treatment (for example, within the first 15 minutes). Both were reduced to 1 / 100th, and at least a 1 / 100th reduction in infections was achieved by prog After contact with the ruminated nuclease, allow to rest for at least 60 minutes (e.g., at least 120, 145 minutes). It is maintained for 180 minutes or longer.

[0203] Optionally, the subject may be a plant, or the substrate may be a metal substrate, plastic substrate, or concrete. The substrates are lite, stone, wood, glass, or ceramic. (Optional) The subject is a fluid (for example, a liquid or a gas).

[0204] Optionally, microorganisms are either bacteria or archaea. Optionally, bacteria are Gram-positive bacteria. The bacteria are, at random selection, Staphylococcus, Streptococcus, Enterococcus, and Legion. lla, Heamophilus, Ghonnorhea, Acinetobacter, Escherichia, Klebsiella, Pseudomona s, or Stenotrophomonas bacteria (e.g., E. coli (e.g., EHEC E. coli), C. dificile, V. c holera, Staphylococcus (e.g., S. aureus or MRSA), Streptococcus pyogenes, Acinet obacter baumannii, Legionella, Pseudomonas aeruginosa, Klebsiella pneumoniae bacteria )

[0205] Optionally, the nuclease can be a Cas nuclease (e.g., Cas3 or Cas9), or a meganuclease. Aase, TALEN (Transcription Activator-like Effector Nuclease), or Zinc Finger Nuclease It's Crease.

[0206] Refer to International Publication No. 2016177682, which describes the microbial corrosion of substrates. The MIC (microfiltration efficiency) or the nature of biofouling has been investigated, and methods for controlling the MIC or biofouling of the substrate have been considered. The law is disclosed. The methods, nucleases, arrays, RNA, etc. disclosed in this document are disclosed. For example, to carry out the method or use of the present invention, the actor and virus are used in the present invention. It can be used, and the disclosure of those parts and the opening in International Publication No. 2016177682 The substrates and bacteria shown may be used in one or more claims of this specification. To potentially provide disclosure of features, these are incorporated herein by reference.

[0207] Optionally, the use of the present invention may be industrial or household (for example, International Publication No. 2016177682). The system disclosed in the brochure (which is incorporated herein by reference) This is for controlling microbial corrosion (MIC) or biofouling of materials. For example, the system is an apparatus (e.g., For example, it includes (for use in industrial processes), and the surface is the surface of the apparatus. Biofouling includes the formation, growth, or maintenance of microbial biofilms and / or sludge. In the example, the microorganisms are sessile. In the example, "controlling" refers to the aforementioned MIC or biocontamination. To prevent, reduce, or eliminate damage, or to prevent the spread of MIC or biocontamination in the system. This includes reducing dispersion. Cell growth or proliferation or maintenance is a characteristic of cell viability, for example. Therefore, in this example, this method reduces microbial growth and / or maintenance.

[0208] Optionally, microorganisms are included by a microbial biofilm in contact with the substrate. In the selection, the surface and host cells are exposed to aqueous liquids (e.g., seawater, freshwater, stagnant water, or drinking water). It is in contact with the fluid.

[0209] Freshwater is found on the Earth's surface in ice sheets, ice caps, glaciers, icebergs, wetlands, ponds, lakes, rivers, and waterways. Furthermore, freshwater is naturally occurring water located underground as groundwater in aquifers and groundwater flows. Generally, it is characterized by low concentrations of dissolved salts and other total dissolved solids. This excludes seawater and brackish water in particular, but includes mineral-rich water such as iron-containing spring water. For example, Freshwater is one of these freshwater types. Drinking water is for humans or animals (e.g., households). (Livestock) Water for consumption. For example, the fluid is industrial cooling water if the system is a cooling system; wastewater if the system is a cooling system. If the system is a treatment or storage system, it refers to wastewater; if the system is a drinking water treatment, storage, transport, or distribution system. If the system is a papermaking or processing system, then the water used is for papermaking; if the system is a swimming pool or swimming If it is a pool water treatment (teatment) or water storage system, then swimming pool water; if the system is a fire extinguishing system If necessary, use a fire extinguisher; or any industrial water for pipes, tanks, pits, ponds, or canals. Selected from Seth water.

[0210] Optional use is for controlling bacterial rancidity of liquids in a reservoir or container. The fluid contains a population of first host cells of a first microbial species that mediates the biocontamination, The notation method is, (i) The population is brought into contact with multiple vectors capable of transforming or transducing cells. Each vector contains a CRISPR array, which is then introduced into host cells. (a) Each CRISPR array contains one or more sequences for expressing crRNA and host cells It contains a promoter for transcribing the sequence inside, (b) Each crRNA hybridizes with a target sequence in the host cell and interacts with the host cell's Cas (e.g., Ca It is possible to introduce an S-nuclease and modify the target sequence (for example, cleave the target sequence). The process involves the target sequence being a gene sequence that mediates host cell viability. The above method enables the expression of the cRNA in the host cell in the presence of Cas, thereby This modifies target sequences in host cells, leading to a reduction in host cell viability and control of biofouling. Includes the process.

[0211] In the example, the fluid is a liquid. In the example, the fluid is a gaseous fluid.

[0212] system Exemplary systems are selected from the following group: petrochemical recovery, processing, and storage. , or transport systems; recovery, processing, storage, or transport systems for hydrocarbons; recovery, processing, storage, or crude oil Transportation systems; natural gas recovery, processing, storage, or transportation systems (e.g., oil wells, oil rigs, oil drilling equipment) Installation, oil pump system, oil pipeline, gas rig, gas extraction equipment, gas pump equipment, Gas pipelines, oil tankers, gas tankers, oil storage facilities, or gas storage facilities); water Processing or storage facilities; water reservoirs (e.g., drinking water reservoirs); air or water quality adjustment (e.g., Cooling or heating devices, such as coolant tubes, condensers, or heat exchangers; medical devices or surgical equipment. Equipment; environmental (e.g., soil, waterways, or air) treatment equipment; papermaking or paper recycling equipment; power plants, e.g. For example, thermal or nuclear power plants; fuel (for example, hydrocarbon fuels, for example, petroleum, diesel, or LPG storage facilities; mining or metallurgy, mineral or fuel recovery systems, e.g., mines or mining equipment; engine Engineering systems; loading equipment; cargo or goods storage facilities (e.g., cargo containers); food or beverages Food manufacturing, processing, or packaging equipment; cleaning equipment (e.g., washing equipment, e.g., washing machines or dishwashers) ); catering (e.g., home or commercial catering) equipment; farm equipment; construction (e.g., (Building, public infrastructure, or road construction) equipment; aircraft equipment; aerospace equipment; transport equipment Places (for example, automobiles (for example, cars, trucks, or vans); railway vehicles; aircraft (for example, airplanes) ) or marine or waterway vessels (for example, boats, ships, submarines, or hovercraft) )); Packaging equipment, for example, consumer product packaging equipment; or food or beverage packaging equipment; electro NICS (for example, computers or mobile phones or their electronic components); or Electronics manufacturing or packaging equipment; dental equipment; industrial or household plumbing ( For example, underwater pipes) or storage tanks (for example, water tanks or fuel tanks (for example, gasoline (e.g., gasoline tanks for vehicles); underground facilities; buildings (e.g., residences or offices) (or commercial premises, factories, or power plants); roads; bridges; agricultural equipment; factory systems; crude oil or natural gas exploration facilities) Equipment; office-type; and household-type.

[0213] In the example, the system is used in industries and businesses selected from the following group: Agriculture, oil and petroleum industry, food or beverage industry, apparel industry, packaging industry, electronics industry Computer industry, environmental industry, chemical industry, aerospace industry, automotive industry, biotechnology Medicine industry, medical industry, healthcare industry, dental industry, energy industry, consumer products industry, Pharmaceutical industry, mining industry, cleaning industry, forestry industry, fisheries industry, leisure industry, recycling industry, cosmetics industry The industry, plastics industry, pulp or paper industry, textile industry, clothing industry, leather or suede or The animal hide industry, the tobacco industry, and the iron and steel industry. In the example, the surface or fluid to be treated is the aforementioned The surface or fluid of the equipment used in the selected industry. For example, the system is used in the crude oil industry. For example, the system is used in the natural gas industry. For example, the system is used in the petroleum industry. For example, the system is a marine container, platform, or rig (for example, used at sea). It is a ship or boat (or an offshore oil or gas platform or rig) used for that purpose. In terms of implementation, such systems are moored at sea, for example, non-temporarily moored at sea. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24 months, or longer (for example, several consecutive months) (Month) Moored at sea. In embodiments, such a system is located in the waters of a nation or state. For example, they exist non-temporarily on the sea in such bodies of water, for example, 1, 2, 3, 4, 5, 6, 7, 8 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 months, or any other month. It is present in the waters of the aforementioned country for an extended period (for example, several consecutive months).

[0214] In the example, the substrate surface to be treated is stainless steel, carbon steel, copper, nickel, brass, Includes aluminum, concrete, plastic, or wood. In the example, the base material is metal solubility. It is a joint or connection. For example, the surface is metal (e.g., steel or iron) or nonmetal (e.g., (Plastic, concrete, asphalt, wood, rubber, or stone) surfaces. So, metals are alloys (for example, stainless steel, brass or nickel alloy, zinc alloy, copper alloy) (Nickel alloy, or aluminum alloy). In the example, the surface is an artificial polymer surface. Yes. In the example, the surface is a substrate coating. In the example, the substrate is soil, freshwater, or seawater. It is in contact with water.

[0215] In the example, the fluid is drinking water; waterway; brackish water; or liquid fuel, such as gasoline or diesel (example). For example, cars or motorized vehicles), LPG, kerosene, alcohol (e.g., ethanol, methamphetamine). (butanol, or ol), liquid hydrogen, or liquid ammonia; for example, the fuel is stored It is a liquid fuel. In the example, the fluid is an oil or a non-aqueous liquid. In the example, the fluid is , waterways, or water, for example, seawater, freshwater, drinking water, rivers, currents, ponds, lakes, reservoirs, stagnant water (for example) (For example, in a water storage tank or cooling system), groundwater, well water, water from rock layers, soil moisture, or It is a liquid contained in a body of rainwater. In this example, the liquid is seawater. In this example, the base material It is in contact with the liquid mentioned in this paragraph. In the example, the fluid or liquid is the following: Selected from the group consisting of: oil, aqueous solutions, hydraulic fracturing fluids, fuels, carbon dioxide, natural gas, Oil / water mixtures, fuel / water mixtures, saltwater, ocean water or seawater, brackish water, freshwater sources, lakes, Rivers, streams, swamps, ponds, wetlands, runoff water originating from melting snow or ice, springs, groundwater, aquifers, It is a liquid at precipitation and ambient temperature (e.g., at RTP), hydrophobic, but soluble in organic solvents. Any substance, hexane, benzene, toluene, chloroform, diethyl ether, vegetable oil Petrochemical oils, crude oil, refined petrochemical products, volatile aromatic oils, fossil fuels, gasoline, carbonized water Raw mixtures, jet fuel, rocket fuel, biofuels. In the example, the fluid is an oil / water mixture. That is the case.

[0216] The terms “microbial corrosion” or “MIC” as used herein, unless otherwise indicated. Furthermore, any element (substrate) of the system is at least one of the microbial communities, such as bacterial communities or archaeal communities. This refers to a process that is structurally disrupted by the action of one member. The term "biofouling" is, When used herein, unless otherwise indicated, microorganisms (bacteria and / or archaea, etc.) , a base in contact with a fluid (e.g., water or aqueous liquid, or hydrocarbons, or petrochemical products) This refers to the process of accumulation on the surface of a material. It also refers to the process of accumulation of fluids (e.g., water or aqueous liquids, or carbon). Undesirable accumulation of microorganisms (bacteria and / or archaea, etc.) in hydrogen ion or petrochemical products and This involves proliferation, or "rancidity" of the fluid. For example, bacteria grow in ship or boat ballast water. It contains bacteria, which are undesirable in the environment. The term "substrate" as used herein means Cells can adhere to it, a biofilm can form and grow, or biofouling can occur (e.g., slime). This refers to any type of surface on which sludge formation may occur. Substrates include petrochemicals, fuels, etc. It may be an "industrial" substrate such as crude oil or the surface of gas pipe system equipment, or kitchen "Non-industrial" materials such as counter or shower base materials, or garden base materials (e.g., household, e.g., household) Alternatively, it may be an office-based material.

[0217] In the alternative form, the population is a population of archaeal cells of the first species, instead of a population of host bacterial cells. That is the case.

[0218] Optionally, the fluid is an aqueous liquid (e.g., seawater, freshwater, stagnant water, or drinking water).

[0219] In the alternative form, the microorganisms are instead algal cells.

[0220] Optionally, the microorganisms are sulfate-reducing bacterial (SRB) cells (e.g., Desulfovibrio or Desulfo These are tomaculum cells. In this example, the cells are selected from the following group: Desulfo tomaculum nigrificans, Desulfacinum infernum, Thermodesulfobacterium mobile, The rmodesulforhabdus norvegicus, Archaeoglobus fulgidus, Desulfomicrobium apsheronu m, Desulfovibrio gabonensis, Desulfovibrio longus, Desulfovibrio vietnamensis, D esulfobacterium cetonicum, Desulfomaculum halophilum, Desulfobacter vibrioformi s, and Desulfotomaculum thermocisternum cells. In the example, the population consists of two of these cell types. Or it contains more than that amount of the mixture.

[0221] Optionally, the surface or fluid may be used for the recovery, processing, storage, or This is included by the transportation equipment. Crude oil is one of the world's most important energy resources. Oil undergoes various technical processes to become gasoline, oil, paraffin oil, lubricants, and asphalt. Refined petrochemicals are refined into consumer products such as household fuel oil, petroleum jelly, and polymers. It is used as a raw material in numerous industries, including industrial production. Oil-derived products are used in many other chemical processes. It is also commonly used in Seth. In alternative forms, the fluid is the above consumer product or The surface is in contact with such consumer products.

[0222] Optionally, the surface is in contact with seawater, fracturing liquid, or well fluid, or the fluid is It is seawater, crushing liquid, or well water.

[0223] Optionally, step (i) of this method involves a population of microbial cells of a second species (second host cell). Prepare the second cell, which contains the vector, and transfer the vector from the second host cell to the first host cell. A process that allows for transition to cells; and combining a second host cell with a first host cell, The process includes the step of introducing a vector into a first host cell. In the example, the second cell is exposed to the environment. (For example, in aquatic or soil environments) environmentally, industrially, or domestically permissible, The first host cell is unacceptable in the environment.

[0224] Optionally, the first host cell is subjected to a mixture of microbial cells before contact with the vector. It is contained (for example, by microbial biofilms), and the mixture contains the fine particles of the second species. It contains cells.

[0225] Optionally, the second species may be Bacillus, nitrate-reducing bacteria (NRB), or nitrate-reducing sulfides. It is a species of oxidation-causing bacteria.

[0226] Optionally, NRBs include Campylobacter sp., Nitrobacter sp., Nitrosomonas sp., and Thiom icrospira sp., Sulfurospirillum sp., Thauera sp., Paracoccus sp., Pseudomonas sp. Selected from the group consisting of , Rhodobacter sp., and Desulfovibrio sp., or the above species Includes at least two.

[0227] Optionally, NRB is Nitrobacter vulgaris, Nitrosomonas europea, Pseudomonas stu tzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Sulfurospirillum deleyi The selection is made from the group consisting of anum and Rhodobacter sphaeroides.

[0228] Optionally, this method involves simultaneously distributing the host cells of the first species with a biocide and the vector. Or, the process may include sequential contact. For example, the composition to be contacted with the host cells is pre-mixed. Vectors and biocides are provided.

[0229] The biocide is optionally selected from the following group: tetrakishydrox methylphosphonium sulfate (THPS), glutaraldehyde, chlorine monoxide, chlorine dioxide, next Calcium chlorite, potassium hypochlorite, sodium hypochlorite, dibromonitor Dibromonitriloproprionamide (DBNPA), methylenebis(thiocyanate) (MBT), 2-(thiocyanomethylthio)benzothiazole (TCMTB), bronopol, 2-bro Mo-2-nitro-1,3-propanediol (BNPD), tributyltetradecylphosphonium chloride (TTPC), taurinamide and its derivatives, phenols, quaternary ammonium salts, chlorine Contains ingredients: quinaldinium salt, lactone, organic dye, thiosemicarbazone, quinone, carba Mate, urea, salicylamide, carboanilide, guanide, amidine, imidazoline, Acetic acid, benzoic acid, sorbic acid, propionic acid, boric acid, dehydroacetic acid, sulfurous acid, vanillin Acid, parahydroxybenzoic acid ester, isopropanol, propylene glycol, benzyl Alcohol, chlorobutanol, phenylethyl alcohol, formaldehyde, iodine and its solution, povidone-iodine, hexamethylenetetramine, noxythioline, 1-(3-c (Lorroaryl)-3,5,7-Triazo-l-Azonia Adamantan Chloride, Taurolidine, Taurol Lutam, N-(5-nitro-2-flufridene)-1-amino-hydantoin, 5-nitro-2-flualdehyde Hydosemicarbazone, 3,4,4'-Trichlorocarbanilide, 3,4',5-Tribromosalicylic acid Lido, 3-trifluoromethyl-4,4'-dichlorocarbanilide, 8-hydroxyquinoline, 1-S Clopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecal Voxylic acid, 1,4-dihydro-1-ethyl-6-fluoro-4-oxo-7-(1-piperazinyl)-3-quino Phosphorus carboxylate, hydrogen peroxide, peracetic acid, sodium oxychlorocene, parachloromethyl Taxylenol, 2,4,4'-trichloro-2'-hydroxydiphenol, thymol, chlorhexyl Xyzidine, benzalkonium chloride, cetylpyridium chloride, silver sulfadiazine, silver nitrate Bromine, ozone, isothiazolones, polyoxyethylene (dimethylimino)ethylene (di Methylimino)ethylene dichloride, 2-(tert-butylamino)-4-chloro-6-ethylamino-5 - Triazine (terbutyrazine), and combinations thereof. For example, the biocide is tetraquinol. The biocide is hydroxymethylphosphonium sulfate (THPS). In this example, the biocide is a quaternary ammonium compound. It is a monoium compound.

[0230] Optionally, the system is used for industrial operations selected from the following group: mining; Shipping; recovery or processing of crude oil, gas, or petrochemical products; hydraulic fracturing; heating of air or water or Cooling; production, storage, or distribution of drinking water; transport of hydrocarbons; and wastewater treatment.

[0231] Optionally, the surface may be the surface of equipment used in the selected industry, or a fluid This is the fluid contained in the equipment used in the selected industry.

[0232] Optionally, the surface is a kitchen, bathroom, or gardening utensil surface, or the fluid is a kitchen, This includes bathroom or gardening equipment. For example, the equipment is used in a household setting.

[0233] Optionally, the fluid is a drinking liquid contained in a container (e.g., a water tank or bottle). The surface is the surface of the container that comes into contact with the liquid.

[0234] Optionally, each vector contains mobile genetic elements (MGEs), which are located at the origin of transposition (oriT) and pre-transposition. The CRISPR array includes the MGE, which further comprises the host cells of the first species and the industrial or household systems. Transfer is possible between microbial host cells. For example, further cells can be transferred in the environment (e.g. For example, in aquatic or soil environments) environmentally, industrially, or domestically permissible, and the first The host cell is unacceptable in the environment. Optionally, oriT is used to create a first and further host cell. It is functional in cells.

[0235] Optionally, the first and further host cells are enclosed by a fluid biofilm in contact with the surface. Or, the cells are contained in the fluid.

[0236] Optionally, each MGE may be an embedded and connected element (ICE) or an embedded and connected element. The vector contains a synthetic element, or each vector contains a phage capable of infecting the host cells of the first species. Each MGE is a phage nucleic acid capable of intercellular transfer. Optionally, each ICE is Transposons, for example, junctional transposons. Optionally, each vector is... A plasmid containing MGE as described herein by choice. By choice, the sequence is: It is contained by the fused transposons of the cells and / or further cells.

[0237] In the example, the above method is used for the recovery, processing, storage, or transportation of crude oil, gas, or petrochemical products. Microbial corrosion of substrates contained in equipment (e.g., crude oil tankers, oil rigs, or oil drilling rigs) A method for controlling food contamination (MIC) or biofouling, wherein the surface of the substrate is a collection of first host cells In contact with the group, the first host cell transmits the first species of sulfur or biofouling to the substrate. These include sulfate-reducing bacteria (SRBs), extracellular polymer-producing bacteria (EPSBs), acid-producing bacteria (APBs), sulfur, or sulfur. Sulfur-oxidizing bacteria (SOB), iron-oxidizing bacteria (IOB), manganese-oxidizing bacteria (MOB), ammonia-producing bacteria (Am PB) or acetic acid-producing cells (AcPB), the surface and cell population are seawater, freshwater, fracturing liquid, Or it is in contact with a liquid selected from the well's liquid (e.g., an oil well or a natural gas well), and the above method The law is, (i) The liquid is mixed with multiple vectors capable of transforming or transducing a first host cell. By mixing, the cell population is brought into contact with the vector, and each vector contains a CRISPR array. As a result, the CRISPR array is introduced into the host cell, (a) Each CRISPR array contains one or more sequences for expressing crRNA and host cells It includes a promoter for transcribing the sequence, (b) Each crRNA hybridizes with a target sequence in the host cell and interacts with the host cell's Cas (e.g., Ca An s-nuclease (e.g., Cas9 or Cfp1) is introduced to modify the target sequence (e.g., target sequence It is possible to cut the sequence, and the target sequence is a gene that mediates host cell viability. It is an array, Each sequence in (c)(a) is used to induce the expression and production of the corresponding crRNA in the first host cell. The formula includes the column R1-S1-R1', where R1 is the first CRISPR repeat and R1' is the second CRISPR repeat R1 or R1' is optional, and S1 is the target sequence of the first host cell and 70, 75. Containing or consisting of nucleotide sequences with 80, 85, 90, 95%, or higher identity. The first CRISPR spacer process, and (ii) Enable the expression of the cRNA in the host cell in the presence of Cas, thereby enabling the expression of the host cell Modify the target sequence of cells to reduce host cell viability and control the MIC or biofouling of the substrate. This method includes the process of bringing about the result. In the embodiment, both R1 and R1' are present.

[0238] In this example, the above method is for controlling bacterial biofouling of ballast water in a ship or boat. The law states that water contains a population of first host cells of a first microbial species that mediates the biopollution. The above method is (i) The population is brought into contact with multiple vectors capable of transforming or transducing cells. Each vector contains a CRISPR array, which is then introduced into host cells. (a) Each CRISPR array contains one or more sequences for expressing crRNA and host cells It includes a promoter for transcribing the sequence, (b) Each crRNA hybridizes with a target sequence in the host cell and interacts with the host cell's Cas (e.g., Ca It is possible to introduce an S-nuclease and modify the target sequence (for example, cleave the target sequence). The process involves the target sequence being a gene sequence that mediates host cell viability, and (ii) Enable the expression of the cRNA in the host cell in the presence of Cas, thereby enabling the expression of the host cell A process to modify the target sequence of cells to reduce host cell viability and control the biofouling. This is a method that includes this.

[0239] Optionally, the first host cell is Vibrio cholerae, E coli, or Enterococci sp. cells. be.

[0240] Optionally, step (i) is to mix the ballast water of a ship or boat hull with the vector. The process includes the following: Optionally, the ship or boat is an ocean vessel and the water is seawater. Alternatively, instead of a ship or boat, ballast water can be used for offshore containers or drilling platforms. This includes, for example, oil platforms or oil rigs. Examples include ships and boats. , containers, platforms, or rigs are moored at sea (i.e., their location (Not just temporarily).

[0241] In the example, the above method is a method for discharging ballast water from a ship or boat, The ballast water discharged includes water treated by the method described above. Optionally, the water is a single unit. To water, for example, the sea, ocean, or waterways (for example, rivers, canals, lakes, or reservoirs), or to containers It is released into Na.

[0242] paragraph The present invention provides the following paragraphs, which are supported by the following embodiments. 1. A program for use in treating the target E coli or C dificile infection. A ram-able Cas (e.g., Cas3 or Cas9) nuclease, wherein the Cas nuclease is the target To cleave target sites contained in the genome of infected E coli or C dificile bacteria It is programmable with guide RNA, thereby killing E coli or C-difficult cells. Alternatively, cell growth or proliferation is reduced, and the treatment method involves contacting the target with Cas nuclease. The process involves the nuclease being exposed to the target site and programmed with guide RNA to cleave it. This includes, thereby, the genome of E coli or C dificile bacteria contained in the target is cleaved. The target infection is detected within the first 30 minutes of treatment (for example, within the first 15 minutes) A programmable Cas nuclease that can be reduced to 1 / 100th of its original level. 2. A program for use in treatment of the target E coli or C dificile infection. A ram-capable Cas (e.g., Cas3 or Cas9) nuclease (optionally, as described in paragraph 1) Cas nuclease is contained in the genome of E coli or C dificile bacteria that infect the target. It can be programmed with guide RNA to cleave the target site, As a result, E coli or C dificile cells die, or their growth or proliferation is slowed. The treatment method involves bringing the target into contact with Cas nuclease, and the nuclease then targets the target site. The process involves being programmed with guide RNA to cleave, thereby containing more of the target The genome of E coli or C dificile bacteria is cut, reducing the incidence of the target infection. A reduction of at least 1 / 100 is achieved after the subject has been brought into contact with the programmed nuclease. It is maintained for at least 60 minutes (for example, at least 120, 145, or 180 minutes), Programmable Cas nuclease. 3. At least 60% of infections occurred when subjects came into contact with programmed nucleases. The nuclease described in paragraph 1 or 2, which is reduced by 0 minutes. 4. A nuclease (e.g., a programmed nuclease) and / or guide RNA are used. The nucleic acid to be administered is given to the subjects at time point 1 (T1) and time point 2 (T2), and T2 is at least as high as T1. The nuclease described in any one of paragraphs 1 to 3, which is also 1 hour later. 5. The above method ensures that the reduction in infection is sustained for 30 minutes immediately after the first 30 minutes of treatment. To that end, the nuclea described in any one of paragraphs 1 to 4, which includes a step to reduce infection Ze. 6. The above method involves applying RNA or a nucleus encoding RNA to express RNA in the target. The process includes an acid administration step, during which the RNA complexes with the nuclease and programs the nuclease. Then, depending on the target, cut the target site of the microorganism contained in the target, as described in one of paragraphs 1 to 5. Nucleases are included. 7. Nucleases are administered to the target simultaneously with or sequentially with RNA or RNA-encoding nucleic acids. The nuclease described in any one of paragraphs 1 to 6. 8. The target is RNA or nucleic acid, and before administration, the nuclease described in paragraph 7 is used. Crease. 9. Multiple viruses (e.g., phages) are administered to a target, and each virus carries RNA. The virus contains copies of nucleic acids, and it infects microorganisms that it targets. A nuclease for delivering nucleic acids, as described in any one of paragraphs 1 to 8. 10. Viruses administered: The ratio of microorganisms included varies depending on the target, from 10 to 150, see paragraph 9. The nucleases listed. 11. The subjects are humans or animals, and are randomly selected to be older than 65 years of age or children. A nuclease described in any one of paragraphs 1 to 10, for patients in the Department of Medicine. 12. The infection is an infection of the lungs, abdomen, or urinary tract, or the subject has undergone surgery. This may include being under immunosuppressant drug therapy and / or suffering from a chronic disease, paragraph 11 The nuclease described in [reference]. 13. Infections may be treated for an optional period of time, from one hour to the first 30 days of treatment. Within minutes (for example, within the first 15 minutes), paragraphs 1 through 12 should be reduced by at least 90%. Any of the nucleases listed in item 1. 14. The above method should treat the infection within the first 30 minutes of treatment (for example, within the first 15 minutes). ), including a process to reduce the infection by at least 1 / 100, After bringing the subject into contact with the programmed nuclease, leave it for at least 60 minutes (for example, a short period of time). Maintained for at least 120, 145, or 180 minutes, in any one of paragraphs 1 through 13 The nucleases listed. 15. The above method treats the target sepsis and / or sepsis (e.g., septic shock) The nucleases described in any one of paragraphs 11 to 14 prevent the following: 16. At the start of treatment, the subject (e.g., human) should have a body temperature <36°C or >38°C and a heart rate >90 beats / min. Number of breaths, respiratory rate >20 breaths / min or PaCO2 <4.3kP, and <4000 / mm² 3 or >12,000 / mm 3 White blood cell count The nuclease described in paragraph 16, having the following characteristics. 17. At the start of treatment, the subject (e.g., human) exhibits abnormal body temperature, abnormal heart rate, abnormal respiratory rate, etc. The presence of two or more abnormal blood gases and abnormal white blood cell counts, The nuclease described in item 15 or 16. 18. The subject is a human or animal, and the microorganism is a bacterium (e.g., E. coli or C. difi). (cile), target blood infections caused by bacteria should be treated within the first 30 minutes of treatment (for example, the first 15 minutes) (By the time of) any one of paragraphs 1 to 17 to be reduced to at least 1 / 100 or 1 / 1000 The nuclease described in [reference]. 19. The blood sample to be used should be 10 immediately before treatment. 7 ~1012 Infection with bacteria at CFU / ml, paragraphs 11-18 A nuclease as described in any one of the following items. 20. A nuclease described in any one of paragraphs 1 to 10, wherein the target is a plant. 21. Bacteria are included in the microbiome, as described in any one of paragraphs 1 through 20. Nuclease. 22. The microbiome includes Lactobacillus and / or Streptococcus bacteria, paragraph The nuclease described in 21. 23. E coli is EHEC E coli, the nuclea described in any one of paragraphs 1 to 22. Ze. 24. Nucleases include Cas nucleases (e.g., Cas3 or Cas9), meganucleases, TALEN (Transcription Activator-like Effector Nuclease), or Zinc Finger Nuclease The nuclease described in any one of paragraphs 1 through 23. 25. Use in conjunction with any of the nucleases described in paragraphs 1 through 24 of the treatment method. Multiple viruses for this purpose (e.g., phages or phagemids for producing phages) Each virus contains a copy of nucleic acid that codes for RNA, and viruses vary depending on the target. Multiple viruses capable of infecting the microorganisms contained within and delivering nucleic acids to them. 26. Program the nuclease described in any one of paragraphs 1 to 24 in the treatment method. A composition comprising multiple nucleic acids for the purpose of, wherein each nucleic acid is defined in any one of paragraphs 6 to 9. A composition that is a nucleic acid. 27. Nucleases described in any one of paragraphs 1 to 26 for use in therapeutic methods. A CRISPR / Cas system including a Cas nuclease (e.g., Cas3 or Cas9) The system encodes one or more guide RNAs or one or more guide RNAs. Each guide RNA contains DNA and is designed to cleave target sites contained within the microbial genome. The CRISPR / Cas system allows for the programming of Cas nucleases. 28. Use in methods to treat target acute microbial infections, such as sepsis or sepsis. Guide RNA or DN encoding guide RNA for use in the system described in paragraph 27 for use A. 29. A nucleic acid vector comprising the guide RNA or DNA described in paragraph 27 or 28. 30. Vectors include phages, phagemids, biiriophages, viruses, and plasmin. A vector, such as a plasmid (e.g., a conjugating plasmid) or a transposon, as described in paragraph 29. 31. Antiseptics for administration to humans or animals to treat sepsis or sepsis Alternatively, an anti-sepsis composition comprising multiple vectors, each vector being described in paragraph 29 or 30. As described, an anti-septic or anti-sepsis composition. 32. A method for treating a target acute microbial infection, any of the methods described in paragraphs 1 to 31. The method as prescribed in paragraph 1. 33. A set for carrying out the treatment methods specified in any one of paragraphs 1 through 32. In the manufacture of the product, the nuclease described in any one of paragraphs 1 to 25 and 27 to 30, The use of multiple viruses, systems, guide RNA, DNA, or vectors, where the target is human or animal. Use of a living organism other than an object. 34. Compositions for carrying out ex vivo methods to treat microbial infections of substrates Nucleases described in any one of paragraphs 1 to 25 and 27 to 30, multiple viruses The use of a system, guide RNA, DNA, or vector, wherein microbial infection is caused by the first species or strain. Caused by microorganisms, the nuclease is contained in the substrate by the genome of the microorganism that infected it. It can be programmed to cut the target site, thereby the first species or strain The microorganisms are killed, or their growth or proliferation is reduced, and the treatment method targets the target. When brought into contact with clease, the nuclease is programmed to cleave the target site. This process involves cutting the genome of microorganisms contained in the target, and the acute microorganisms of the substrate. Use for treating object infections. 35. Compositions for carrying out ex vivo methods to treat microbial infections of substrates The use of a programmable nuclease, wherein microbial infection is caused by the first species or strain Caused by microorganisms, the nuclease is contained in the substrate by the genome of the microorganism that infected it. It can be programmed to cut the target site, thereby the first species or strain The microorganisms are killed, or their growth or proliferation is reduced, and the treatment method targets the target. When brought into contact with clease, the nuclease is programmed to cleave the target site. This process involves cutting the genome of microorganisms contained in the target, and the acute microorganisms of the substrate. Use for treating object infections. 36. Recognition of nucleases (e.g., programmed nucleases) and / or target sites. And the nucleic acid programmed to cleave the nuclease is used at the first time point (T1) and the second time point. (T2) is administered to the subject or substrate, and T2 is at least 1 hour after T1, paragraphs 33, 34, 35 Use as described in any one of the items. 37. Infection occurs within the first 30 minutes of treatment (for example, within the first 15 minutes), at least once. The use described in any one of paragraphs 33 to 36, reduced to 1 / 00th. 38. Infection reduction occurs when the subject is exposed to the programmed nuclease and then... Both are maintained at at least 1 / 100th for 60 minutes (for example, at least 120 minutes). or use as described in any one of paragraphs 33 to 37. 39. The reduction in infection lasts for 30 minutes immediately after the first 30 minutes of treatment, as described in paragraphs 33 to 38. Use as described in any one of the items. 40. The above method involves generating RNA in or on the target or substrate. The process includes administering a nucleic acid encoding RNA to a target or substrate, wherein the RNA is a nucleic acid It complexes with an enzyme and programs the nuclease to contain microorganisms depending on the target or substrate. The use described in any one of paragraphs 33 to 39, for cutting a target portion of an object. 41. Nucleases are administered to the target or substrate simultaneously with or sequentially with RNA or nucleic acids. Use as described in paragraph 40. 42. The subject or substrate contains a nuclease before administering RNA or nucleic acid, as described in paragraph 40. Use. 43. Multiple viruses (e.g., phages) are administered to a target or substrate, and each virus The virus contains copies of nucleic acids, and it infects microorganisms contained in the target or substrate. Use as described in any one of paragraphs 40 to 42, for delivering nucleic acids. 44. The ratio of viruses to microorganisms administered is 10 to 150, as described in paragraph 43. 45. Infection may be detected by choice within the first 30 minutes of treatment (for example, within the first 15 minutes). Any of paragraphs 33 to 44, which are reduced by at least 90% over a period of time or longer. Use as described in item 1. 46. ​​Infection occurs within the first 30 minutes of treatment (for example, within the first 15 minutes), at least once. Reduced to 1 / 00th, and infection reduced to at least 1 / 100th, the target or substrate, program After contact with the nuclease, leave for at least 60 minutes (for example, at least 120, 145, or The use described in paragraph 44 or 45 is maintained for 180 minutes. 47. Is the subject a plant, or is the substrate a metal substrate, plastic substrate, or concrete? The substrate is a stone substrate, a wood substrate, a glass substrate, or a ceramic substrate, as specified in paragraphs 33 to 46. Use as described in any one of the items. 48. The microorganism is a bacterium, as used in any one of paragraphs 33 to 47. 49. The bacteria are Gram-positive bacteria, as described in paragraph 48. 50. Bacteria include Staphylococcus, Streptococcus, Enterococcus, Legionella, and Heamophi. lus, Ghonnorhea, Acinetobacter, Escherichia, Klebsiella, Pseudomonas, or Stenot Rophomonas bacteria (e.g., E. coli (e.g., EHEC E. coli), C. dificile, V. cholera, Staph ylococcus (e.g., S aureus or MRSA), Streptococcus pyogenes, Acinetobacter bauma (Bacteria) nnii, Legionella, Pseudomonas aeruginosa, Klebsiella pneumoniae, paragraph Use as described in 48 or 49. 51. Nucleases include Cas nucleases (e.g., Cas3 or Cas9), meganucleases, TALEN (Transcription Activator-like Effector Nuclease), or Zinc Finger Nuclease The use described in any one of paragraphs 33 through 50.

[0243] Treatment of pathogenic bacterial infections Infectious complications are a pathological condition and death in cancer patients, especially those with underlying hematological malignancies. This is a serious cause of death, and autopsy studies have shown that approximately 60% of deaths are related to infectious diseases. It has been proven that there is less data on infection mortality in patients with solid organ tumors. Although not a known cause of death, approximately 50% of these patients experience either a primary or related cause of death. It is suspected that the patient has an infectious disease (as described in "Infectious Complications in Cancer Patients"). “Epidemiology of Infections in Cancer Patients” Springer International Publishers (Switzerland, 2014). Most cases are bacterial infections. These infectious complications are still common. This remains a significant limitation of cancer treatment modalities.

[0244] The adverse effects of classical antibiotic treatment using broad-spectrum antibiotics are due to immune checks. This has been demonstrated in cancer patients treated with point inhibitors (ICIs). Routy et al. studied the gut microbiome. However, this research will investigate how it affects the effectiveness of PD-1-based immunotherapy for epithelial tumors. (Routy et al., Science 2018, Vol. 359, pp. 91-97). In this study, the authors found one or Progressive NSCLC, receiving antibody ICI against PD-1 / PD-L1 interaction after several previous therapies. Infections / antibiotics in patients with (n=140), renal cell carcinoma (n=67), or urothelial carcinoma (n=42). The usage datasets were also analyzed. Among these patients, some patients received the first dose of PD-1 / PD-L1 mAb. Within two months prior to administration or within one month after administration, broad-spectrum antibiotics (beta-lactam + / - inhibitors) A harmful drug (fluoroquinolone, or macrolide) was prescribed. The patient generally had a common indication. Antibiotics were taken orally for the following conditions (dental infection, urinary tract infection, and lung infection). ICI therapy was administered. Adverse effects have been observed when treating infections in cancer patients with classic broad-spectrum antibiotics. See Figure 8, which shows that antibiotic treatment during ICI therapy has a fatal outcome. In contrast to the 9.8-month overall survival with biomaterial therapy, the median survival rate in the absence of antibiotic therapy was lower. Overall survival was 21.9 months. Therefore, the median overall survival for patients treated with classical antibiotics was: The median overall survival rate for patients not receiving antibiotic treatment is <50% (or >12 months shorter).

[0245] A study by Gopalakrishnan et al. found that "healthy" micro-oncological outcomes in immuno-oncological therapy Another recent example supporting the importance of biomes (Gopalakrishnan et al., Science 2018) (Vol. 359, pp. 97-103). See Figures 9A and 9B. The intestinal microbiome is related to melanoma. Modulation of the effectiveness of anti-PD-1 inhibition in patients was observed.

[0246] Several other studies have shown a significant correlation between the microbiome and immuno-oncological outcomes. An expanded evidence-based approach to sex has been added. · “Microbiota: a key orchestrator of cancer therapy”, Nat. Rev. Cancer 201 Year 7, Volume 17, pages 271-285 Matson et al., Science 2018, Vol. 359, pp. 104-108. · L. Derosa et al., Annals of Oncology 2018 (epub March 30, 2018) M. Vetizou et al., Science, 2015, Vol. 350, pp. 1079-1074. • Sivan et al., Science 2015, Vol. 350, pp. 1084-1089

[0247] Another report indicates that among patients receiving immune checkpoint blockers for cancer therapy... It claims to be the first systematic reinvestigation of infectious diseases, and that immune checkpoint inhibitors (C Severe infections in melanoma patients treated with TLA-4, PD-1, and / or PD-L1 have been studied. (M. Del Castillo et al., Clin. Infect. Dis. 2016, Vol. 63, pp. 1490-1493). Severe symptoms. Infections were defined as infections requiring hospitalization or parenteral antimicrobial therapy. 740 patients Of the 898 patients who underwent immune checkpoint blockade, 54 developed serious infections. (7.3%). Nine patients (17%) were found to have died from the infection. Some patients had >1 infection. The total number of infectious diseases that developed was 58. The vast majority of these infectious diseases were of bacterial origin. (Approximately 80%; i.e., bacterial infections: 7.3% of 80% of patients: 5.8%). Pneumonia and blood infections are the two main causes. It was a bacterial infection.

[0248] Immune checkpoint blockers have immune-related adverse effects (irAEs) that affect the upregulation of the immune system. Related. Complications are managed with immunosuppressants such as steroids (immunosuppression is necessary for subsequent days (A risk factor for Wami infections). Of the 740 patients, 46% received steroids during the course of treatment. I received it. The risk of serious infection is associated with receiving corticosteroids or infliximab. In the selected cohort, the figure was 13.5% (compared to 7.3% in the overall population).

[0249] Another report has investigated the potential for increased infectious disease in lung cancer patients receiving ICI therapy. Of the 84 NSCLC patients who received nivolumab (PD-1 inhibitor), 20 patients (23.8%) %) developed infectious diseases. Bacterial infections accounted for 75% of infections. In other words, 1% of patients developed infectious diseases. 8% were bacterial infections. The most common type of bacterial infection was pneumonia. K. Fujita et al., E See ur. Resp. J. 2017, Vol. 50, OA1478.

[0250] Gram-negative bacilli E. coli are one of the most common causes of bacteremia in cancer patients. The all-cause 30-day mortality rate against the active ingredient is high (approximately 15%) (YE Ha et al., Int. J. Antimicr. Agen. 20 (2013, Vol. 42, pp. 403-409). Publication of 30-day all-cause mortality rates for patients with E. coli bacteremia (cancer / non-cancer). The estimates ranged from approximately 10% to 35% (JK Abernethy et al., Clin. Microbiol. Infe). ct. 2015, Vol. 21, 251.e1-251.e8), the high burden associated solely with this pathogen was clearly highlighted. Overall, the pathogens causing bacteremia were found in >100 cases of bacteremia among cancer patients. According to the studies examined, the bacteria were mainly Gram-negative (65%), with E. coli (18.3%) and P. aeruginosa. (18.3%) and K. pneumoniae (17.3%) are the most common organisms encountered, and these three diseases When combined with other pathogens, they account for 54% of bacteremia cases (G. Samonis et al., Support Care Cancer 2013). (Volume 21, pp. 2521-2526). The in-hospital mortality rate in this study was 26.2%. Similar figures were observed elsewhere. For example, neutropenia and non-neutropenia with blood infections can be found. In a study of adult cancer patients, blood infections were investigated in 399 cases among 344 cancer patients. The major causative pathogens were Gram-negative bacilli (45%). Among clinical isolates, E. coli (35%) ) is the most common cause among Gram-negative bacteria, followed by K. pneumoniae (20%) and P. aeruginosa (19%) (E. Velasco et al., Eur. J. Clin. Microbiol. Infect. Dis. 2006, Vol. 25, 1) (Pages 1-7). These three pathogens together account for 33% of bacteremia cases (or 7% of Gram-negative cases). 4%). The overall 30-day mortality rate was 32% in this study. Two other reports showed that solid tumors The causative agents of blood infections in patients were investigated, and Gram-negative bacteria were found to be the dominant pathogen type. It was found that this was the case (47-55% of infections across several hundred patients) (M. Marin et al., Medi cine 2014, Vol. 93, pp. 143-149; M. Anatoliotaki et al., Infection 2004, Vol. 32, pp. 65-71 See also C. Gudiol et al., Virulence 2016, Vol. 7, pp. 298-308. In larger studies (where the individual pathogen levels are more robust), three main groups within the Gram-negative group were found. In this case, the pathogens were again E. coli (55%), P. aeruginosa (18%), and Klebsiella spp. 11%). This corresponds to 92% of Gram-negative cases or to the total of 528 bloodborne infections studied. This corresponds to 51%.

[0251] The above data regarding specific causative infectious pathogens in cancer patients can be found in Table 5 below. This is summarized in 34).

[0252] Therefore, available data on bloodborne infections in cancer are for Gram-negative pathogens. However, it is involved in 45-65% of infection cases, and three important pathogens are involved: E. coli, K. pneumoniae, and P. This indicates that *Aeruginosa* is responsible for the majority of Gram-negative cases (73-92%).

[0253] Therefore, the inventors have systematically described the dilemma faced by oncologists. • Reduced effectiveness of cancer therapy is due to the decrease in microbiome diversity caused by antibiotic therapy. It is highly likely that this is due to reduction. • At least one-third of patients under checkpoint inhibitors (inhibitors) experience serious, life-threatening complications. To contract an infectious disease related to the condition. • If these infections are not treated, they can lead to death (1~ (In two weeks). Treatment with classic antibiotics has resulted in a reduction in the 4-year progression-free survival rate from >40% to approximately 10%. To be startled. • The choice is based on the immediate need for treatment that poses a risk of seriously harming cancer therapy and is potentially lethal. It is to treat infectious diseases that need to be addressed.

[0254] Therefore, the inventors have developed a method to minimize interference with cancer therapy by eliminating bacterial pathogenicity in a different way. We understood the need for a method that can treat infectious diseases. The need is for other therapies in which microbiome compositions can modulate therapeutic outcomes. I understand that this will be useful in setting up legal frameworks, for example, in setting up porting configurations.

[0255] While we do not wish to be bound by any particular theory, the inventors of this invention use This aims to mitigate the adverse effects of conventional antibiotic therapy on overall survival (survivability) in ICI patients. In some embodiments, this could lead to a doubling (or >12 months) of overall survival. Capturing the treatment effect over several months in terms of median values ​​is a very essential achievement in this field. In fact, this magnitude of effect size is typical of ICI trials (i.e., the benefits are usually seen in months, not years). The outcomes are comparable to those reported (measured by [method / tool]). In addition, PD-1 / PD-L1 drugs dominate the ICI market. It is predicted that in 2023, PD-1 / PD-L1 will account for 94% of ICI's $46 billion USD global sales. (CTLA-4 blockers account for only 6%). Source: Landscape & Fore "cast: Immune Checkpoint Inhibitors," Decision Resources, December 2017. This improves treatment using PD-1 or PD-L1 immune checkpoint inhibitors (inhibitors). The need for this is particularly urgent in medicine, and the inventors of this invention are specifically focused on this point. We believe that this will lead to profits.

[0256] In this example, the method involves using a classic antibiotic such as a broad-spectrum antibiotic (or any other antibiotic disclosed herein). Eliminates the need to administer other substances. In another example, the present invention treats infectious diseases. Classical antibiotics such as broad-spectrum antibiotics (or those disclosed herein) administered to the target for this purpose Reduce the amount or frequency of administration of any other substance. For example, a low-dose broad-spectrum antibiotic (e.g., For example, administering 50%, 40%, 30%, 20%, 10% or less of the conventional dose while inducing a nucleic acid Aase cleavage can be used, and therefore, infections are treated in this setting. This invention This applies to patients under immunosuppressant therapy, such as cancer patients, transplant patients, or those with viral infections (e.g., H2). IV (Human Immunodeficiency Virus), CMV (Cytomegalovirus), or RSV (Respiratory Syncytial Virus (RSV) This may be particularly beneficial for patients suffering from ory synctial virus infection.

[0257] The term "broad-spectrum antibiotic" refers to two major groups of bacteria: Gram-positive bacteria and Gram-negative bacteria. This refers to antibiotics that act on a specific group of bacteria, or any antibiotic that acts against a wide range of disease-causing bacteria. This is possible. Such drug treatment is used when a bacterial infection is suspected, but the bacterial group is unknown. It is used (also called empirical therapy) or when an infection caused by multiple bacterial groups is suspected. Strong broad-spectrum antibiotics, in particular, can interfere with naturally occurring and normal bacteria and lead to the development of antimicrobial resistance. There are specific risks. Examples of commonly used broad-spectrum antibiotics include aminoglycosides (S (Excluding treptomycin), ampicillin, amoxicillin, amoxicillin, Clavula Calcium phosphate (Augmentin), carbapenems (e.g., imipenem), piperacillin, tazobacter Mu, quinolones (e.g., ciprofloxacin), tetracyclines, chloramphenicol These are ticarcillin, trimethoprim, and sulfamethoxazole (Bactrim). In medicine, examples include amoxiclav (for example, in small animals) and penicillin. These include streptomycin, oxytetracycline, and reinforced sulfonamides.

[0258] Clause Therefore, in one aspect, the present invention relates to pathogenicity using programmed nucleases. The following provisions regarding the treatment of bacterial infections are provided. 1. A pathogen affecting humans or animals caused by the first species or strain of bacteria (the first bacterium). A method for treating sexually transmitted bacterial infections, comprising a target region contained in the genome of a first bacterium. By cutting the site, the first bacteria contained in the target are selectively killed, and the cutting is Using a programmable nuclease programmed to cleave the target site The process includes steps taken to carry out the procedure, and the subjects are those suffering from other diseases or conditions in addition to pathogenic bacterial infections. The above method involves administering therapy to a subject in order to treat or prevent further diseases or conditions. Nucleases, including the nutrient, treat infections, and the therapy involves programmed nucleases. A method that is effective in treating or preventing a disease or condition in the presence of other factors.

[0259] In the example, item 1 provides the following: Pathogenic bacterial infections in cancer patients caused by the first species or strain of bacteria (first bacterium) A method for treating the disease, comprising cutting a target site contained in the genome of a first bacterium. This selectively kills the primary bacteria present in the target, and the cutting cuts the target site. This is performed using a Cas nuclease programmed by a guide RNA to interrupt the process. The above method includes a step of administering immunotherapy to a target in order to treat the patient's cancer. Nucleases treat infections, and immunotherapy involves the presence of programmed nucleases. Below are some methods that are effective in treating cancer. Pathogenic bacterial infections in cancer patients caused by the first species or strain of bacteria (first bacterium) A method for treating the disease, comprising cutting a target site contained in the genome of a first bacterium. This selectively kills the primary bacteria present in the target, and the cutting cuts the target site. This is performed using a Cas nuclease programmed by a guide RNA to interrupt the process. The above method includes a step of administering immunotherapy to a target in order to treat the patient's cancer. Nucleases treat infections, and immunotherapy involves the presence of programmed nucleases. It is effective in treating cancer, (a) Immunotherapy may be optional, including pembrolizumab (or KEYTRUDA®) and nivolumab (or This includes the step of administering an anti-PD-1 antibody selected from OPDIVO (trademark) to a patient. (b) Cancers include metastatic melanoma; renal cell carcinoma; bladder cancer; solid tumors; non-small cell lung cancer (NSCLC); frontal cancer Squamous cell carcinoma of the pelvis and neck (HNSCC); Hodgkin lymphoma; overexpression of PD-L1 and spikes in EGFR or ALK. Cancer without natural mutations; selected from colorectal cancer and hepatocellular carcinoma. (c) The first bacterium is Pseudomonas aeruginosa, Klebsiella pneumoniae, E coli, Salmone lla (for example, S typhimurium), Clostridium dificile, Staphylococcus (for example, S aureu (S or S epidermis), Streptococcus (e.g., S viridans or S thermophilus), Pneumoco A method for selecting bacteria from Ccus and Enterococcus. Pathogenic bacterial infections in cancer patients caused by the first species or strain of bacteria (first bacterium) A method for treating the disease, comprising cutting a target site contained in the genome of a first bacterium. This selectively kills the primary bacteria present in the target, and the cutting cuts the target site. This is performed using a Cas nuclease programmed by a guide RNA to interrupt the process. The above method includes a step of administering immunotherapy to a target in order to treat the patient's cancer. Nucleases treat infections, and immunotherapy involves the presence of programmed nucleases. It is effective in treating cancer, (a) Immunotherapy may be optional, including atezolimmab (or TECENTRIQ®), Selected from velumab (or BAVENCIO®) and durvalumab (or IMFINZI®) The procedure includes the step of administering an anti-PD-L1 antibody, (b) Cancers include metastatic melanoma; renal cell carcinoma; bladder cancer; solid tumors; non-small cell lung cancer (NSCLC); frontal cancer Squamous cell carcinoma of the cervix and neck (HNSCC); Merkel cell carcinoma; Hodgkin lymphoma; overexpressing PD-L1, E Cancer without GFR or ALK mutations; selected from colorectal cancer and hepatocellular carcinoma. (c) The first bacterium is Pseudomonas aeruginosa, Klebsiella pneumoniae, E coli, Salmone lla (for example, S typhimurium), Clostridium dificile, Staphylococcus (for example, S aure us or S epidermis), Streptococcus (e.g., S viridans or S thermophilus), Pneumo A method for selecting bacteria from *Coccus* and *Enterococcus*. Pathogenic bacterial infections in cancer patients caused by the first species or strain of bacteria (first bacterium) A method for treating the disease, comprising cutting a target site contained in the genome of a first bacterium. This selectively kills the primary bacteria present in the target, and the cutting cuts the target site. This is performed using a Cas nuclease programmed by a guide RNA to interrupt the process. The above method includes a step of administering immunotherapy to a target in order to treat the patient's cancer. Nucleases treat infections, and immunotherapy involves the presence of programmed nucleases. It is effective in treating cancer, (a) Immunotherapy involves administering an anti-CD52 antibody, or optionally alemtuzumab (or CAMPATH®) to the patient. Including the administration step, (b) The cancer is B-cell chronic lymphocytic leukemia (CLL), (c) The first bacterium is Pseudomonas aeruginosa, Klebsiella pneumoniae, E coli, Salmone lla (for example, S typhimurium), Clostridium dificile, Staphylococcus (for example, S aure us or S epidermis), Streptococcus (e.g., S viridans or S thermophilus), Pneumo A method for selecting bacteria from *Coccus* and *Enterococcus*. Pathogenic bacterial infections in cancer patients caused by the first species or strain of bacteria (first bacterium) A method for treating the disease, comprising cutting a target site contained in the genome of a first bacterium. This selectively kills the primary bacteria present in the target, and the cutting cuts the target site. This is performed using a Cas nuclease programmed by a guide RNA to interrupt the process. The above method includes a step of administering immunotherapy to a target in order to treat the patient's cancer. Nucleases treat infections, and immunotherapy involves the presence of programmed nucleases. It is effective in treating cancer, (a) Immunotherapy may include an anti-CD20 antibody, optionally ofatumumab (or ARZERRA®), or The process includes administering rituximab (or RITUXAN®) to a patient. (b) Cancer is B-cell chronic lymphocytic leukemia (CLL) (e.g., refractory CLL) or non-Hodgkin lymphoma It is a tumor, (c) The first bacterium is Pseudomonas aeruginosa, Klebsiella pneumoniae, E coli, Salmone lla (for example, S typhimurium), Clostridium dificile, Staphylococcus (for example, S aureu (S or S epidermis), Streptococcus (e.g., S viridans or S thermophilus), Pneumoco A method for selecting bacteria from Ccus and Enterococcus. Pathogenic bacterial infections in cancer patients caused by the first species or strain of bacteria (first bacterium) A method for treating the disease, comprising cutting a target site contained in the genome of a first bacterium. This selectively kills the primary bacteria present in the target, and the cutting cuts the target site. This is performed using a Cas nuclease programmed by a guide RNA to interrupt the process. The above method includes a step of administering immunotherapy to a target in order to treat the patient's cancer. Nucleases treat infections, and immunotherapy involves the presence of programmed nucleases. It is effective in treating cancer, (a) Immunotherapy includes the step of administering an anti-KIR antibody, optionally lirilumab, to the patient. (b) The cancer is, at the discretion of the party, acute myeloid leukemia or head and neck squamous cell carcinoma (SCCHN), (c) The first bacterium is Pseudomonas aeruginosa, Klebsiella pneumoniae, E coli, Salmone lla (for example, S typhimurium), Clostridium dificile, Staphylococcus (for example, S aureu (S or S epidermis), Streptococcus (e.g., S viridans or S thermophilus), Pneumoco A method for selecting bacteria from Ccus and Enterococcus. Pathogenic bacterial infections in cancer patients caused by the first species or strain of bacteria (first bacterium) A method for treating the disease, comprising cutting a target site contained in the genome of a first bacterium. This selectively kills the primary bacteria present in the target, and the cutting cuts the target site. This is performed using a Cas nuclease programmed by a guide RNA to interrupt the process. The above method includes a step of administering immunotherapy to a target in order to treat the patient's cancer. Nucleases treat infections, and immunotherapy involves the presence of programmed nucleases. It is effective in treating cancer, (a) Immunotherapy may be optional, including axicabtagene ciloleucel. Anti-C selected from (or YESCARTA®) and tisagenlecleucyl (or KYMRIAH®) The process includes administering a D19 CAR-T to a patient, (b) Cancer is a type of B-cell lymphoma (e.g., non-Hodgkin lymphoma (NHL); erosive large B cell lymphoma) Lymphoma (DLBCL); primary mediastinal large B-cell lymphoma; or high-grade B-cell lymphoma); B-cell acute Lymphoblastic leukemia (ALL); or central nervous system lymphoma, selected from the above. (c) The first bacterium is Pseudomonas aeruginosa, Klebsiella pneumoniae, E coli, Salmone lla (for example, S typhimurium), Clostridium dificile, Staphylococcus (for example, S aureu (S or S epidermis), Streptococcus (e.g., S viridans or S thermophilus), Pneumoco A method for selecting bacteria from Ccus and Enterococcus.

[0260] Instead, CAR-T is anti-CD30, CD38, or CD22 CAR-T. In the example, cancer is less common. It is large B-cell lymphoma after failure of at least two other types of treatment. For example, These include high-grade B-cell lymphoma and DLBCL arising from follicular lymphoma. In the example, cancer This is relapsed / remittent B-cell acute lymphoblastic leukemia. In this case, the cancer is primary central nervous system It is a type of lymphoma.

[0261] In this example, the nuclease treats the infection without causing a reduction in the effectiveness of the therapy. In the embodiment, "without causing a reduction in the effectiveness of the therapy" means a plurality of different species including a first species By administering a broad-spectrum antibiotic (or an antibiotic disclosed herein) that kills the species, the patient This refers to the effectiveness of the therapy compared to the reduction caused by the therapy. In the embodiment, "effectiveness of the therapy" "Without causing a reduction in efficacy" refers to the application of therapy in the absence of treatment for pathogenic bacterial infections and In comparison (or patients suffering from a disease or condition and receiving the aforementioned treatment for that purpose) Compared to therapies typically achieved in [location], the effectiveness of the therapy is greater than 70, 80, 90, or 95%. This means that it will not be reduced. For example, the progression-free survival period of the subject or the disease or By determining the duration of treatment for symptoms, or the overall survival of the subject, and / or the disease or condition This can be evaluated by determining the reduction of one or more of the symptoms.

[0262] In one example, the infection is cured completely or substantially completely. In another example, the infection is low Reduced (for example, determined by infectious disease markers or symptoms, at least 80, 90, or 95%). Markers are, for example, after the method is performed, for example, when the method is performed. Within 24 hours, for example, 1 to 12 hours or 1 to 24 hours after implementing this method, or In elephants, RNA or RNA-coding DNA is administered to program nucleases. One to twelve hours or one to twenty-four hours later, the first seed per 1 ml of blood sample taken from the patient Alternatively, it may be a CFU of the bacterial strain. For example, RNA is a guide RNA and a nuclease. This is Cas (e.g., Cas3 or Cas9). Reduction is performed by taking samples from the subject immediately before the start of this method. The sample may be compared with a stool sample, saliva sample, or urine sample. That's good too.

[0263] For example, the present invention increases the overall survival rate of human subjects (those who have the same cancer and undergo the same cancer treatment). Therapeutic treatment (e.g., nivolumab, pembrolizumab, or other antibodies disclosed herein) (compared to the median overall survival rate of people who received the same immune checkpoint inhibitor) For example, any composition or other product of the present invention as described herein may be used in such therapeutic methods. Provided for use.

[0264] In this example, the method was performed on a human population, and the median overall survival rate for the population was the same for the same cancer. If you have been diagnosed with cancer and have received the same cancer therapy (e.g., nivolumab, pembrolizumab, or as described herein), Overall survival of humans who received the same immune checkpoint inhibitor (such as another antibody) The median rate is 120-250% (e.g., 150-200%). For example, any of the present inventions as described herein. A composition or other product is provided for use in such a therapeutic method.

[0265] "Pathogenic bacterial infections" are infectious diseases that threaten the health of the target individual, such as life-threatening infections. Yes. In this embodiment, pathogenic bacterial infections are infections requiring hospitalization or parenteral antimicrobial agents. It is a disease. The infection may be an acute bacterial infection, such as a systemic infection or a localized infection. Bacterial pathogens often cause infections in specific areas of the body. Others cause generalized infections. It is a generalist pathogen. Pathogenic bacterial infections are caused by symbiotic intestinal bacteria, etc., in this case, bacteria. Infections caused by symbiotic bacteria that do not immediately threaten health or cause life-threatening situations are treated differently. It is illuminating.

[0266] The infection (or its symptoms) may be any of the following: • Bacterial vaginosis: This is caused by bacteria that displace lactobacilli species that maintain a healthy vaginal microbiome. It is caused by the overgrowth of bacteria and alters the vaginal microbiome. • Bacterial meningitis: This is a bacterial inflammation of the meninges, that is, the protective membranes covering the brain and spinal cord. ru. • Bacterial pneumonia: This is a bacterial infection of the lungs. • Urinary tract infection: This is mainly caused by bacteria. Symptoms include severe and frequent Symptoms include a urge or sensation of urination, pain during urination, and cloudy urine. The main causative factor is Escher It is Ichia coli. The bacteria can ascend to the bladder or kidneys, causing cystitis and nephritis. There is. • Bacterial gastroenteritis: This is caused by pathogenic bacteria in the intestines. These pathogenic species These are usually different from the normally harmless bacteria of a healthy gut microbiota. However, different bacteria of the same species Some bacterial species can be pathogenic. • Bacterial skin infections: Examples of bacterial skin infections include the following: • Impetigo is a highly contagious bacterial skin infection commonly seen in children. It is caused by Staphylococcus aureus and Streptococcus pyogenes. Erysipelas is an acute streptococcal bacterial infection that spreads through the lymphatic system to deeper layers of the skin. Cellulitis is a diffuse inflammation of connective tissue involving severe inflammation of the dermis and subcutaneous layers of the skin. Cellulitis can be caused by a normal skin bacterial flora or by contagious contact. Typically, this includes open skin, cuts, blisters, cracked skin, insect stings, animal bites, burns, and surgical wounds. It occurs via wounds, intravenous drug injection sites, or intravenous catheter insertion sites. In most cases, The skin of the face or lower extremities is usually affected, but cellulitis can also occur in other tissues.

[0267] In this example, the first bacterium is Streptococcus, and the patient has a chest infection, cellulitis, or tonsil infection. The patient has adenitis. In this example, the primary bacterium is Enterococcus, and the patient has a bladder infection. Or the patient has sepsis. In this example, the first bacterium is Pseudomonas aeruginosa, and the patient The person is suffering from diarrhea. In this example, the first bacterium is E coli, and the patient is suffering from diarrhea. They are doing it. 2. The method described in paragraph 1, wherein the subject is cancer patients and the therapy is cancer therapy. 3. Therapies include hematopoietic stem cell transplants, chemotherapy agents, immune checkpoint inhibitors, and immune cells. Checkpoint agonists, or immune cell (e.g., T cells and / or NK cells) enhancers Administration of; adoptive cell therapy (e.g., CAR-T therapy); radiation; or surgery, as described in paragraph 2. method.

[0268] In this example, the therapy is immunotherapy. A suitable example of immunotherapy is adoptive cell therapy (e.g., CAR-T). Therapies), immune checkpoint inhibitors, immune checkpoint agonists, or immune cells ( For example, administration of T cell and / or NK cell enhancers. For example, ipilimumab (if (YERVOY®), tremelimumab, nivolumab (or OPDIVO®), pembrolili Zumab (or KEYTRUDA®), pidilizumab, BMS-936559, durvalumab, and Administration of antibodies selected from atezolizumab, etc., for anti-CTLA4, PD-1, PD-L1, PD-L2, LAG3, 0 X40, CD28, BTLA, CD137, CD27, HVEM, KIR, TIM-3, VISTA, ICOS, GITR, TIGIT, or This involves administration of SIRPa antibodies, or axicapbutagen siroyleucell (Yescarta®) or tisagen. CAR-T therapy such as Lecleucel (Kymriah®).

[0269] In this example, the immune enhancer is interleukin-2 (IL-2) or its fragments or deletions. Includes natural variants.

[0270] In this example, surgical procedures include the removal of necrotic or cancerous tissue.

[0271] In the example, chemotherapy includes the administration of platinum-containing chemotherapy drugs. In the example, chemotherapy is gefi This includes the administration of tinib.

[0272] In this example, the therapy involves cyclophosphamide, methotrexate, and 5-fluorouracil (C MF); or doxorubicin and cyclophosphamide (AC); docetaxel, doxorubicin, and cyclophosphamide (TAC); or doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD); or mustine, vincristine, procarbazine, and Prednisolone (MOPP); cyclophosphamide, doxorubicin, vincristine, and Prednisolone (CHOP); bleomycin, etoposide, and cisplatin (BEP); epirubic Syn, cisplatin, and 5-fluorouracil (ECF); or epirubicin, cisplatin, and capecitabine (ECX); methotrexate, vincristine, doxorubicin, and cis Platin (MVAC); cyclophosphamide, doxorubicin, and vincristine (CAV); or 5 - This includes administering fluorouracil, folinic acid, and oxaliplatin (FOLFOX). nothing.

[0273] In the example, the cancer is breast cancer, and the therapy involves administering CMF or AC. The diagnosis is Hodgkin lymphoma, and the treatment includes administering TAC, ABVD, or MOPP. In the example, the cancer is non-Hodgkin lymphoma, and the therapy includes administering CHOP. In this case, the cancer is germ cell carcinoma, and the therapy involves administering BEP. The patient has gastric cancer, and the treatment involves administering ECF or ECX. In another example, the cancer is bladder cancer. Yes, the therapy includes administering MVAC. In the example, the cancer is lung cancer, and the therapy is CA This includes administering V. In an example, the cancer is colorectal cancer, and the therapy involves administering FOLFOX. This includes doing so. 4. The therapy is an immune checkpoint inhibitor antibody, as described in Section 3.

[0274] The antibodies selected are, at the discretion of the patient, anti-CTLA4, PD-1, PD-L1, PD-L2, LAG3, 0X40, CD28, BTLA, and CD137. These are CD27, HVEM, KIR, TIM-3, VISTA, ICOS, GITR, TIGIT, or SIRPa antibodies. In the example, the antibody is an anti-PD-1 antibody. In the example, the antibody is a PD-L1 antibody. In the example, the antibody is an anti-C This is a TLA4 antibody. 5. Therapies include ipilimumab (or YERVOY®), tremelimumab, nivolumab (or OPD) IVO(trademark), pembrolizumab (or KEYTRUDA(trademark)), pidilizumab, BMS-936559, The person described in paragraph 3 is the one who receives an antibody selected from ulvalumab and atezolizumab. Law.

[0275] Optionally, the antibody (e.g., anti-PD-L1 antibody) may be an anti-CTLA4 antibody (e.g., ipilimumab or It is administered together with tremelimumab.

[0276] In this specification, anti-PD-1 antibodies include nivolumab, pembrolizumab, and pidilizumab. (pidillizumab), OPDIVO (registered trademark), KEYTRUDA (registered trademark), AMP-514, REGN2810, CT-011 The following are selected: BMS 936559, MPDL3280A, and AMP-224.

[0277] For example, in this specification, anti-CTLA4 antibodies are tremelimumab, YERVOY®, and ipi Selected from limumab.

[0278] In this example, the therapy involves administering an anti-KIR antibody, such as lirilumab.

[0279] In this example, checkpoint inhibitors include CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, and B7. An inhibitor of H3, B7H4, TIM3, KIR, or A2aR is selected. In certain embodiments, the immunosuppressant Checkpoint inhibitors are human programmed cell death 1 (PD-1) system-binding antagonists. In some embodiments, PD-1 system-binding antagonists are PD-1 binding antagonists, PD-L1 binding antagonists Selected from a group consisting of antagonists and PD-L2 binding antagonists. In some cases, a PD-1 binding antagonist is a PD-1 binding antagonist. PD-1-binding antagonists inhibit the binding of PD-1 to PD-L1 and / or PD-L2.

[0280] In some embodiments, the immune checkpoint inhibitor is also known as MEDI4736. durvalumab, also known as MPDL3280A, atezolizumab, or MSB00010118C It is a PD-L1 antagonist, such as avelumab, which is also known as a PD-L1 antagonist. In certain aspects, Immune checkpoint inhibitors are PD-L2 antagonists such as rHIgM12B7. So, immune checkpoint inhibitors are LAG-3 antagonists such as IMP321 or BMS-986016. Immune checkpoint inhibitors, such as PBF-509, target the adenosine A2a receptor (A2aR). It's fine to be a tagonist.

[0281] In some embodiments, the antibodies described herein (anti-PD-1 antibody, anti-PD-L1 antibody, or anti-PD-L2) are used. The antibody (or similar) further comprises a human or mouse constant region. In a further embodiment, the human constant region is The group is selected from IgG1, IgG2, IgG2, IgG3, and IgG4. In one embodiment, the human constant region is IgG1. In another embodiment, the mouse constant region is IgG1. The antibody is selected from the group consisting of IgG2A, IgG2B, and IgG3. In a more specific embodiment, the antibody It has reduced or minimal effects functionality. In further specific embodiments, Minimal effector function occurs in production in prokaryotic cells, CHO cells, Cos cells, or HEK cells. This is due to... Furthermore, in more specific forms, the minimum effect function is "no effect function" This is caused by an "Fc mutation" or deglycosylation.

[0282] For example, therapies include hematopoietic stem cell transplantation, for example, bone marrow transplantation (for patients with cancer, for example) (For example, if the patient has blood cancer or leukemia.)

[0283] For example, therapies include stem cell transplantation, skin transplantation, or organ transplantation, such as heart transplantation or liver transplantation. , including kidney transplants or lung transplants. 6. The therapy is tissue transplantation, organ transplantation, or cell transplantation, as described in paragraph 1 or 2. . 7. Treatment of bacterial infections shall be carried out simultaneously with administering therapy to the subject, as described in paragraphs 1 through 6. The method described in either of the above terms. 8. Treatment of bacterial infections shall be carried out immediately before administering therapy to the subject, as described in paragraphs 1 through 6. The method described in any one of the items.

[0284] For example, treatment of bacterial infections is performed before treating further diseases or conditions, 7, 6, 5, 4, 3, 2. Alternatively, it may be carried out within 1 day, or within 24, 12, 6, 5, 4, 3, 2, 1, or 0.5 hours. For example, treatment of bacterial infections is followed by treatment of further diseases or conditions in 7, 6, 5, 4, 3, and 2. Alternatively, it may be carried out within 1 day, or within 24, 12, 6, 5, 4, 3, 2, 1, or 0.5 hours. .

[0285] The treatment and application of therapies for infection may be carried out simultaneously or sequentially. 9. Treatment of bacterial infections shall be carried out immediately after the subject has received treatment, as described in paragraphs 1 through 6. The method described in either of the above terms. 10. The above method involves applying RNA (e.g., gRNA) to the target, or RNA used to express RNA in the target. The process includes administering nucleic acids that encode RNA, and the RNA complexes with a nuclease, and the nuclease The enzyme is programmed to cleave the target site of the first bacterium contained in the target, thereby The method according to any one of paragraphs 1 through 9, for killing the first bacterium.

[0286] RNA or nucleic acids can be administered, for example, orally, by intravenous injection, subcutaneous injection, or inhalation. It is administered to the target or patient. 11. The process includes administering a vector (e.g., a phage or plasmid) to a target. The term is one of the terms from 1 to 10, which encodes a programmable nuclease. Methods used.

[0287] Nucleases can be administered, for example, orally, by intravenous injection, subcutaneous injection, or inhalation. It is administered to the target or patient. 12. Programmable nucleases are the endogenous nucleases of the first cell (e.g., Cas The method according to any one of the items 1 to 10, wherein the nuclease is... 13. The effectiveness of therapy in the presence of programmed nucleases is compared to broad-spectrum antibiotics. The method according to any one of paragraphs 1 through 12, which is greater than the effectiveness of the therapy in the presence of the other substance.

[0288] In this example, greater effectiveness is measured by progression-free survival or the duration of treatment for the disease or symptoms. To determine and / or to determine the reduction of one or more symptoms of a disease or condition. This is evaluated by, for example, the disease or condition and the presence of a bacterial infection. The disease is treated with therapy and antibiotics (not by killing the nuclease of the first bacterium according to the present invention). This will be compared to similar decisions made in other patients. 14. The effectiveness of therapy in the presence of programmed nucleases is demonstrated by methicillin, ban. Comycin, linezolid, daptomycin, quinupristin, dalfopristin; Teiko Planin; Cephalosporin; Carbapenem; Fluoroquinolone; Aminoglycoside; Coris Chin; Erythromycin; Clindamycin; Beta-lactam; Macrolide; Amoxicillin Azithromycin; Penicillin; Ceftriaxone; Azithromycin; Ciprofloxa Syn; isoniazid (INH); rifampicin (RMP); amikacin; kanamycin; capreomycin Syn; Trimethoprim; Itrofurantoin; Cephalexin; Amoxicillin Select from phosphorus; metronidazole (MTZ); cefixime; tetracycline; and meropenem. Any of the following conditions, from item 1 to 13, is more effective than the treatment in the presence of the antibiotic. The method described in section [section number]. 15. The first bacteria are (i) methicillin, vancomycin, linezolid, daptomycin, Resistance to antibiotics selected from quinupristin, dalfopristin, and teicoplanin. (ii) Staphylococcus aureus; cephalosporins, carbapenems, fluoroquinolones Pseudomonas resistant to antibiotics selected from aminoglycosides and colistins aeuroginosa; (iii) Klebsiella species resistant to carbapenems; (iv) erythromycin, cri Ndamycin, beta-lactam, macrolide, amoxicillin, azithromycin, and Streptoccocus species resistant to antibiotics selected from penicillin; (v) ceftriaxo Sa is resistant to antibiotics selected from azithromycin and ciprofloxacin. (vi) Shigella species resistant to ciprofloxacin or azithromycin; (vii ) Isoniazid (INH), Rifampicin (RMP), Fluoroquinolone, Amikacin, Canamycetin Antibiotics selected based on resistance to syn, capreomycin, and azithromycin Mycobacterium tuberculosis is resistant to vancomycin; (viii) Enterococcus is resistant to vancomycin. s species; (ix) Entero resistant to antibiotics selected from cephalosporins and carbapenems Bacteriaceae species; (x) Trimethoprim, Itrofurantoin, Cephalexin, and Amoxicillin E coli (xi) metronidazole (MTZ), which is resistant to antibiotics selected from sisillin, Clostridium species resistant to oroquinolone or carbapenem; (xii) cefixime, ceph Resistance to antibiotics selected from triaxone, azithromycin, and tetracycline. Neisseria gonnorrhoea; (xiii) beta-lactam, meropenem, and carbapenem Acinetoebacter baumannii is resistant to antibiotics selected from (xiv) ciprofloxaminol; as well as (xiv) ciprofloxaminol. Selected from Campylobacter species resistant to xacin or azithromycin, from item 1 The method described in any one of paragraphs 14. 16. Treatment of infectious diseases in the target population includes vaginitis, meningitis, pneumonia, urinary tract infections, cystitis, and kidney infections. A condition selected from inflammation, gastroenteritis, skin infections, impetigo, erysipelas, dental infections, and cellulitis. The method described in any one of paragraphs 1 through 15 for treating or preventing a disease. 17. Treatment of infections shall be to treat or prevent the sepsis or sepsis of the subject, paragraphs 1 through 16. The method described in any one of the items.

[0289] In this example, the infection is a bloodborne infection. 18. Further diseases or conditions include cancer; autoimmune diseases or conditions; viral infections or GI duct diseases. The method described in any one of paragraphs 1 through 17, relating to a disease or condition. In this example, the cancer is metastatic. In this example, the cancer is melanoma. In this example, the cancer is melanoma. This is a solid tumor with repair defects or microsatellite instability. In this example, the cancer is NSCL. It is C. In the example, the cancer is HNSCC. In the example, the cancer is Hodgkin lymphoma. In the example, In one example, the cancer is urothelial carcinoma. In another example, the cancer is lung cancer. In yet another example, the cancer is head and neck cancer. In this example, the cancer is head cancer. In this example, the cancer is cervical cancer. In this example, the viral infection is HIV, CMV, or RSV infection. 19. The target is one or more strains or species of bacteria (second bacteria) that are different from the first strain or species. The genome of the second bacterium includes, and does not contain the target site, and the genome of the second bacterium is in the target, The second bacterium is not cleaved by programmed nucleases, and thereafter, in the patient, It survives in the presence of programmed nucleases, and the therapy is effective in the presence of a second bacterium. A method according to any one of paragraphs 1 through 18. 20. Reduction of secondary bacteria in patients (e.g., in the gut microbiome) indicates the effectiveness of the therapy. The method described in paragraph 19 relating to reduction.

[0290] Optionally, the therapy is effective in the presence of a second bacterium in the target gut.

[0291] Optionally, the first and / or second bacteria are present in the subject's intestine immediately before performing this method. ru.

[0292] Optionally, the first and / or second bacteria are present in the blood sample immediately before performing this method. do.

[0293] Optionally, immediately before performing this method, the first bacterium is present in the subject's blood, and the second bacterium is It is present in the target intestine.

[0294] Optionally, immediately before performing this method, the first bacterium is present in the target intestine, and the second bacterium is present in the target intestine. It is found in elephant blood.

[0295] The primary bacteria in the target blood are killed through optional selection.

[0296] By random selection, the bacteria are Gram-positive bacteria. By random selection, the bacteria are Gram-negative bacteria. be.

[0297] Optionally, both the first and second bacteria can be killed with the same antibiotic. Optionally, this method does not include the step of administering antibiotics. In the example, the antibiotic is Methicillin, vancomycin, linezolid, daptomycin, quinupristin, dalfo Pristin; Teicoplanin; Cephalosporin; Carbapenem; Fluoroquinolone; Amino Glycoside; colistin; erythromycin; clindamycin; beta-lactam; macrola Ido; Amoxicillin; Azithromycin; Penicillin; Ceftriaxone; Azithromycin n; ciprofloxacin; isoniazid (INH); rifampicin (RMP); amikacin; kanamy Syn; Capreomycin; Trimethoprim; Itrofurantoin; Cephalexin; Amoxi Select from cillin; metronidazole (MTZ); cefixime; tetracycline; and meropenem. Selected. For example, antibiotics include aminoglycosides, ampicillin, amoxicillin, and Moxicillin or clavulanic acid, carbapenems (e.g., imipenem), piperacillin or Tazobactam, quinolone (e.g., ciprofloxacin), tetracycline, chloramfen Enicol, ticarcillin, trimethoprim or sulfamethoxazole, penicillin, Selected from streptomycin, oxytetracycline, and enhanced sulfonamides. In the example, the first bacterium is aminoglycoside, ampicillin, amoxicillin, amoxi Clavulanic acid or clavulanic acid, carbapenems (e.g., imipenem), piperacillin or tazoba Kutam, quinolone (e.g., ciprofloxacin), tetracycline, chloramphenicol Calcium, trimethoprim or sulfamethoxazole, penicillin, streptococcus Antibiotics selected from ptomycin, oxytetracycline, and enhanced sulfonamides It is resistant to quality. In alternative forms, antibiotics include beta-lactams, fluoroquinolones, and It is selected from macrolides.

[0298] By arbitrary selection, the first and second bacteria are of the same species, but are different strains of that species. .

[0299] By arbitrary selection, the first and second bacteria belong to the same genus, but are different species within that genus. be.

[0300] By arbitrary selection, the first and second bacteria belong to the same family, but to different genera within that family. be.

[0301] By optional selection, the first and second bacteria are Gram-positive bacteria.

[0302] By arbitrary selection, the first and second bacteria are Gram-negative bacteria.

[0303] Optionally, the therapy is effective in the presence of a second bacterium.

[0304] In optional selection, a reduction in secondary bacteria in the patient is associated with a reduction in the effectiveness of the therapy. Reducing the secondary bacteria in the patient reduces the effectiveness of the therapy.

[0305] On an optional basis, the presence of a second bacterium in the patient is associated with enhanced therapeutic efficacy. The presence of a second bacterium in the patient enhances the effectiveness of the therapy. For example, the enhanced efficiency is due to the presence of a second bacterium. Compared to therapy in the absence or reduced presence of a second bacterium, such as in the presence of antibiotics that kill the bacteria. They are all effective.

[0306] In this example, the therapy is effective in the presence of the second bacterium and is effective against the target disease or condition (or its Symptoms are reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 95%. In the example, The law is effective in the presence of the second bacterium and does not affect the progression of the disease or condition (or its symptoms) in question. This is reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 95%. In the example, the therapy is It is effective in the presence of a second bacterium and prevents the disease or condition (or its symptoms) from progressing. The duration is reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 95%. For example, The law is effective in the presence of the second bacterium and does not affect the continuation of the disease or condition (or its symptoms). The duration is reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 95%. For example, The law is effective in the presence of the second bacterium and in the severity of the disease or condition (or its symptoms) in question. The degree is reduced by at least 20, 30, 40, 50, 60, 70, 80, 90, or 95%. For example, therapy It is effective in the presence of a second bacterium, and the patient's disease or condition (or its symptoms) is less severe. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 days or for at least 1, 2, 3, 4, 5, 6, or 12 months, at least 20, 30 The reduction is 40, 50, 60, 70, 80, 90, or 95%. For example, the therapy is performed when there is a second bacterium present. It is effective under the following conditions, and the patient's disease or condition (or its symptoms) is at least 1, 2, 3, 4, 5 , over a period of 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 days, or at least 1 , for a period of 2, 3, 4, 5, 6, or 12 months, at least 20, 30, 40, 50, 60, 70, 80 90% or 95% are treated. In some cases, the therapy is effective in the presence of a second bacterium, and the patient The disease or condition (or its symptoms) is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , over a period of 12, 13, 14, 21, or 28 days, or at least 1, 2, 3, 4, 5, 6, if For a period of 12 months, at least 20, 30, 40, 50, 60, 70, 80, 90, or 95% testing was conducted. It is impossible to obtain. 21. The second type of bacteria is Akkermansia, Alistipes, Bacteroides, Barnesiella, and Bifidobact. erium, Clostridium, Collinsella, Enterococcus, Fusobacterium, Lactobacillus, Pro pionibacterium, Ruminococcus, Segmented filamentous bacteria (SFB); from the group consisting of Veillonella, Prevotella, Escherichia, and Streptococcus bacteria The method described in paragraph 19 or 20, as selected.

[0307] In the example, a second bacterium that produces short-chain fatty acids (e.g., a butyrate-producing bacterium). Specific aspects So, bacterial species produce butyrates. For example, the second bacterium is Clostridiales. Clostridiales bacteria may be or may include bacteria that are substantially in spore form. In certain aspects, the second bacterium belongs to the families Ruminococcaceae, Christensenellaceae, and Clostridium. It belongs to the family Diaceae, or the family Coriobacteriacease. In some embodiments, Clostridiales (e.g.) For example, Clostridium bacteria include a first family and a second family. In some embodiments, the first family The families include Ruminococcaceae, Christensenellaceae, Clostridiaceae, and Coriobacteriaceae. Selected from a group consisting of ease, the second family is not identical to the first family. In the example, the second bacterium is , Faecalibacterium prausnitzii, Ruminococcus albus, Ruminococcus bromii, Ruminoc occus callidus, Ruminococcus flavefaciens, Ruminococcus champanellensis, Ruminoc occus faecis, Ruminococcus gauvreauii, Ruminococcus gnavus, Ruminococcus hanseni i, Ruminococcus hydrogenotrophicus, Ruminococcus lactaris, Ruminococcus luti, Ru minococcus obeum, Ruminococcus palustris, Ruminococcus pasteurii, Ruminococcus p roductus, Ruminococcus schinkii, Ruminococcus torques, Subdoligranulum variabile , Butyrivibrio fibrisolvens, Roseburia intestinalis, Anaerostipes caccae, Blauti a obeum, Eubacterium nodatum, or Eubacterium oxidoreducen In certain embodiments, the second bacterium is Faecalibacterium prausnitzii. The second bacterium is Firmicutes.

[0308] In a particular embodiment, the first bacterium is Bacteroidia or Prevotellaceae, for example, Ba They are either cteroidetes or Bacteroides.

[0309] In embodiments, the treatment targets the microbiome (e.g., the gut microbiome and It brings about or maintains the (biome / or blood microbiome), and it is an immune checkpoint It is beneficial for inhibition or other therapies. For example, the microbiome is the phylum Firmicutes, Clos. Class tridia, order Clostridiales, family Ruminococcaceae, genus Ruminococcus, Hydrogenoanaerobac genus terium, genus Faecalibacterium, phylum Actinobacteria, class Coriobacteriia, Coriaobacteria Order les, Family Coriobacteriaceae, Domain Archaea, Phylum Cyanobacteria, Euryarchaeo Includes one or more bacterial species with high relative abundance derived from the phylum ta or the family Christensenellaceae. In addition to or instead of that, the microbiome includes the genus Dialister, Veillonellace Derived from the family Ae, phylum Bacteroidetes, class Bacteroida, order Bacteroidales, or family Prevotellaceae. It contains bacteria in low relative abundance. Therefore, the preferred microbial profile is Firmicut es, class Clostridia, order Clostridiales, family Ruminococcaceae (famiy), genus Ruminococcus, H Genus ydrogenoanaerobacterium, phylum Actinobacteria, class Coriobacteria, Coriaobacteriales Order, Family Coriobacteriaceae, Domain Archaea, Phylum Cyanobacteria, Phylum Euryarchaeota, or Having one or more bacterial species of the family Christensenellaceae in a higher relative abundance. It becomes and / or Dialister genus, Veillonellaceae family, Bacteroidetes phylum, Bacteroida order, One or more of the reduced abundances derived from the order Bacteroidales and / or the family Prevotellaceae It contains a species of fungus.

[0310] For example, the microbiome is composed of Bacteroidetes, Bactero Ida, Bactero Idales, or Pr Compared to evotellaceae, it contains Firmicutes with higher relative abundances. For example, microbi Ohm is compared to Bacteroidetes, Bacteroida, Bacteroidales, and Prevotellaceae. , including Firmicutes with higher relative abundance.

[0311] The second bacterium, chosen at random, is Akkermansia muciniphila;Alistipes shahii;Bacteroides fragilis;Bacteroides uniformis;Barnesiella intestinihominis;Bacteroides dorei;Bi fidobacterium adolescentis;Bifidobacterium brief;Bifidobacterium longum;Clostrid ium orbiscindens;Clostridium novii;Clostridium perfringens;Collinsella aerofacie ns;Enterococcus hirae;Fusobacterium nucleatum;Lactobacillus casei Shirota;L. cas ei AO47;Lactobacillus rhamnosus;Propionibacterium granulosum;Ruminococcus gnavus ;Segmented filamentous bacterium (SFB);Veillonella;Lactobacilli;Bacteroids;Clost ridia;Prevotella;E. coli Nissle;Lactobacillus plantarum;Lactobacillus delbruecki i(Bulgaricus species);Lactobacillus paracasei;Lactobacillus acidophilus;Bifi dobacterium infantis; and Streptococcus salivarius (also called Thermophilus). The microbiome in cancer immunotherapy: Diagnostic tools and therapeutic strategies; Lawrence Zitvogel; 382 , 1366~1370; DOI:10.1126 / science.aar6918.

[0312] In this example, the second bacterium is a symbiotic bacterium in humans.

[0313] In the example, the first bacterium is found in the gut microbiota, skin microbiota, oral microbiota, throat microbiota, and hair microbiota. Biological flora, axillary flora, vaginal flora, rectal flora, anal flora, ocular flora, nasal flora Microbiota, tongue microbiota, lung microbiota, liver microbiota, kidney microbiota, reproductive microbiota, penile microbiota Microbiota, scrotal microbiota, mammary microbiota, ear microbiota, urethral microbiota, labia microbiota, organs It is contained in the microbial flora, or dental microbial flora.

[0314] In the example, the second type of bacteria is found in the gut microbiota, skin microbiota, oral microbiota, throat microbiota, and hair microbiota. Biological flora, axillary flora, vaginal flora, rectal flora, anal flora, ocular flora, nasal flora Microbiota, tongue microbiota, lung microbiota, liver microbiota, kidney microbiota, reproductive microbiota, penile microbiota Microbiota, scrotal microbiota, mammary microbiota, ear microbiota, urethral microbiota, labia microbiota, organs It is contained in the microbial flora, or dental microbial flora.

[0315] In this example, the first and / or second bacteria are bloodborne bacteria. 22. The first bacteria are Staphylococcus, Streptococcus, Enterococcus, Helicobacter, L egionella, Heamophilus, Ghonnorhea, Acinetobacter, Escherichia, Klebsiella, Pseu Selected from the group consisting of bacteria domonas or Stenotrophomonas, any of items 1 to 21 The method described in any one of the items.

[0316] H pylori has been suggested to be involved in gastric cancer and gastric ulcers. Therefore, in this example, The first bacterium is H pylori, and the disease is, at will, cancer such as stomach cancer. Therapy is either chemotherapy or therapy with immune checkpoint inhibitors (e.g., antibodies). In this example, the first bacterium is H pylori, and the disease is a gastric ulcer. In this embodiment, three gastric ulcers Drug therapy is administered to the patient.

[0317] In this example, the first bacterium is a Gram-negative bacterium, selected randomly, and the infection is a bloodborne infection. In the example, the first bacterium was selected from E. coli, P. aeruginosa, and K. pneumoniae. By choice, the infectious disease is a bloodborne infection. 23. The first bacteria are E coli (e.g., EHEC E coli), C dificile, V cholera, and Staphylococcus. Coccus (e.g., S. aureus or MRSA), Streptococcus pyogenes, Helicobacter pylori, A cinetobacter baumannii, Legionella, Pseudomonas aeruginosa, and Klebsiella pneum The method described in item 22, selected from the group consisting of oniae bacteria.

[0318] In the example, the subject is either receiving immunosuppressant drugs or immunosuppressant drugs, for example The patient is currently undergoing treatment with corticosteroids or other steroids. 24. Programmable for use in the method described in any one of paragraphs 1 through 23 Nuclease. 25. Nucleases include Cas nucleases (e.g., Cas3 or Cas9), meganucleases, TALEN (Transcription Activator-like Effector Nuclease), or Zinc Finger Nuclease A nuclease, which is a method described in any one of paragraphs 1 to 24. 26. The nuclease is a Cas nuclease (e.g., Cas3 or Cas9), and the system is one or Each includes multiple guide RNAs (gRNAs) or DNA encoding one or more guide RNAs, The guide RNA cleaves the target site contained within the first bacterial genome as a Cas nucleus. The method described in any one of paragraphs 1 to 23, which makes it possible to program the -se A CRISPR / Cas system containing the nuclease described in paragraph 24 or 25 for use in [the specified application]. 27. Guide RNA or DNA encoding guide RNA for use in the system described in paragraph 26. 28. Methods for treating pathogenic bacterial infections as described in any one of paragraphs 1 through 23. A guide RNA or DNA encoding a guide RNA for use, wherein the guide RNA is a nucleus It is possible to program the rease, and the nuclease is Cas nuclease (for example) A guide RNA or guide (Cas9, Cas3, Cas13, CasX, CasY, or Cpf1 nuclease) DNA that codes for drug RNA. 29. Nucleic acid vectors containing guide RNA or DNA as described in any one of paragraphs 26 to 28. . 30. Nucleases as described in paragraph 24 or 25 and, optionally, guide RNA as described in paragraph 29. A nucleic acid vector that codes for this. 31. Vectors include phages, phagemids, and plasmids (e.g., conjugating plasmids). The vectors described in paragraph 29 or 30, which are transposons.

[0319] Phages are capable of infecting the first bacterium, and phagemids are helper phages. It is possible to produce such phages in the presence of other phages. 32. A first nucleic acid vector (or so) encoding the nuclease described in paragraph 24 or 25. (multiple of the above), and a second nucleic acid vector (or multiple thereof) encoding the guide RNA described in Section 29. A pharmaceutical composition comprising a pharmaceutically acceptable diluent, excipient, or carrier. A pharmaceutical composition. 33. CRISPR / Cas systems as described in Section 26 and pharmaceutically acceptable diluents, excipients, or carriers. A pharmaceutical composition containing the following: 34. Including the vectors described in Section 31 and pharmaceutically acceptable diluents, excipients, or carriers. A pharmaceutical composition.

[0320] Prevention of disease or condition as used herein means, for example, reducing the risk of disease or condition in a subject or patient. It may be reduced.

[0321] In an alternative form, the infection is caused by a first archaea instead of a first bacterium, and In this embodiment, instead, the features and other configurations of the method of the present invention relating to the killing of the first bacteria are described. All of this relates to the death of the first archaea, with the necessary modifications.

[0322] In this embodiment, the method is for treating acute microbial infections as described herein. The method includes the step of carrying out the method, and therefore the features of the method as described herein are disease This method for treating pathogenic bacterial infections (that is, pathogenic bacterial infections, in the first method) It can be combined with (in the case of acute microbial infection). In the embodiment, this method is specified herein. This includes the process of implementing methods for the sustained treatment of microbial infections as described above. Therefore, the features of the method described herein are for treating pathogenic bacterial infections. This method (i.e., when the pathogenic bacterial infection is a microbial infection in the first method) and combination It is possible to do so. Any of the optional features of the first method described herein can be modified as necessary. This method can be applied to the treatment of pathogenic bacterial infections.

[0323] manner Accordingly, the present invention provides the following embodiments, which are any selection of the above provisions. It is a sign. 1. The infection rate should be reduced to at least 1 / 100th within the first 30 minutes of performing process (b). The method described in any one of paragraphs 1 through 23. Optionally, the infectious disease is carried out in step (b). The risk is reduced to at least 1 / 1000th within the first 30 minutes after administration. Optionally, infection reduction is also possible. The reduction lasts for 30 minutes immediately after the first 30 minutes following the implementation of process (b). For example, reduction (i) in a sample (e.g., a blood sample) taken from the subject immediately before the start of this method and (ii) in the treatment The first type of sample (of the same type as the sample in (i), for example, a blood sample) collected from the subject at 30 minutes past the time. Alternatively, it can be evaluated by determining the difference in the number of bacteria in the strains. For example, the sample is, For example, plate the sample onto agar in a corresponding Petri dish and incubate under the same conditions. The difference in colony-forming units (CFU) / ml samples in the following case may also be evaluated. In another example, Microscopic counting of microorganisms in the sample or other routine methods known to those skilled in the art may be used. 2. The target bloodborne infection caused by the first bacterium should be minimal within the first 30 minutes of performing step (b). The amount is reduced to at least 1 / 100 or 1 / 1000, as specified in any one of paragraphs 1 through 23. Law. 3. Blood should be taken 10 minutes immediately before treatment. 5 ~10 12 (For example, 107 ~10 12 ) Infected with the first bacterium at CFU / ml The method according to embodiment 2. 4. The process includes administering nucleic acids (e.g., RNA) and nucleases as the target, wherein the nucleic acids are nucleases. It complexes with an enzyme and programs the nuclease to target the first bacterium contained within the target. The method according to any one of paragraphs 1 through 23, for cutting a target site. 5. The nuclease is administered to the target simultaneously with or sequentially with the nucleic acid, as described in Embodiment 4. Law. 6. The method according to embodiment 4, wherein the substance includes a nuclease before administering nucleic acids as the target. 7. Multiple phages are administered to the target, and each phage contains a copy of nucleic acid. This involves infecting the first bacteria contained in the target and delivering nucleic acid to it, according to any of embodiments 4 to 6. The method described in any one of the items. 8. Phages administered: The ratio of the first bacterium contained in each phage varies from 10 to 150 depending on the target, aspect 7 The method described above. For example, the ratio is 10 to 100, i.e., a multiple of infection (MOI) of 10 to 100.

[0324] The ratio is, for example, derived from a sample from a human or animal subject immediately before treatment (e.g., a blood sample or intestinal sample). The bacterial count can be determined by using the sample and determining the number of bacteria per 1 ml of blood or intestinal sample. Subsequently, the amount of phage to be administered is calculated according to the determination made based on the use of the sample. It is possible. 9. An infectious disease is an infection of the lungs, brain, skin, abdomen, or urinary tract, as described in paragraphs 1 through 23. The method described in either of the above terms. 10. The subjects are those who have undergone surgery, are receiving immunosuppressant drug treatment, or have burns. If you have diabetes, cancer, or any other chronic illness A person suffering from one of the conditions described in any one of paragraphs 1 through 23 or any one of the conditions described in any one of the descriptions 1 through 9. Law. 11. The subjects are individuals aged 65 or older, or pediatric patients, as described in paragraphs 1 through 23. The method described in any one of the items or any one of the embodiments 1 to 10. 12. Treat or prevent the target sepsis, any one of paragraphs 1 through 23 or aspect 1 The method described in any one of items 11 to 11. 13. At the start of treatment, the subject (e.g., human) should have a body temperature <36°C or >38°C and a heart rate >90 beats / min. Number of breaths, respiratory rate >20 breaths / min or PaCO2 <4.3kP, and <4000 / mm² 3 or >12,000 / mm 3 White blood cell count The method described in paragraph 12, having the following characteristics. 14. At the start of treatment, the subject (e.g., human) exhibits abnormal body temperature, abnormal heart rate, and abnormal respiratory rate. The first is characterized by the presence of two or more abnormal blood gases and abnormal white blood cell counts. The method described in paragraph 2 or paragraph 13.

[0325] Immune checkpoint modulation The immune checkpoint of the present invention adjusts the signal (e.g., a co-stimulatory molecule) upward or Alternatively, the signal may be adjusted downwards. In this invention, immune checkpoint mode Inhibitory immune checkpoint molecules that can be targeted by julation include: adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocytes Tenuator (BTLA), cytotoxic T lymphocyte-associated protein (CTLA-4, also known as CD152) (It is present), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KI) R), Lymphocyte Activation Gene-3 (LAG3), Programmed Cell Death 1 (PD-1), T Cell Immunoglobulin May The mucin domain 3 (TIM-3), and the V domain Ig suppressor (VISTA) for T cell activation. These include, in particular, immune checkpoint inhibitors that target the PD-1 system and / or CTLA-4. do.

[0326] Immune checkpoint inhibitors are small molecules, ligands, or recombinant receptors, or It may also be a drug such as a human antibody (for example, International Publication No. 2015016718 pamphlet) Pardoll, Nat Rev Cancer, Vol. 12 (No. 4): pp. 252-254, 2012. Both references are provided by the original source. (To be included in the detailed description). Known inhibition of immune checkpoint proteins or their analogues. Agents can be used, in particular by using the chimera, humanization, or human form of antibodies. This is possible. As those skilled in the art will know, alternative and / or equivalent names are available in this disclosure. It may be used in certain antibodies mentioned. Such alternative and / or equivalents The names can be used synonymously in the context of the present invention. For example, lambrolizumab It is publicly known that it is also known by the alternative and equivalent names MK-3475 and pembrolizumab. be.

[0327] There are no known immune checkpoint inhibitors in this field that stimulate the immune response. It is intended that all of these can be used. This directly targets antigen-specific T lymphocytes. or inhibitors that indirectly stimulate or enhance immune checkpoints. However, although not limited to them, immune checkpoints involved in PD-L2, LAG3, BTLA, B7H4, and TIM3 Examples include agents that target inhibitor proteins and pathways. LAG3 inhibitors include soluble LAG3 (IMP321, or International Publication No. 2009044273). (LAG3-Ig disclosed in) and mice or humanized antibodies that block human LAG3 (for example) If, then, IMP701) disclosed in International Publication No. 2008132601, or blocking human LAG3 Examples include fully human antibodies (such as those disclosed in European Patent No. 2320940). Another example This includes, but is not limited to, antibodies that block human BTLA interactions with their ligands. Provided by the use of an inhibitor against BTLA (as described in International Publication No. 2011014438). (As shown in 4C7, etc.) Another example is the relationship between human B7H4 and (International Publication No. 2013025779 pamphlet) (Disclosed in International Publication No. 2013067492) or soluble recombinant B7H4 Antibodies against morphology (such as those disclosed in U.S. Patent Application Publication No. 20120177645) This is provided by the use of an agent that neutralizes B7H4, including but not limited to these. An example is an antibody that neutralizes human B7-H3 (for example, in U.S. Patent Application Publication No. 20120294796, B Neutralizing B7-H3, including but not limited to MGA271 and its derivatives disclosed as RCA84D. It is provided by an agent that does this. Yet another example is an antibody that targets human TIM3 (e.g., international Disclosed in Pamphlet No. 2013006490 A2, or Jones et al., J Exp The anti-human TIM3 blocking antibody F38-2E2) disclosed in Med. 2008; Vol. 205 (No. 12): pp. 2763-2779 is included. However, these are not limited to TIM3-targeting agents.

[0328] A. PD-1 series antagonist T cell dysfunction or anergy is caused by inhibitory receptors, programmed death 1 polypeptide (PD-1). Expression induction and maintenance occur simultaneously. Therefore, in this specification, PD-1 and programmed death Ligand 1 (PD-L1) and programmed-dead ligand 2 (PD-L2), among others, interact with PD-1. Therapeutic targeting of other molecules that signal signaling is provided. PD-L1 is overexpressed in many cancers. This is often associated with a poor prognosis (Okazaki T et al., Intern. Immun. 2007, Vol. 19). (Issue 7): p. 813). Therefore, in this specification, the modulation of the microbiome and combined An improved method for treating cancer by inhibiting PD-L1 / PD-1 interaction in combination has been provided. It can be done.

[0329] For example, PD-1 system-binding antagonists include PD-1 binding antagonists and PD-L1 binding antagonists. Examples include antagonists and PD-L2-binding antagonists. These are alternative names for "PD-1". Examples include CD279 and SLEB2. Alternative names for "PD-L1" include B7-H1, B7-4, and CD Examples include 274 and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD273. Examples include: In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and It is PD-L2.

[0330] In some embodiments, a PD-1 binding antagonist interacts with its ligand-binding partner. It is a molecule that inhibits the binding of PD-1. In a specific embodiment, the PD-1 ligand binding partner is , PD-L1 and / or PD-L2. In another embodiment, the PD-L1-binding antagonist is its binding This molecule inhibits the binding of PD-L1 to its binding partner. In a specific embodiment, it inhibits PD-L1 binding. The partner is PD-1 and / or B7-1. In another embodiment, a PD-L2-binding antagonist This is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific embodiment, The PD-L2 binding partner is PD-1. The antagonist is the antibody, its antigen-binding fragment. This may be an immunoadhensin, a fusion protein, or an oligopeptide. The antibodies are described in U.S. Patent No. 8735553, U.S. Patent No. 8354509, and U.S. Patent No. 8008. This is described in Specification No. 449. All of these references are incorporated herein by reference. Other PD-1 derivative antagonists for use in the manner provided herein are published in the U.S. Patent Publication No. U.S. Patent Application No. 20140294898, U.S. Patent Application No. 2014022021, and U.S. Patent Application Publication The documents described in Specification No. 20110008369, etc., are publicly known in the art. This is incorporated herein by reference.

[0331] In some embodiments, the PD-1 conjugated antagonist is an anti-PD-1 antibody {e.g., a human antibody, etc.} (A t-modified antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is nivolumab. Selected from the group consisting of pembrolizumab and CT-011. In some embodiments, PD-1 is used. Synthetic antagonists include immunoadhensins (e.g., in the constant region (e.g., immunoglobulin compounds) Immunoadhen containing extracellular or PD-1 binding moieties of PDL1 or PD-L2 fused to the Fc region of the column. Syn) is. In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolta The product is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO (registered trademark). It is a known anti-PD-1 antibody, described in international publication no. 2006 / 121168. Brolizumab is also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and S Also known as CH-900475, it is an anti-PD-1 virus described in International Publication No. 2009 / 114335. It is an antibody. CT-011 is also known as hBAT or hBAT-1, and is listed in International Publication No. 2009 / 101611. This is the anti-PD-1 antibody described in the pamphlet. AMP-224 is also known as B7-DCIg. This information is provided in International Publication No. 2010 / 027827 and International Publication No. 2011 / 066342. It is a PD-L2-Fc fusion soluble receptor. Additional PD-1 binding antagonists include CT-011 and Pidilizumab, also known as AMP-514, MEDI0680, and REGN2 810 is listed.

[0332] In some embodiments, the immune checkpoint inhibitor is also known as MEDI4736. durvalumab, also known as MPDL3280A, atezolizumab, or MSB00010118C It is a PD-L1 antagonist, such as avelumab, which is also known as a PD-L1 antagonist. In certain aspects, Immune checkpoint inhibitors are PD-L2 antagonists such as rHIgM12B7. Therefore, immune checkpoint inhibitors include, but are not limited to, IMP321 and BMS-986016. These are LAG-3 antagonists. Immune checkpoint inhibitors include adenoviruses such as PBF-509. It may also be a syn-A2a receptor (A2aR) antagonist.

[0333] In some embodiments, any antibody described herein (anti-PD-1 antibody, anti-PD-L1 antibody, or anti-PD-L1 antibody) may be used. The PD-L2 antibody (etc.) further comprises a human or mouse constant region. In a further embodiment, the human constant region The region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In one embodiment, the human constant region is IgG1. In another embodiment, the mouse constant region is Ig The group is selected from G1, IgG2A, IgG2B, and IgG3. In further specific embodiments, The antibody has reduced or minimal effector function. In further specific embodiments... The minimal effector function is due to production in prokaryotic cells. Furthermore, further specific states In this case, the minimum effect function is "no effect Fc mutation" or deglycosylation. This is due to glycosylation. Antibody glycosylation is typically either N-linked or O-linked. N-linking refers to the attachment of the carbohydrate portion to the side chain of an asparagine residue. (Tripeptide sequence) Asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) The anoic acid is a recognition sequence that enzymatically attaches the carbohydrate portion to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences in the polypeptide This creates potential glycosylation sites. O-linked glycosylation is linked to sugar N-acetylgal. One of the compounds, cutosamine (N-aceylgalactosamine), galactose, or xylose, is hydroxylated. The term "cyamino acid" most commonly refers to its attachment to serine or threonine, but it also refers to its attachment to 5-hydro Xyproline or 5-hydroxylysine may also be used. Glycosylation from antibodies The removal of the site is conveniently done by removing the aforementioned tripeptide sequence (for the N-linked glycosylation site). This is achieved by altering the amino acid sequence so that one is removed. The alteration is performed by glyc Replace an asparagine, serine, or threonine residue within the cosylation site with another amino acid residue. This may be done by (for example, glycine, alanine, or a conservative substitution).

[0334] The antibody or its antigen-binding fragment is prepared by a method known in the art, for example, the previously described anti-PD. - Encodes either L1, anti-PD-1, anti-PD-L2 antibody, or an antigen-binding fragment in a form suitable for expression. The process involves culturing host cells containing nucleic acids under conditions suitable for the production of such antibodies or fragments. It can be manufactured using a method that includes a step of recovering antibodies or fragments.

[0335] B. CTLA-4 Another immune checkpoint that can be targeted by the methods provided herein is CD This is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as 152. (Human CTL) The complete cDNA sequence of A-4 has Genbank acceptance number L15006. CTLA-4 is found on the surface of T cells. Furthermore, when it binds to CD80 or CD86 on the surface of antigen-presenting cells, it acts as an "off switch." CTLA4 is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. It is a member of the epidemic globulin superfamily. CTLA4 is a T cell costimulatory protein (CD). Similar to 28, both molecules are found on antigen-presenting cells, also known as CD, B7-1 and B7-2, respectively. It binds to 80 and CD86. CTLA4 sends inhibitory signals to T cells, while CD28 sends inhibitory signals to T cells. It transmits signal. Intracellular CTLA4 is also found in regulatory T cells and is important for their function. This may be necessary. T cell activation by T cell receptors and CD28 is inhibited by the B7 molecule. This leads to increased expression of CTLA-4.

[0336] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., human anti-CTLA-4 antibody). (body, humanized antibody, or chimeric antibody), its antigen-binding fragment, immunoadhensin, fusion antibody It is an protein or oligopeptide.

[0337] Suitable anti-human CTLA-4 antibodies (or VH and / or VL antibodies derived therefrom) for use in this method The main component can be generated using methods well known in the art. Alternatively, Anti-CTLA-4 antibodies recognized in this field can be used. For example, see the following references. The anti-CTLA-4 antibody disclosed herein can be used in the method disclosed herein: U.S. Patent No. 8,119,129, International Publication No. 01 / 14424, International Publication No. 98 / 4275 Pamphlet No. 2; International Publication No. 00 / 37504 (CP675,206; as tremelimumab) Also known as; formerly tisilimmab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998 Year. The teachings of each of the aforementioned publications are incorporated herein by reference. We also use antibodies that compete for binding to CTLA-4 with any of the antibodies recognized in the field. This is possible. For example, humanized CTLA-4 antibody is described in International Publication No. 2001014424, International This is described in Publication No. 2000037504 and U.S. Patent No. 8017114. All of these references are incorporated herein by reference.

[0338] Exemplary anti-CTLA-4 antibodies include ipilimumab (10D1, MDX-010, MDX-101), and Yervoy (Registered Trademark). (also known as the standard) or its antigen-binding fragments and variants (e.g., international public disclosure) (See Brochure No. 01 / 14424). In other embodiments, the antibody is the heavy chain of ipilimumab and It includes a light chain CDR or VR. Therefore, in one embodiment, the antibody is the VH region of ipilimumab. The CDR1, CDR2, and CDR3 domains of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab Contains . In another embodiment, the antibody is the same epinephrine of CTLA-4 as the antibody mentioned above. Competing for and / or binding to the tope. In another embodiment, the antibody is as mentioned above. The antibody has at least approximately 90% variable region amino acid sequence identity (for example, ipilimumab and at least It also has variable region identity of approximately 90%, 95%, or 99%.

[0339] Other molecules for modulating CTLA-4 are all incorporated herein by reference. This includes U.S. Patent No. 5844905, U.S. Patent No. 5885796, and International Publication No. 19 The following may be found in Pamphlet No. 95001994 and International Publication No. 1998042752, etc. Soluble CTLA-4 ligands and receptors, as incorporated herein by reference, U.S. 83 Examples of immunoadhensins include those described in Specification No. 29867.

[0340] C. Killer immunoglobulin-like receptor (KIR) Another immune checkpoint inhibitor for use in this invention is an anti-KIR antibody. Anti-human KIR antibodies (or VH and / or VL domains derived therefrom) suitable for use in law This can be generated using methods well known in the art.

[0341] Alternatively, anti-KIR antibodies recognized in this technology can be used. Antibodies may cross-react with multiple inhibitory KIR receptors, and one or more of these receptors may cross-react. It enhances the cytotoxicity of NK cells that maintain their numbers. For example, anti-KIR antibodies include KIR2D2DL1 and KIR2DL 2, and KIR2DL3, which bind to each of these KIRs, and NK cells mediated by any or all of these KIRs By reducing, neutralizing, and / or reversing cytotoxic inhibition, NK cell activity is enhanced. This is possible. In some embodiments, the anti-KIR antibody does not bind to KIR2DS4 and / or KIR2DS3. For example, monoclonal antibody 1-7F9 (IPH21) described in International Publication No. 2006 / 003179 You can use 01, 14F1, 1-6F1, and 1-6F5. The instructions are incorporated herein by reference. Such antibodies recognized in the art Antibodies that compete for binding to KIR with any of the others can also be used. Additional anti-KIR antibodies recognized in the art include, for example, International Publication No. 2005 / Pamphlet No. 003168, International Publication No. 2005 / 009465, International Publication No. 2006 / 072625 Pamphlet No. 2006 / 072626, Pamphlet No. 2007 / 042573 Fret, International Publication No. 2008 / 084106 pamphlet, International Publication No. 2010 / 065939 pamphlet See also International Publication No. 2012 / 071411 and International Publication No. 2012 / 160448. Examples of those disclosed include:

[0342] An exemplary anti-KIR antibody is lirirumab (also known as BMS-986015 or IPH2102). Other In this embodiment, the anti-KIR antibody is used to determine the heavy and light chain complementarity-determining regions (CDRs) or variable regions of lirilumab. The region (VR) is included. Therefore, in one embodiment, the antibody is the heavy chain variable (VH) region of lirilumab. CDR1, CDR2, and CDR3 domains, as well as CDR1, CDR2, and light chain variable (VL) region of lirilumab It contains the CDR3 domain. In another embodiment, the antibody contains lirilumab and at least about 90% It possesses amino acid sequence identity in the variant region.

[0343] Examples of cancers targeted for treatment include: lung cancer, head and neck cancer. Cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, digestive Tubocarcinoma, lymphoma, preneoplastic lesions of the lung, colon cancer, melanoma, metastatic melanoma, basal cell carcinoma Cancer, squamous cell carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, Kaposi's sarcoma, keratitis Toacanthoma, spindle cell tumor, sebaceous carcinoma, microcystic adnexal carcinoma, Paget's disease of the breast, atypical Fibroxanthomas, leiomyosarcomas, and angiosarcomas, lentigo malignant, lentigo malignant melanoma, and superficial spreading melanoma. Tumors, nodular melanoma, acral lentiginous melanoma, adhesive melanoma, and bladder cancer.

[0344] In some embodiments, the subject is resistant to one or more anticancer therapies. (It has been demonstrated that) the patient has cancer. In some embodiments, resistance to anticancer therapy is This includes recurrence of cancer or refractory cancer. Recurrence is cancer at the original site or a new site after treatment. In some embodiments, resistance to anticancer therapy may refer to the reappearance of [unclear]. This includes the progression of cancer during treatment by the law. In some embodiments, the cancer is in the early or late stages. This is the stage. The target is those that express the PD-Ll biomarker (for example, those that express it in diagnostic tests). The patient may have cancer (as shown). In some embodiments, the patient's cancer is low PD-L It expresses a biomarker. In some embodiments, the patient's cancer is high in PD-Ll biomarkers. - expresses. PD-Ll biomarkers are detected by FACS, Western blot, ELISA, immunoprecipitation, Immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, Surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-q PCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY techniques, and It can be detected in the sample using FISH and methods selected from combinations thereof. ru.

[0345] In some embodiments, the cancer has a low level of T cell infiltration. In some embodiments, The cancer does not have detectable T cell infiltration. In some embodiments, the cancer is non-immunogenic. (For example, non-immunogenic colorectal cancer and / or ovarian cancer.)

[0346] For example, therapeutically effective or sufficient amounts of immune checkpoint inhibitors, such as antibodies, can be used in humans. Administered, whether in one dose or multiple doses, at a dose of approximately 0.01 to approximately 50 mg / kg patient body weight It would be within a certain range. In some embodiments, the antibody used is, for example, about 0.01 to about 45 mg / day kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, Approximately 0.01 to 20 mg / kg, approximately 0.01 to 15 mg / kg, approximately 0.01 to 10 mg / kg, approximately 0.01 to 5 mg / kg, or 0 The antibody is administered at approximately 0.01 to 1 mg / kg. In some embodiments, the antibody is administered at 15 mg / kg. However, However, other drug regimens may be useful. In one embodiment, the anti- PD-L1 antibody is administered at approximately 100 mg, 200 mg, 300 mg, 400 mg, and 500 mg doses on day 1 of a 21-day cycle. g, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, Alternatively, it is administered to humans in doses of approximately 1400 mg. The dose can be administered as a single dose or in multiple doses, such as by infusion. It may be administered as (for example, 2 or 3 doses). The progression of this therapy is easily facilitated by conventional techniques. It will be monitored.

[0347] Anti-cancer therapy and other therapies In some embodiments, immune checkpoint inhibitors are used as at least one additional therapeutic agent. It may be administered in combination with other therapies. Additional therapies include radiation therapy, surgery, chemotherapy, and genetic therapy. DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal Cancer therapies such as nal antibody therapy or a combination of the above may also be used. Additional therapies may include A This may take the form of adjuvant therapy or neoadjuvant therapy.

[0348] For example, cancer (regardless of whether or not immune checkpoint inhibitors are administered) Therapy for (not present) or any other disease (e.g., viral infection or autoimmune disease) is radiation Treatment, surgery, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, Bone marrow transplantation, nanotherapy, or monoclonal antibody therapy may be used. The therapy is as described above. A combination of these may be used. Additional therapies may be administered.

[0349] In some embodiments, the therapy (or additional cancer treatment) involves small molecule enzyme inhibitors or anti-metastatic agents. This is an administration. In some embodiments, additional therapy is an agonist that limits side effects (e.g., anti- (Antiviral agents, such as nausea-inducing agents, that are intended to reduce the occurrence and / or severity of side effects of treatment.) This is the administration of [the substance].

[0350] In some embodiments, the therapy (or additional cancer therapy) is radiotherapy. In some embodiments, the therapy (or additional cancer treatment) is surgical intervention. Additional cancer therapies are a combination of radiation therapy and surgery. In some embodiments, The therapy (or additional cancer therapy) is gamma ray irradiation. In some embodiments, the therapy (or additional Cancer treatments in Canada include therapies targeting the PBK / AKT / mTOR pathway, HSP90 inhibitors, and tubulin inhibitors. These are agents, apoptosis inhibitors, and / or chemopreventive agents. Therapies (or additional cancer therapies) are It may be one or more chemotherapeutic agents known in the art.

[0351] Administration of any compound or therapy of this embodiment to a patient may, if applicable, result in toxicity of the agonist. Taking this into consideration, we will follow the general protocol for administering such compounds. Therefore, in some embodiments, there is a step to monitor toxicity resulting from combination therapy. ru.

[0352] The therapy may or may not include administering to any of the following: It's fine if you do that.

[0353] 1. Chemotherapy Various chemotherapeutic agents can be used according to this embodiment. "Chemical therapy" refers to the use of drugs to treat cancer. "Chemotherapy agents" are drugs administered to treat cancer. It is used to mean a compound or composition that acts in a certain way. Such agents or drugs are, for example, Regardless of the stage at which they affect the cell cycle, they are classified according to the mode of intracellular activity. Instead, the agonist either crosslinks directly with the DNA and intercalates into the DNA, or Based on its ability to induce chromosomal and genetic division abnormalities by affecting nucleic acid synthesis. It can be characterized.

[0354] Examples of chemotepas include: thiotepa and cyclosphospham Alkylating agents such as cyclosphosphamide; busulfan, improsulfan, and pip Alkyl sulfonates such as sulfan; benzodopa, carbocone, metsuredo Aziridines such as meturedopa and uredopa; altoretamine, triethyl Lenmelamine, triethylenephosphoramide, triethylenethiophosphoramide (triethiyl Contains enethiophosphoramide and trimethylolomelamine. Ethyleneimines and methylamelamines; acetylenins (especially, b Ratacin and bratacinone; Camptothecin (including the synthetic analog topotecan); Briosta Chin; Callistatin; CC-1065 (for example, its adzelesin, karzelesin, and biselesin) Synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dora Statins; duocalmycin (e.g., synthetic analogs, KW-2189 and CB1-TM1); erythrovitamin N; Pancratistatin; Sarcodictiin; Spongistatin; Chlorambucil, Chlorna Fasin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, Mechloretamine oxide hydrochloride, melphalan, nobuenvicin, phenesterine, pre Nitrogen masters such as donimustine, trophosphamide, and uracil mustard Do; carmustine, chlorozotosine, fotemustine, lomustine, nimustine, and lan Nitrosureas such as ranimnustine; engine antibiotics (for example, Calicheamicin, especially calicheamicin gamma II and calith Antibiotics such as amycin omega II (calicheamicin omegall); dynemicin A and other antibiotics Sewing machines; bisphosphonates such as chlordronate; Espera sewing machines; and neocalcinostomy Chromophore and related pigment proteins enediine antibiotic chromophore, acrasinomycin (acl acinomysins, actinomycin, authrarnycin, azacerin, bre Omycin, kakutinomycin, carabicin, carminomycin, cardino Phylin, chromomycinis, dactinomycin, daunorubicin, detox Rubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (e.g., morpholino-d Xorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and Oxidoxorubicin, Epirubicin, Esolubicin, Idarubicin, Marceloma Mitomycin C, mycophenolic acid, nogalarnycin ), olibomycin, peplomycin, potfiromycin, puromycin Icin, Queramycin, Rodorubicin, Streptonigrin, Strep Tozocin, tubercidine, ubenimex, dinostatin, and zolubicin; methotrexate Antimetabolites such as 5-fluorouracil (5-FU); denopterin, pteropterin, and Folic acid analogs such as trimethrexate; fludarabine, 6-mercaptopurine, thiamipri Purine analogs such as thioguanine; ancitabine, azacitidine, 6-azauridine Carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and pyrimidine analogs such as phloxuridine; carsterone, dromostan propionate Androgens such as lon, epithiostanol, mepithiostane, and testactone; Anti-adrenal agents such as galvanic acid and trilostane; folic acid supplements such as frolinic acid; acetaminophen Graton; Aldophosphamide glycoside; Aminolevulinic acid; Enyluracil; Amsacri N; Best Love Sil; Bisantrene; Edatraxate; Defofamin amine; demecoltin; diaziquan; elformithine; eriptinine acetate Mu; Epothiron; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Ronidynin (l onidainine; mytansinoids such as mytansin and anthamitosin; mitogazone; mi Toxanthrone; Mopidanmol; Nitraerine; Pentostat n; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethyl hydrazide; Procarbazine; PSK polysaccharide complex; Lazoxane; Rhizoxin; Schizophyllan; Spin Germanium; tenuazonic acid; triadiquane; 2,2',2''-trichlorotriethylamine; Trichothecenes (e.g., T-2 toxin, beracrine A, loridine A, and anguidin); urethanes Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobro Manganese; gacytosine; arabinoside ("ara C"); cyclophosphamide; taxois For example, paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercapto Purines; platinum-coordinate compounds such as cisplatin, oxaliplatin, and carboplatin; bins Blastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine Vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopter Phosphate; Xeloda; Ibandronate; Irinotecan (e.g., CPT-11); Topoisomela -ase inhibitor RFS2000; difluorometholornithine (DMFO) Retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, proxyco Plicomycin, gemcitabien, navelbine, farnesirtan tansferase inhibitors, trans platinum, and pharmaceutically acceptable salts, acids, is one of the above derivatives.

[0355] 2. Radiation therapy Other factors that cause DNA damage and are widely used include gamma rays, X-rays, and Examples include those known as and / or directed delivery of radioisotopes to tumor cells. Microwave, proton beam irradiation (US Patent No. 5,760,395 and No. 4,870,287) and Other forms of DNA damage factors, such as ultraviolet irradiation, are also being considered. All of these factors affect DNA. to the precursor of DNA, to DNA replication and repair, and to the construction and maintenance of chromosomes. It is most likely to affect widespread damage. The X-ray dose range is long-term (3-4 weeks) From a daily dose of 50-200 roentgens over a period of time, to a single dose of 2000-6000 roentgens. This is the range. The dose range of radioactive isotopes is wide and varied, and the half-life of the isotope and the amount of radiation emitted It depends on the intensity and type of radiation, as well as uptake by newly formed cells.

[0356] 3. Immunotherapy Those skilled in the art may use immunotherapy in combination with or in conjunction with the methods described herein. You will understand that it is possible. In the context of cancer treatment, immunotherapy agents are generally, It relies on the use of molecules to target and destroy immune effector cells and cancer cells. Rituximab (RITUXAN®) is an example of immunotherapy. An immune effector is, for example... Alternatively, the antibody may be specific to a marker on the surface of tumor cells. The antibody alone may be used as a therapeutic agent. It may also function as an effector, or it may actually kill other cells. They may be mobilized. Also, antibodies are drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, etc.) It may also be conjugated with (Lera toxin, pertussis toxin, etc.) and used as a targeted agonist. It may also serve the role of [another function]. Alternatively, the effector may directly or indirectly target tumor cells. Lymphocytes carrying surface molecules that interact via either contact or other means. Various F Examples of catalytic cells include cytotoxic T cells and NK cells.

[0357] For example, immunotherapy involves CAR-T therapy, such as anti-CD19 or CD20 CAR-T therapy, which involves adoptive cells. Includes therapy.

[0358] For example, immunotherapy involves IL-2 (e.g., cleaved IL-2 or pegylated IL-2 or F The treatment includes or consists of administering (c) fused IL-2.

[0359] Antibody-drug conjugates are emerging as a groundbreaking method in the development of cancer treatment agents. Body-drug conjugates (ADCs) are monoclonal cells covalently linked to a cell-killing drug. This method includes a multi-antibody (MAb). This technique utilizes the high specificity of MAb to the antigen target to produce very strong, finely detailed results. When combined with a cytotoxic agent, the payload (drug) is delivered to tumor cells with abundant levels of antigen. This brings the "armed" MAb to be delivered. Also, targeted delivery of the drug involves normal tissue coming into contact with it. This minimizes the risk of side effects, resulting in reduced toxicity and improved therapeutic index. Two ADC drugs, 2011 ADCETRIS (registered trademark) (brentuximab vedotin) in 2013 and KADCYLA (registered trademark) in 2013 With the FDA's approval of rastuzumab emtansine (T-DM1), this method has become available. It has been proven. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment. Yes, antibody engineering and linker-payload optimization are becoming increasingly mature, and new ADCs are emerging. The discovery and development of this method involves identifying and validating new targets suitable for this technique, as well as target-directed MAb It is becoming increasingly dependent on the generation of [this substance]. Two criteria for ADC targets are that their expression is upregulated in tumor cells. The key features are a high level of performance and robust internal migrations.

[0360] In one form of immunotherapy, tumor cells are easily targeted, meaning they are not present in the vast majority of other cells. It is necessary to maintain certain markers. There are many tumor markers, and they are In all cases, the conditions of this embodiment may be suitable for targeting. As a general tumor marker These include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, and sialyl Lewis antibody. Examples include the primordial, MucA, MucB, PLAP, laminin receptor, erb B, and pi55. These are alternatives to immunotherapy. The alternative approach involves combining the anti-cancer effect with the immunostimulatory effect. IL-2, IL-4, IL-12, G Cytokines such as M-CSF and gamma-IFN, chemokines such as MIP-1, MCP-1, and IL-8, and FLT3 There are also immunostimulatory molecules, including growth factors such as Gandr.

[0361] 4. Surgery Cancer or other diseases or conditions may be treated by surgical intervention in this invention.

[0362] Approximately 60% of individuals with cancer will undergo a certain type of surgery. Surgical options include prophylactic, diagnostic, or staging, curative, and palliative surgical procedures. As a curative surgical procedure, all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Destroyed, treatment of this embodiment, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy Examples include surgical excision, which can be used in conjunction with other therapies such as therapies and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, external Surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery. One example is laparoscopic surgery (Morse procedure).

[0363] When cancer cells, tissue, or part or all of a tumor are removed, a cavity is formed in the body. There is a possibility of this occurring. Treatment is achieved by local perfusion, direct injection, or topical application in addition to anticancer therapy. Such treatment may be performed, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or 1 Every 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months Repeat treatment is acceptable. Similarly, these treatments may involve varying dosages.

[0364] 5. Bacterial transplantation In the embodiment, the therapy involves bacterial transplantation, for example, fecal microbiota transplantation containing defined bacteria. This includes administering to elephants. For example, the transplants are disclosed in International Publication No. 2018064165. Any composition that is present and disclosed (in particular, the compositions within it) is conceivable in this invention. For application, the entirety is incorporated herein by reference. For example, the transplant is as follows: This is based on one of the paragraphs (table numbers and sequence numbers are from International Publication No. 2018064165). Refer to the table and arrangement of the labels. They are for the possible uses in the claims. (as expressly incorporated herein). 1. Ruminococcaceae, Clostridiaceae, Lachnospiraceae, Micrococcaceae, and / or at least one isolated or purified specimen belonging to one or more of the Veilonellaceae families A composition containing a population of bacteria. 2. Ruminococcaceae, Clostridiaceae, Lachnospiraceae, Micrococcaceae, and / or at least two isolated or purified species belonging to one or more families of Veilonellaceae A composition containing a population of bacteria. 3. Each of the bacterial populations present in the composition at a concentration of at least 10^3 CFU, paragraph 1 or The composition described in paragraph 2. 4. The composition described in paragraph 1 or 2, which is a live bacterial product or a biotherapy product. 5. At least one isolated or purified bacterial population or at least two isolated or purified Or the purified bacterial population is provided as bacterial spores, with the composition described in paragraph 1 or paragraph 2. thing. 6. A population of at least one bacterium or a collection of at least two isolated or purified bacteria. The group belongs to Clostridiales family XII and / or Clostridiales family XIII, as described in paragraph 1 or paragraph 2. The composition of the material. 7. At least one isolated or purified bacterial population or at least two isolated or purified The purified bacterial population belongs to the families Ruminococcaceae and / or Clostridiaceae. The composition described in paragraph 1 or paragraph 2. 8. The group of bacteria belonging to the family Ruminococcaceae is the group of bacteria belonging to the genus Ruminococcus. The compositions described in paragraph 1 or paragraph 2, which are further defined as having the following characteristics. 9. The group of bacteria belonging to the genus Ruminococcus is a collection of bacteria belonging to the species Ruminococcus bromii. The composition described in paragraph 8, which is further defined as a compound. 10. The group of bacteria belonging to the family Ruminococcaceae is a collection of bacteria belonging to the genus Faecalibacterium. The composition described in paragraph 1 or paragraph 2, which is further defined as a compound. 11. The group of bacteria belonging to the genus Faecalibacterium includes the species Faecalibacterium prausnitzii. The composition according to paragraph 10, further defined as a group of bacteria belonging to it. 12. The group of bacteria belonging to the family Micrococcaceae is the group of bacteria belonging to the genus Rothia. Further defined, the composition described in paragraph 1 or paragraph 2. 13. Porphyromonas pasteri sp., Clostridium hungatei sp., Phascolarctobacterium fa Further including a population of bacteria belonging to the species *ecium*, the genus *Peptoniphilus*, and / or the class *Mollicutes*, The composition described in paragraph 1 or paragraph 2. 14. Paragraph 1 or Paragraph 2 that does not essentially include a population of bacteria belonging to the order Bacteroidales. The composition described above. 15. At least one isolated or purified bacterial population or at least two isolated or purified bacteria Alternatively, the purified bacterial population has an enrichment index (ei) greater than 0.5 (Table 1-29) ) belonging to one or more species, subspecies, or bacterial strains selected from the group consisting of the species, paragraph 1 Or the composition described in paragraph 2. 16. At least one isolated or purified bacterial population or at least two isolated or purified bacteria Alternatively, is the purified bacterial population a group consisting of species from Table 1 (Tables 1-29) where "ei" is equal to 1? A composition selected from paragraph 1 or paragraph 2. 17. At least one isolated or purified bacterial population or at least two isolated or purified bacteria Alternatively, the purified bacterial populations are classified by NCBI taxonomy IDs: 717959, 587, 758823, 649756, 44749, 6 71218, 1264, 1122135, 853, 484018, 46503, 54565, 290052, 216931, 575978, 433321 ,1796646,213810,228924,290054,1509,1462919,29375,337097,1298596,487174 ,642492,1735,1297424,742766,46680,132925,411467,1318465,1852367,184185 7, 169679, 1175296, 259063, 172901, 39488, 57172, 28118, 166486, 28133, 1529, 69 4434, 1007096, 84030, 56774, 102148, 626947, 216933, 1348613, 1472417, 100176, 8 24, 1471761, 1297617, 288966, 1317125, 28197, 358743, 264639, 1265, 1335, 66219 ,69473,115117,341220,1732,873513,396504,1796619,45851,2741,105841,863 32, 1349822, 84037, 180311, 54291, 1217282, 762984, 1185412, 154046, 663278, 154 3、398512、69825、1841867、1535、1510、84026、1502、1619234、39497、1544、29343 、649762、332095、536633、1033731、574930、742818、177412、1121308、419208、1673 717、55779、28117、626937、180332、1776382、40519、34062、40518、74426、1216062 、293826、850、645466、474960、36835、115544、1515、88431、216932、1417852、3949 2、1583、420247、118967、169435、37658、138595、31971、100886、1197717、234908、 537007、319644、168384、915173、95159、1816678、626940、501571、1796620、888727 、1147123、376806、1274356、1267、39495、404403、1348、253314、258515、33033、11 18061、357276、214851、320502、217731、246787、29371、649764、901、29374、33043 、39778、682400、871665、160404、745368、408、1584、333367、47246、1096246、5334 2、438033、351091、1796622、1776384、817、48256、720554、500632、36849、301302、 879970、655811、264463、1532、285、995、242750、29539、1432052、622312、1796636 、1337051、328814、28446、1492、820、39496、52786、1549、1796618、582、46507、10 9327、1531、1382、33039、311460、230143、216935、539、35519、1681、328813、21485 3、89014、1121115、1585974、29466、1363、292800、270498、214856、142877、133926 、209880、179628、1121102、105612、1796615、39777、29353、1579、163665、53443、2 61299、1302、1150298、938289、358742、471875、938278、1796613、1118057、1077144 、1737、218205、1121298、684066、433659、52699、204516、706562、253257、328812、 1280、147802、58134、1335613、891、585394、1582、235931、308994、1589、1682、 1736、28129、178001、551788、2051、856、118562、101070、515619、40215、187979 、82979、29363、1776391、1285191、84112、157688、38304、36850、341694、287、7561 2、818、371674、338188、88164、588581、676965、546271、1236512、178338、862517、 157687、158、51048、1583331、529、888745、394340、40545、855、553973、938293、93 063、708634、179995、1351、476652、1464038、555088、237576、879566、1852371、742 727、1377、35830、997353、218538、83771、1605、28111、131109、46609、690567、462 06, 155615, 51616, 40542, 203, 294, 1034346, 156456, 80866, 554406, 796942, 1002 367, 29347, 796944, 61592, 487175, 1050201, 762948, 137732, 1211819, 1019, 27254 8, 1717, 384636, 216940, 2087, 45634, 466107, 1689, 47678, 575, 979627, 840, 166 0, 1236517, 617123, 546, 28135, 82171, 483, 501496, 99656, 1379, 84032, 39483, 1 107316, 584, 28124, 1033744, 657309, 536441, 76123, 1118060, 89152, 76122, 303, 1541, 507751, 515620, 38302, 53419, 726, 40324, 1796610, 988946, 1852370, 1017, 1168289, 76936, 94869, 1161098, 215580, 1125779, 327575, 549, 1450648, and 478 16S rRNA nucleotide sequences of bacteria identified by NCBI taxonomic IDs selected from a range of groups. and are at least 90% identical (for example, at least 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical). (i) a set of 16S ribosomal RNA (rRNA) nucleotide sequences as described in paragraph 1 or paragraph 2 A finished product. 18. At least one isolated or purified bacterial population or at least two isolated or purified bacteria Alternatively, the purified bacterial population is at least 80% identical to the sequences of sequence numbers 1-876 (for example, Species, subspecies, or bacterial fungi containing 16S rRNA gene sequences that are at least 85%, 90%, 95%, or 98% identical. The composition described in paragraph 1 or paragraph 2, which is a strain. 19. At least one isolated or purified bacterial population or at least two isolated or purified bacteria Alternatively, the purified bacterial population includes Bacteroides coagulans, Clostridium aldenense, Clos tridium aldrichii, Clostridium alkalicellulosi, Clostridium amygdalinum, Clostri dium asparagiforme, Clostridium cellulosi, Clostridium citroniae, Clostridium cl ariflavum DSM 19732, Clostridium clostridioforme, Clostridium colinum, Clostridi um fimetarium, Clostridium hiranonis, Clostridium hungatei, Clostridium hylemona e DSM 15053, Clostridium indolis, Clostridium lactatifermentans, Clostridium lep tum, Clostridium methylpentosum, Clostridium oroticum, Clostridium papyrosolvens DSM 2782, Clostridium populeti, Clostridium propionicum, Clostridium saccharoly ticum, Clostridium scindens, Clostridium sporosphaeroides, Clostridium stercorar ium, Clostridium straminisolvens, Clostridium sufflavum, Clostridium termitidis 、Clostridium thermosuccino genes、Clostridium viride、Clostridium xylanolyticum 、Desulphomaculum guttoideum、Eubacterium rectale ATCC 33656、Eubacterium dolic hum, Eubacterium eligens ATCC 27750, Eubacterium hallii, Eubacterium infirmum, E. Eubacterium siraeum, Eubacterium tenue, Ruminococcus torques, Acetanaerobacterium elongatum, Acetatifactor muris, Acetivibrio cellulolyticus, Acetivibrio ethanol gignens、Acholeplasma brassicae 0502、Acholeplasma parvum、Acholeplasma vituli、 Acinetobacter junii、Actinobacillus porcinus、Actinomyces bowdenii、Actinomyces dentalis、Actinomyces odontolyticus、Acutalibacter muris、Aerococcus viridans、A Eromicrobium fastidiosum, Alistipes finegoldii, Alistipes obesi, Alistipes onder donkii、Alistipes putredinis、Alistipes shahii、Alistipes shahii WAL 8301、Alist ipes timonensis JC136、Alkalibacter saccharofermentans、Alkaliphilus metalliredi gens QYMF, Allisonella histaminiformans, Allobaculum stercoricanis DSM 13633, Al loprevotella rava、Alloprevotella tannerae、Anaerobacterium chartisolvens、Anaer Obiospirillum thomasii, Anaerobium acetethylicum, Anaerococcus octavius ​​NCTC 981 0、Anaerococcus provenciensis、Anaerococcus vaginalis ATCC 51170、Anaerocolumna jejuensis, Anaerofilum agile, Anaerofustis stercorihominis, Anaeroglobus geminat us、Anaeromassilibacillus senegalensis、Anaeroplasma abactoclasticum、Anaerorhab dus furcosa, Anaerosporobacter mobilis, Anaerostipes butyraticus, Anaerostipes c accae, Anaerostipes hadrus, Anaerotruncus colihominis, Anaerovorax odorimutans, Anoxybacillus rupiensis、Aquabacterium limnoticum、Arcobacter butzleri、Arthrosp Ira platensis, Asaccharobacter celatus, Atopobium parvulum, Bacteroides caccae, Bacteroides caecimuris, Bacteroides cellulosilyticus, Bacteroides clarus YIT 120 56、Bacteroides dorei、Bacteroides eggerthii、Bacteroides finegoldii、Bacteroides s fragilis, Bacteroides gallinarum, Bacteroides massiliensis, Bacteroides oleici plenus YIT 12058, Bacteroides plebeius DSM 17135, Bacteroides rodentium JCM 1649 6、Bacteroides thetaiotaomicron、Bacteroides uniformis、Bacteroides xylanisolven s XB1A, Bacteroides xylanolyticus, Barnesiella intestini hominis, Bedouin massili ensis, Bifidobacterium bifidum, Bifidobacterium dentium, Bifidobacterium longum subsp. infantis, Blautia caecimuris, Blautia coccoides, Blautia faecis, Blautia glucerasea、Blautia hansenii DSM 20583、Blautia hydrogenotrophica、Blautia luti 、Blautia luti DSM 14534、Blautia wexlerae DSM 19850、Budvicia aquatica、Butyric icoccus pullicaecorum, Butyricimonas paravirosa, Butyrivibrio crossotus, Caldico probacter oshimai、Caloramator coolhaasii、Caloramator proteoclasticus、Calorama tor quimbayensis、Campylobacter gracilis、Campylobacter rectus、Campylobacter ur eolyticus DSM 20703、Capnocytophaga gingivalis、Capnocytophaga leadbetteri、Capn Ocytophaga sputigena, Casaltella massiliensis, Catabacter hongkongensis, Catenib acterium mitsuokai、Christensenella minuta、Christensenella timonensis、Chryseob Acterium taklimakanense, Citrobacter freundii, Cloacibacillus porcorum, Clostridium ioides difficile ATCC 9689 = DSM 1296、Clostridium amylolyticum、Clostridium bow manii, Clostridium butyricum, Clostridium cadaveris, Clostridium colicanis, Clos tridium gasigenes, Clostridium lentocellum DSM 5427, Clostridium oceanicum, Clos tridium oryzae, Clostridium paraputrificum, Clostridium pascui, Clostridium perf ringens、C...

Claims

1. Programmable NUCs for use in methods to treat target acute microbial infections Rease, wherein the microbial infection is caused by a first species or strain of microorganism. The nuclease cuts the target site contained in the genome of the microorganism that infected the target. It is programmable to interrupt the process, thereby killing the first species or strain of microorganism. The growth or proliferation of the microorganism is reduced, and the treatment method is performed on the subject before The step of bringing the marked target site into contact with the nuclease which is programmed to cut the marked target site. Including, thereby the genome of the microorganism contained by the subject is cut, and the subject An acute microbial infection is treated, and the method is used to eliminate the infection within the first 30 minutes of treatment. A programmable nuclease that includes a step to reduce the amount by at least 1 / 100th.

2. Programmable for use in methods for the sustained treatment of target microbial infections A nuclease wherein the microbial infection is caused by a first species or strain of microorganism. The nuclease is a target site contained in the genome of the microorganism that infected the target. It is programmable to cut, thereby allowing the first species or strain of microorganism to possess The microorganisms are subsequently killed, or their growth or proliferation is reduced, and the treatment method is the same as above. The object is brought into contact with the nuclease, which is programmed to cleave the target site. The process includes a step which causes the genome of the microorganism contained in the target to be cut. a programmable nuclease that provides continuous treatment for the aforementioned microbial infection.

3. The nuclease recognizes and / or cleaves the target site. The nucleic acid that programs is administered to the subject at a first time point (T1) and a second time point (T2), and T2 is The nuclease according to claim 2, which is at least 1 hour after T1.

4. The method reduces the infection to at least 1 / 100th within the first 30 minutes of the treatment. The nuclease according to claim 2 or 3, comprising the step of [doing something].

5. The method involves bringing the target into contact with the programmed nuclease, and then at least Claims 1 to 4 each include a step of maintaining a reduction of at least 1 / 100th of the infection for 60 minutes. A nuclease as described in any one of the following items.

6. The method described above ensures that the reduction of infection is sustained for 30 minutes immediately after the first 30 minutes of treatment. To that end, the Nuc according to any one of claims 1 to 5, which includes a step to reduce the infection. Lease.

7. The above method involves coating the target with RNA or RNA for expressing the RNA in the target. The process includes administering a nucleic acid, wherein the RNA complexes with the nuclease, and the RNA Program the crease to cleave the target site of the microorganisms contained in the subject. , the nuclease according to any one of claims 1 to 6.

8. a. The nuclease is administered to the target simultaneously with or sequentially to the RNA or nucleic acid. Administered, or b. The subject contains the nuclease before administering the RNA or nucleic acid to the subject. 、 The nuclease according to claim 7.

9. Multiple viruses are administered to the subject, and each virus contains a copy of the nucleic acid, The virus infects the microorganisms contained in the aforementioned target and transmits the nucleic acid to the microorganisms. The nuclease according to claim 7 or 8, which reaches the target.

10. Virus administered: The ratio of microorganisms contained in the subject is 10 to 150, claim 9. The nuclease described in [reference].

11. The subject is a human or animal, and the infectious disease is an infection of the lungs, abdomen, or urinary tract. Yes, or the subject has undergone surgery and is under immunosuppressant drug therapy, Alternatively, the nuclease according to any one of claims 1 to 10, which is used by individuals suffering from a chronic disease. 。

12. The nuclease is caused by the first species or strain of bacteria (first bacterium) This is used in a method for treating pathogenic bacterial infections in elephants, and the method is the same as By cleaving the target site contained in the genome of the first bacterium, the target contained in the subject The process includes a step of selectively killing a first bacterium, wherein the cutting cuts the target site. This is carried out using the programmable nuclease which is programmed to do so, The subject is suffering from a disease or condition other than the aforementioned pathogenic bacterial infection, and the method , including the step of administering therapy to the subject in order to treat or prevent the further disease or condition, The nuclease treats the infection, and the therapy is the programmed nuclease Any of claims 1 to 11, which is effective in treating or preventing the aforementioned disease or condition in the presence of -se. The nuclease described in item 1.

13. (i) The subject is a cancer patient, and the therapy is a hematopoietic stem cell transplant, a chemotherapy agent, an immunotherapy agent. Epidemic checkpoint inhibitors, immune checkpoint agonists, or immune cell enhancers (ii) administration of a sensor; adoptive cell therapy; radiation; or surgery, or (ii) the said therapy is combined The nuclease according to claim 12, wherein the transplant is a tissue transplant, organ transplant, or cell transplant.

14. The aforementioned treatment for the aforementioned infections includes the aforementioned vaginitis, meningitis, pneumonia, urinary tract infections, cystitis, and nephritis. A condition selected from gastroenteritis, skin infections, impetigo, erysipelas, cellulitis, sepsis, or sepsis. Nucleo according to any one of claims 1 to 13, for treating or preventing the condition in the subject. Arze.

15. The aforementioned infection lasts for one hour or longer, and is optionally treated during the first three treatments. By 0 minutes (optionally, by the first 15 minutes), it is reduced by at least 90%, as per claim 1. A nuclease as described in any one of items 14.

16. The method described above includes, within the first 30 minutes (optionally, within the first 15 minutes) of the treatment, The process includes reducing the infection to at least 1 / 100th of the original amount. The subject is brought into contact with the programmed nuclease for at least 60 minutes. A nuclease according to any one of claims 1 to 15, which is maintained over time.

17. The method described above treats or prevents sepsis and / or sepsis in the subject, and the subject is The subject has a body temperature of <36°C or >38°C; a heart rate of >90 beats / minute; Respiratory rate >20 breaths / min or PaCO2 <4.3kP 2 ; and <4000 / mm 3 or >12,000 / mm 3 The white blood cell count The nuclease according to any one of claims 1 to 16.

18. At the start of the aforementioned treatment, the subject exhibits abnormal body temperature, abnormal heart rate, abnormal respiratory rate, and abnormal The blood gas and two or more abnormal white blood cell counts as described in claim 17. Nuclease.

19. The subject is a human or an animal, the microorganism is a bacterium, and the subject is the bacterium. The incidence of blood infection in elephants is reduced to at least 1 / 100 or 1 / 1000 within the first 30 minutes of the aforementioned treatment. A nuclease according to any one of claims 1 to 18, which is reduced.

20. The nuclease according to any one of claims 1 to 19, wherein the microorganism is a bacterium.

21. The aforementioned nucleases include Cas nucleases, meganucleases, and TALENs (transcription activators). Claims 1 to 20, which are effector nucleases or zinc finger nucleases. A nuclease as described in any one of the following items.

22. Used in the aforementioned treatment method together with the nuclease described in any one of claims 1 to 21. Multiple viruses, phages, or phagemids for producing phages Each virus, phage, or phagemid is defined in any one of claims 7 to 9. The virus or phage contains a copy of the nucleic acid, and the virus or phage contains the minute amount of the substance. Multiple viruses capable of infecting living organisms and delivering the nucleic acid to the microorganisms, Phage, or phagemid.

23. In the aforementioned treatment method, the nuclease described in any one of claims 1 to 21 is programmed A composition comprising multiple nucleic acids for the purpose of, wherein each nucleic acid is any one of claims 7 to 9 A composition that is a nucleic acid as defined by [the relevant regulations].

24. Nuclease according to any one of claims 1 to 21 for use in the aforementioned treatment method A CRISPR / Cas system comprising, wherein the nuclease is a Cas nuclease, and the system is , comprising one or more guide RNAs or DNA encoding one or more guide RNAs Each guide RNA cleaves the target site contained in the genome of the microorganism, as described in the Cas. The CRISPR / Cas system allows for the programming of nucleases.

25. Methods for treating the aforementioned acute microbial infections, and optionally sepsis or sepsis. A guide RNA or a guide RNA encoding for use in the system described in claim 24. DNA that is a guide RNA or DNA encoding a guide RNA, wherein the target is a human or an animal. 。

26. A nucleic acid vector comprising guide RNA or DNA as described in claim 24 or 25.

27. The aforementioned vectors include phages, phagemids, biiriophages, viruses, and plasmids. The vector according to claim 26, which is a transposon.

28. Antiseptic or anti-sepsis for administration to humans or animals to treat sepsis or septicemia A sepsis composition comprising a plurality of vectors, each vector being as described in claim 26 or 27 An antiseptic or anti-sepsis composition.

29. A method for treating a target acute microbial infection, any one of claims 1 to 28 The method as prescribed in the section.

30. Composition for carrying out the treatment method specified in any one of claims 1 to 29 Nucleases, multiple viruses according to any one of claims 1 to 27, in the manufacture of a product Use of phages, phagemids, systems, guide RNA, DNA, or vectors.

31. The nuclease recognizes and / or cleaves the target site. The nucleic acid that programs the substance is administered to the subject or substrate at the first time point (T1) and the second time point (T2). The method according to claim 29 or claim 30, wherein T2 is at least one hour after T1. use.

32. The infection is reduced within the first 30 minutes of treatment (optionally, within the first 15 minutes). The method or use according to any one of claims 29 to 31, which reduces both to 1 / 100th.

33. The reduction of the infection is achieved by bringing the subject into contact with the programmed nuclease, Maintained at least 1 / 100 for at least 60 minutes, any one of claims 29 to 32 The method or use described in paragraph 1.

34. The reduction in infection is said to last for 30 minutes immediately following the first 30 minutes of treatment, claim. The method or use described in any one of paragraphs 29 to 33.

35. The microorganism is either E. coli (optionally, EHEC E. coli) or C. difficle bacteria. Nucleases, viruses, phages, and phagemids listed in any one of the requirements 1 through 34 , system, guide RNA, DNA, vector, composition, method or use.

36. The aforementioned microorganisms are E. coli bacteria (optionally, EHEC E. coli), Klebsiella, or Pseudomonas bacteria. The bacterium, the subject is a human, and the infectious disease is a lung infection, according to claims 1 to 35. Nucleases, viruses, phages, phagemids, systems, or guide RNs listed in any one of the items. A, DNA, vectors, compositions, methods or uses.

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